extfieldsofstudy: Biology Medicine

text:
Effect of lysophosphatidic acid on the follicular development and the expression of lysophosphatidic acid receptor genes during in vitro culture of mouse ovary.

Lysophosphatidic acid (LPA) known as a serum-derived growth factor, is involved in several cell physiological functions in the female reproductive system including: oocyte maturation, in vitro fertilization and embryo implantation by its transmembrane G protein-coupled receptors. The aim of the present study was to examine the effect of LPA on in vitro follicular development of mouse ovarian tissue. Neonatal mouse ovarian tissues were cultured in five different concentrations of LPA (0, 5, 10, 20 and 40 µM). The developmental competence and the function of cultured ovarian tissue were assessed by morphological study using hematoxylin and eosin staining and hormonal analysis. The expression of LPA receptor (LPAR 1-4) genes were analyzed by real-time RT-PCR. The proportion of preantral follicles and the level of E2 hormone were significantly higher in the 20 µM LPA-treated group than those in the other treatment groups. There was a significant difference in the expression of LPAR 1-4 genes in 20 µM LPA treated group in comparison with 0 µM LPA (control group) treated and non-cultured groups. In addition, the expression of LPAR1 gene was higher than other receptor genes in all studied groups. In conclusion supplementation of the media with 20 µM LPA, could improve the survival and developmental potential of follicles and it had positive effects on cell function and stimulation of E2 synthesis in mouse whole ovarian tissues.

Introduction
In vitro culture (IVC) of ovarian tissue has been introduced as an alternative approach for follicular development in the field of reproductive biology. [1][2][3][4] However, the low survival rate of follicles, maturation rate of oocytes and embryo development was published and up to now no pregnancy was recorded in human. 5 Therefore, the improvement of in vitro culture of ovarian tissue is challenging because it isn't defined the optimal maturation condition (physical and biochemical condition) for IVC of mouse ovarian tissue. 6,7 Many researchers have also been committed to trigger the activation of primordial follicles and oocyte maturation by adding some supplements to culture media such as growth factors, antioxidants, gonadotropins, nutrients. 5,[8][9][10] Lysophosphatidic acid (LPA) is a phospholipid with 430-480 Da molecular weight 11 that was detected in the blood serum. It is produced by cell components of follicle in all stages of follicular development. 12 It is known as a serum-derived growth factor, involving in several physiological functions of the cells in the female reproductive system [13][14][15][16] including: oocyte maturation, in vitro fertilization and embryo implantation. 17-20 LPA mediates these functions by transmembrane G proteincoupled receptors. 21 It has been shown that following LPA binding to its receptors, multiple signaling pathways can be activated via variety heterotrimeric G proteins subtypes. 20,[22][23][24][25] Previous studies demonstrated that the specific cell surface receptors of LPA (LPARs) are expressed in oocyte, cumulus cells, endometrial cells and mast cells. 18,[25][26][27][28][29] Available data regarding LPA receptor in the ovarian tissues and cells demonstrated that mRNA of LPAR1, LPAR2 and LPAR4 was detected in mouse ovarian tissues, 30,31 while the expression of LPAR3 is not demonstrated in mouse ovary. 31,32 It may be happened because LPAR3 participates in the initiation of downstream signaling cascades though main cellular mechanisms. 27,[33][34][35][36][37][38][39][40] To the best of our knowledge, there is not sufficient information regarding to the effects of different concentrations of LPA on the follicular development of mouse ovarian tissue. Since the biological effects of LPA may be concentration dependent, therefore, in the present study, the survival and development of mouse ovarian follicles were investigated using different concentrations of LPA.
The results of this work can be used to provide better culture conditions for in vitro culture of human primordial follicles. Furthermore, the present study updates our knowledge on the effect of LPA on the expression of LPA receptor genes in neonatal and postnatal mouse ovaries.

Materials and Methods
Chemicals. All supplements were acquired from Sigma-Aldrich (Dusseldorf, Germany) except otherwise indicated.
Animals and ovarian tissue. In this experimental study, the ovaries were collected from neonatal (7-dayold) National Medical Research Institute (NMRI) mice that were kept under a controlled condition (22.00 ± 2.00 ˚C, 12 hr light: 12 hr dark and 40 to 50% humidity) in the animal house of Tarbiat Modares University. These experiments were performed according to the ethical guidelines for the Care and Use of Laboratory Animals in Tarbiat Modares University (Ref No: 52.8188). The mice (n = 45) were sacrificed by cervical dislocation, and their ovaries were removed and dissected mechanically and washed in alpha-minimal essential medium (α-MEM; Invitrogen, Paifley, UK) supplemented with 5.00% fetal bovine serum (Invitrogen), 100 IU mL -1 penicillin (Invitrogen), 100 µg mL -1 streptomycin (Invitrogen), 0.23 mMol sodium pyruvate (Sigma-Aldrich) and sodium bicarbonate (Sigma-Aldrich).
Experimental design. The collected ovaries were randomly divided into six groups (n = 15 in each group and in three repeats). The ovaries were cultured in α-MEM supplemented with different concentrations of LPA (0, 5, 10, 20 and 40 µM). 19 One week after in vitro culture of mouse ovaries, the morphology of cultured ovaries were studied using hematoxylin and eosin staining and the expression of genes related to folliculogenesis and LPA receptors were evaluated by real time RT-PCR.
In vitro culture of ovarian tissue. The whole ovaries in all studied groups (n= 15 in each group) were cultured on inserts (Millicell-CM, 0.4-m pore size; Millipore Corp, Billerica, USA) in 24-well plates including 400 µL of basic culture medium consisted of α-MEM supplemented with 1% insulin, transferrin and selenium (ITS; Gibco), 5.00% fetal bovine serum, 100 mIU mL -1 recombinant FSH (rFSH Serono. Laboratories, Aubonne, Switzerland) and 5, 10, 20 and 40 µM LPA at 37 ˚C in a 5.00% CO2 for one week. Half of the culture medium (200 µL) was renewed with fresh medium every other day and the collected media were reserved at -20 ˚C for evaluation of 17-βestradiol hormone.
Histological evaluation. Histological features of the ovarian tissues (n = 5 in each group) were evaluated by hematoxylin and eosin staining. The ovaries were fixed in Bouin's fixative for eight hr. After fixation, the ovaries were embedded in paraffin and the tissue sections were prepared serially at 5 µm-thickness and subsequently mounted on slides with 5 th intervals and stained with routine hematoxylin and eosin method. The morphology of ovarian tissues and the number of ovarian follicles were assessed under light microscope field by field. Classification of ovarian follicles (primordial, primary, preantral and antral) was performed according to the previously described method. 41 The follicles containing germinal vesicle in the oocyte were counted before and after seven days in vitro culturing. The normal follicles had intact oocyte and granulosa cells and the degenerated follicles had pyknotic oocyte nuclei and disarranged granulosa cells.
Ovarian surface area. The micrographs of each ovary at the same magnification during culture period (on days 0, 3, 5 and 7 of culture) were captured and the area of each ovary was analyzed and determined in cubic micrometers (µm 2 ) by Digimizer software (MedCalc Software bvba, Acacialaan, Belgium).
RNA extraction and cDNA synthesis. Total RNA was extracted from 0 and seven days cultured ovaries (n = 3 in each group in three repeats) using the trizol reagent (Invitrogen) according to procedure described by the manufacturer's instructions RNeasy Mini Kit (Qiagen, Hilden, Germany). Briefly, the ovaries were homogenized in 0.5 mL of trizol reagent then, 0.2 mL of chloroform was added per one mL of trizol and the samples were centrifuged at 12000 g for 10 min. The upper colorless aqueous phase was transferred to a fresh two mL microtube and 700 μL of 100% isopropanol (Sigma-Aldrich) were added and centrifuged at 12000 g for 10 min. Then 1.5 mL 70% ethanol (Merck, Dusseldorf, Germany) per one mL of trizol reagent was added. RNA concentration was determined using spectrophotometry (Eppendorf, Hamburg, Germany).
The cDNA was synthesized using cDNA synthesis kit (ThermoFisher Scientific, Lithuania, USA) according to the manufacturer's instructions. Oligo(dT) was used for cDNA synthesis and reverse transcriptase reaction was incubated at 42 ˚C for 60 min. After inactivation of the reverse transcriptase enzyme, the resultant product was stored at -20 ˚C.
Real-time RT-PCR. The primers for LPAR1-4 genes were designed using Primer-BLAST tool in NCBI ( Table 1). The designed primers were analyzed using Oligo-Analyser. 42 In the present study β-actin gene was considered as housekeeping gene. Real-time RT-PCR was performed using QuantiTect SYBR Green RT-PCR kit (Qiagen). Thermal program of the real-time RT-PCR was set with the following parameters: the initial holding stage for 5 min at 95 ˚C, 40 cycles cycling stage of 15 sec at 95 ˚C, 58 ˚C for 30 sec, and 72 ˚C for 15 sec, that was continued by a melt curve stage at 95 ˚C for 15 sec, 60 ˚C for 1 min, and 95 ˚C for 15 sec. After completing the PCR run, the melt curve was obtained using the amplicon. For determining of relative quantitation of target genes the Pfaffl method was used. 43 Hormonal assay. For the evaluation of the endocrine function of ovaries, the concentration of 17-β-estradiol (E2), was measured in the collected media obtained from cultured ovaries on days 3 and 7 (n = 15 per groups in three repeats) using microplate enzyme immunoassay kit (Monobind Inc., Lake Forest, USA) with sensitivity = 6.5 pg mL -1 ). 44 Statistical analysis. Statistical analysis was performed using SPSS (version 21.0; IBM Corp., Armonk, USA). Values are given as mean ± SE. One-way ANOVA and post hoc Tukey was used to compare differences in follicular count, ovarian area and mRNA expression. The data of hormone level was analyzed with Student's t-test. A p value less than 0.05 was considered as statistically significant at the 95.00% confidence level.

