extfieldsofstudy: Biology Medicine

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Biology
Agricultural And Food Sciences

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Vaccination of goats with a combination Salmonella vector expressing four Brucella antigens (BLS, PrpA, Omp19, and SOD) confers protection against Brucella abortus infection

Salmonella is an intracellular pathogen with a cellular infection mechanism similar to that of Brucella, making it a suitable choice for use in an anti-Brucella immune boost system. This study explores the efficacy of a Salmonella Typhimurium delivery-based combination vaccine for four heterologous Brucella antigens (Brucella lumazine synthase, proline racemase subunit A, outer-membrane protein 19, and Cu/Zn superoxide dismutase) targeting brucellosis in goats. We inoculated the attenuated Salmonella delivery-based vaccine combination subcutaneously at two different inoculation levels; 5 × 109 colony-forming unit (CFU)/mL (Group B) and 5 × 1010 CFU/mL (Group C) and challenged the inoculations with virulent Brucella abortus at 6 weeks post-immunization. Serum immunoglobulin G titers against individual antigens in Salmonella immunized goats (Group C) were significantly higher than those of the non-immunized goats (Group A) at 3 and 6 weeks after vaccination. Upon antigenic stimulation, interferon-γ from peripheral blood mononuclear cells was significantly elevated in Groups B and C compared to that in Group A. The immunized goats had a significantly higher level of protection as demonstrated by the low bacterial loads in most tissues from the goats challenged with B. abortus. Relative real-time polymerase chain reaction results revealed that the expression of Brucella antigens was lower in spleen, kidney, and lung of immunized goats than of non-immunized animals. Also, treatment with our combination vaccine ameliorated histopathological lesions induced by the Brucella infection. Overall, the Salmonella Typhimurium delivery-based combination vaccine was effective in delivering immunogenic Brucella proteins, making it potentially useful in protecting livestock from brucellosis.

Introduction
Brucellosis is a severe and acute febrile disease caused by infection with a Brucella species, a gram-negative bacterium of the genus Brucella [5,21]. It remains one of the important worldwide zoonotic diseases [3]. In 1955, bovine brucellosis was reported among dairy cattle imported to the Republic of Korea [17,25]. Thereafter, a total of 85,521 Brucella reactor animals have been identified in 14,215 outbreaks between 2001 and 2011. The numbers of brucellosis cases at both the individual animal and farm levels increased after 2003, peaked in 2006, and slightly decreased thereafter because of implementation of a 'test-and-slaughter' policy [16,23].
It is important to control brucellosis in animal population because humans can be directly or indirectly infected by infected animals; thus immunization against Brucella in animals has a critical role in human health. Brucella abortus S19 and Brucella melitensis Rev. 1 vaccines have been widely used in developed countries. However, those vaccines have induced abortions in pregnant animals [18,22]. Moreover, they elicit anti-Brucella antibodies that interfere with serodiagnosis [13]. Antigen delivery systems become necessary when antigens are inefficiently transported to appropriate sites or presented to the immune system [20]. Brucella protective immunogens can be delivered to critical immunological sites by using a Salmonella-based vector. Therefore, an intracellular pathogen, such as an attenuated Salmonella strain, can be licensed as a Asd + , pBR ori, -lactamase signal sequence-based periplasmic secretion plasmid, 6× His tag [8] Omp19, outer-membrane protein 19; BLS, Brucella lumazine synthase; PrpA, proline racemase subunit A; SOD, superoxide dismutase.
vector to deliver Brucella antigens. As Salmonella can produce infection in a manner similar to that of Brucella, it is a pragmatic choice for an anti-Brucella vaccine-delivery platform. The attenuated Salmonella Typhimurium delivery-based Brucella vaccine used in this study was previously developed and has proven to be a suitably vectored Brucella vaccine when applied through different routes of immunization in mice [11]. That vaccine's protective efficiency was improved by combining four heterologous Brucella antigens: Brucella lumazine synthase (BLS), proline racemase subunit A (PrpA), outer-membrane protein 19 (Omp19), and Cu/Zn superoxide dismutase (SOD) proteins [9]. However, a livestock vaccine trial to show the actual response of the natural host to the Brucella vaccine delivered by an attenuated Salmonella vector has not been reported. Although large-animal experimental trials are uncommon because of limited resources, they are important for characterization of the equivalent immune responses in livestock hosts (natural host) in order to consider the commercial use of a developed vaccine. Therefore, this study reports on the use of a novel Salmonella system delivering four Brucella antigens (SOD, BLS, PrpA, and Omp19) to determine whether combinations of individual vaccines can efficiently regulate brucellosis in goats.

Ethics and biosafety statement
All animal experimental procedures were approved (CBNU 2016-98) by the Chonbuk National University Animal Ethics Committee in accordance with the guidelines of the Korean Council on Animal Care and Korean Animal Protection Law (2007, Article 13: Experiments with animals). All goats used in the study were housed and maintained humanely. The biosafety level-3 organism B. abortus strain 544 was handled with the required safety precautions and under the supervision of the Ministry of Health & Welfare, South Korea.

Bacteria strains, plasmids, and construction of vaccine
B. abortus strain 544 (ATCC23448) was used as the virulent challenge strain [7,12]. The bacterial strains, plasmids, and primers used in this study are listed in Table 1. Construction and validation of Salmonella Typhimurium strains expressing Brucella immunogenic frames were undertaken as previously described [8,9,11]. The challenge strain (B. abortus strain 544) was prepared for the challenge experiment by brief culture in Brucella broth (Becton, Dickinson and Company, USA) at 37 o C for 24 h and then resuspended to approximately 5 × 10 8 CFU/mL. Goats were conjunctively challenged with 100 L of the challenge strain in saline.

Immunization and challenge of B. abortus biotype 1
A total of 10-month-old goats (n = 18) that were seronegative for brucellosis based on a Rose-Bengal plate agglutination test were used in the study. The animals were divided into 4 groups, fasted for 24 h, and immunized according to the scheme in Table  2. Upon vaccination, animals were monitored for immunization-induced morbidity or mortality and underwent immunological profiling. The negative control (NC) group (n = 3) was subcutaneously (SC) inoculated with 0.1 mL of saline. Group A (n = 5), as the vector control group, was inoculated SC with approximately 5 × 10 9 colony-forming units (CFU/mL) of Salmonella Typhimurium delivery strain containing pJHL65 only in 1 mL. Group B (n = 5) and Group C (n = 5) were immunized SC using the Salmonella Typhimurium system with inoculations of approximately 5 × 10 9 CFU/mL and 5 × 10 10 CFU/mL, respectively, of mixtures containing four different delivery strains in 1 mL. At 6 weeks post-immunization, Groups A, B, and C were conjunctively challenged with goat-passaged virulent B. abortus strain 544 at a dosage of 5 × 10 8 CFU/mL in 100 L (50 L of inoculum per eye). Animals were kept in a restricted large-animal isolation facility with no contact between groups and were observed daily.

Humoral immune responses of the vaccinated goats
To determine the level of anti-Brucella antibodies generated, the humoral response of the control and immunized goats were investigated by performing goat immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA). Serum samples were collected at 3-week intervals after immunization. A standard ELISA was carried out in serum to evaluate the immune response against the BLS, PrpA, Omp19, and SOD antigens in goats according to the modified method previously reported [6]. Briefly, 96-well microtiter plates (Nunc, Denmark) were coated overnight with pre-titrated recombinant BLS (5 g/mL), Omp19 (5 g/mL), PrpA (5 g/mL), or SOD (5 g/mL) proteins in phosphate-buffered saline (PBS), blocked for 30 min using PBS containing 1% bovine serum albumin (diluent; 200 L/well), and washed with PBS containing 0.05% Tween-20. Serum samples were diluted at 1:100 in diluent. The plate was treated with horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG-Fc detection antibody (1:150,000; Bethyl Lab, USA). For IgG samples, colorimetric changes resulting from the action of HRP on o-phenylenediamine (Sigma-Aldrich, USA) were measured through an automated ELISA spectrophotometer (Multiskan GO; Thermo Fisher Scientific, Finland) at 492 nm, 10 min after development. The values for binding of IgG to the respective antigens in each group were expressed as the mean optical density (OD) ± SE.

Interferon (IFN)- quantification by goat IFN- ELISA kit
Measurement of IFN- from peripheral blood mononuclear cells (PBMCs) was done at 6 weeks post-immunization. PBMCs were prepared by modifying a previously described method [4]. PBMCs were prepared using Ficoll sodium diatrizoate gradient (Sigma-Aldrich), washed twice with PBS and resuspended in RPMI 1640 medium to a level of 2.5 × 10 6 viable cells per milliliter as determined by trypan blue dye exclusion. The PBMCs (5 × 10 5 cells/well) were stimulated in vitro with BLS, PrpA, SOD, or Omp19 antigen (4 g/well) for 24 h. The supernatants were collected and used for cytokine measurement as previously described [1,9]. Antigen-specific induction of IFN- was measured by using an IFN- ELISA kit with biotin-conjugated anti-IFN- primary antibody. HRP-avidin (1:1,000; Cusabio Biotech, China) was used to bind the primary antibody. The OD of each well was measured by using a microplate reader set to 450 nm.

Bacterial culture
At 8 weeks post-infection, the experimental animals were humanely sacrificed, and tissues from each animal were freshly collected. Sampled tissues included spleen, liver, lung, kidney, heart, testis, epididymis, mandibular lymph node (LN), parotid LN, retropharyngeal LN, superficial cervical LN, bronchial LN, portal LN, and mesenteric LN. Each sample (5 g) was collected aseptically in 5 mL of PBS and immediately homogenized with a sterile wooden applicator. Inoculum (200  The systemic humoral responses elicited by the four vaccine strains were investigated by measuring the relative levels of plasma immunoglobulin G (IgG) from samples obtained from non-immunized negative control (NC) group, Group A (vector control containing pJHL65), Group B (Salmonella delivery vaccine inoculated at 5 × 10 9 CFU/mL; CFU, colony-forming unit), and Group C (Salmonella delivery vaccine inoculated at 5 × 10 10 CFU/mL). Serum IgG titers against Brucella lumazine synthase (BLS), Brucella superoxide dismutase (SOD), Brucella outer-membrane protein 19 (Omp19), and Brucella proline racemase subunit A (PrpA) in Group C goats were significantly increased compared to those of the non-immunized NC and vector control groups at 3 and 6 weeks post-immunization. Antibody levels are expressed as mean optical density at 492 nm (OD492) ± SE values. Bars labeled with a different letter on the graph are significantly different from each other (p ＜ 0.05) based on results of one-way ANOVA with Duncan's multiple range test. L) was seeded and spread on Brucella agar media containing an antibiotic supplement and incubated. Cultures were examined daily for presence of colonies and counted after incubation for 7 days at 37 o C in a 5% CO2 atmosphere.

