Association between Fas/FasL gene polymorphism and musculoskeletal degenerative diseases: a meta-analysis

Background It was reported that Fas (rs1800682, rs2234767) and FasL (rs5030772, rs763110) gene polymorphism might be related to the risk of musculoskeletal degenerative diseases (MSDD), such as osteoarthritis (OA), intervertebral disc degeneration (IVDD) and rheumatoid arthritis (RA). However, data from different studies was inconsistent. Here we aim to elaborately summarize and explore the association between the Fas (rs1800682, rs2234767) and FasL (rs5030772, rs763110) and MSDD. Methods Literatures were selected from PubMed, Web of Science, Embase, Scopus and Medline in English and VIP, SinoMed, Wanfang and the China National Knowledge Infrastructure (CNKI) in Chinese up to August 21, 2017. All the researches included are case-control studies about human. We calculated the pooled odds ratios (ORs) with 95% confidence intervals (95% CI) to evaluate the strengths of the associations of Fas (rs1800682, rs2234767) and FasL (rs5030772, rs763110) polymorphisms with MSDD risk. Results Eleven eligible studies for rs1800682 with 1930 cases and 1720 controls, 6 eligible studies for rs2234767 with 1794 cases and 1909 controls, 3 eligible studies for rs5030772 with 367 cases and 313 controls and 8 eligible studies for rs763110 with 2010 cases and 2105 controls were included in this analysis. The results showed that the G allele of Fas (rs1800682) is associated with an increased risk of IVDD in homozygote and recessive models. The G allele of Fas (rs2234767) is linked to a decreased risk of RA but an enhanced risk of OA in allele and recessive models. In addition, the T allele of FasL (rs763110) is correlated with a reduced risk of IVDD in all of models. However, no relationship was found between FasL (rs5030772) and these three types of MSDD in any models. Conclusions Fas (rs1800682) and FasL (rs763110) polymorphism were associated with the risk of IVDD and Fas (rs2234767) was correlated to the susceptibility of OA and RA. Fas (rs1800682) and Fas (rs2234767) are more likely to be associated with MSDD for Chinese people. FasL (rs763110) is related to the progression of MSDD for both Caucasoid and Chinese race groups. But FasL (rs5030772) might not be associated with any types of MSDD or any race groups statistically. Electronic supplementary material The online version of this article (10.1186/s12891-018-2057-z) contains supplementary material, which is available to authorized users.