Results
Morphology of ovaries and follicular count. The morphological observations of cultured ovaries under invert microscope in different groups of study were shown in Figure 1. Our results demonstrated that the follicles were grown more on the periphery of ovarian tissues and they had a large and clear antral cavity. The growth of follicles was parallel with increasing in the size of ovaries during the culture period. However, in the central parts of the ovaries the density of the tissue was higher than marginal and central parts of tissue was dark.
Light microscopy. The light microscopic observations of examined groups using H&E staining are shown in Figure 2. Before in vitro culturing, the primordial follicles were mainly seen and located in the peripheral part of the ovaries. After seven days of ovaries culture, the primordial, primary and antral follicles were prominent within the ovarian tissue (Fig. 2). The healthy follicles with normal structure were visible in all examined groups especially in 20 µM LPA supplemented group (Fig. 2: D1-D3). The granulosa cells of follicles arranged well around the oocyte. The damage follicles (pyknotic nuclei oocytes and disarranged granulosa cells) were prominent (Fig. 2: A1-A3) especially in the central area of ovaries that were cultured in the absence of LPA (control group; Fig. 2). In addition, in 40 µM LPA-treated group some premature antral follicles were seen (Fig. 2: E1-E3).  The number of normal follicles in the examined groups. The percentages of normal and atretic follicles at different developmental stages at the end of seven days of culture were summarized in Table 2. Before in vitro culturing, the proportion of primordial follicles were higher than other stages of follicles but after seven days culture, the percentage of primordial and primary follicles were decreased and the percentages of preantral follicles were significantly increased in all groups (p < 0.05). The percent of preantral follicles was significantly higher in the concentration of 20 µM LPA than other groups (p < 0.05; Table 2).
Ovarian surface area. The analysis of ovarian surface area data in all groups of study during in vitro culture was shown in Figure 3. The mean surface area was significantly increased during seven days culture (Fig. 3) and the surface area was markedly higher in the 20 µM LPAtreated group than those of other groups (p < 0.05). Real time RT-PCR results. The expression ratio of the LPA receptors genes to β-actin gene in non-cultured, 20 µM LPA-treated and without LPA cultured (control) groups were demonstrated and compared in Figure 4A to 4D. As the results shown, the expression ratio of LPAR1-4 genes in non-cultured group were 0.27 ± 0.04, 0.21 ± 0.04, 0.21 ± 0.03, 0.15 ± 0.01 in control cultured groups were 0.20 ± 0.03, 0.21 ± 0.01, 0.31 ± 0.01, 0.22 ± 0.01 and in 20 µM LPA-treated cultured group were 0.35 ± 0.02, 0.26 ± 0.01, 0.43 ± 0.01, and 0.35 ± 0.02, respectively (p < 0.05).
There was no significant difference between control cultured group and non-cultured group. However, there was a significant difference between 20 µM LPA treated cultured group and other two groups (p < 0.05). The results demonstrated that the expression of LPAR1 gene was higher than other genes in all examined groups (p < 0.05).
Hormonal assay. The concentrations of E2 hormone obtained from collected media in different groups on days three and seven of culturing period are presented in Table 3. There was a significant difference in the level of E2 hormone on day three of culture between two groups (p < 0.05). The level of E2 hormone significantly increased in 20 µM LPA-treated group in comparison with control group (p < 0.05).

Discussion
The findings of the present study confirmed that the supplementation of the mouse ovarian organ culture medium with 20 µM LPA could lead to achieve the higher percentages of follicular development (up to preantral stage), increase the mean surface area of ovaries and enhance the function of granulosa and theca cells by production of higher levels of 17-β-estradiol in comparison with the other groups. These results revealed that the LPA could stimulate the growth of primordial and primary follicles to preantral follicle stage and it seems these effects of LPA are dose dependent. The follicular growth rate was gradually improved during in vitro culture up to 20 µM concentration of LPA then this rate was decreased at concentrations of 40 µM of LPA. The previous studies postulated that LPA involved in the cell survival, activation of entire primordial follicle pool and promotion nuclear and cytoplasmic maturation of mouse oocytes by binding to its receptors and involving in the MAPK (mitogenactivated protein kinase)/p38 and PI3K (phosphoinositol 3-kinase)/Akt pathways. 16 23) The percentage of follicles at different developmental stages ovaries before and after culturing in the presence of different doses of LPA. a indicates significant differences with before cultured ovaries in different developmental stage (p < 0.05) and b indicates significant with other groups after culture (p < 0.05). that treatment of mouse oocyte culture media with 30 µM LPA, could significantly enhances the maturation rate of oocyte, the fertilization rate and subsequently the developmental rate of blastocyst. 19 According to Boruszewska et al., the supplementation of bovine oocyte maturation medium with LPA increases mRNA abundances of genes involved in bovine oocyte maturation (FST and GDF9). 46 They confirmed that LPA plays a potential autocrine and/or paracrine role between the bovine oocytes and granulosa cells, via several LPARs. However, they did not see any significant influence of LPA on maturation rate of oocytes. They concluded that mRNA level the usage of low concentration of LPA can affect the expression of gene related to maturation of oocytes. 46 In this study, we examined the expression of several LPA receptors genes in cultured mouse ovaries. Our results showed that in the ovarian tissues of this strain of NMRI mouse, all four types of LPARs (1-4) were expressed at the mRNA level. But there are controversial reports related to the expression of LPA receptors in mouse ovarian tissue. 32 32 While our study is the first to demonstrate that the four types of LPA receptors were expressed before and after in vitro culture (in the presence and absence of LPA) in mouse ovarian tissue. These different observations may be due to the mouse strain which have been used.
The LPA-LPARs binding activates various types of Gproteins and thereby induces signal intracellular transduction cascade and stimulates many vital pathways. 23 These signaling key pathways regulate many essential processes including cell proliferation, survival and growth, cytoskeletal rearrangement and primordial follicles activation through multiple downstream molecules. 24,27,48,49 In addition, in this study it was demonstrated that LPA stimulated granulosa and theca cell to produce 17-βestradiol (functional marker) in mouse ovarian tissues that may promotes ovarian follicular development. Moreover, the levels of 17-β-estradiol was significantly enhanced in 20 µM LPA supplemented group. Boruszewska et al. postulated that LPA stimulated 17-βestradiol synthesis in bovine granulosa cells that correlated with increasing in the follicle stimulating hormone receptor and 17-β-hydroxy steroid dehydrogenase mRNA expression. In addition, they documented this stimulatory effect of LPA enhanced in the presence of follicle stimulating hormone and thereby, induced the expression of cytochrome P450 aromatase genes that convert the androgens to 17-β-estradiol in thecal cells. 50 In conclusion, this study demonstrated that the addition of LPA to the IVC media, especially at 20 µM, could improve the survival and developmental potential of follicles in mouse whole ovarian tissues after 7 days of culture. Furthermore, the present study updates our knowledge on gene expression of LPA receptors in neonatal and postnatal mouse whole ovaries that may Fig. 4. A, B, C, and D) The mRNA expression of LPA receptor genes (LPAR 1, LPAR 2, LPAR 3, LPAR 4) before and after in-vitro culture (0 and 20 µM LPA-treated groups) of mouse ovarian tissues. * indicates a significant difference with non-cultured group.
have positive effects on cell function and stimulation of 17β-estradiol synthesis.


extfieldsofstudy: Medicine Chemistry

text:
Trans-Ferulic Acid-4-β-Glucoside Alleviates Cold-Induced Oxidative Stress and Promotes Cold Tolerance

Trans-ferulic acid-4-β-glucoside (C16H20O9, TFA-4β-G) is a monomer extracted from the Chinese medicine called radix aconiti carmichaeli (Fuzi). To date, research on this substance is lacking. Here, we found that trans-ferulic acid-4-β-glucoside effectively promoted cold acclimatization in mice via increased heat production and alleviation of oxidative stress in a cold environment. Thus, our work indicates that ferulic acid-4-β-glucoside is a potential therapeutic candidate for prevention and treatment of cold stress injury.

Introduction
When exposed to a cold environment, the body activates metabolic organs through a series of different metabolic pathways to increase heat production and reduce heat loss, thus maintaining a relatively constant temperature and protecting the cells from cold stress injury. Generally, short-term cold exposure firstly induces skeletal muscle tremors to increase energy expenditure. By shivering, heat production can increase by five times compared with the baseline level [1,2]. Besides shivering thermogenesis, non-shivering thermogenesis (NST) is essential for increasing energy expenditure, especially during long-term cold exposure. Non-shivering thermogenesis causes higher energy expenditure mediated by burning brown adipose tissue and increasing mitochondrial uncoupled protein 1 (UCP1), gluconeogenesis, glycogen consumption in liver, and so on [3]. Furthermore, white adipose tissue stores triglycerides in the form of large fat droplets to increase energy storage. Conversely, with a multi-lacunar vesicle structure, brown adipose tissue has many more mitochondria and highly expresses UCP1 protein, thus uncoupling the oxidative phosphorylation pathway to interrupt ATP synthesis and releasing a large amount of energy to produce heat [4,5]. Moreover, liver-one of the most important organs in the human body-is largely involved in the metabolic process. Hepatic glucose production accounts for 90% of the endogenous glucose production [6], which is critically important to maintain the balance of glucose metabolism. Gluconeogenesis is a process that contributes to maintaining blood glucose homeostasis: the liver transforms glycerol, glycogenic amino acid, and other non-glucose substances into glucose, and this process is closely related to liver lipolysis [7].
Prolonged or intermittent repeated exposure to low temperatures enhances the body's ability to acclimate to cold, meaning that the proportion of shivering thermogenesis decreases, while the proportion of non-shivering thermogenesis increases [8]. In 1961, Davis found the phenomenon in the human body: Prolonged regular cold exposure raised the level of non-shivering thermogenesis and resulted in cold adaptation [9]. One of the characteristics of cold acclimation is the increase of heat production induced by cold, usually accompanied with higher skin temperature but with relatively normal rectal temperature [10].
Oxidative stress is a common phenomenon that occurs in organisms faced with certain external stimuli. Once oxidation and antioxidation are out of the homeostatic state, reactive oxygen species (ROS) are overproduced. Excessive ROS-i.e., beyond the cell's antioxidation capacity-causes oxidative stress in cells [11], and even causes other metabolic-related diseases [12]. Short-term exposure to cold can induce oxidative stress in warm-blooded mammals [13,14]. Lomakina found that the level of MDA (malondialdehyde) in the brain increased after exposure to cold for 24 h in rats [15]. In addition, Kaushik and Kaur found that rats' brain, heart, liver, kidney, and small intestine emerged from oxidative stress after exposure to 7-8 • C with their levels of MDA increased significantly [16]. Other studies found that exposure to cold for 2 days could significantly raise HPS (hydroperoxides) levels in rats' liver, skeletal muscle, and myocardium [17]. All these results indicate that cold exposure causes oxidative stress in many organs, further revealing that oxidative stress is widespread after cold stimulation.
The Chinese medicine radix aconiti carmichaeli (Fuzi) promotes sweating and defends the body from cold. However, the underlying mechanism remains unclear. To determine the key substance involved, we conducted purification experiments and obtained trans-ferulic acid-4-β-glucoside (C 16 H 20 O 9 ), which has been relatively little studied. Our research found that trans-ferulic acid-4-β-glucoside promotes energy consumption and improves cold tolerance under low-temperature (4 • C) conditions. Furthermore, this substance may mainly be involved in liver and adipose tissue metabolism. Trans-ferulic acid-4-β-glucoside can significantly prevent cold-induced oxidative stress in liver and upregulate the protein level of UCP1 in brown fat tissue.