Fig. 2.
Bar graphs of the enzyme-linked immunosorbent assay analysis results for the levels of goat interferon (IFN)- in peripheral blood mononuclear cells re-stimulated with each antigen at 6 weeks after immunization. The levels of IFN- (pg/mL) from the non-immunized negative control (NC) group, Group A (vector control containing pJHL65), Group B (Salmonella delivery vaccine inoculated at 5 × 10 9 CFU/mL; CFU, colony-forming unit), and Group C (Salmonella delivery vaccine inoculated at 5 × 10 10 CFU/mL) were significantly elevated in Group C, and Group B levels were higher than those of Group A and the NC group. Optical density of each well was measured by using a microplate reader set to 450 nm. Values are presented as mean ± SE. Bars labeled by different letters on the graph are significantly different from each other (p ＜ 0.05) based on one-way ANOVA and Duncan's multiple range test. BLS, Brucella lumazine synthase; Omp19, outer-membrane protein 19; PrpA, proline racemase subunit A; SOD, superoxide dismutase.

DNA extraction and relative real-time polymerase chain reaction (PCR) measurement of B. abortus antigens in tissue samples
DNA was extracted from tissue by using the GeneAll genomic DNA (gDNA) extraction kit (Seoul, Korea) according to the manufacturer's recommendation. The total gDNA concentration and purity (A260/A280) were measured by using an e-spect Malcom spectrophotometer (ES-2 model; Malcom, Japan) at a 250 nm wavelength and using the Nanodrop method. An A260/A280 absorbance ratio of 1.8 to 2.0 was regarded as indicating pure gDNA. A 20 L reaction mixture containing 2 L gDNA was used as a template in the assay, which used 500 nmol B. abortus-specific primers. Real-time PCR conditions were set as follows: denaturation and polymerase activation step at 50 o C for 2 min and 95 o C for 10 min, followed by 40 cycles of 95 o C for 15 sec and 60 o C for 1 min, concluding with derivation of dissociation curves on a CFX96 real-time PCR detection system (Bio-Rad Laboratories, USA) using SYBR green I as the double-strand DNA-specific binding dye. After reaction completion, specificity was verified by performing melting curve analysis. Quantification was performed by comparing cycle threshold (Ct) values of each sample with that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Sequences of the real-time PCR primers are summarized in Table 3. The oligonucleotide primers used in the study were purchased from Bioneer (Korea).

Microscopic observation of tissue sections
Tissues in 10% neutral buffered formalin were routinely processed by using a Shandon Citadel 1000 tissue processor and were embedded in paraffin. Tissue sections (5 m) were obtained by using a microtome (HM-340E; Thermo Fisher Scientific, USA) and placed on glass slides. Hematoxylin and eosin staining was performed according to standard techniques [2]. A microphotograph was taken by using an Olympus microscope (BX53F; Olympus, Japan) and digital imaging software (Olympus). Goat liver sections were examined for hepatic microgranulomas by counting the number of foci in 10 microscopic fields in each tissue sample at 100× magnification.

Statistical analysis
All data are expressed as mean ± SE values. Differences between multiple groups were compared by using one-way analysis of variance (ANOVA) by using SAS (ver. 9.1; SAS Institute, USA). Individual comparisons were made by applying Duncan's multiple range test.

Brucella antigen-specific humoral immune responses increased in vaccinated goats
The systemic humoral immune responses elicited by the combined vaccine strains were investigated. As shown in Fig. 1, all immunized goats (Groups B and C) developed serum IgG antibodies production against the individual antigens, including Brucella BLS, Brucella SOD, Brucella Omp19, and Brucella PrpA. Serum IgG titers against the individual antigens in Group C were significantly higher than those in the non-immunized NC group and vector control Group A at 3 and 6 weeks after the vaccine injection. Individual antigen-specific serum IgG titers in Group B were also significantly higher than those of the non-immunized NC group and vector control Group A at 6 weeks after vaccine injection, but only the SOD and Omp19 antigen-specific IgG levels were significantly increased at 3 weeks after vaccine injection. However, relative serum IgG titers against all antigens in Group C were not statistically different from those of Group B at 3 and 6 weeks after the vaccine injection. There were no statistical differences between the non-immunized NC group and the vector control Group A.
These results indicate that treatment by our combination vaccine at each of the two dosages tested can fully induce humoral immune responses targeting individual Brucella

Treatment with the combination vaccine increased the secretion of IFN- from PBMCs
Antigen-specific induction of IFN- production from PBMCs stimulated with individual antigens (BLS, Omp19, PrpA, or SOD) was measured by using an ELISA at 6 weeks post-vaccination. As shown in Fig. 2, significantly higher IFN- production occurred in PBMCs of goats in Groups B and C than in the non-immunized NC group and vector control Group A (p ＜ 0.05). Although levels of IFN- were higher in Group C than in Group B after stimulation with the individual antigens, no significant differences were noted between those two groups. These results indicate that treatment with our combination vaccine at two different dosages can fully induce cell-mediated immune responses against individual Brucella antigens at 6 weeks after immunization.

Salmonella delivery vaccine immunization protected against the challenge of B. abortus
The protective efficacy of immunization with the Salmonella Typhimurium-based Brucella antigen vaccine was investigated in a goat model. At 8 weeks after infection, the magnitude of the challenge bacteria load in the spleen, heart, kidney, liver, lung, testis, epididymis, parotid LN, portal LN, retropharyngeal LN, superficial cervical LN, mandibular LN, mesenteric LN, and bronchial LN reflected the efficacy of the immunization.
Compared to the Group A vector control goats, the immunized goats showed significantly higher protection and a lower bacterial load in most of the above-mentioned tissues of goats challenged with B. abortus virulent strain 544 (Fig. 3). Generally, the degree of protection in the Group C goats was higher than that in the Group B goats, but the bacteria load in Group B was still lower in most of the collected tissue types than that of Group A. As expected the non-infected NC group showed no bacterial loads.
Relative real-time PCR technique was used to compare the presence of B. abortus antigens in all collected samples. As shown in Fig. 4, expression of B. abortus antigens was lower in the spleen, liver, kidney, and lung of Groups B and C than those of Group A, although no significant difference was noted between Groups B, C, and the NC group. Such a pattern of decreased antigen expression in the vaccine-treated groups was not observed in the other collected tissues.

Treatment with combination vaccine ameliorated histopathological lesions induced by B. abortus strain 544 infection
At 8 weeks after infection with B. abortus strain 544, aggregations of inflammatory cells that consisted predominantly of lymphocytes (microgranulomas) in the liver and distinctly visible trabeculae in the spleen were microscopically observed in the examined goats (panel A in Fig. 5). As shown in panel B in Fig. 5, the incidence of microgranuloma foci lesions was significantly low in the livers of goats in Groups B and C. Levels of distinct visibility of trabeculae in the spleen were lower in Groups B and C than that in Group A, indicating lower septicemia associated with the vaccination. These results suggest that the combination vaccine treatment ameliorated B. abortus infection-related lesions.

Discussion
Immunization of animals and humans has been successfully practiced as a means to control infectious diseases for centuries [7]. In order to avoid the residual virulence of Brucella live vaccines, an alternative approach of delivering Brucella subunit components via live vaccine vectors has been used. Safety is the major concern when using attenuated live vaccine vectors delivering Brucella antigens compared to the use of live attenuated Brucella vaccine. Attenuated Salmonella Typhimurium live vector strains delivering various heterogeneous antigens to the immune system can be used as a combination vaccine vehicle against a variety of targeted pathogens at a relatively low cost [10].
The success of immunization with a mixture of antigens using attenuated Salmonella Typhimurium live vaccine was anticipated based on the results of previous studies. However, the immune response in livestock was based much on the precept that live attenuated Salmonella vaccine expressing recombinant rBL protein can induce significant protective effects in a mouse model [27]. In this study, the attenuated Salmonella Typhimurium Δlon, ΔcpxR, and Δasd bacterial delivery vector was used with the pBP65 plasmid encoding the asd gene to deliver the Brucella antigens at two different dosages. The goats inoculated with 5 × 10 10 CFU/mL (Group C) showed significantly increased serum IgG titers against all antigens when compared to the vector control group. However, the lower inoculant concentration (5 × 10 9 CFU/mL) should not be overlooked, as it also produced higher serum IgG titers than that in Group A. Humoral response results of the two inoculation dosages showed the effectiveness of delivering antigens by the attenuated Salmonella Typhimurium vaccine. Furthermore, IFN- concentration was evaluated in supernatants of PBMCs following re-stimulation with heat-inactivated B. abortus antigens of goats immunized with vaccine candidates. The levels of IFN- were significantly elevated in both Group C (5 × 10 10 CFU/mL) and Group B (5 × 10 9 CFU/mL) over that in Group A. The results suggest that a Th1-type immune response is strongly induced by both inoculation dosages. It seems logical to favor the higher inoculation for sufficient protection, but the lower inoculation dose also produces a significant immune response. In addition, economics must be considered because the lower dosage (5 × 10 9 CFU/mL) may offer a cost-effective alternative for the production of effective vaccines against brucellosis.
To examine cross-reactivity, Brucella spp. have been identified by performing amplification of a specific region of its genome, as has been done for other closely related bacteria [14,19]. The IS711 region of the Brucella genome has been used to identify B. abortus, B. melitensis, and Brucella suis biovar 1. Similar primers and probes exploiting the IS711 repetitive element have been used for real-time detection in some studies [14,15]. This study explored using real-time PCR and SYBR green I, a double-stranded DNA intercalating dye [14]. Real-time PCR was used as a confirmatory diagnostic alternative to problematic culturing of Brucella spp. The high detection sensitivity of real-time PCR can be explained by the fact that it detects DNA from bacteria that are damaged or nonviable and which therefore cannot be isolated by applying conventional culture techniques. In addition, relative real-time PCR can indicate which tissues have higher/lower levels of bacterial expression. Our results showed that the relative expression of B. abortus antigens was more decreased in spleen, kidney, and lung of immunized goats than in the non-immunized group. However, in contrast to the bacteria load recovery results, such a decrease in antigen expression in the vaccine groups was not observed in other collected tissues, especially not in the various LNs that were examined. Based on these results, we speculate that many nonviable Brucella in the LNs of immunized goats were measured by real-time PCR, since macrophages phagocytose antigens and then migrate to draining LNs. Therefore, we believe that the challenge strain-tissue recovery model is a gold standard for evaluating the efficacy of our combination vaccine, and that results obtained by using real-time PCR should be used for reference purposes only.
In this study, the experimental goats did not show any gross lesions, including splenomegaly, when necropsy was performed, thus indicating that severe brucellosis did not develop as a result of the experimental infection. Even though few gross lesions were observed, distinctly visible trabeculae was microscopically evident in the spleen of non-immunized goats after Brucella infection. This suggests that the spleen can respond to septicemia through active hyperemia [26], but the hyperactive spleen may not always be enlarged [8]. In addition, the experimentally infected goats revealed aggregations of inflammatory cells (microgranulomas), and fewer neutrophils were observed in the liver. The recruitment of lymphocytes in the liver was evident based on the formation of microgranulomas, supporting the observations in a previous study that showed an inflammatory response in the liver of mice [24]. These two microscopic changes after Brucella infection were decreased in our vaccine-treated groups, indicating that such histopathological parameters can be used for evaluating the severity of brucellosis and the efficacy of a vaccine in goat.
In conclusion, we have shown that SC vaccination with an attenuated Salmonella Typhimurium live vector strain delivering a mixture of four Brucella immunogens (BLS, PrpA, Omp19, and SOD) can induce a protective immune response against infection by B. abortus. It was apparent that vaccination at the higher dose (Group C) was more efficacious than that at the lower dose (Group B); regardless, vaccination at the lower dose also had a significant protective effect. It is likely that a Salmonella-based delivery system could be adapted to develop multivalent recombinant Salmonella vaccines against brucellosis. Further work is needed to compare the efficacy of our combination vaccine with that of vaccines already in use, such as RB51.