Background
Degenerative disease is the consequence of a successive process resulted from degenerative cell changes, influencing tissues or organs, which will gradually deteriorate over time. The most common degenerative diseases in musculoskeletal systems include intervertebral disc degeneration (IVDD), osteoarthritis (OA) and rheumatoid arthritis (RA). IVDD, which results from ageing, small injuries and natural daily compression on intervertebral disc (IVD), has been regarded as one of the main causes to low back pain and motor deficiency. OA, a common type of joint disease, is owing to the destruction of joint cartilage and subchondral bone and is traditionally considered to be associated with articular cartilage degeneration [1]. RA, a long term autoimmune dysfunction that primarily affects joints, is also considered to be a degenerative rheumatoid and arthritis [2,3]. All of the three diseases are of high prevalence in the society and exert huge burdens to the global medical care [4]. And they were all have been found to be related to gene alternations or heredity by recent studies [5][6][7][8].
Apoptosis represents a physiological procedure in order to remove harmful, damaged, or unwanted cells [9]. Fas is a cell-surface receptor referring to apoptotic signaling in various cell types and interacts with the natural ligand Fas ligand (FasL) to start the death signal cascade, which can contribute to apoptotic cell death [10,11]. Fas/FasL genetic polymorphisms have been reported to be related to the development or progression of several common diseases such as cancer, systemic lupus erythematosus [12][13][14]. Fas (− 670 G > A rs1800682, − 1377 G > A rs2234767) and FasL (IVS2nt-124 A > G rs5030772, − 844 T > C rs763110) are the most commonly studied sites in Fas/FasL gene recently.
Although the exact etiology of OA is still unclear, current researches have explored an association between chondrocyte apoptosis and the progression of OA [15,16]. RA, which is characterized by synovial cells proliferation and T lymphocyte collection inside the synovial tissue, is partly due to the inhibition of T cell death by which Fas/FasL participated in [17,18]. One of the main processes in the initiation and development of IVDD is the decrease in disc cells, leading to decline in ability of synthesizing and repairing extracellular matrix [19]. Recent studies have observed a significantly higher expression levels of Fas and FasL in disc cells of the herniated lumbar disc tissues, which may result in a rapid apoptosis of resident disc cells [20,21]. From the evidences above, we hypothesize that there may be an association of Fas and FasL gene polymorphisms with musculoskeletal degenerative diseases (MSDD). A few previous researches have reported that Fas and FasL variations were associated with these MSDD risks but came to a contradictory published results [19,[22][23][24][25][26][27][28][29][30][31][32]. However, no meta-analysis has investigated the association between IVDD or OA and Fas/FasL polymorphism up to now. Two meta-analyses, Zhu et al. (published in 2016) [31] and Lee et al. [33] have analyzed the association between RA and Fas/FasL recently. For Zhu et al.(published in 2016) [31] it only included and analyzed Chinese patients for Fas (rs2234767) site. For Lee et al. [33], it only analyzed Fas polymorphism and there are some mistakes in data extractions for some studies included, such as, Huang et al. [26], Lee et al. [28] and Coakley et al. [25] So we performed a comprehensive meta-analysis containing three MSDD (OA, RA and IVDD) and enrolling all races of populations besides Chinese. Also we corrected the mistakes of the previous meta-analysis, Lee et al. [33] and added a new study, Zhu et al. (published in 2016) [31] when analyzing. This meta-analysis is designed to explore the association of MSDD (OA, RA and IVDD) with Fas/FasL polymorphism, which could assist to forecast the susceptibility of MSDD for specific individuals or conduct the clinical treatment for 'high-risk' individuals.

Strategy for literature search
To identify all literatures that studied the association of Fas and FasL genes polymorphisms with MSDD, we searched nine electronic databases including PubMed, Web of Science (WOS), Embase, Scopus and Medline in English and VIP, SinoMed, Wanfang and the China National Knowledge Infrastructure (CNKI) in Chinese. The search period for all these nine databases was up to August 21, 2017. The search strategy to explore all potential studies involved the use of the following terms: "Intervertebral Disk Degeneration" or "IDD" or "Disc Degeneration" or "disc herniation" or "low back pain" or "IVDD", "Osteoarthritides" or "Osteoarthrosis" or "Arthritis, Degenerative" or "Degenerative Arthritis" or "Osteoarthrosis Deformans", "Rheumatoid Arthritis", "CD95 antigen, human" or "Fas" or "tumor necrosis factor receptor superfamily, member 6 protein, human" or "CD95L" or "Fas Ligand" or "FasL Protein" or "tumor necrosis factor ligand superfamily member 6" or "CD178 Antigens" or "CD95 Antigen Ligand" or "TNFRSF6 protein, human" or "Fas1 protein, human" or "rs1800682" or "rs2234767" or "rs5030772" or "rs763110", "polymorphism" and "SNP".

Inclusion and exclusion criteria
To be included in this meta-analysis, studies should satisfy the following inclusion criteria: (1) evaluated the association of Fas and FasL genes polymorphisms with IVDD, OA and RA; (2) case-control studies; (3) offered sufficient data to calculate an odds ratio (OR) with 95% confidence interval (CI). What's more, the following exclusion disciplines were also applied: (1) non-casecontrol studies; (2) repeated publications; (3) the study only concerned with a case group; (4) comment or review; and (5) not relevant to MSDD. Two investigators (Xiao and Huang) independently evaluated the articles in accord with the inclusion and exclusion criteria. Any inconsistency was solved by discussion. If these 2 authors could still not reach the uniformity, senior authors (Ma and Deng) were asked to resolve the disputes.

Data extraction
For each study, the following characteristics were collected: (1) name of the first author; (2) year of publication; (3) country of enrollment; (4) ethnicity, age range and gender of the study population; (5) diagnosis and diagnostic criteria for MSDD cases; (6) genotyping methods; (7) source of controls; (8) matching criteria. (9) number of subjects under MSDD cases and controls; and (10) the HWE among the controls. Data were extracted cautiously from all eligible articles independently by 2 authors (Xiao and Huang). For conflict resolution, the accordance was realized by discussion.