Trans-Ferulic Acid-4-β-Glucoside Improves Cold Tolerance in Mice under Cold (4 • C) Condition
We established an animal model ( Figure 1A,B) and found that the rectal temperature of mice exposed to cold decreased and was maintained at a relatively low level with great fluctuation. Mice treated with trans-ferulic acid-4-β-glucoside had a greatly increased rectal temperature, which was maintained at a relatively high level in a cold environment ( Figure 1C, p < 0.05). Long-term cold exposure dramatically increased food intake in untreated mice ( Figure 1D, p < 0.05). However, there was no difference in food intake between mice treated with trans-ferulic acid-4-β-glucoside and the control group ( Figure 1D,E, p > 0.05). The body weight and blood biochemical parameters did not significantly alter, indicating that trans-ferulic acid-4-β-glucoside had no toxicity toward the liver and kidneys ( Figure 1F and Figure S1F, p > 0.05). Intermittent-cold-exposed mice with this treatment showed the same trend ( Figure S1A

Trans-Ferulic Acid-4-β-Glucoside Alleviates Cold-Induced Oxidative Stress in Liver
The indicators of oxidative stress, ROS, include the superoxide anion (O − 2 ), hydroxyl radical (•OH), and hydrogen peroxide (H 2 O 2 ). At the same time, the organism itself can combat the occurrence of oxidative stress using an antioxidant enzyme system, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and so on. We detected oxidative stress injury indicators in liver, and the results revealed that long-term cold exposure increased the level of H 2 O 2 in liver, while reducing the levels of SOD and GSH-Px. Moreover, trans-ferulic acid-4-β-glucoside could significantly increase the level of antioxidation, and thus alleviate oxidative stress (Figure 2A, p < 0.05). However, this phenomenon was not found in the heart and kidney ( Figure 2B,C, p > 0.05).

Trans-Ferulic Acid-4-β-Glucoside Alleviates Cold-Induced Oxidative Stress in Liver
The indicators of oxidative stress, ROS, include the superoxide anion (O − 2), hydroxyl radical (•OH), and hydrogen peroxide (H2O2). At the same time, the organism itself can combat the occurrence of oxidative stress using an antioxidant enzyme system, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and so on. We detected oxidative stress injury indicators in liver, and the results revealed that long-term cold exposure increased the level of H2O2 in liver, while reducing the levels of SOD and GSH-Px. Moreover, trans-ferulic acid-4-β-glucoside could significantly increase the level of antioxidation, and thus alleviate oxidative stress (Figure 2A, p < 0.05). However, this phenomenon was not found in the heart and kidney ( Figure 2B,C, p > 0.05).

Trans-Ferulic Acid-4-β-Glucoside Activates Brown Adipose Tissue and Promotes Thermogenesis
Cold exposure increases the protein levels of UCP1 in brown adipose tissue, as well as induces thermogenesis to fight cold. The results of real-time PCR and Western blot consistently indicated that trans-ferulic acid-4-β-glucoside administration upregulated the expression of UCP1 in brown adipose tissue under cold conditions ( Figure 4B, p < 0.05; Figure 4C, UCP1, p < 0.01; PGC1α, p < 0.001), which is not obvious in inguinal WAT ( Figure S1I,J). At the same time, HE staining showed that the lipid droplets of the brown fat were smaller with treatment ( Figure 4A). Intermittent-cold-exposed mice treated with trans-ferulic acid-4-β-glucoside showed the same trend ( Figure S1G,H). These results suggest that trans-ferulic acid-4-β-glucoside promotes the activation of brown adipose tissue and increases heat production.

Trans-Ferulic Acid-4-β-Glucoside Activates Brown Adipose Tissue and Promotes Thermogenesis
Cold exposure increases the protein levels of UCP1 in brown adipose tissue, as well as induces thermogenesis to fight cold. The results of real-time PCR and Western blot consistently indicated that trans-ferulic acid-4-β-glucoside administration upregulated the expression of UCP1 in brown adipose tissue under cold conditions ( Figure 4B, p < 0.05; Figure 4C, UCP1, p < 0.01; PGC1α, p < 0.001), which is not obvious in inguinal WAT ( Figure S1I,J). At the same time, HE staining showed that the lipid droplets of the brown fat were smaller with treatment ( Figure 4A). Intermittent-cold-exposed mice treated with trans-ferulic acid-4-β-glucoside showed the same trend ( Figure S1G,H). These results suggest that trans-ferulic acid-4-β-glucoside promotes the activation of brown adipose tissue and increases heat production.  (C) protein level of genes related to heat production in BAT. * p < 0.05, ** p < 0.01, and *** p < 0.001 as indicated.

Discussion
As a common stimulus in the environment, cold exposure causes a series of metabolic changes in the body [18]. These changes play an important role in the formation of cold adaptation. The ability, or lack thereof, to adapt to cold determines the living conditions of mammals to a great extent, particularly in cold environments [19]. Recent studies have revealed that cold acclimation is related to the concomitant increase in brown adipose tissue (BAT) activity [8,20], as well as liver metabolism [21]. As a traditional Chinese medicine, radix aconiti carmichaeli (Fuzi) promotes heat production and protects from cold [22,23]. As the major ingredient from radix aconiti carmichaeli (Fuzi), the aconitum alkaloids mainly produce their pharmacological effects by regulating heart and blood vessel functions [24]; however, the key active ingredients from radix aconiti carmichaeli (Fuzi) which regulate BAT activity and liver metabolism in cold acclimation are still unknown. In order to elucidate the key substance that plays a role in this effect, we got a new active ingredient which is purified from Fuzi. The chemical structure is made up of ferulic acid and glucoside. Li Xia et al. found that sodium ferulate improves energy metabolism and plays a protective role after myocardial ischemia reperfusion injury, while Wu et al. suggested that ferulic acid protects the heart from lesion by inhibiting the apoptosis of mitochondria [25]. Pan Yiou et al. found that ferulic acid inhibits lipid deposition in obese mice by mediating the expression of some important lipid metabolism genes. These findings suggest that ferulic acid may participate in the process of energy metabolism and increase the body's energy expenditure to fight obesity, however, these studies did not assess its role in liver and adipose tissue. In our study, the active ingredient trans-ferulic acid-4-β-glucoside promotes cold acclimation with increased fatty acid oxidation in liver and heat production in adipose tissue By establishing and performing analysis on an animal model, we found that trans-ferulic acid-4-β-glucoside resulted in the maintenance of a relatively high rectal temperature level upon exposure to cold, indicating that trans-ferulic acid-4-β-glucoside promotes the process of cold acclimatization. Furthermore, we found that the underlying mechanism is the alleviated oxidative stress in liver and increased thermogenesis in brown adipose tissue. The genes acox1 and acadm are related to fatty acid beta-oxidation and degradation of medium-chain fatty acids, respectively, and we found that trans-ferulic acid-4-β-glucoside increased the expression of acox1 and acadm upon cold exposure, indicating the promotion of lipid oxidation. Sepa-Kishi et al. found that fatty acid oxidation was increased with cold acclimation [21], indicating that trans-ferulic acid-4-β-glucoside promotes cold acclimation with increased fatty acid oxidation. The genes nrf1, tfam, and tfb2m are