Expression of microRNAs in the plasma of patients with acute gouty arthritis and the effects of colchicine and etoricoxib on the differential expression of microRNAs

Introduction To determine the microRNA (miRNA) expression profiles in the plasma of acute gouty arthritis (AGA) patients and investigate the effects of colchicine and etoricoxib treatment on the differential expression of miRNAs. Material and methods Exiqon miRCURYLNA microRNA Array was used for miRNA expression profiling in AGA. Two of the 21 differentially expressed miRNAs were confirmed using quantitative real-time polymerase chain reaction (qRT-PCR). A randomized, double-blind, parallel-controlled design was used to divide 160 AGA patients into colchicine and etoricoxib groups. Changes in 2 differentially expressed miRNAs, interleukin-1 (IL-1) β, cyclooxygenase-2 (COX-2) and joint pain scores were detected. Results Compared with normal subjects and asymptomatic hyperuricemia (HUA) patients, plasma of AGA contained 21 differentially expressed miRNAs. qRT-PCR indicated specific downregulation of miR-223-3p and miR-451a in AGA. There were no statistically significant differences in the baseline characteristics between colchicine and etoricoxib groups. Furthermore, no significant difference in joint pain scores after 5- and 10-day treatment were found between groups (p > 0.05). Comparison of differences between pre- and 5-day post-treatment values confirmed that the upregulation of miR-223-3p and downregulation of IL-1β induced by colchicine were more significant than etoricoxib (p < 0.05). However, the latter outperformed the former in the upregulation of miR-451a and downregulation of COX-2 (p < 0.05). After 10-day treatment, the magnitude of miR-223-3p upregulation and IL-1β downregulation in the colchicine group was significantly higher than in the etoricoxib group, while the etoricoxib group had higher expression of miR-451a and lower expression of COX-2 than the colchicine group (p < 0.05). Conclusions In AGA patients, 21 differentially expressed miRNAs were detected in the plasma. Colchicine could upregulate miR-223-3p and downregulate IL-1β in the plasma, while etoricoxib may treat AGA by upregulating miR-451a and downregulating COX-2.

Introduction
Gout is a type of arthritis, which may present as chronic tophaceous gout due to the deposition of monosodium urate (MSU) crystals in the joints or tissues of patients with purine metabolism disorders. It is believed that the pathogenesis of acute gouty arthritis (AGA) is closely related to the NLRP3 inflammasome, interleukin-1 (IL-1) β and cyclooxygenase-2 (COX-2) [1,2]. MicroRNAs (miRNAs) are a kind of non-coding RNA in eukaryotes, which has been proved to play a key role in the regulation of inflammatory diseases [3,4]. However, the miRNA expression profile in AGA has rarely been studied [5,6]. Therefore, it is possible to further clarify the pathogenesis and treatment of AGA from the perspective of miRNAs.
Non-steroidal anti-inflammatory drugs (NSAID) and colchicine are the first-line drugs in the treatment of AGA. Etoricoxib, a specific COX-2 inhibitor, has been recommended by the American College of Rheumatology (ACR) as class A evidence for the treatment of AGA [7]. Colchicine may act by inhibiting the MSU crystal-induced production of NLRP3 inflammasomes, thus reducing IL-1β to treat AGA [8]. However, it is not yet certain whether colchicine and etoricoxib ameliorate AGA by specifically regulating miRNAs.
Therefore, we utilized a miRNA array to detect miRNAs differentially expressed in the plasma of patients with AGA. Differentially expressed miRNAs were verified using quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, miRNAs differentially expressed before and after colchicine and etoricoxib treatment were observed.

Patients
In this study, 160 newly diagnosed AGA patients receiving outpatient therapies in the Gout and Endocrinology Department of the Affiliated Hospital of Qingdao University were selected as the screening subjects. The study complied with the Declaration of Helsinki, and the ethics committee of Qingdao Medical University approved the study protocol. Informed consent was provided by all patients.
The diagnostic criteria were as follows: patients who met the 2015 ACR and EULAR diagnostic criteria for AGA [9], and the asymptomatic HUA diagnostic criteria. The inclusion criteria were as follows: age 18-70 years; newly diagnosed AGA; an onset of AGA duration of less than 48 h and no administration of colchicine, NSAID or glucocorticoids; no administration of medications affecting uric acid metabolism over the last 3 months; without diseases affecting uric acid metabolism.
The exclusion criteria were as follows: repeated or intermittent onset of AGA, or acute onset of chronic gout; chronic tophaceous gout, rheumatoid arthritis, traumatic arthritis or other types of arthritis; gouty nephropathy; pregnancy and lactation; diabetes; hypertension; severe dyslipidemia; severe liver damage, alanine aminotransferase (ALT) or aspartate aminotransferase (AST) 2 times higher than upper limit of normal; severe cardiovascular and cerebral disease; malignancies.
The termination criteria were as follows: joint pain and swelling cannot be alleviated in the course of treatment; and serious adverse drug reactions that patients were not able to tolerate.
There were 25 patients who did not meet the inclusion criteria and 13 patients who did not agree to join the study. Therefore, the final eligible sample size was 122 ( Figure 1). From 122 patients recruited, 60 AGA patients were randomly selected to identify differentially expressed miRNAs in the plasma. Thirty asymptomatic HUA patients and 30 healthy subjects who were ageand gender-matched and treated in the same period were included. We performed high-throughput sequencing using miRNA microarray to obtain miRNA expression profiles in 12 volunteers. Then, the differential expression of miRNAs was detected using qRT-PCR in the plasma of 60 AGA cases, 30 cases with asymptomatic HUA and 30 normal subjects, including the volunteers.
Additionally, we conducted a randomized double-blind and parallel-controlled study. A co-investigator randomized 122 patients in two-member blocks to the colchicine or etoricoxib group on the basis of a computer generated random number table previously established by a statistician. The colchicine group had 52 patients (excluding 2 patients with severe diarrhea, 2 patients with liver damage, 4 patients with unrelieved joint pain and swelling, and 1 patient lost to follow-up), and the etoricoxib group included 53 patients (excluding 1 patient with severe vomiting, 5 patients with unrelieved pain and 2 patients lost to follow-up) ( Figure 1).

RNA extraction
The blood samples were collected from peripheral veins using EDTA tubes. Plasma was obtained by centrifugation at 2000 g for 20 min, and 250 μl of plasma was used to isolate RNA samples. Total RNA was harvested using TRI reagent BD (MRCgene, TB-126) according to the manufacturer's instructions, which efficiently recovered all RNA species, including miRNAs. RNA quality and quantity were measured using a NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies).

miRNA array
The 12 RNA samples from 4 AGA patients, 4 asymptomatic HUA and 4 healthy controls (age-and gender-matched) were labeled using the miRCURY Hy3/Hy5 Power labeling kit and hybridized on the miRCURYTM LNA Array (v.18.0) (Exiqon, Vedbaek, Denmark). The reaction mix for each microfluidic card was prepared by adding 360 μl of H 2 O and 450 μl of TaqMan Universal PCR Master Mix 2× to 90 μl of diluted and preamplified cDNA. The product of this reaction was incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The real-time PCR instrument ViiATM7 was used to perform the reactions. Then the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA). The data were extracted from scanned images using GenePix Pro 6.0 software (Axon).
Verifying miRNA array data by qRT-PCR cDNA was synthesized from total RNA using microRNA-specific, stem-loop RT primers and reagents from the TaqMan microRNA Reverse Transcription Kit (ABI, Foster City, USA), following the manufacturer's protocol. Real-time PCR was performed in triplicate on the ViiATM 7 Real-time PCR System (ABI, Foster City, USA). The SYBR Green PCR Master Mix (ABI) was used to confirm the relative expression levels of differentially expressed microRNA. The miRNA primers were designed using Primer 5.0 software. Each plasma sample had 3 replicates, and the average of the 3 replicates was taken as the final result. Data analysis for qRT-PCR was performed by the comparative threshold cycle (Ct) method. MiR-39 is a synthetic Caenorhabditis elegans miR-39 miRNA mimic (cel-miR-39, Qiagen, Hilden, Germany) that served as the spike-in control to monitor the effectiveness of RT-PCR and quantitative PCR. Cel-miR-39 demonstrated both stable and moderate abundance Ct values (27.45 ±0.52) in all subjects. The relative expression of each miRNA after normalization to cel-miR-39 was determined by the 2 -ΔCt method.

Methods of drug intervention
All patients were given a low-purine diet and forbidden from smoking and drinking alcohol, and were required to drink enough water, 2500-3000 ml daily. All patients were also given sodium bicarbonate 1.0 g three times daily orally to alkalinize the urine. Meanwhile, all patients were required to stay in bed and avoid overtiring, cold and tension. Patients in the colchicine group received colchicine 0.5 mg orally (Xishuangbanna Pharmaceutical Co., Ltd.), 3 times daily, for 5 days, later changed to once daily. Patients in the etoricoxib group received etoricoxib 120 mg orally (Hangzhou MSD Pharmaceutical Co., Ltd), once daily, for 5 days, later changed to 60 mg orally once daily. Differentially expressed miRNAs, IL-1β and COX-2 were detected at the beginning of the experiment and at 5 days and 10 days after treatment. The total courses of treatment were 10 days. The primary outcome measure was changes of expression of 2 differential miRNAs between groups before and after 10-day treatment. The secondary end-points included changes of expression of 2 differential miRNAs after 5-day treatment, and changes of joint pain scores, IL-1β and COX-2 after 5-and 10-day treatment.
Before and after treatment, all patients underwent measurements of height, weight and blood pressure. The joint pain scores were recorded in patients' diaries. The differential expression of miRNAs was detected according to the methods mentioned above. ELISA technique was used to detect IL-1β and COX2. The ELISA kit was purchased from CloudSeq Biotech Inc. and used according to the manufacturer's instructions. Biochemical indicators including white blood cells (WBC), fasting plasma glucose (FPG), blood lipids, ALT, and AST were detected. The above detected indexes were measured by two experienced technicians, who were unaware of treatment allocation.