Methodological quality assessment
The qualities of all the included studies were assessed by two investigators (Xiao and Huang) separately using the Clark scores system, which includes 10 items [34]. Scores under 5 represent low quality; while 5-7 scores denote moderate quality and 8-10 scores indicate high quality [34].

Statistical analysis
The PRISMA checklists and their guidelines were carefully followed in the whole process of this study [35]. The HWE in control groups for all the studies were calculated by χ 2 test before statistical analysis and P < 0.05 was thought to indicate significant disequilibrium. We examined Fas (rs1800682, rs2234767) and FasL (rs5030772, rs763110) genotypes using the allele (G vs. The strength of the association between Fas (rs1800682, rs2234767) and FasL (rs5030772, rs763110) polymorphism and MSDD was assessed by the pooled ORs and 95% CI. Subgroup analyses were conducted to find whether diagnosis of MSDD or race groups was also related to the value of the pooled ORs and 95% CI. The statistical heterogeneity was verified by I 2 statistics. Fixed-effects model was applied to estimate the ORs and 95% CI when heterogeneity was low (I 2 < 50%); instead, the randomeffects was used when heterogeneity was high (I 2 > 50%) [36]. Sensitivity analyses were carried out by removing one study each time to test the stability of the results.
Publication bias was evaluated by the Begg's test [37] and the Egger's test [38] (P < 0.05 was considered to be statistically significant). All statistical analyses were managed using STATA 14 (Stata, College Station, TX). All P-values were two-sided.

Characteristics of the studies
A flow chart showing the exclusion/inclusion of literatures is presented as Fig. 1. The comprehensive publications search screened 1761 potentially relevant articles, of which 267 articles were excluded for duplication and 1469 articles were omitted after browsing the title and/or abstract due to obvious irrelevance to MSDD or Fas/FasL gene we studied. Eight articles were deleted because they did not study MSDD or single nucleotide polymorphisms (SNPs) unrelated to the object of our study; 1 article was excluded on account of no detailed data; and 4 articles were wiped off because they were reviews. Finally, 12 case-control studies [19,[22][23][24][25][26][27][28][29][30][31][32] were identified for meta-analysis based on the inclusion criteria. As shown in Table 1, 4 eligible studies for IVDD, 1 eligible study for OA, 7 eligible studies for RA. Also, 5 eligible studies for Chinese, 5 eligible studies for Caucasoid and 2 eligible studies for other race groups.
As shown in Table 2, 11 eligible studies for rs1800682 with 1930 cases and 1720 controls, 6 eligible studies for rs2234767 with 1794 cases and 1909 controls, 3 eligible studies for rs5030772 with 367 cases and 313 controls and 8 eligible studies for rs763110 with 2010 cases and 2105 controls were included in this analysis. The characteristics of all the included studies are also listed in the Table 1 and Table 2, including the year, country and continent of studies, the ethnicity, age and gender of subjects, the type of MSDD, the diagnosis methods, genotyping methods, source of controls, matching items of cases and controls, the number of subjects in control/ case group and Hardy-Weinberg equilibrium (HWE) in each studies. The genotype distributions for all of the control groups were consistent with the HWE, except Sezgin et al. [22]. The quality assessment of study was listed in Table 3.

Association between Fas (rs1800682) polymorphism and MSDD risk
No significant heterogeneity was noted among the studies of rs1800682 in the overall analysis, subgroup analysis leveled by diagnosis or recessive model of subgroup analysis leveled by race groups. Thus, the fixed-effects model was used for analysis in these models mentioned above. And other models used the random-effects model. However, no significant associations were found in any models for overall analysis.
The results of subgroup analyses leveled by diagnosis were listed below: For OA subgroup, no significant relationship was found in any models. For IVDD subgroup, significant associations were noted in GG vs. AA, OR = 1.388, 95% CI: 1.062-1.812, P = 0.016; in GG vs. GA + AA, OR = 1.357, 95% CI: 1.063-1.731, P = 0.014 (Fig. 2). However, no significant relationship was observed in other models. For RA subgroup, no significant relationship was found in any models. (Additional file 1: Table S1) The results of subgroup analyses leveled by race groups were listed below: For Caucasoid subgroup, no significant relationship was found in any models. For Chinese subgroup, significant associations were noted in GG vs. AA, OR = 1.388, 95% CI: 1.062-1.812, P = 0.016; in GG vs. GA + AA, OR = 1.357, 95% CI: 1.063-1.731, P = 0.014 (Fig. 3). However, no significant relationship was observed in other models. (Additional file 2: Table S2).