Discussion
As a common stimulus in the environment, cold exposure causes a series of metabolic changes in the body [18]. These changes play an important role in the formation of cold adaptation. The ability, or lack thereof, to adapt to cold determines the living conditions of mammals to a great extent, particularly in cold environments [19]. Recent studies have revealed that cold acclimation is related to the concomitant increase in brown adipose tissue (BAT) activity [8,20], as well as liver metabolism [21]. As a traditional Chinese medicine, radix aconiti carmichaeli (Fuzi) promotes heat production and protects from cold [22,23]. As the major ingredient from radix aconiti carmichaeli (Fuzi), the aconitum alkaloids mainly produce their pharmacological effects by regulating heart and blood vessel functions [24]; however, the key active ingredients from radix aconiti carmichaeli (Fuzi) which regulate BAT activity and liver metabolism in cold acclimation are still unknown. In order to elucidate the key substance that plays a role in this effect, we got a new active ingredient which is purified from Fuzi. The chemical structure is made up of ferulic acid and glucoside. Li Xia et al. found that sodium ferulate improves energy metabolism and plays a protective role after myocardial ischemia reperfusion injury, while Wu et al. suggested that ferulic acid protects the heart from lesion by inhibiting the apoptosis of mitochondria [25]. Pan Yiou et al. found that ferulic acid inhibits lipid deposition in obese mice by mediating the expression of some important lipid metabolism genes. These findings suggest that ferulic acid may participate in the process of energy metabolism and increase the body's energy expenditure to fight obesity, however, these studies did not assess its role in liver and adipose tissue. In our study, the active ingredient trans-ferulic acid-4-β-glucoside promotes cold acclimation with increased fatty acid oxidation in liver and heat production in adipose tissue By establishing and performing analysis on an animal model, we found that trans-ferulic acid-4-β-glucoside resulted in the maintenance of a relatively high rectal temperature level upon exposure to cold, indicating that trans-ferulic acid-4-β-glucoside promotes the process of cold acclimatization. Furthermore, we found that the underlying mechanism is the alleviated oxidative stress in liver and increased thermogenesis in brown adipose tissue. The genes acox1 and acadm are related to fatty acid beta-oxidation and degradation of medium-chain fatty acids, respectively, and we found that trans-ferulic acid-4-β-glucoside increased the expression of acox1 and acadm upon cold exposure, indicating the promotion of lipid oxidation. Sepa-Kishi et al. found that fatty acid oxidation was increased with cold acclimation [21], indicating that trans-ferulic acid-4-β-glucoside promotes cold acclimation with increased fatty acid oxidation. The genes nrf1, tfam, and tfb2m are related to mitochondrial biogenesis, and we found that trans-ferulic acid-4-β-glucoside increased the expression of these genes. Bruton et al. found stimulated mitochondrial biogenesis in muscle fibers of cold-acclimated mice [26], which also supports our results to some extent. Meanwhile, we found the expression of genes related to thermogenesis in brown adipose tissue, especially UCP1, increased with trans-ferulic acid-4-β-glucoside treatment, which could partially explain the improved cold tolerance in mice.
Besides, some researchers have proved that ferulic acid removes excess free radicals in the body, playing an antioxidant role. Also, sodium ferulate plays a major role in antiplatelet aggregation, neuroprotection, and so on [27]. Our study found that trans-ferulic acid-4-β-glucoside significantly increased antioxidant enzymes, thus reducing the oxidative stress injury in liver. For other important internal organs, such as heart and kidney, we found that the effect was not obvious. We suspect that it may be due to the following reason: As a metabolic organ, the liver may experience oxidative stress earlier than other organs, like heart or kidney. Some studies have found that acute cold exposure causes oxidative stress and organ damage in the liver of mice, but this phenomenon was not found in kidney, skeletal muscle, and brown adipose tissue [28]; which supports our conjecture.
Improved energy metabolism and alleviated oxidative stress are vital for the formation of cold acclimation [13,29]. Our results have shown that trans-ferulic acid-4-β-glucoside promotes cold acclimation through the above process. Some studies have focused on the relation between energy metabolism and oxidative stress and reported that oxidative stress induces autophagy in cells [30]; autophagy can reduce the damage caused by oxidative stress and thus protect cells. Meanwhile, others have reported that autophagy is closely related to energy metabolism processes, such as brown adipose tissue thermogenesis [31,32]. Therefore, whether there is a certain intrinsic link between oxidative stress and energy metabolism, and whether trans-ferulic acid-4-β-glucoside affects this link and thus plays a protective role, will be the next issue on molecular mechanisms to be addressed with further research.

Animals and Treatments
Eight-week-old C57BL/6 male mice, purchased from the experimental animal center of The Fourth Military Medical University, were kept at room temperature, with a 12 h light-dark cycle and regular chow diet. All procedures and experiments on mice were approved by Ethics Committee of Air Force Military Medical University (05 Dec 2017). We randomly divided these C57BL/6 mice into four groups to establish the animal model: one group lived at room temperature and received water, another group lived at room temperature and received trans-ferulic acid-4-β-glucoside, while the other two groups received water or trans-ferulic acid-4-β-glucoside in a cold environment (4 • C), respectively. We gave water or trans-ferulic acid-4-β-glucoside through intragastric administration at the fixed time 9:30 a.m. Each day, we measured the weight and food surplus before administration. After administration, we put the mice in 4 • C or room temperature environments and recorded the rectal temperature after 2 h. We repeated these operations for the next 5 consecutive days. On the 6th day, the mice were sacrificed, and the tissues were extracted and preserved at −80 • C. In addition, we established a short-time intermittent cold exposure model: We randomly divided the C57BL/6 mice into two groups, administering water or trans-ferulic acid-4-β-glucoside, respectively, in a 4 • C environment. The mice received cold exposure for 4 h and then were put at room temperature. The operation was repeated for 4 consecutive days. We examined the same index as mentioned above. On the 5th day, the mice were sacrificed, and the tissues were preserved.

Q-PCR
We used Trizol (Invitrogen, Carlsbad, CA, USA) to extract RNA from mouse liver, fat, and other tissues. With reverse transcription (TAKARA, Kusatsu, Japan), we obtained cDNA.

Western Blot
The brown adipose tissue homogenate of mice was mixed with RIPA lysate (Beyotime Biotechnology, shanghai, China), protease inhibitor (Roche, Basel, Switzerland), and PMSF (Beyotime Biotechnology, shanghai, China) (100:4:1), and the mixture was placed on ice for 15 min to carry out cell lysis. Then, we centrifuged the mixture and obtained the supernatant. Next, we measured protein concentration and added 5× loading buffer (Cwbiotech, Beijing, China), boiled the sample for 7 min, and loaded the proteins for electrophoresis. After electrophoresis, we transferred the proteins to a PVDF membrane. The membrane was blocked at room temperature in 5% BSA for 2 h, and the membrane was incubated with primary antibodies (UCP1 primary antibody, rabbit 1:1000, Abcam, Cambridge, UK; PGC1α primary antibody, rabbit, 1:1000, Abcam, Cambridge, UK; ACTIN primary antibody, mouse, 1:1000, Cambridge, UK; TUBULIN primary antibody, rabbit, 1:1000, Cambridge, UK) overnight at 4 • C. The following day, the membrane was washed in TBST three times, 10 min each, and the membrane was incubated with secondary antibodies for 2 h. The protein bands were visualized by chemiluminescence (Bio-Rad, Hercules, CA, USA) [34].

Blood Biochemistry Analysis
Serum was obtained by centrifugation at a speed of 3000 rpm/min for 5 min. Then, blood glucose, triglycerides, and total cholesterol were measured [35], and the damage-related indexes, such as creatinine, CK (Creatine Kinase), AST (Aspartate aminotransferase), ALT (Alanine aminotransferase), and LDH (lactate dehydrogenase) were measured.

Liver Glycogen Staining
For conventional PAS staining (Periodic Acid-Schiff stain) [36], liver tissues fixed with 4% polyoxymethylene were paraffin-embedded, sliced, and dewaxed. Then, the slices were stained in periodate dye solution for 15 min. After washing with tap water and distilled water two times, they were stained with Schiff dye in the dark for 30 min. After washing with running water, they were stained in hematoxylin dye for 3-5 min. After differentiation, dehydration and sealing, they were observed under the microscope.

Brown Adipose Tissue HE Staining
Brown adipose tissue fixed by 4% polyoxymethylene was paraffin-embedded by conventional techniques [37], sliced, and dewaxed. They were stained in hematoxylin dye for 3-5 min. After washing with tap water and differentiation, they were dehydrated in 85% and 95% alcohol successively for 5 min, and stained in eosin dye for 5 min. After dehydration and sealing, they were observed under the microscope.

Oxidative Stress Index of Liver Tissue
T-SOD was obtained according to the T-SOD test kit protocol (Nanjing Jiancheng, Nanjing, China). Briefly, an appropriate amount of liver tissue was grinded with saline to obtain the supernatant extract. Setting up a blank tube, we mixed 0.05 mL samples of from the different groups, 1 mL reagent one and 0.1 mL each of reagent two, three, and four. Then, the mixture was placed in a water bath for 40 min at 37 • C, after which we added the chromogenic agent and measured the absorbance at a wavelength of 550 nm. Briefly, an appropriate amount of liver tissue was grinded with saline to obtain the supernatant extract. Setting up a blank tube, 0.1 mL samples or standards were mixed with 1 mL each of reagent one and reagent two, and the absorbance was measured at 405 nm wavelength.

Measurement of GSH-px
GSH-px was obtained according to the GSH-px test kit protocol of (Nanjing Jiancheng, Nanjing, China). Briefly, an appropriate amount of liver tissue was grinded with saline to obtain the supernatant extract. A blank tube, standard tube, enzyme tube, and non-enzyme tube were set up. The reagents were mixed together according to the recommended quantities and the absorbance was measured at a wavelength of 412 nm.

Statistical Analyses
The data were expressed as mean ± standard error of the mean (SEM). The results were tested by one-way ANOVA with Tukey's post hoc t tests among the groups, and the differences between the two groups were analyzed using Student's t-test. p values <0.05 were considered statistically significant. All these analyses were performed using SPSS20.0 software.

Acknowledgments:
We would like to thank Zhenlong Xin, Yuankang Zou, Peng Su and Xiuquan Wu for their suggestions on article submission, and thank the roommates for their spiritual support during the trial.

Conflicts of Interest:
There are no financial or other interests with regard to the paper that represent a conflict of interest.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy:
Biology
Computer Science
Physics
Chemistry

text:
Glycosaminoglycans and Proteoglycans

In this editorial to MDPI Pharmaceuticals special issue “Glycosaminoglycans and Proteoglycans” we describe in outline the common structural features of glycosaminoglycans and the characteristics of proteoglycans, including the intracellular proteoglycan, serglycin, cell-surface proteoglycans, like syndecans and glypicans, and the extracellular matrix proteoglycans, like aggrecan, perlecan, and small leucine-rich proteoglycans. The context in which the pharmaceutical uses of glycosaminoglycans and proteoglycans are presented in this special issue is given at the very end.