Statistical analysis
Statistical significance was accepted at α = 0.05 and β = 0.2 (power of a test 80%). Based on the literature [10], it was predicted that the differentially expressed miRNAs in the colchicine group would change about 30% as compared with the etoricoxib group. With a drop-out rate of approximately 20%, at least 48 subjects would be needed in each group. Considering poor efficacy and unrelieved pain, 160 total subjects were included.
Data were analyzed using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA). Normality of distributions was evaluated by the Shapiro-Wilk statistic. The normally distributed data were expressed as means ± standard deviation (SD). Continuous variables were analyzed using Student's t test. Pearson's correlation coefficient was used to analyze the correlation between variables. Two differentially expressed miRNAs were compared at different time points by repeated measures analysis of variance (ANOVA). The intention-to-treat (ITT) analysis and the per-protocol (PP) analysis Expression of microRNAs in the plasma of patients with acute gouty arthritis and the effects of colchicine and etoricoxib on the differential expression of microRNAs Arch Med Sci 4, July / 2019 1051 were carried out on the main outcome measure. Statistical significance was considered at p < 0.05.

Intergroup comparison of baseline characteristics
No statistically significant differences in baseline characteristics were found between groups (p > 0.05, Table I).

Verification of differentially expressed miRNAs by qRT-PCR
To verify the miRNA microarray results, we tested 2 miRNAs that were presumed to play a role in AGA, namely, miR-223-3p [11,12] and miR-451a [13,14]. The results showed that qRT-PCR was consistent with the microRNA microarray, which further verified the accuracy of microRNA array screening results (Figure 2).

Intragroup comparison of evaluation indicators before and after treatment
Compared with the baseline before treatment, joint pain scores were significantly decreased in both groups after 5 and 10 days of treatment (p < 0.05). After 5-day treatment, miR-223-3p ex-pression was significantly upregulated (p < 0.05), IL-1β declined considerably, while miR-451a and COX-2 did not change significantly (p > 0.05) in the colchicine group. In the etoricoxib group, miR-451a was obviously upregulated and COX-2 was significantly decreased (p < 0.05). After 10 days of colchicine treatment, miR-223-3p increased and IL-1β decreased dramatically compared to pre-treatment (p < 0.05). Additionally, COX-2 decreased slightly, accompanied by a slight upregulated expression of miR-451a (p < 0.05). In the etoricoxib group, miR-451a was upregulated and COX-2 declined significantly (p < 0.05). Meanwhile, there was slight upregulation of plasma miR-223-3p and downregulation of IL-1β after etoricoxib treatment (p < 0.05, Table III).

Intergroup comparison of evaluation indicators after treatment
The results of the repeated-measures ANOVA test for comparing the variation of 2 differentially expressed miRNAs showed that miR-451a in the etoricoxib group was significantly higher than the colchicine group (F = 7.382, p < 0.05), and miR-223-3p in the colchicine group was upregulated in comparison to the etoricoxib group (F = 3.127, p = 0.080). Further comparison of differences between pre-and 5-day post-treatment confirmed that the upregulation of plasma miR-223-3p and downregulation of IL-1β induced by colchicine were more significant than etoricoxib (p < 0.05). However, the latter outperformed the former in the upregulation of miR-451a and downregulation of COX-2 (p < 0.05, Table III). When comparing the difference after treatment for 10 days, the ITT analysis showed that the magnitude of miR-223-3p upregulation in the colchicine group was significantly higher than the etoricoxib group (p < 0.05), while the etoricoxib group had higher expression of miR-451a than the colchicine group (p < 0.05, Table IV). The PP analysis was concordant with the ITT analysis. Additionally, comparison of therapy-related differences showed that the colchicine group had greater downregulation of IL-1β than the etoricoxib group, but the downregulation of COX-2 was more significant in the etoricoxib group than the colchicine group.

Correlation analysis
We performed a linear correlation analysis for the expressions of 2 miRNAs in the plasma, IL-1β and COX-2. The results showed that miR-223-3p expression was negatively correlated with IL-1β in AGA patients (r = -0.796, p < 0.05), and miR-451a was negatively correlated with COX-2 (r = -0.633, p < 0.05).

Adverse events
One patient in the colchicine group suffered from mild vomiting with gradual remission. Four patients in the colchicine group had diarrhea during the treatment. Two patients had severe diarrhea and were withdrawn from the trial, and 2 patients had mild diarrhea with mild symptoms lasting less than 3 days, thereby not affecting the test. Additionally, 2 patients from the colchicine group had obvious abnormal liver function, and they were required to terminate the trial. Three patients in the etoricoxib group had vomiting during the treatment. One patient suffered from severe vomiting and asked to stop the treatment. Two patients had mild vomiting and were allowed to complete the trial. Additionally, 2 patients in the etoricoxib group reported mild diarrhea with mild symptoms, thereby not affecting the trial.

Discussion
In this study, we identified 21 differentially expressed miRNAs in AGA patients compared with normal controls and asymptomatic HUA, and obtained AGA-specific miRNAs expression profiles. Meanwhile, the results of qRT-PCR verified that miR-223-3p and miR-451a were specific downregulation in the AGA patients, which was consistent with gene chip by high-throughput sequencing technology.
According to TargetScanHuman (http://www. targetscan.org), miR-223-3p regulates NLRP3 expression by directly targeting the 3'-UTR of its mRNA, and then reduces IL-1β by inhibiting the inflammasome [11,12]. Meanwhile, in this study, we found that miR-223-3p had a negative correlation with IL-1β, thus making contributions to the occurrence of AGA. MiR-451a is downregulated in many cancers and acts as a tumor suppressor gene. Previous studies have indicated that several miRNAs including miR-451a can target COX-2 [13]. Zhang et al. [14] found that miR-451a targeted CUG triplet repeat-binding protein 2, a ubiquitously expressed RNA-binding protein, known to interact with COX-2 3'-UTR and inhibit its translation. Additionally, in the present study, we found specific downregulation of miR-451a in the plasma of AGA patients. Correlation analysis suggested a significant negative correlation between miR- 451a and COX-2. It was inferred that COX-2 may be the target of miR-451a in AGA patients. The first-line drugs used to treat AGA include NSAID and colchicine. A previous study showed that COX-2 played an important role in the pathogenesis of a variety of diseases [15]. COX-2 in AGA was dramatically upregulated, further promoting progression of gout-related inflammation. As a specific inhibitor of COX-2, etoricoxib has been recommended by the ACR to treat AGA [7]. Colchicine is still a specific drug for AGA, which can inhibit the MSU crystal-induced production of NLRP3 inflammasomes and reduce the production of IL-1β. Our results showed that joint pain treated by colchicine and etoricoxib decreased considerably. In addition, the number of drop-out cases due to poor efficacy did not differ between groups. Therefore, both colchicine and etoricoxib were considered ideal for treating AGA.
The effect of medications on miRNA expression in AGA patients is not well understood. According to the corresponding reference [10], serum inflammatory cytokines such as IL-1β and COX-2 peaked at 3-5 days after the onset of AGA. Meanwhile, according to 2011 recommendation for the diagnosis and management of gout and hyperuricemia made by the European League Against Rheumatism (EULAR), oral colchicine and NSAID should be necessary to continue treatment for an additional 10 days for AGA [16]. Therefore, we treated the AGA patients with etoricoxib and colchicine for 10 days and changes of biochemical indices were characterized starting from 5 days. We found that colchicine was more effective in upregulating miR-223-3p and reducing IL-1β than etoricoxib, while etoricoxib was more effective in upregulating miR-451a and decreasing COX-2 than colchicine. It is suggested that colchicine may play an important role in the treatment of AGA by upregulating plasma miR-223-3p and decreasing IL-1β, while etoricoxib may treat AGA by upregulating miR-451a and downregulating COX-2.
It should be pointed out that the effects of colchicine and etoricoxib therapy on miRNAs in AGA patients may be complex. As is well known, AGA is a self-limited disease. A previous study showed that colchicine did not have an inhibitory effect on COX-2 [17]. However, in the present study, COX-2 decreased after 10 days of colchicine treatment, accompanied by slightly upregulated expression of miR-451a. Therefore, miR-451a was likely to be involved in the self-healing process of AGA. Additionally, there was mild upregulation of plasma miR-223-3p and downregulation of IL-1β after etoricoxib treatment. This also suggested that miR-223-3p may play an important role in the self-healing process of AGA. Meanwhile, previous studies have shown that COX-2 can promote the generation of IL-1β by regulating the NLRP3 inflammasome [18]. According to Daniels et al. [19], NSAID can suppress the activity of the NLRP3 inflammasome. Whether etoricoxib regulates the NLRP3 inflammasome by upregulating miR-223-3p remains unclear. At the same time, AGA heals spontaneously probably through the upregulation of anti-inflammatory cytokines such as transforming growth factor-β (TGF-β) 1 [20]. However, we are still uncertain about which miRNAs are involved in the spontaneous healing of AGA.
Several limitations of this study must be taken into account. Firstly, due to the small number of cases and short duration of follow-up, these conclusions need to be further confirmed by largescale clinical trials. Secondly, in spite of repeated measurements and analysis of variance to characterize the differentially expressed miRNAs, the dynamic changes in miRNA expression still cannot be fully reflected due to the low detection frequency. Thirdly, the specific mechanism of miRNAs in AGA is not clear. For example, the secretion of IL-1β and IL-18 requires the activation of caspase-1, which is regulated by NLRP3 [21]. Therefore, IL-18 may also be the target gene of miR-223-3p. Additionally, Moriya et al. [22] found that IL-17 was one of the targets of miR-223-3p. However, we did not verify whether there was targeting regulation of miR-223-3p and IL-17 in AGA patients. Therefore, the role of miR-223-3p and miR-451a in the pathogenesis of AGA needs further study.
In conclusion, 21 differentially expressed miRNAs were detected in the plasma in AGA patients compared with normal subjects and asymptomatic HUA patients. Colchicine could upregulate miR-223-3p and downregulate IL-1β in the plasma, while etoricoxib may treat AGA by upregulating miR-451a and downregulating COX-2.


extfieldsofstudy: Chemistry Medicine

text:
Neuroglobin mediates neuroprotection of hypoxic postconditioning against transient global cerebral ischemia in rats through preserving the activity of Na+/K+ ATPases

Hypoxic postconditioning (HPC) is an innovative neuroprotective strategy with cytoprotective effects on the hippocampal neurons against transient global cerebral ischemia (tGCI) in adult rats. However, its molecular mechanisms have not yet been adequately elucidated. Neuroglobin (Ngb) is an endogenous neuroprotectant with hypoxia-inducible property, and its role in experimental stroke has been increasingly attractive. Hence, the purpose of this study is to explore the involvement of Ngb in HPC-mediated neuroprotection and to further investigate its underlying molecular mechanism. We found that HPC increased Ngb expression in CA1 subregion after tGCI. Also, the inhibition of Ngb expression with Ngb antisense oligodeoxynucleotide (AS-ODNs) eliminated the neuroprotective effect mediated by HPC, whereas overexpression of Ngb ameliorated neuronal damage in CA1 after tGCI, indicating that HPC conferred neuroprotective effects via upregulation of Ngb. We further showed that HPC increased the membranous level of Na+/K+ ATPases β1 subunit (Atp1b1) in CA1 after tGCI. Furthermore, we demonstrated that Ngb upregulation in CA1 after HPC maintained the membranous level of Atp1b1 through Ngb–Atp1b1 interaction and reduced the glutathionylation of membranous Atp1b1 via suppression of reactive oxygen species (ROS), ultimately preserving the activity of NKA. Taken together, these data indicate that Ngb is involved in the neuroprotection of HPC against tGCI via maintenance of NKA activity in the hippocampal CA1.