Association between Fas (rs2234767) polymorphism and MSDD risk
Significant heterogeneity was observed among the studies of rs2234767 in the allele and recessive models for overall analysis and subgroup analysis leveled by diagnosis and allele, heterozygote and recessive models of subgroup analysis leveled by race groups. Thus, the random-effects model was chosen to assess the connection between rs2234767 polymorphism and MSDD risk in these models mentioned above. And other models used the fixed-effects model. Significant associations were noted in GG vs. AA, OR = 0.771, 95% CI: 0.608-0.976, P = 0.031. However, no significant relationship was observed in other models.

Association between FasL (rs5030772) polymorphism and MSDD risk
Significant heterogeneity was explored among the studies of rs5030772 in the allele model of overall and subgroup leveled by diagnosis analysis. Thus, the random-effects model was used to evaluate the association between rs5030772 polymorphism and MSDD risk in allele model. The other models used the fixed-effects   model. However, no significant associations were observed in any models for overall analysis. The results of subgroup analyses leveled by diagnosis were listed below: For OA subgroup, no significant relationship was found in any models. For RA subgroup, no significant relationship was found in any models. (Additional file 3: Table S3) The results of subgroup analyses leveled by race groups were listed below: For Caucasoid subgroup, no significant relationship was found in any models. (Additional file 4: Table S4).

Association between FasL (rs763110) polymorphism and MSDD risk
Significant heterogeneity was observed among the studies of rs763110 in the allele, homozygote and dominant models for overall analysis and subgroup analyses stratified by diagnosis and all the models stratified by race groups. So the random-effects model was applied to assess the association between rs763110 polymorphism and MSDD risk in models mentioned above. Other models used the fixed-effects model. Significant associations were noted in all models: in the allele model, C vs.  vs. CT + TT, OR = 0.694, 95% CI: 0.576-0.837, P = 0.001 (Fig. 6). For RA subgroup, no significant relationship was found in any models. (Additional file 3: Table S3) The results of subgroup analyses leveled by race groups were showed below: For Caucasoid subgroup, significant associations were explored in C vs. T, OR = 0.777, 95% CI: 0.626-0.964, P = 0.022; in CC vs. CT + TT, OR = 0.647, 95% CI: 0.465-0.901, P = 0.010 (Fig. 7). However, no significant relationship was observed in other models. For Chinese subgroup, significant associations were explored in C vs. T, OR = 0.772, 95% CI: 0.603-0.989, P = 0. 041; in CC vs. CT + TT, OR = 0.786, 95% CI: 0.618-0.998, P = 0.048 (Fig. 7). However, no significant relationship was observed in other models. (Additional file 4: Table S4).

Sensitivity analysis
Sensitivity analysis was performed to assess the influence set by one study on the pooled ORs for Fas (rs1800682, rs2234767) and FasL (rs5030772, rs763110) polymorphism by deleting one study each turn in every genetic model.
We observed that the pooled ORs significantly differed when we deleted Huang et al. [26]

Publication bias
The Begg funnel plot (Fig. 8) and the Egger's test were conducted to evaluate the publication bias in selected literature. No evidence of publication bias was noted in this study for Fas rs1800682 (Begg's test: P = 0.436, Egger's test: P = 0.576 for allele model; Begg's test P = 0. 640, Egger's test P = 0.609 for homozygote model; Begg's test P = 0.876, Egger's test P = 0.694 for heterozygote model; Begg's test: P = 0.640, Egger's test: P = 0.965 for dominant model; Begg's test: P = 1.000, Egger's test: P = 0.508 for recessive model). (Table 4) Because of the limited number (below 10) of studies included in Fas (rs2234767) and FasL (rs5030772, rs763110), publication bias was not evaluated in these sites.