Introduction
This short article is intended to provide a brief introduction to the structures of glycosaminoglycans (GAGs) and proteoglycans (PGs) to set the articles in this special issue of Pharmaceuticals on "Proteoglycans and Glycosaminoglycans" into context. The class of glycosylated proteins known as PGs is represented in the pharmaceutical world chiefly by its carbohydrate constituents. These are polysaccharides known as GAGs, such as heparin (Hp) [1] and chondroitin sulfate (CS) [2]. When attached to their native protein cores these polysaccharides form the glycoconjugates known as PGs. Whole PGs are less often proposed as therapeutic agents, though recently, particularly in the context of regenerative medicine, the concept of PG mimetics, 'neoproteoglycans', is becoming more familiar [3]. The development of PG-and GAG-based medicines is beginning to take into account the way the GAGs are organized and presented by attachment to their PG cores, as well as the sequences and covalent structures of the compounds themselves. In this special issue, contributed articles will cover the current pharmaceutical uses of GAGs and their mimetics, with others describing the involvement of GAGs in processes, such as cell growth and differentiation, morphogenesis, inflammation, and healing; all of which are likely to give rise to future therapeutic uses of GAGs and PGs.
The disaccharides of HS and Hp are both composed of alternating 4-linked UA and 4-linked α-GlcN units [4,5]. Structurally, HS and Hp differ only in the relative proportions of their monosaccharide and disaccharide substructures. HS has β-D-GlcA as its major UA type while Hp has α-L-IdoA. GlcA in HS usually alternates with GlcNAc units, but lower amounts of N-sulfated glucosamine (GlcNS) and rare amounts of unsubstituted GlcN can also occur. Hp is conversely predominantly composed of 2-sulfated IdoA units (IdoA2S) together with N,6-di-sulfated GlcN units (GlcNS6S). These disaccharides are reflected in the three-dimensional structures of HS/Hp tetrasaccharides shown in Figure 1A,B. They are experimental structures from the Protein Databank (PDB), and clearly adopt fairly linear shapes, though in solution they may twist and fold to varying extents depending on the exact monosaccharide sequence [10].  Rarely, 3-O-sulfation at the GlcNS6S units can also occur (GlcNS3S6S) in both HS and Hp but is more common in the Hp chains; a pentasaccharide containing this unusual monosaccharide residue confers high affinity for antithrombin and, thereby, high anticoagulant activity, on Hp [1].
Since the length of GAG chains can vary widely, both among different GAG types and among chains of the same GAG type, the range of MWs in GAGs is very broad, ranging from a few kDa to over a hundred kDa. It is also true that manufacturing processes can alter the molecular weight (MW) of GAGs; for example, unfractionated heparin (UFH) and low-molecular weight heparin (LMWH) have chains whose average MWs are respectively ~15 kDa (~25 disaccharide units) and less than 8 kDa (~12 disaccharide units or fewer). The measurement of MWs for LMWH is not straightforward, as Hp consists of a mixture of sequence types as described above, each occurring in Rarely, 3-O-sulfation at the GlcNS6S units can also occur (GlcNS3S6S) in both HS and Hp but is more common in the Hp chains; a pentasaccharide containing this unusual monosaccharide residue confers high affinity for antithrombin and, thereby, high anticoagulant activity, on Hp [1].
Since the length of GAG chains can vary widely, both among different GAG types and among chains of the same GAG type, the range of MWs in GAGs is very broad, ranging from a few kDa to over a hundred kDa. It is also true that manufacturing processes can alter the molecular weight (MW) of GAGs; for example, unfractionated heparin (UFH) and low-molecular weight heparin (LMWH) have chains whose average MWs are respectively~15 kDa (~25 disaccharide units) and less than 8 kDa (~12 disaccharide units or fewer). The measurement of MWs for LMWH is not straightforward, as Hp consists of a mixture of sequence types as described above, each occurring in domains with particular hydrodynamic and conformational properties. The contribution of Jian Liu and co-workers, introducing the concept of homogeneous structurally defined MW markers for Hp is a real step forward [11].
CS disaccharides are essentially composed of alternating 4-linked β-D-GlcA and 3-linked GalNAc units. Various CS subtypes exist; for example, CS-A is mostly 4-sulfated at the GalNAc units ( Figure 1C), while CS-C is predominantly 6-sulfated. CS-B, widely known as DS ( Figure 1D), has α-L-IdoA units rather than β-D-GlcA. The IdoA units in DS may occasionally bear 2-sulfation while the GalNAc units are mostly 4-sulfated [6]. KS disaccharides are composed of alternating 3-linked β-D-Gal and 4-linked β-D-GlcNAc units [8]. These KS disaccharides can bear sulfation at the 6-position of either unit ( Figure 1E), although sulfation at GlcNAc occurs more often [7,8]. The typical average MW of KS is around 20 kDa, thus, around 45 disaccharide units assuming the MW of the commonest structure. HA is the only GAG type which is not sulfated; it is composed of repeating disaccharide units of alternating 4-linked β-D-GlcA and 3-linked β-D-GlcNAc ( Figure 1F) [9], and has the longest chain among all GAG types. The MW of HA is usually above 100 kDa and the degree of polymerization of HA is, therefore, in the range of at least 255 disaccharide units/chain, ranging upwards to MW of several million [12]. This very high MW polysaccharide has high viscosity at low concentration [12].
The structures of all these GAG families are represented in Figure 1. The GAG structural variations and heterogeneities associated with their high sulfation content (except HA) and their common occurrence at the extracellular matrix (ECM) or at the surface of cells are all contributing factors to the diversity of their biomedical roles because they give GAGs the capacity of binding to multiple extracellular proteins [13,14] whose actions are spread in various pathophysiological events ( Figure 2). domains with particular hydrodynamic and conformational properties. The contribution of Jian Liu and co-workers, introducing the concept of homogeneous structurally defined MW markers for Hp is a real step forward [11]. CS disaccharides are essentially composed of alternating 4-linked β-D-GlcA and 3-linked GalNAc units. Various CS subtypes exist; for example, CS-A is mostly 4-sulfated at the GalNAc units ( Figure 1C), while CS-C is predominantly 6-sulfated. CS-B, widely known as DS ( Figure 1D KS disaccharides are composed of alternating 3-linked β-D-Gal and 4-linked β-D-GlcNAc units [8]. These KS disaccharides can bear sulfation at the 6-position of either unit ( Figure 1E), although sulfation at GlcNAc occurs more often [7,8]. The typical average MW of KS is around 20 kDa, thus, around 45 disaccharide units assuming the MW of the commonest structure. HA is the only GAG type which is not sulfated; it is composed of repeating disaccharide units of alternating 4-linked β-D-GlcA and 3-linked β-D-GlcNAc ( Figure 1F) [9], and has the longest chain among all GAG types. The MW of HA is usually above 100 kDa and the degree of polymerization of HA is, therefore, in the range of at least 255 disaccharide units/chain, ranging upwards to MW of several million [12]. This very high MW polysaccharide has high viscosity at low concentration [12].
The structures of all these GAG families are represented in Figure 1. The GAG structural variations and heterogeneities associated with their high sulfation content (except HA) and their common occurrence at the extracellular matrix (ECM) or at the surface of cells are all contributing factors to the diversity of their biomedical roles because they give GAGs the capacity of binding to multiple extracellular proteins [13,14] whose actions are spread in various pathophysiological events ( Figure 2).  , nitrogen (blue), hydrogen (light grey); oxygen (red) and sulfur (yellow). In the proteins, the alpha-helices, beta-sheets, loops, and random coils are represented, respectively, in red, blue, green and grey. The pathophysiological systems in which these complexes play a role are indicated by grey fonts in the panel.

Pharmaceutical Applications of GAGs and PGs
GAGs interact with proteins in many biological systems, and as a consequence they have numerous biological and therapeutic functions [15][16][17][18][19][20][21]. In fact, GAGs can be considered the most exploited carbohydrates in the pharmaceutical market [14]. The use of GAGs as therapeutic agents is dominated by the potent anticoagulant and antithrombotic GAG Hp [1], isolated from mast-cell rich tissues such as intestinal mucosa of pigs and cattle [22]. Hp is the by far the most widely used GAG type and perhaps the most common therapeutic carbohydrate worldwide [23]; it is used in treatments and prophylaxis of thromboembolic disorders [1,24,25]. The pharmaceutical analysis of Hp preparations is rarely simple; in this issue, a radical new approach to molecular weight measurements using synthetic calibrant and computational extrapolation [11] is proposed. The potent anticoagulant activity of Hp occasionally requires an antidote in clinical use. Hogwood and co-workers address the use of protamine to neutralize Hp from different species and tissues [26]. The heterogeneity of Hp, and other consequences of its origins as a natural mammalian product, has led to the development of synthetic and semi-synthetic Hp mimetics, as described by Mohamed and Coombe in this issue [27].
CS [2] and HA [28] are also exploited as pharmaceutical ingredients. CS can be used as an alternative therapeutic in cases of osteoarthritis [29], and sometimes even osteoporosis [30], because of its essential roles in cartilage and other connective tissues, though some degree of caution in interpretation of trials may be wise [30,31]. The beneficial use of CS in arthritic disease is usually associated with the use of GlcN, another key constituent of cartilage tissues. Santos and coworkers have in this issue described a systematic process based on electrophoresis, liquid-chromatography and NMR for assessment and control of pharmaceutical preparations of CS combined with GlcN [32]. KS can be employed as active ingredient in eye drops for treatment of certain visual dysfunctions; KS is one of principal functional components in cornea [7,8]. The best-known physicochemical property of HA is its capacity of forming gels in solution. This property enables HA to be used as a vehicle to make specific hydrogel formulations for regenerative medicine [33,34]. HA-based medium can be employed in cosmetics to soften and smooth skin owing to its inherent regenerative and hydrating properties; HA is an important functional component of the ECM of skin [35,36].

Proteoglycans
There are fewer than 50 distinct PG genes, though many more proteins due to alternative splicing [37]. Fewer than 20 distinct mammalian HS-PG core proteins have so far been identified [38]. This apparently limited repertoire of structures is responsible for numerous structural and functional properties of animal cells and ECM. Though many PGs are also glycoproteins, bearing Nand O-glycans, the defining type of glycosylation for PGs is the presence of one or more-sometimes many more-O-linked GAG chains. These GAGs make their contribution to the biological functions of PGs in many ways, and in some extreme cases the protein core may simply act as a scaffold for the presentation of the biologically-active GAG.
Most PGs act predominantly in the extracellular space, either as structural elements or as ligands for the many small protein growth factors, cytokines, chemokines, and morphogens that regulate embryonic development, inflammatory responses to pathogens and injury, and communication between cells [16,37]. PGs are often large proteins, heavily glycosylated and attached to membranes at the cell surface or in the ECM.
Descriptions of PGs have often used simple schematic diagrams, such as those shown in Figure 3 and in [39] depicting cartoon schematics for two major cell-surface PGs, syndecan, and glypican, and several PGs of the ECM (aggrecan and the small leucine rich PGs (SLRPs) such as decorin, lumican and biglycan). They are useful to show the rough relative sizes of PGs, with number and approximate attachment sites of GAG chains. These simple diagrams can give a clue as to function; where GAG chains are closely spaced they are likely to have a space-packing function, in the extracellular matrix in the case of aggrecan, and in the mast cell secretory granules in the case of serglycin. Where the GAG chains are less dense, such as on the cell surface attached to glypican or syndecan, their role is more likely to be in signaling, or in tissue organization. In this short article we will introduce only a few of the best known PGs; more comprehensive descriptions can be found elsewhere [37]. where GAG chains are closely spaced they are likely to have a space-packing function, in the extracellular matrix in the case of aggrecan, and in the mast cell secretory granules in the case of serglycin. Where the GAG chains are less dense, such as on the cell surface attached to glypican or syndecan, their role is more likely to be in signaling, or in tissue organization. In this short article we will introduce only a few of the best known PGs; more comprehensive descriptions can be found elsewhere [37].