Introduction
Transient global cerebral ischemia (tGCI) may result from cardiac arrest, serious hypertension, asphyxia, shock, or brain injuries, etc. The hippocampal CA1 pyramidal neurons are essential for cognitive functions and are selectively vulnerable to tGCI 1,2 . Although tremendous work has been directed to the molecular basis of the selective vulnerability of CA1, until now, there have been few effective therapies to this delayed neuronal damage after brain ischemia/reperfusion. Previously, we had demonstrated that hypoxic postconditioning (HPC) with 8% O 2 for 60-120 min significantly reduced cell death in the CA1 subregion after 10 min of tGCI. HPC was effective only when applied 1-2 days after ischemia. The maximum protective effect of HPC was achieved with 120 min of hypoxia and 1-day interval between hypoxia and tGCI 3 . HPC has attracted great attention, since it holds the promise as an innovative endogenous neuroprotective strategy against tGCI 44,5 . Despite the identification of several triggers and mediators of HPC 3,6-8 , the precise mechanisms by which HPC protects the brain against tGCI remain poorly understood.
Neuroglobin (Ngb) was originally identified in the human and the mouse brain. It is predominantly expressed in the neurons and the astrocytes 9 . Sharing little similarity in the amino-acid sequence, Ngb is distinct from myoglobin and hemoglobin in evolution and function 10 . Ngb may serve as a hypoxic sensor and initiate signal transduction involving in oxidative and hypoxic pathway, cell survival/apoptotic pathway, and ATP pathway in neuronal cells 11 . It has also been proposed that the neuroprotection of Ngb at the mitochondrial level is not only related to its activities to scavenge a variety of free radicals, but also to its ability to interact with cytochrome C (Cyc) in the mitochondria 12,13 . In recent years, the neuroprotective role of Ngb, particularly in hypoxic and ischemic conditions, has been explored [14][15][16] . Li et al. demonstrated that limb ischemic preconditioning upregulates Ngb in the hippocampal CA1 to diminish mitochondrial dysfunction induced by tGCI 17 . Similar result was also reported in vitro that the overexpression of Ngb could suppress ROS production and ameliorate the neurotoxicity induced by oxygen-glucose deprivation (OGD) in primary cortical neurons 18 . However, the neuroprotection of Ngb upregulation in cerebral ischemia was challenged by a few studies 19,20 . Research from Raida et al. revealed that the infarct size was reduced in Ngb-null mice after permanent MCAO, compared to the wild-type mice 20 . In fact, both ischemia and hypoxia exhibit regulatory functions in Ngb induction, leading to either promotion or suppression of Ngb expression in different tissues and distinct cell types 21,22 . To fill in these knowledge gaps, we examined the expression of Ngb after tGCI with or without hypoxia in CA1. Afterwards, we tested whether the alternation of Ngb expression is associated with the neuroprotective effect mediated by HPC after tGCI.
Na + /K + ATPases (NKA), constituted from a catalytic α (α1, α2, α3, and α4) subunit and a regulatory β (β1, β2, and β3) subunit, has been proven to be a vital energy transducing ion pumps in maintaining intracellular ion homeostasis and excitability 23,24 . The dysfunction of the ionic pumps, particularly the NKA, fails to maintain the ionic gradient across the neuronal membrane, resulting in massive cytoplasmic Na + and Ca 2+ accumulation, swelling, and degeneration of organelles, and eventually leading to cell dissolution after ischemia [25][26][27] . No less importantly, the β subunit is critical for the ion transport activity and the modulation of K + and Na + affinity of NKA 28,29 . Among the three isoforms of β subunit, Na + /K + ATPases β1 subunit (Atp1b1) is one of the Ngbinteracting proteins recently identified by the yeast twohybrid screening system that has the highest positive clone number 30 . Atp1b1 contains seven cysteine (Cys) residues. Six of them locate in the extracellular domain 31 , only Cys-46 locates in the lipid bulk phase of the membrane and is expected to prevent from accessing to the cytosolic, hydrophilic glutathione 32 . Additionally, oxidative stress can induce a reversible suppression on the NKA activity [33][34][35] , which is mediated by glutathionylation of its β1 subunit at Cys-46 36 . Intriguingly, as a scavenger of nitric oxide and reactive oxygen species (ROS), Ngb plays an important role against oxidative stress 13,37 . Therefore, Ngb is likely to be associated with the regulation of Atp1b1 directly or indirectly, which might explain how Ngb involves in the neuroprotection of HPC against tGCI.
Considering the hypoxia-inducible feature and the antioxidant property of Ngb, we hypothesized that the alteration of Ngb in CA1 subregion contributes to the neuroprotection of HPC against tGCI by reducing the ROS production, subsequently upregulating membranous Atp1b1, suppressing the glutathionylation of Atp1b1, and eventually preserving the activity of NKA.

Results
The upregulation of Ngb induced by HPC in the hippocampal CA1 contributed to the neuroprotection against tGCI Immunohistochemical assay was conducted to examine the distribution of Ngb in the brain. The results showed a wide distribution of Ngb-positive cells in the Sham brains, including cerebral cortex, hippocampus, and subcortical structures (Fig. 1A, a-d). These cells have deep staining nuclei with several short dendrites in the parietal external granular layer (layer II) (Fig. 1A, b) and large soma with elongated axon in the temporal internal pyramidal layer (layer V) (Fig. 1A, c). Similar cells were also found in the parietal internal pyramidal layer (layer V) of tGCI and HPC rats at 26 h after reperfusion (Fig. 1A, e, f, i, j). In CA1, Ngb-positive cells mainly existed in the pyramidal cell layer with round and regular nuclei (Fig 1A, d). Double-fluorescent immunohistochemistry revealed that they were neuronal nuclei (NeuN)-positive ( Fig. 2A, a-c) and only a few were colocalized with glial fibrillary acidic protein (GFAP) ( Fig. 2A, d-f), indicating predominant neuronal localization of Ngb in the Sham brains. Notably, Ngb was expressed mainly in cells with polymorphic somata and processes. These cells were dispersedly distributed in the stratum radiatum and the oriens layer at 168 h of reperfusion after tGCI (Fig. 1A, m and n). The double-labeled immunofluorescence studies further revealed that these cells were mainly GFAP-positive ( Fig. 2A, g-l). In contrast, the majority of Ngb-positive cells in HPC rats exhibited similar appearance to neurons (Fig. 1A, o and p). Also, these cells were shown to be NeuN-positive ( Fig. 2A, m-o). The quantitative analysis showed that the number of Ngb-positive neuron-like cells in CA1 decreased at 26 h, then increased to the basal expression level at 50 h, and significantly reduced at 168 h after tGCI, while kept increasing consistently in the HPC groups (Fig. 1B). Meanwhile, Ngb-positive glia-like cells sharply increased at 168 h of reperfusion after tGCI, instead of the early phase of reperfusion, and a reverse trend was observed in the HPC group (Fig. 1C). These observations were further confirmed by the colocalization of Ngb with NeuN or GFAP (Fig. 2B, C). Also, Ngb level gradually decreased at 0-26 h after reperfusion of tGCI and increased back to the basal level at 50 h, while in the HPC groups, Ngb expression dramatically increased right after ischemia (Fig. 1D).  Fig. 3B, C). However, the administration of Ngb AS-ODNs, rather than S-ODNs, significantly reduced the Ngb expression after HPC (Fig. 3E). Also, compared with HPC rats, the neuronal damage was markedly aggravated in the CA1 of HPC rats that were preadministrated with Ngb AS-ODNs (Fig. 3A, q-t), accompanied by a decrease in surviving and NeuNpositive cells, as well as an increase in Fluoro-Jade B (FJ-B)-positive cells (Fig. 3B-D).
Opposite results were obtained after the administration of Ngb-carried lentivirus into the bilateral hippocampus (Fig. 4). Rats were randomly subjected to Sham operation or tGCI with or without hypoxia 14 days after the administration of Ngb-carried lentivirus (referred to as Lenti-Ngb) or scrambled lentivirus vector (referred to as Lenti-control) (Fig. 4A). As shown by immunofluorescence, Ngb was colocalized with GFP in Lenti-Ngb-administrated rats at 7 days after reperfusion (Fig. 4C). Similarly, no neurotoxic effects were observed on the pyramidal neurons in the CA1 of Sham-operated rats with lentivirus administration (Fig. 4D, a-h). Also, Ngb expression significantly increased in Sham-operated and tGCI rats after Lenti-Ngb administration (Fig. 4G). Meanwhile, Lenti-Ngb administration markedly ameliorated cell damage and neuronal loss, compared to Lenti-control administration after tGCI (Fig. 4D, i-p; Fig. 4E, F). However, no additive effect on neuroprotection was observed when either Lenti-Ngb or HPC was applied (Fig. 4D, q-x; Fig. 4E, F).
The upregulation of Ngb induced by HPC increased the expression of membranous Atp1b1 in the hippocampal CA1 after tGCI As shown in Fig. 5A, compared with the Sham-operated group, the tGCI groups with or without hypoxia showed no significant differences in the level of total Atp1b1. We thereafter further measured the membranous Atp1b1 expression. Notably, membranous Atp1b1 exhibited a similar trend compared to Ngb level, whereas there were no significant differences in the level of cytoplasmic Atp1b1 among the Sham-operated, hypoxia, tGCI, and HPC groups (Fig. 5B). Although the level of total Atp1b1 had no remarkable change at 0-50 h of reperfusion of tGCI with or without hypoxia, the number of Atp1b1immunopositive cells significantly reduced at 168 h after tGCI. However, HPC induced an increase in the number of Atp1b1-positive cells (Fig. 5D). These Atp1b1-positive cells with astrocytes-like appearance were seen to be scattered over the stratum radiatum and oriens layer (Fig. 5C). Double-label immunofluorescence studies also confirmed that these cells were mainly colocalized with GFAP (Fig. 5E).
We further examined the interaction between Ngb and membranous Atp1b1 in rats after tGCI with or without HPC by co-immunoprecipitation assay. The results revealed a detectable interaction between Ngb and membranous Atp1b1 after tGCI, though the strength of this interaction was weaker than that in the Shamoperated group. Moreover, HPC enhanced the strength of Ngb-Atp1b1 interaction, compared to the tGCI group (Fig. 6A). These observations were further confirmed by the proximity ligation assay (PLA) (Fig. 6B, C). Intriguingly, PLA signal was not only observed at the membrane, but also in the cytoplasm of the cells with typical neuronal appearance in the pyramidal cell layer of CA1. Afterwards, the regulatory effects of Ngb on the membranous and the cytoplasmic Atp1b1 were tested when Ngb expression was downregulated or upregulated. The results showed that the downregulation of Ngb with AS-ODNs in HPC rats led to a decrease in membranous Atp1b1, contrary to an increase in membranous Atp1b1 when Ngb was overexpressed with Lenti-Ngb in tGCI rats, whereas the regulation of Ngb expression had no impact on the level of cytoplasmic Atp1b1 (Fig. 6D, E).