Intracellular Proteoglycans
The PG from which Hp is derived is called serglycin [41], and it is unusual as a PG in that it is found only in the granules of mast cells and related cell types, not, like other PGs, in the ECM or on the cell surface. The narrowly spaced Hp chains on the small peptide backbone of serglycin accommodate positively charged proteins, such as the proteolytic enzymes found in mast cell secretory granules, allowing much closer packing by neutralizing their charge [42]. Hp is partially depolymerized on degranulation of mast cells, to give the MW distributions characteristic of Hp  [40]; (A) the intracellular PG serglycin, bearing closely packed Hp (or oversulfated chondroitin) chains, on a small peptide core; (B) cell-surface PGs syndecan and glypican; the cell membrane is shown in black; (C) the complex between aggrecan and HA, mediated by Link protein, that forms the structural basis for cartilage elasticity; and (D) a generic diagram of a SLRP, such as biglycan or decorin. Between the globular regions near the N-and C-termini the leucine-rich repeats (LRRs) form a curved structure; the dimers can form by interaction between the two offset concave faces of monomers.

Intracellular Proteoglycans
The PG from which Hp is derived is called serglycin [41], and it is unusual as a PG in that it is found only in the granules of mast cells and related cell types, not, like other PGs, in the ECM or on the cell surface. The narrowly spaced Hp chains on the small peptide backbone of serglycin accommodate positively charged proteins, such as the proteolytic enzymes found in mast cell secretory granules, allowing much closer packing by neutralizing their charge [42]. Hp is partially depolymerized on degranulation of mast cells, to give the MW distributions characteristic of Hp sodium in clinical use [43]. Further deliberate depolymerization gives rise to the range of LMWH products, such as enoxaparin, described by Arnold et al. in this issue [11].

Cell Surface PGs: Syndecans and Glypicans
The cell-surface PG groups of glypicans are modulators of morphogens such as Wnt, bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs) and Sonic Hedgehog (Shh) [44]. There are six mammalian glypican genes, giving rise to core proteins glypican-1 to glypican-6, all of them attached to the outer surface of the cell membrane by a C-terminal glycosylphosphatidylinositol (GPI) anchor [44]. The 'cartoon' schematics shown in Figure 3 depict the glypicans as globular proteins, with an extended sequence containing HS attachment sites between the globular region of the protein and the GPI anchor [45]. The glypican-1 protein measures about 120 Å along its long axis [46], whereas a 50-100 residue long HS chain could extend to 200-400 Å. The HS attachment site is a few amino acid residues away from the globular protein core, with more than 50 amino acids between it and the GPI anchor, allowing considerable freedom in the orientation of protein and GAG [46], both of which may be available for interactions with extracellular proteins simultaneously.
By contrast, the four mammalian syndecans [45,47] hold their GAG side-chains out further from the cell surface, around 200 amino acids from the cell surface, though we have no information on the 3D structure of syndecan ectodomains. Syndecans have a transmembrane domain, and a cytodomain that interacts with kinases and the actin cytoskeleton [47].
Cell-surface GAGS attached to these PGs play a part in the early stages of viral invasion of the host cell. As an example, Kim and co-workers in this issue describe the recognition of cell surface GAGs by the envelope proteins of Flaviviruses, including dengue and yellow fever viruses [17]. In addition, viral carbohydrates are involved in interaction with the cell surface receptor dendritic cell-specific ICAM-grabbing non-integrin, DC-SIGN [17].
GAGs are also involved in the cell uptake of potentially useful drug-delivery systems. Takechi-Haraya and associates have in this issue described the contribution of GAGs in the non-endocytic direct cell membrane translocation of arginine-rich cell-penetrating peptides by studying the cell-permeation of octaarginine monitored through real-time in-cell 19 F NMR spectroscopy [21].
Damage to the cell surface glycocalyx, of which GAGs form an important part, is a feature of disease states of the vascular endothelium. Wodicka and coworkers have developed a PG mimetic designed to bind to inflamed endothelium and prevent platelet binding to create a more quiescent endothelial state [20].

Aggrecan in the Extracellular Matrix
The most abundant PG in ECM rich tissues such as cartilage is aggrecan, which forms very large aggregates with HA, mediated by the link protein [18]. These aggregates are held in a collagen network to form strong but elastic articular cartilage structure, a target tissue for regenerative medicine due to the ubiquity of osteoarthritis in the aging population [18]. The close packing of CS chains attached to the aggrecan core gives the molecule a 'bottle-brush' appearance, not only in the simplified diagram in Figure 3C, but also in atomic force microscopy images [48].

Perlecan at the Basement Membrane
No 3D structure has been published for the large, multi-domain PG perlecan, found at the basement membrane and in the pericellular space. As an HS-bearing PG in the space between cells, perlecan can act as an extracellular storage device for growth factors such as FGFs and is involved in angiogenesis through its interactions with vascular endothelial growth factor (VEGF) [49].

Small Leucine-Rich Proteoglycans
The SLRPs exemplified by decorin have ordering functions in tissues; decorin in tendons appears to wrap round the D-band of collagen fibrils forming a ring-mesh of GAG [50]. SLRPs are also important for the structure of cornea, for which transparency requires an absolutely regular structure. The SLRP lumican is substituted with three N-linked KS chains [51] and its absence leads to a loss of order in the array of collagen fibrils, resulting in opacity of the cornea [52]. The importance of the SLRPs and their DS chains provides the focus of Mizumoto and coworkers, who, in this issue, have described mutations in human genes encoding the glycosyltransferases, epimerases, and sulfotransferases responsible for the biosynthesis of DS chains and their effects on connective tissue disorders, including some forms of Ehlers-Danlos syndrome [19].

The Context of Glycosaminoglycans, Proteoglycans, and Their Pharmaceutical Uses in the Special Issue "Glycosaminoglycans and Proteoglycans"
This special issue on "Glycosaminoglycans and Proteoglycans" contains a diverse collection of articles, illustrating the range of biological systems in which GAGs and PGs operate and can be considered in the design of pharmaceutical interventions. They range from the analysis of GAG pharmaceutical products [32] and Hp neutralization [26] through Hp mimetics [27], GAG-mediated uptake of cell penetrating peptides [21], to the development of PG mimics for endothelial repair [20]. Review articles cast a new light on regenerative medicine [18], chemokine-GAG interactions [16], the DS-PG related human genetic disorders, and Flavivirus interactions with host cells [17].
Some puzzles, vital to the progress of GAG and PG pharmaceuticals research, remain. The complex substitution patterns that GAGs are capable of displaying allow a remarkably large repertoire of structural motifs for recognition by proteins at the cell surface and between cells. We know that HS epitopes vary through processes of stem cell differentiation and embryonic development [18], and that the fine structure of GAGs adds to the intricacies of interactions between chemokines and their receptors [16]. We still, however, have no clear strategy to determine the exact preference of a protein for a specific sequence within a GAG polysaccharide chain, as we are hampered by a lack of experimental tools for the task. The development of synthetic GAG mimetics [27] may, in the future, offer libraries of homogenous GAG-like compounds that will allow detailed identification of protein ligand motifs within long GAG chains, leading to the possibility of the rational design of a whole new class of pharmaceuticals.

Conflicts of Interest:
The authors declare no conflict of interest.


s2fieldsofstudy: Biology Chemistry

text:
Reduced Cyclic Adenosine Monophosphate Level in Hippocampal CA1 Participates in Propofol Induced Amnesia in Rats

Propofol inhibits long-term potentiation (LTP) in the hippocampal CA1 region and impedes episodic memory formation. However, the molecular mechanisms involved in the effect of propofol are still poorly understood. It had been reported that propofol inhibited cAMP response element binding protein signaling, which was proposed to contribute to memory retention impairment in rats. Here, we first demonstrated that propofol perfusion could inhibit forskolin induced LTP in the rat hippocampal CA1 slices. Propofol also reduced the level of cAMP, which could be reversed by non-selective PDE inhibitor IBMX. We further discovered that propofol could increase both PDE4 activity and PDE4AX protein expressions in the hippocampal CA1 region. Furthermore, pretreatment of rolipram, a PDE4 inhibitor, rescued propofol induced inhibition of CA1 LTP and the impairment of hippocampus-dependent memory formation in rats. Thus, our results suggest that reduced levels of cAMP by increasing PDE4 activity and PDE4AX protein expressions in the hippocampal CA1 region plays an important role in the propofol-induced amnesia.

INTRODUCTION
Propofol, the most widely used intravenous general anesthetic, can impede episodic memory formation even at sedative doses (Pang et al., 1993;Veselis et al., 2004;Zhang et al., 2013). That is to say patients could preserve conscious awareness when subanesthetic propofol is present, but have no recollection of these events after the fact. It is known that the formation of episodic memories depends on the hippocampus, a region of the brain critical to memory formation (Wixted et al., 2014;Moscovitch et al., 2016). However, the change induced by propofol in the hippocampus related to the memory formation remains unknown.
Long-term potentiation (LTP), which is the long-lasting increase in synaptic strength following trains of stimuli, has been proposed to be as a cellular mechanism of memory formation in the brain (Eichenbaum, 1996;Miller and Mayford, 1999). Previous researches from ourselves and others found that propofol could inhibit theta burst stimulation induced hippocampal CA1 LTP in both rats and mice (Nagashima et al., 2005;Takamatsu et al., 2005;Li et al., 2014), but had little effect on the CA1 LTP evoked by high frequency stimulations (Li et al., 2014). It has been proved that different protocols induced LTP have different underlying molecular mechanisms (Nguyen and Kandel, 1997;Staubli et al., 1999;Costa and Grybko, 2005;Li et al., 2014). For example, theta burst stimulation induced LTP in hippocampal CA1 region is reported cAMP signaling dependent and sensitive to be affected by GABA receptor activation (Nguyen and Kandel, 1997;Staubli et al., 1999;Costa and Grybko, 2005). On the contrary, high frequency stimulation induced LTP is cAMP signaling independent and not sensitive to being affected by GABA receptor activation. Thus one of the differences, such as cAMP signaling and effect by the GABA receptor activation between these two forms of LTP, could likely be a contributor to this differential effect of propofol on LTP as well as memory formation.
Many findings support the view that cAMP signaling pathway mediates synaptic plasticity, such as hippocampal LTP, in rodents which is responsible for learning and memory (Wong et al., 1999;Nguyen and Woo, 2003;Wang et al., 2004). cAMP levels in cells are positively regulated by adenylyl cyclases (ACs) and negatively affected by cyclic nucleotide phosphodiesterases (PDEs). An increase in cAMP level activates protein kinase A (PKA) that, in turn, phosphorylates target proteins including kinases and transcriptional factors, such as cAMP response element-binding protein (CREB) (Kandel, 2012). Our previous study had reported that propofol could suppress the CREB phosphorylation and the expression of brain-derived neurotropic factor and activityregulated cytoskeleton-associated proteins in the hippocampus of rats (Zhang et al., 2013). Additionally, it was reported that GABA(A) receptors could modulate the effect of cAMP on synaptic transmission and therefore determine the direction of synaptic plasticity (Yu et al., 2001). These findings indicated that propofol could either directly or indirectly affect the downstream of cAMP signaling and its functions.
Based on these findings, we hypothesized that amnesia caused by propofol might through suppress cAMP signaling in the hippocampal CA1 region. In this aspect, we had performed electrophysiological, biochemical and behavioral experiments, and discovered that propofol inhibited cAMP signaling by enhancing PDE 4 activation and PDE4AX expression in the hippocampus.