The upregulation of Ngb induced by HPC alleviated the levels of ROS and the glutathionylation of Atp1b1 in CA1 after tGCI
To elucidate whether the free radical scavenging capacity of Ngb is involved in the neuroprotective mechanisms of HPC against I/R injury, we examined the ROS level in CA1 with 2′,7′-dichlorofluorescein diacetate (DCFH-DA).
(see figure on previous page) Fig. 2 Cellular localization of Ngb in the CA1 after tGCI with or without HPC. A Representative photomicrographs of fluorescent double staining show that Ngb (green) is mainly distributed in the cytoplasm and the surrounded NeuN (red)-positive nuclei in CA1 (a-c) with a few colocalization of Ngb (green) with GFAP (red) (d-f) in the Sham-operated group. However, Ngb (green)-positive cells were mainly colocalized with GFAP (red) (j-l) rather than NeuN (red) (g-i) in the CA1 at 168 h after tGCI. Alternatively, Ngb (green) located mainly in NeuN (red) (m-o) and partially in GFAPpositive cells (p-r) in the CA1 at 168 h after HPC. Scale bar: 50 μm. B, C Quantitative analysis of Ngb-positive neurons and Ngb-positive astrocytes in the CA1, respectively (n ≥ 3 in each group). Each bar represents the mean ± S.D. *p < 0.05 vs. Sham-operated animals and # p < 0.05 vs. tGCI group at the same time point The results showed a low level of ROS production at baseline in the Sham-operated group (Fig. 7A, a, e, i). However, ischemic stimuli were clearly observed to have triggered a robust generation of ROS. As shown in Fig. 7B, DCFH-DA signal in CA1 increased progressively and peaked at 7 days of reperfusion after tGCI. On the contrary, HPC efficiently decreased the ROS level, compared to the tGCI groups ( Fig. 7A, B). Even so, the effect of HPC on the reduction of ROS was attenuated by the knockdown of Ngb with Ngb AS-ODNs ( Fig. 7A, d, h, l, and Fig. 7B).
Given that the alteration of intracellular ROS affected the NKA activity by regulating the glutathionylation of Atp1b1, we measured the glutathionylation of Atp1b1 in the CA1 of tGCI and HPC groups with or without Ngb downregulation, respectively. As expected, the glutathionylation of Atp1b1 was upregulated after tGCI and downregulated by HPC (Fig. 7C), concomitant with the dynamics of ROS in the CA1 subregion. Moreover, the suppression of HPC on the glutathionylation of membranous Atp1b1 was impeded after downregulating Ngb expression with Ngb AS-ODNs (Fig. 7D).
The upregulation of Ngb mediates the neuroprotection of HPC against tGCI via preserving the activity of Na + /K + ATPases Next, we examined the activity of NKA after tGCI with or without HPC and the effect of Ngb on the activity of NKA. No differences were observed in the NKA activity in Sham-operated and Sham-operated hypoxia-treated groups. Nevertheless, compared to Sham-operated rats, the NKA activity in the tGCI groups significantly reduced at 4 h after reperfusion and maintained a gradual decrease until 7 days (Fig. 8A). In contrast, HPC significantly preserved the NKA activity compared with tGCI rats, which was abrogated when Ngb was downregulated via Ngb AS-ODNs administration (Fig. 8B). Meanwhile, the Ngb overexpression with Lenti-Ngb reversed the inhibition of NKA activity in tGCI rats (Fig. 8C).

Discussion
This study demonstrates that HPC activated endogenous neuroprotective mechanisms through upregulating Ngb expression in the CA1 subregion after tGCI. Ngb has been proven to be essential, highly conserved, and beneficial to neurons 38 . However, some previous studies reported that Ngb exerts no neuroprotective effect in mild traumatic brain injury 39 and Ngb deficiency reduces cerebral infarct volume after permanent cerebral artery occlusion 20 . In this study, HPC induced upregulation of the Ngb expression in the CA1 and mediated neuroprotective effects after tGCI. To further confirm the involvement of Ngb in HPC-mediated neuroprotection, we silenced Ngb expression in CA1 with Ngb AS-ODNs and overexpressed Ngb with Ngb-carried lentivirus. As expected, Ngb overexpression effectively ameliorated post-ischemic neuronal death in CA1, whereas the silencing of Ngb expression abolished the neuroprotection of HPC. This neuroprotective role of Ngb after cerebral ischemia may depend on several distinct factors. First, there are significantly different expression levels of Ngb in different conditions. Physiologically, the concentration of Ngb in the brain is quite low (<0.01% of the total protein) 10 . Therefore, neuroprotection of the endogenous expressed Ngb is negligible 40 . However, under cerebral ischemia, the expression of Ngb in CA1 is reduced, while the upregulation or overexpression of Ngb is reported to promote neuronal survival 17,41 . Consistent with the previous studies, our results revealed that Ngb upregulation induced by HPC ameliorated neuronal injury in the CA1 after tGCI. Second, the distinct cell types may have an impact on the expression of Ngb. Our study demonstrated that Ngb level decreased at 26 h in the neurons after tGCI. However, activated astrocytes induced the upregulation of Ngb at 168 h after tGCI, which may have contributed to delayed cell death after ischemia. In contrast, HPC enhanced the expression of Ngb in the neurons, which consequently promoted neuronal survival after ischemia. Last, the anatomical and subcellular distribution of Ngb was clearly revealed to be associated with its neuroprotection. In a study from Shang et al., Ngb expression increased in the cerebral cortex and decreased in the hippocampus after transient global forebrain ischemia in the gerbil brain 42 . Differently, neither the tGCI nor the HPC group exhibited a significant change in the number of Ngb-positive cells in the cortex. These results (see figure on previous page) Fig. 3 Effects of Ngb knockdown on the protein expression of Ngb and neuronal cells damage in CA1 after ischemia with or without HPC. A Representative microphotographs of cresyl violet staining, NeuN immunostaining, and FJ-B staining in the hippocampus at 7 days after tGCI with or without ODNs administration. Sham group (a-d); tGCI group (e-h); HPC group (i-l); Sham + AS-ODNs group (m-p), injection with AS-ODNs without ischemia or hypoxia; HPC + AS-ODNs group (q-t), injection with AS-ODNs before tGCI and HPC. Scale bar: a, e, i, m, q: 250 μm, b-d, f-h, j-l, n-p, r-t: 75 μm. B-D Quantitative analyses of survival cells, NeuN-positive cells and FJ-B-positive cells in the CA1 (n = 6 in each group). Each bar represents the mean ± S.D. *p < 0.05 vs. Sham-operated group, # p < 0.05 vs. tGCI group, and & p < 0.05 vs. HPC group. E Effects of Ngb ODNs on the expression of Ngb in the CA1 of Sham-operated and HPC rats using immunoblot analysis at 26 h after reperfusion (n > 3 in each group). The values are expressed as mean ± S.D. *p < 0.05 vs. Sham-operated animals and # p < 0.05 vs. HPC group with S-ODNs administration suggested that the hippocampus is more vulnerable than the cortex to tGCI. High expression of Ngb could protect the neurons from ischemic damage. Furthermore, Ngb was recognized only in the cytoplasm previously. However, with immune localization, cell fractionation, and electron microscopy, Ngb has been found in the cytosol, inner wall of the mitochondria, and nuclei of the neurons 12,43 . Noteworthily, Ngb can shift between cellular organelles to change its compartmentation and concentration upon physiopathological situation 12,44 .
The upregulation of Ngb gene expression under hypoxia was reported to be regulated by several transcription factors such as nuclear factor κB (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf-2), and hypoxia-inducible factor-1α (HIF-1α) 45,46 . Intriguingly, our previous research showed a significant increase of HIF-1α and its downstream target vascular endothelial growth (VEGF) in the HPC groups, compared with the tGCI rats 6 . Considering this point, we speculated that the upregulation of Ngb protein by HPC might be associated with the regulation of HIF-1α and VEGF. Acting as Ngb inducers, both HIF-1α and VEGF can enhance the expression of Ngb 47,48 . Haines et al. further confirmed this connection between HIF-1α and Ngb induction. Under the condition of knockdown or overexpression of HIF-1α, the expression of Ngb decreased or increased in cultured neural cells 47 . In addition, the overexpressed Ngb can inhibit the apoptotic cascade by promoting the activation of the pro-survival Akt pathway and regulating the induction of the anti-apoptotic Bcl-2 protein network 11,49 . Previously, we had reported that the activation of Akt/FoxO pathway by HPC contributed to the induction of neuroprotection against tGCI 3 , suggesting that the upregulation of Ngb mediated by HPC may be related to the activation of Akt pathway.
Several possible mechanisms of neuroprotection conferred by Ngb have been proposed, including oxygen sensing, ROS scavenging, regulation of signal transduction, and maintenance of the mitochondrial function 37,50-52 . Mitochondrial impairment resulted from cerebral ischemia, and reperfusion was reported to cause rapid increase of ROS 53 , and the capability of Ngb in relieving oxidative stress has been well documented 11,38 . On one hand, Ngb can be bound to the mitochondrial complex III subunit Cyc1, a component of mitochondrial respiratory chain and a major source of ROS production. The interaction between Ngb and Cyc1 can lead to the inhibition of the mitochondrial complex III activity and the suppression of subsequent ROS generation 18,40 . On the other hand, Ngb could bind to O 2 with higher affinity and change the oxidation state of its heme iron from ferric to oxygenated ferrous form to efficiently scavenge a variety of ROS 51 . Our study showed the gradually increased level of ROS in the CA1 over time after the reperfusion of tGCI and reduced level in the HPC groups. Furthermore, downregulation of the Ngb expression with Ngb AS-ODNs reversed the effect of HPC on the suppression of the ROS level.
Oxidative stress can activate pro-apoptotic signaling pathway and initiate cell damage cascade 52,54,55 . Notably, oxidative stress played a role in the regulation of NKA that is essential for a number of vital cell functions 35,56,57 . Previous research showed that oxidation regulated the activity of NKA through inducing reversible glutathionylation of Atp1b1 36 . In the current study, we found that glutathionylation of Atp1b1 gradually enhanced after tGCI, but reduced in HPC rats, exhibiting the same trend as the ROS level. Additionally, the effect of HPC on the reduction of Atp1b1 glutathionylation was dampened when Ngb was downregulated. Consequently, the reduction of Atp1b1 glutathionylation after HPC might be caused by Ngb upregulation and subsequent ROS suppression. Noteworthily, the interaction between ROS and NKA causes a conformational change of NKA 58 . Meanwhile, the production of ROS inhibits the activity of NKA. In turn, the activation of NKA signaling cascade stimulates more generation of ROS with an amplification loop 59 . Previously, we have observed a reduction in the activity of NKA and the protein level of NKA α1 subunit (see figure on previous page) Fig. 4 Effect of Ngb overexpression on Ngb expression and neuronal cells damage in CA1 after ischemia with or without HPC. A Design of experiments in which rats were stereotaxically injected bilaterally with Ngb lentiviral vectors in the dorsal CA1 pyramidal layer and subjected to either Sham-operation or tGCI with or without hypoxia. B Phase contrast and fluorescent images from the coronal sections of CA1 following injection of Ngb lentiviral vectors in Sham-operated animals. C Representative photomicrographs show the co-localization of Ngb (red), GFP (green), and 4′,6diamidino-2-phenylindole (DAPI) (blue) in the brain sections from animals at 7 days after tGCI with Lenti-Ngb injection. D Cresyl violet stained and NeuN-immunostained hippocampal sections from rats administered bilaterally with either Lenti-control or Lenti-Ngb at 7 days after reperfusion with or without hypoxia. Boxes indicate the magnified regions displayed in the right panel. E, F Quantitative analyses of the survival cells and NeuNpositive cells in the CA1. Each bar represents the mean ± S.D. *p < 0.05 vs. Sham-operated animals with injection of Lenti-control, # p < 0.05 vs. tGCI group with injection of Lenti-control (n = 6 in each group). G Representative immunoblots of Ngb expression in the CA1. The histogram presents the quantitative analyses of Ngb levels. Data are expressed as percentage of value of the Sham-operated animals. Each bar represents the mean ± S.D. *p < 0.05 vs. Sham-operated animals, # p < 0.05 vs. Sham-operated group with injection of Lenti-control, and & p < 0.05 vs. HPC group with injection of Lenti-control (n ≥ 3 in each group) after tGCI. Furthermore, hypoxic preconditioning enhanced the recovery of NKA activity by preserving NKA α1 subunit, thereby reduced DNA fragmentation after tGCI 60 . The current study reconfirmed the suppressed activity of NKA in the CA1 after ischemia, accompanied by an increase of ROS production. Also, HPC maintained the activity of NKA and reduced ROS through the upregulation of Ngb in CA1, thus exerting neuroprotection against tGCI.
Proteins that interact with Ngb, including those involved in ionic homeostasis maintenance, energy metabolism, mitochondrial function, and signaling pathways for cell survival and proliferation, may provide important clues for the exploration of the functions played by Ngb 40 . Interestingly, Atp1b1 is one of the various putative interacting partners of Ngb and their interaction had been verified in the cultured mouse cortical neuron 30 . Consistent with the expression pattern of Ngb, the level of membranous Atp1b1, rather than total or cytoplasmic Atp1b1, decreased in the CA1 after tGCI, but evidently increased with HPC. These results suggested that the increase of Atp1b1 in the membranous fraction did not result from the relocalization from cytoplasmic fraction. Additionally, the overexpression of Ngb with Lenti-Ngb increased membranous Atp1b1 in the CA1 after tGCI, whereas Ngb downregulation with Ngb AS-ODNs impeded the augment of membranous Atp1b1 and the rescue of the NKA activity resulting from HPC. Although the physiological role of Ngb-Atp1b1 interaction remains to be investigated, we speculate that the role of Ngb in maintaining membranous Atp1b1 may result from its direct interaction with Atp1b1. In our research, we did observe the interaction between Ngb and Atp1b1 in CA1, which was significantly enhanced by HPC after tGCI. It was reported that increased ROS can oxidize NKA α/β subunits, thereby promoting its susceptibility to degradation 35,56 . Hence, the maintenance of membranous Atp1b1 after HPC is more likely to be the inhibition of the degradation of Atp1b1, which may depend on HPCinduced protection of Atp1b1 from ROS. Notably, with the binding properties and (pseudo-)enzymatic properties, Ngb is able to releases O 2 to bind ROS and reactive nitrogen radicals (RNS) during cerebral ischemia 11,61 . Therefore, in addition to suppressing the ROS level, Ngb in the Ngb-Atp1b1 complex may bind ROS and shield Atp1b1 to block the oxidation of β subunit derived from ROS, thereby preserving membranous Atp1b1.
In conclusion, we have demonstrated the central role of Ngb in HPC-mediated neuroprotection against tGCI for the first time. Ngb effectively scavenged the overproduced ROS after cerebral ischemia and in turn prevented the inhibition of NKA activity that resulted from oxidative stress-modulated Atp1b1 glutathionylation. In addition, the direct interaction of Ngb with Atp1b1 in vivo may help maintain membranous Atp1b1, which is responsible for preservation of the NKA activity. Since Ngb can act as an endogenous neuroprotectant, in-depth identification of its functions and potential mechanisms would open up new areas of a non-pharmacological therapeutic intervention against cerebral ischemia.