Animals and Drugs
The animal experiments were approved by the Institutional Review Board for animal research and performed according to the guidelines for animal use in laboratories established by Fudan University and Second Military Medical University. All rats, purchased from SLAC laboratory animal company (Shanghai, China), were maintained on a 12 h light/dark cycle with food and water provided ad libitum.

Hippocampal Slice Preparation
Sprague-Dawley rats (21-28 days old) were decapitated following diethyl ether anesthesia. The brain was rapidly removed and placed in cold normal ACSF, saturated with 95% O2/5% CO 2 mixed gas. The collected hippocampal tissue (350 um) was immediately sliced using a vibrotome (Lecia, Nussloch, Germany). Slices were recovered in an incubation chamber for 30 min at 32 • C. They were placed at room temperature for at least 60 min, before being transferred to a recording chamber perfused with gassed ACSF.

Electrophysiological Recordings
The detailed protocol for recording field excitatory post-synaptic potentials (fEPSPs) had been described previously (Li et al., 2014). The brain slices were perfused with normal oxygenated ACSF, which contained (in mM): 126 NaCl, 2.5 KCl, 1.0 NaH 2 PO 4 , 26 NaHCO 3 , 2.5 CaCl 2 , 1.3 MgCl 2 , and 10.0 glucose, with the pH adjusted to 7.4. To obtain fEPSPs from the s. radiatum of the CA1 region, bipolar stimulating electrodes were placed in the Schaffer collateral pathway, and 5-8 MO glass electrodes filled with normal ACSF were placed on the s. radiatum for recording. The stimulus intensity was adjusted to evoke 40-50% of the maximum amplitude of fEPSPs. In all experiments, the baseline of synaptic transmission was recorded steadily for at least 30 min before drug administration or delivery of the stimulus. For LTP induction, we utilized FSK perfusion protocol by perfusing 50 uM FSK for 20 min, and theta-burst stimulation (TBS) protocol, which consisted of 10 bursts of four pulses at 100 Hz, applied at 5 Hz. The strength of synaptic transmission was determined by measuring the maximum slope of the fEPSPs.
cAMP Concentration Assay in the CA1

Region of Hippocampal Slices
There were three small experiments in this part and each experiment had two groups: control group (1% DSMO) and propofol group (50 uM). The slices were incubated at normal ACSF in the experiment 1.

PDE4 Activity Assay in Rats' Hippocampal CA1 Region
Twenty-four rats, aged 10-12 weeks, were randomly divided into four groups: control group at 30 min (intralipod, 5 ml/kg, i.p.), propofol group at 30 min (25 mg/kg, i.p.), control group at 120 min and propofol group at 120 min. Rats were decapitated 30 min and 120 min after the propofol injection respectively. The CA1 region of hippocampal tissues was harvested in cold ACSF and homogenated in a protein extraction reagent. Protein concentrations were determined by the BCA method according to BCA Protein Assay protocol. PDE4 activity assay was according to the instructions of PDE Activity Assay Kit (Abcam, Shanghai, China).

Contextual Fear Conditioning
The protocol of fear conditioning experiment is similar to the previous described (Curzon et al., 2009). Briefly, on the training of day 1, rats were give a 120 s habituation period before cue and shock trails begins. Then, a tone cue is presented at a level of 80 dB for 15 s. A foot shock (1 mA) was administered during the last 2 s of the tone presentation. After 120 second intervals, the tone and shock presented again 4 times. On the task test of day 2, rats were gently placed into the conditioning chamber for 180 s for contextual memory at the same time and then moved to an altered chamber to test non-contextual memory. After 60 min, rats were transferred to a new location for cue test. Rats were allowed to habituate for 3 min and then the same tone cue was given for 15 s. After 60 s intervals, the tones were repeated for 2 times. Computer-controlled cameras captured the behavior of rats during the experiment course. Memory function was assessed by analysis of freezing time.

Data and Statistical Analysis
Field EPSPs were recorded and analyzed by the pCLAMP software system (Molecular Devices, Sunnyvale, CA, USA). The slope of the fEPSPs during the 5 min prior to induction of LTP was taken as the baseline, and all values were normalized to this baseline.
Data collected in this study were expressed as the mean ± SEM. Statistical significance was determined by a two-tailed unpaired Student's t-test or one-way or two-way ANOVA with a post-hoc Tukey test using SPSS 20.0 (IBM Corporation, Armonk, NY, USA). P < 0.05 was considered to be significant.

Propofol Inhibits FSK Induced LTP in Hippocampal CA1 Region
Our previous reports showed that treatment with propofol significantly suppressed hippocampal CA1 LTP induced by TBS, a cAMP signaling dependent LTP induction protocol, but not HFS, a cAMP signaling independent LTP induction protocol. In current study, we investigated whether the differentiation of propofol on two different protocols induced LTP was due to modulation of the cAMP levels in hippocampal neurons. FSK is an activator of the enzyme adenylyl cyclase of which activation could increase intracellular cAMP levels. FSK perfusion (50 uM) for 20 min induced a long lasting and significant increase of the fEPSP in the CA1 region of the hippocampus. The slope of fEPSP increased to 139 ± 8% of baseline (n = 6, p < 0.001) at 60 min after FSK perfusion and lasted for at least 100 min. However, propofol (50 µM) pretreatment for 20 min significantly inhibited the FSK induced increases of the fEPSP slope (108 ± 8% (n = 6) of baseline vs. 139 ± 8% (n = 6) in FSK alone group, p < 0.05) (Figure 1). These results suggest that propofol is likely to influence the cAMP pathway during the LTP induction in the hippocampal CA1 region.
cAMP Level Decreased by Propofol Was Due to the Increase of Both PDE Activity and Protein Level Propofol decreases the cAMP level and inhibits cAMP signaling, but its underlying mechanism is little to be known. Cell cAMP level is positively regulated by adenylyl cyclases (ACs) and negatively affected by cyclic nucleotide phosphodiesterases (PDEs). In this aspect, we performed experiments to test whether stimulating ACs by FSK, or inhibition of PDEs by IBMX, would affect the cAMP level in either normal or propofol treatment conditions. Our experimental results showed that FSK treatment significantly increased the cAMP level to 184 ± 12% (n = 6) of ACSF control level (P < 0.01 compared with ACSF group) in hippocampal CA1 cells, and there was a further increase of cAMP level to 233 ± 15% (n = 6) of ACSF control level while IBMX was added on top of the FSK treatment (P < 0.01 compared with ACSF control and P < 0.05 compared with FSK alone group) (Figure 2A). This result suggested that both FSK, by increasing the cAMP synthesize, and IBMX, by enhancing cAMP degradation, could influence the intracellular cAMP level in hippocampus. Next, we tested whether FSK or IBMX could reverse propofol induced decrease of cAMP in hippocampus. Similar as previous reported, propofol alone significantly decreased cAMP concentration in the hippocampal CA1 region (75 ± 7% of control, n = 6, p < 0.05) (Figure 2B). In the slices of FSK treatment, propofol could also decrease cAMP concentration (71 ± 7% of control n = 6, p < 0.05, Figure 2B). In contrast, when co-treatment FSK together with IBMX, the cAMP concentration in the presence of propofol was no longer significantly reduced (87 ± 6% of ACSF control, p = 0.19) (Figure 2B). These results indicated that blockade of cAMP synthesize with FSK could not, but suppressing cAMP degradation by inhibiting PDE activity, could reverse propofol induced cAMP concentration deduction. Since PDEs are enzymes that provide the sole route for cAMP degradation in cells and PDE4 has a major role in the brain cells. Therefore, we speculated that the reduction of the cAMP level in the hippocampal CA1 region by propofol was related to PDE, particularly PDE4, pathway. Indeed by measuring the PDE4 activity in the hippocampus using Enzyme Immunoassay, we observed that PDE4 activity in the hippocampus was significantly enhanced in the presence of propofol. The PDE4 activity in the hippocampus was increased to 164 ± 13% (n= 6, P < 0.01) and 147 ± 15% (n = 6, P < 0.05) of ACSF control level at either 30 or 120 min after propofol administration, respectively (Figure 3).
Next, we investigated the PDE4 isoforms protein expression in the hippocampal CA1 region by western blot. PDE4 AX, D1, D3, and A1 were detected by using a pan-PDE4 antibody. PDE4 AX level in the hippocampal CA1 region was found significantly increased in propofol group (146 ± 9% of control, n = 6, P = 0.01), but not all the other subtypes (Figure 4). These results suggested that propofol suppressing the cAMP level in the Frontiers in Neuroscience | www.frontiersin.org hippocampus is likely due to the increase of both the activity and the protein level of the PDE4, and in turn to reduce the cAMP degradation rate.

Rolipram Reversed Propofol Induced LTP Inhibition in the Hippocampal CA1 Region
Based on the above result that propofol enhanced the PDE 4 activity in the hippocampal CA1 region, we further investigated whether increased PDE4 activity was responsible for propofol induced inhibition of LTP in the hippocampal CA1 region. Similar to our previous findings, propofol 30 µM) significantly inhibited TBS induced hippocampal CA1 LTP (110 ± 6% of the baseline in propofol group vs. 146 ± 6% of the baseline in control group at 60 min after TBS, n = 6 in each group, p < 0.01; Figure 5). However, rolipram perfused together with the propofol, significantly reversed the suppression of FIGURE 3 | Propofol increases the PDE4 activity in the hippocampal CA1 of rats. Bar histograms showing the ELISA measured PDE4 activity in the hippocampal CA1 area significantly increased 30 min and 120 min after propofol injection (50 mg/kg, i.p.) in rats. *p < 0.05, **p < 0.01 compared with vehicle control group by Student's t-test. LTP by propofol in the hippocampal CA1 slices. Rolipram increased TBS induced hippocampal CA1 LTP from 110 ± 6% (n = 6) of the baseline in the propofol group to 133 ± 5% (n = 6) of the baseline in the propofol plus rolipram group (p < 0.01; Figure 5). This result showed that PDE4 inhibitor could rescue propofol induced inhibition of LTP in the hippocampal CA1 region, which suggested that PDE4 pathway impairment was responsible for propofol induced LTP inhibition.

Rolipram Rescued Contextual Fear Memory Impairment Caused by Propofol in Rats
Contextual fear conditioning was employed to exam hippocampal dependent memory formation in this experiment. During the memory tasks, freezing behavior was used as an indicator of rats' recognition of a potentially aversive stimulus, and thus an indicator of memory function. Thirty-six male rats (10-12 weeks) were randomly divided into four groups. Rats received propofol treatment (25 mg/kg, i.p.), rolipram treatment (1 mg/kg, i.p.), or vehicle (2% DMSO in 0.9% saline) respectively at 30 min before undergoing behavior test.
The baseline of freezing behavior indicated by the freezing percentage before training on day 1 was similar among the four groups (p > 0.05, Figure 6). During the task tests on day 2 in the contexture test, the percentage of freezing time in propofol group was significantly lower than that of control group (14 ± 6% vs. 51 ± 6%, n = 9, p < 0.01, Figure 6). However, combined injection of rolipram with propofol rescued the decreased freezing percentage induced by propofol (44 ± 9%, n = 9, p < 0.05, Figure 6). There was no significant difference of freezing behavior among the four groups on the non-contextual tests on day 2 (p > 0.05, Figure 6). The ability of rolipram to rescue propofol induced impairment in hippocampus dependent memory formation demonstrated that propofol increasing of PDE4 activities and inhibiting cAMP signaling in the hippocampal CA1 region is likely, at least in

DISCUSSION
In this study, we demonstrated that propofol inhibited cAMP signaling dependent LTP in the CA1 region of rats' hippocampus through suppressing cAMP levels as a result of increased PDE4 FIGURE 6 | Rolipram rescued contextual fear memory impairment caused by propofol in rats. Fear conditioning tested the context specific memory of rats. The freezing percentage before training was similar between four groups. During the context test, the percentage of freezing in propofol group was significantly lower than it in control group (p < 0.01). Combined rolipram injection with propofol rescued the percentage of freezing decrease induced by propofol (p < 0.05). **p < 0.01 compared with Control group, # p < 0.05 compared with propofol group by one way ANOVA with post-hoc test.
activity and PDE4AX protein level. And treatment of PDE4 inhibitor rolipram rescued propofol induced CA1 LTP inhibition and memory formation impairment in rats. These results showed that the suppression of cAMP signaling by increasing PDE4 activity and protein expression in the hippocampal CA1 region played a major role in the propofol induced amnesia.
Animal studies have shown that cAMP-CREB pathway in the hippocampus is crucial for the LTP and memory formation (Suzuki et al., 2011;Kida, 2012;Kida and Serita, 2014). It had been reported that propofol inhibited the phosphorylation of the CREB, a transcriptional activator, in the hippocampus and down regulated the BDNF and Arc protein expression (Zhang et al., 2013). In the present study, we obtained several sets of data about the role of the key molecular cAMP in the propofol induced LTP inhibition and memory impairment. First, consistent with the results that propofol inhibited cAMP dependent LTP in the hippocampal CA1 region (Nagashima et al., 2005;Li et al., 2014), propofol inhibited the FSK induced LTP in the hippocampal CA1 region. It further endorsed the assumption that propofol inhibited cAMP signaling induced CA1 LTP. Second, propofol reduced the cAMP level in the rats' hippocampus, which provided direct evidence that propofol could inhibit cAMP signaling in the rats' hippocampus. Third, PDE4 inhibitor could rescue propofol induced cAMP dependent LTP inhibition in the hippocampal CA1 region. Taken together, these results indicated that propofol inhibited cAMP signaling in the hippocampal CA1 region was involved in its effects on LTP formation.
Many factors could modulate cAMP levels in cells mainly through two series of enzymes ACs and PDEs. For example, numerous agents such as FSK and pertussis toxin increased the cAMP levels in brain cells by activating AC directly or indirectly (Ahuja et al., 2004;Vecsey et al., 2009). PDE activity enhanced or inhibited also affects the cAMP levels in neurons (Kleppisch, 2009;Xu et al., 2011). In this study, the reduced cAMP levels by propofol could be partly reversed by a broadspectrum PDEs inhibitor IBMX, but not AC activator FSK,which suggested that the effect of propofol on reduced cAMP levels was due to PDE inhibition. The cAMP-specific PDE4 in the brain played an important role in regulating cAMP signaling (Houslay and Adams, 2003;Kleppisch, 2009). Then we found that propofol could increase the PDE 4 activity in the hippocampus in rats. Additionally, the PDE 4 selective inhibitor rolipram could rescue cAMP dependent LTP inhibition and memory formation by propofol treatment. These results may be taken to endorse the hypothesis that propofol reduced the cAMP levels in the hippocampal CA1 region and inhibited the LTP and memory formation are at least in part by increased PDE4 activity.
Fear conditioning was employed in this study to assess the ability of memory formation in rats. The test paradigm included contextual fear conditioning which required normal hippocampal function (Rudy et al., 2004;Alvarez et al., 2008) and cued fear conditioning which relied heavily on the amygdala (Johansen et al., 2011;Bergstrom et al., 2013). During the contextual fear conditioning test, we found that both propofol and rolipram alone had little effects on learning course. However, propofol (25 mg/kg, i.p.) significantly reduced the freezing time during the task test, indicated that even subanesthesia dose propofol impaired contextual memory formation. Rolipram alone couldn't enhance the memory of normal animals in our study, which agreed with the reported study by Barad and his colleagues (Barad et al., 1998). It may be related to that rolipram had no significant effect on basal cAMP concentration. However, similar to reported results in water maze experiments (Zhang et al., 2013), rolipram treatment before propofol administration rescued the contextual memory function impaired by propofol in rats. There are two factors which may contribute to this effect in vivo. On one hand, propofol induced cAMP reduce can be reversed by IBMX. It could be speculated that rolipram also could in part reversed the cAMP reduction. On the other, many reports had shown that rolipram could facilitate LTP induction and transform early LTP to late LTP. In this study, it rescued propofol induced LTP inhibition in the hippocampal slice (Barad et al., 1998;Navakkode et al., 2004).
In addition, we found the similar results in the cued fear conditioning experiment. Propofol also impaired the memory formation and reduced by rolipram in rats. Because amygdala, but not hippocampus, plays more of an exclusive role in cue fear conditioning (Johansen et al., 2011;Bergstrom et al., 2013), these results suggested that propofol and rolipram also worked on other areas in the brain such as amygdala. Indeed, it had reported that amygdala was involved in the propofol-induced amnesia (Ren et al., 2008(Ren et al., , 2015. And administration of rolipram into the amygdala at a specific time interval after training enhances memory persistence for novel object recognition in rats (Werenicz et al., 2012). All these showed that it should be paid attention to some other memory related areas in the brain that may also play an important role in the effect of propofol and rolipram.
There are some limitations in the study. First, although PDE4 seems to be the most promising target in the brain, there are many other PDEs such as PDE 7 and PDE 8 could play a role in propofol induced cAMP reduction (Heckman et al., 2015). Second, we investigated four PDE4 isoforms by western blot but we did not know whether other isoforms changed. In addition, rolipram was intraperitoneally injected but not locally in behavior tests. As PDE4 was ubiquitously expressed in the brain regions such as amygdala, prefrontal cortex, and basal forebrain, might be also contributed to the effects of rolipram. These possibilities should be included for consideration when interpreting our results.
This study and our previous findings provided further information about the cellular and molecular mechanisms of propofol produced amnesia. It revealed the major role of cAMP signaling and PDE4 activity in the hippocampal CA1 region on propofol induced changes of hippocampal function as well as memory formation. Except the theoretical significance, it also showed practical values in the clinical experiences. Although a great number of patients underwent surgery benefited from propofol-produced amnesia, it would be an adverse effect if some patients underwent short examinations such as gastrointestinal endoscopy recovers slowly. It was reported that memory remain depressed for several hours in these patients after cessation of propofol administration (Korttila et al., 1992). What we found in this study suggested that modification of PDE4 activity and cAMP signaling should have a potential effect to promote memory recovery after propofol administration.
In the current study, we demonstrated that propofol could increase the activity of PDE4 and also the protein level of PDE4AX, which resulted in the decrease of cAMP level in the hippocampal CA1 region. The decrease of cAMP level in the hippocampal CA1 may contribute to the propofol induced amnesia. This was supported by the results that rolipram, a selective PDE4 inhibitor, rescued propofol induced LTP inhibition in the hippocampal CA1 region and amnesia in rats. In summary, our current results in this paper discovered that PDE4 in the hippocampal CA1 region plays a key role in the propofol induced amnesia in rats.

AUTHOR CONTRIBUTIONS
WL, LY, YW, and HX conceived the idea and designed the experiments. WL, LY, XY, LC, LW, and YL performed the experiments and interpreted the experimental results. WL, LY, YW, and HX participated in the study design and data analysis. WL, LY, and YW wrote the manuscript. All authors read and approved the current version of the manuscript.