Materials and methods
This study was performed with the approval of the Animal Care and Use Committee of Guangzhou Medical University. Adult male Wistar rats weighting 220 to 280 g (Southern Medical University, Guangdong, China) were treated in accordance with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. Animals were housed in a temperature-controlled (22°C ± 1°C) and 12 h light/dark cycle environment, fed with ad libitum food and water. All efforts had been made to minimize both the number of animals used and the suffering of the animals.
In this study, 719 rats were used, 20 of which in the tGCI group and 8 in the HPC group died during the procedure of tGCI, 8 in the tGCI group and 9 in the HPC group died during reperfusion after tGCI. Also, 9 died after intracerebroventricular injection of ODNs; another 6 died after lentiviral administration; two died during the procedure of hypoxia; another 6 rats died after anesthesia. Thirteen rats that convulsed during ischemia and 7 during 72 h after tGCI were also excluded.  6). E Representative photomicrographs of fluorescent double staining show that Atp1b1 (green) was mainly colocalized with GFAP (red) (d-f) rather than NeuN (red) (a-c) in the CA1 at 168 h after tGCI. However, Atp1b1 colocalized with NeuN (g-i), but not GFAP (red) (j-l) at 168 h after tGCI with hypoxia. Scale bar: 50 μm

Experimental models
Transient global cerebral ischemia model was produced by a four-vessel occlusion method 62 . Concisely, animals were anesthetized intraperitoneally with 10% chloral hydrate (350 mg/kg). Preoperation skin preparation was performed around the incision. Bilateral vertebral arteries were electrocauterized and blocked after exposing the alar foramen. Common carotid arteries were isolated and assembled with a teflon/silastic occluding device without blocking the carotid blood flow. Global ischemia was induced in awake animals 24 h after surgery by occluding both common carotid arteries for 10 min. Rats that found mydriasis and lost righting reflex within 1 min were enrolled for later experiments. Rectal temperature was maintained at~37-38°C throughout the whole procedure. Animals in the Sham group received the same surgical process, except occluding common carotid arteries. All operations were conducted under aseptic conditions. Rats were exposed to systemic hypoxia 24 h after tGCI for the postconditioning process 63 . Briefly, rats were placed in a sealed plastic chamber of 9000 cm 3 with a mixed gas composed of 8% O 2 and 92% N 2 for 120 min at the temperature of 23-25°C.

Immunohistochemistry
The animals were sacrificed at 0, 4, 26, 50, and 168 h after reperfusion with or without HPC (n = 6 in each group), respectively. Single-label immunohistochemistry was detected by the avidin-biotin-peroxidase complex (ABC) method 63 . Briefly, free-floating sections were soaked in 3% H 2 O 2 for 30 min and then 5% normal serum for 1 h, followed by incubation overnight at 4°C with rabbit anti-Ngb primary antibody (diluted 1:300; Proteintech Group, Inc. Chicago, IL) and a mouse antibody to Atp1b1 (diluted 1:100; Millipore, Bedford, MA). Afterwards, the sections were incubated with biotinylated secondary immunoglobulin G antibody for 2 h at room temperature. Subsequently, the sections were treated with ABC for 30 min at room temperature. Immunoreaction was visualized using 0.05% diaminobenzidine and 0.01% H 2 O 2 . Immunopositive cells, in which the reaction product was present within a clear and regular-shaped cytoplasmic or nuclear border, were quantified under a light microscope (×660). The total number of immunoreactive cells was counted by the total number of four nonrepeated random fields (0.037 mm 2 per field × 4 = 0.148 mm 2 total) in the CA1 regions.
Double-fluorescent immunohistochemistry was conducted to examine the cell types and the subcellular location of the expression of Ngb and Atp1b1. MAP-2, NeuN, and GFAP were used to identify the neuronal cell bodies and the dendrites, nuclei of neurons and astrocytes, respectively. Double-fluorescent immunohistochemistry was performed as described previously 63 . Antibodies used in this part include the mouse and rabbit anti-NeuN (diluted 1:3000; Millipore, Bedford, MA, USA), mouse and rabbit anti-GFAP (diluted 1:4000; Millipore), Cy3-conjugated goat anti-mouse and anti-rabbit IgG antibody (diluted 1:100; Millipore, Bedford, MA), FITCconjugated goat anti-rabbit antibody (diluted 1:100; Abcam, Inc., Cambridge, UK), and FITC-conjugated goat anti-mouse antibody (diluted 1:100; Millipore, Bedford, MA). Slides were analyzed with a confocal laser microscope (XP8, Leica Microsystems, Wetzlar, Hessen, Germany). The total number of Ngb-positive neurons or astrocytes was also counted within the four non-repeated random fields (0.037 mm 2 per field × 4 = 0.148 mm 2 total) in the CA1 regions.

Western blotting
The animals were sacrificed at 0, 4, 26, and 50 h after reperfusion with or without HPC (n = 3 in each group), respectively. Rats were killed under chloral hydrate anesthesia. The brain tissue was incised into 2-mm coronal slices with a brain matrix, and the CA1 regions of bilateral hippocampi were quickly divided under the stereomicroscope. The protein extraction and western blotting procedure were performed as described previously 63 . In order to detect the level of Atp1b1, membranous and cytosol protein were extracted with a membrane and cytosol protein extraction kit (Beyotime (see figure on previous page) Fig. 6 The regulation of Ngb to membrane protein of Atp1b1 in the CA1 after tGCI with or without HPC. A Immunoprecipitation blots showing the Ngb-Atp1b1 complex in the CA1 of the tGCI and the HPC groups. Atp1b1 was immunoprecipitated (IP) using anti-Atp1b1 antibody. Ngb was detected by western blot (WB). The experiments were repeated twice (n = 3 in each group). B Representative photomicrographs of proximity ligation assay showed the interaction between Ngb and Atp1b1 in the Sham-operated group (a), 26 h after reperfusion of the tGCI groups (b), and 26 h after reperfusion of the HPC groups (c). Dots (red) indicate Ngb-Atp1b1 complexes. Scale bar: 25 μm. C Quantitative analysis of PLA signal in CA1. Each bar represents the mean ± S.D. *p < 0.05 vs. Sham-operated animals and # p < 0.05 vs. tGCI group at the same time point. D Immunoblot analysis of Atp1b1 in the cytoplasmic and the membrane fraction from CA1 of Sham and HPC groups with injection of Ngb ODNs. The values are expressed as percentage of value of Sham-operated animals. *p < 0.05 vs. Sham-operated animals and # p < 0.05 vs. HPC group with S-ODNs administration (n ≥ 3 in each group). E Immunoblot analysis of Atp1b1 in the cytoplasmic and the membrane fraction from Sham-operated and tGCI group with injection of Lenti-control or Lenti-Ngb. Data are expressed as percentage of the value of Sham-operated animals. *p < 0.05 vs. Sham-operated animals, # p < 0.05 vs. Sham-operated group with injection of Lenti-control, and & p < 0.05 vs. HPC group with injection of Lenti-control (n ≥ 3 in each group)  2000; Abcam, Inc., Cambridge, UK). Densitometric analysis for the quantification of the bands was performed with the image analysis software (Quantity One, Bio-Rad Laboratories, Inc., Hercules, CA). Relative optical densities of protein bands were calibrated with GAPDH or Caveolin-1 and normalized to those in the Sham-operated rats.

Assessment of cellular damage
Animals were sacrificed at 7 days after tGCI, followed by perfusing intracardially with 0.9% saline and 4% paraformaldehyde in phosphate-buffered saline (PBS). The brain tissues were postfixed in 10, 20, and 30% sucrose in the same fixative for cytoprotection. After postfixation, the brains were frozen at −20°C and sliced into coronal 30-μm-thick sections with cryotome (Leica, Wetzlar, Hessen, Germany). Sections selected from the dorsal hippocampus (between AP 4.8 and 5.8 mm, interaural or AP −3.3 to 3.4 mm, bregma) were used for Nissl, NeuN, and FJ-B staining, which was carried out according to a protocol described in our previous study 3 .
Nissl-and NeuN-stained sections were observed under a light microscope with ×660 magnification. The survived cells displayed well-stained Nissl bodies, whereas the damaged cells were either swollen with loss of stainable Nissl material or necrotic with deeply staining dendrites fragmented. FJ-B- Fig. 8 Effect of HPC on the activity of Na + /K + -ATPase in CA1 after tGCI. A Quantitative analysis of the NKA activity in the CA1 subregion in Sham, tGCI, and HPC groups. B Quantitative analysis of the NKA activity in the Sham and the HPC groups with or without ODNs administration. C Quantitative analysis of the NKA activity in the Sham and the tGCI groups with or without lentiviral administration. Data are expressed as percentage of the value of Sham-operated animals. Each bar represents the mean ± S.D. *p < 0.05 vs. Sham-operated animals, # p < 0.05 vs. tGCI group, & p < 0.05 vs. HPC group with injection of S-ODNs, and △ p < 0.05 vs. tGCI group with injection of Lenti-control (n ≥ 4 in each group) stained images were observed with a fluorescence microscope (Leica Microsystems, Wetzlar, Hessen, Germany). Cell counts were conducted by densities, as previously described 64 . Cells in the CA1 pyramidal layer were quantitatively analyzed within three non-repeated rectangular areas of 0.037 mm 2 . Data were quantified bilaterally in sections from each brain and assessed double-blindedly. Also, four sections for each animal were evaluated.

Drug administration
Sequences for Ngb AS-and S-ODNs were referenced to the report by Sun et al. 41 . The sequence of Ngb AS-ODNs was: 5′-TCCGGGCGCTCCAT-3′ (nucleotides 90-77) and Ngb S-ODNs was: 5′-ATGGAGCGCCCGGA-3′ (nucleotides 77-90). AS-ODNs and S-ODNs were synthesized with a phosphorothioate backbone, purified with ULTRAPAGE (Sangon, Inc. Shanghai, China), and dissolved with aCSF. Solution in volume of 8 µl (16 µmol/l) was injected over a 10-min period each time intracerebroventricularly 30 min before tGCI and HPC. For Sham-operated rats, ODNs or aCSF was administrated twice at the same time point in 2 days after operation.

Lentivirus construction and lentiviral administration
Plasmids containing the sequence of rat Ngb (GenBank  accession number NM_85382), CGCCACCATGTGCGT  TGACGCCGTGCCCACGCCTCGAGGGTCCCATCAC  TGCGTCCCGCGACTCTCCTGGGAGAGAGAGAGT  ATGGAGCGCCTAGAGTCAGAGCTGATCCGGCAG  AGCTGGCGGGCAGTGAGCCGCAGCCCTCTGGA  ACATGGCACTGTCCTGTTCTCCAGGCTCTTCGCC  CTGGAACCCAGCCTGCTGCCTCTCTTCCAGTACA  ATGGCCGCCAGTTCTCCAGTCCTGAGGACTGTCT  CTCCTCTCCAGAATTCCTGGACCACATTAGGAAG  GTTATGCTTGTGATTGATGCTGCTGTGACCAACG  TAGAGGACCTGTCTTCACTGGAGGAGTACCTGG  CCACCTTGGGCAGGAAGCATCGGGCAGTGGGAG  TGAGGCTCAGCTCCTTCTCGGTGGGTAGTGGAAC  AGTAGGTGAGTCCCTGCTCTACATGCTGGAGAAG  TGCCTGGGTCCCGACTTTACGCCGGCTACAAGGA  CCGCCTGGAGCCAACTCTACGGAGCTGTGGTGCA AGCCATGAGCCGAGGCTGGGACGGGGAG, and a negative control sequence were designed by Genechem (Shanghai, China). The sequence was inserted into AgeI and BamHI sites of the Ubi-MCS-3FLAG-CMV-EGFP (GV365) lentiviral vector, according to the manufacturer's instruction. The shuttle vector and viral packaging system were cotransfected into HEK293T cells to produce recombinant lentiviruses using Lipofectamine 2000 (Invitrogen, San Diego, CA, USA). Then HEK293T cells were used for viral infection. The infection efficiency was greater than 80%, as monitored with GFP protein expression. After 48 h of infection with Lenti-Ngb, the cells were harvested and the total RNA were extracted to examine the expression of Ngb mRNA. The titers were approximately 1 × 10 9 TU/ml.
A total of 5 μl volume (1.25 μl virus diluted by 3.75 μl enhanced solution) containing 1.25 × 10 9 TU/ml of particles was injected into the bilateral hippocampal CA1 region (3.5 mm posterior to bregma, 2.3 mm lateral to bregma, and 2.6 mm below the dura) using the RWD syringe and WPI infusion pump at a speed of 30 μl/h. Rats were accepted for four-vessel occlusion surgery or Shamoperation 14 days after administration.

Membrane protein extraction
The animals were sacrificed at 0, 4, 26, and 50 h after reperfusion with or without HPC, respectively. Tissue samples of the hippocampal CA1 subregion were isolated as described previously 8 . Membrane protein of the CA1 subregion was extracted via the tissue membrane protein prep kit (GenMed Scientifics Inc. USA), according to the manufacturer's protocol. After washed by Reagent A, 1 g tissues were homogenized with 4 ml working solution containing 4 ml of Reagent B and 5 μl Reagent C, followed by centrifugation at 1000 g for 10 min at 4°C. Then 1 ml of Reagent B was added in the precipitates, followed by centrifugation again. Supernatants after twice centrifugation were incubated with 5 ml of Reagent E on ice for 60 min, followed by centrifugation at 20,000 × g for 60 min at 4°C. After vibrated for 15 s with 1 ml of Reagent F, the precipitates were stored at -80°C before the use in co-immunoprecipitation assay.

Co-immunoprecipitation
For co-immunoprecipitation assay, 150 μg membrane proteins were incubated with mouse anti-Atp1b1 monoclonal antibody (diluted 1:200; Millipore, Bedford, MA) at 4°C overnight. The protein/antibody complex was added to packed protein G agarose beads (Millipore, Bedford, MA, USA). Following 4 h of incubation at 4°C, the complex was washed five times with PBS containing 1% Tween. Then immunocomplexes were separated by incubation with protein G agarose beads and were resolved by SDS-PAGE. Immunoblot assay was performed with rabbit anti-Ngb antibody (diluted 1:200; Proteintech Group) and mouse anti-Atp1b1antibody (diluted 1:5000; Millipore, Bedford, MA).

Proximity ligation assay
Proximity ligation assay was carried out with Duolink® In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma-Aldrich), according to the manufacturer's protocol. Briefly, the sections selected from the dorsal hippocampus were soaked in blocking solution for 1 h and then incubated overnight at 4°C with rabbit anti-Ngb antibody (diluted 1:100; Proteintech Group, Inc. Chicago, IL) and mouse anti-Atp1b1 (diluted 1:20; Millipore, Bedford, MA). After labeling with PLUS and MINUS PLA probes for 1 h, the sections were incubated with Ligation-Ligase solution and Amplification-Polymerase solution at 37°C. PLA signals were detected with a fluorescence microscope (Leica Microsystems, Wetzlar, Hessen, Germany) and the images were analyzed with ImageJ.

ROS measurement
A slight modification of ROS measurement described by Matsushima et al. was used 65 . The animals were sacrificed at 0, 4, 26, 50, and 168 h after reperfusion with or without HPC and 168 h after HPC with Ngb AS-and S-ODNs administration (n = 6 in each group), respectively. After harvest, the brain tissues were immediately stored at −80°C for 2 h. Then the unfixed frozen brains were cut into 30-µm-thick sections and placed on glass slides. The oxidation-sensitive fluorescent dye DCFH-DA (4.44 μg/ ml) (Molecular Probes, Inc. Oregon, USA) was applied to each tissue section, and then the sections were coverslipped. The slides were incubated in a light-protected humidified chamber at 37°C for 35 min. Green ROS fluorescence was observed with a fluorescence microscope (Leica Microsystems, Wetzlar, Hessen, Germany).
Glutathionylation of Na + /K + ATPase β 1 subunit The glutathionylation of Atp1b1 was detected according to the method in the previous research 34 . Briefly, the membrane proteins were mixed with anti-GSH antibody (diluted 1:100; Virogen, Watertown, MA) at 4°C overnight to isolate the glutathionylated proteins. Then, the proteins were mixed with packed protein G agarose beads (Millipore, Bedford, MA, USA) which had been pre-washed by cold PBS (12,000 × g, 3 min, 3 times, 4°C). After incubation at 4°C for 4 h, the beads were washed with cold PBS (12,000 × g, 5 min, 5 times, 4°C). Then 15 μl of loading buffer was added to the beads and the mixture was heated at 37°C in a water bath for 15 min to be used for immunoblotting. Last, Atp1b1 in the immunoprecipitate was identified with western blotting to show the glutathionylation of Atp1b1.

Measurement of the Na + /K + -ATPase activity
With minor modifications, the Na + /K + -ATPase activity was measured according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), as described previously 60 . Briefly, the tissues from the CA1 subregion of rats sacrificed at 0, 4, 26, 50, and 168 h after reperfusion with or without HPC, Sham-operated or 26 h after reperfusion in the HPC group with Ngb AS-or S-ODNs administration, and Sham-operated or 26 h after reperfusion in the tGCI group with Lenti-control or Lenti-Ngb administration were homogenized in 0.86% sodium chloride solution. After centrifugation at 1000 rpm for 5 min, the supernatant was used for the reaction of Na + /K + -ATPase assay. All samples were run in duplicate. Specific activity of the enzyme was expressed as nmol Pi released per min per mg of protein. For the final calculation, the activity of Na + /K + -ATPase in each group was expressed as a percentage of the Sham-operated control.

Statistical analysis
Data analysis was performed with Statistical Package for Social Sciences for Windows, version 13.0 (SPSS, Inc, Chicago, Illinois, USA). All variables were expressed as mean ± standard deviation. The statistical significance was determined by one-way analysis of variance (ANOVA) or two-tailed Student's t-test. The differences were considered statistically significant when p < 0.05.