Discussion
MSDD are common and one of the most clinically vital somatic disorders. A large number of genetic factors have been discovered among the crucial causes of IVDD [39], RA [40] and OA [41,42] Several studies have reported the Fas/FasL genetic polymorphisms to be related to MSDD, but with conflicting results. In order to offer insight into the connection between Fas/FasL gene and diseases, large sample studies about predisposing gene polymorphisms are required. A meta-analysis, critically reviewing 11 studies on Fas (rs1800682), 6 studies on Fas (rs2234767), 3 studies on FasL (rs5030772) and 8 studies on FasL (rs763110), was performed to assess the association of Fas/FasL genetic polymorphisms with the risk of MSDD. Its strength came from the accumulation of published data, offering more information to evaluate significant differences.
In current meta-analysis, the main findings were that the G allele of Fas (rs2234767) was linked to a decreased risk of MSDD only in homozygote model and the T allele of FasL (rs763110) was associated with a reduced risk of MSDD in all of the comparison models. Besides that, subgroup analyses leveled by diagnosis suggested that the G allele of Fas (rs1800682) was associated with an increased risk of IVDD in homozygote and recessive models. The G allele of Fas (rs2234767) was linked to a decreased risk of RA but an enhanced risk of OA in allele and recessive models. In addition, the T allele of FasL (rs763110) was correlated with a reduced risk of IVDD in all of models. However, no relationship was found between FasL (rs5030772) and these three types of MSDD in any models. In addition, subgroup analyses leveled by race groups showed that the G allele of Fas (rs1800682) was associated with an increased risk of MSDD in homozygote and recessive models only in Chinese people. The G allele of Fas (rs2234767) was linked to a decreased risk of MSDD in homozygote model for Chinese people. What's more, the T allele of FasL (rs763110) was correlated with a reduced risk of MSDD in allele and recessive models for both Caucasoid and Chinese race groups. However, no relationship was found between FasL (rs5030772) and these two race groups of MSDD in any models. Our results have several differences with the previous meta-analyses of RA recently published [31,33]. Compared with Lee et al. 33], we observed no significance between FasL rs763110 and RA but it got an opposite result. A possible explanation for this phenomenon is that we include one more study with large participants, Zhu et al. (published in 2016) [31] Although Lee et al. [33] got the same result with us in Fas (rs1800682) site, it has some errors in data extraction of articles as mentioned above. We corrected the mistakes and analyzed again. Compared with Zhu et al.
(published in 2016) [31], the small differences of result in Fas (rs2234767) for RA might be due to a new study [30] that we added in analysis. Furthermore, these Fas/ FasL polymorphisms influencing the risk of MSDD can be explained partly by that these mutations can remarkably alter the percent of resident cells in tissues, such as disc cells in IVD and T-lymphocyte in synovial tissue, gradually causing occurrence of these MSDD [17,19]. In addition, the same single nucleotide polymorphism exerted disproportionate levels of influence on different MSDD. These might be interpreted by various histology constitutions among IVDD, OA and RA. Finally, significant heterogeneity was noted in allele and recessive models of Fas (rs2234767), in allele model of FasL (rs5030772) and in allele, homozygote and dominant models of FasL (rs763110). Discrepancy among three types of MSDD might contribute to these heterogeneities. Other factors, such as ethnicity, sex distribution,  What's more, the genotype distributions of controls in all of models were in accordance with HWE, except Sezgin et al. [22] in Fas (rs2234767) and Seyfi et al. [30] in FasL (rs5030772). However, the association was not significant change when ruled out these two studies by excluding one at a time. The quality assessment indicated that the enrolled studies were credible. No evidences of publication bias were observed by either Begg's or Egger's test in Fas (rs1800682). In order to analyze the stability of the overall results, sensitivity analysis by deleting each included studies was managed in this meta-analysis. Only one or two studies influenced the result of analysis in some models for Fas (rs1800682, rs2234767) and FasL (rs763110) and no study affects the results for FasL (rs5030772), suggesting the results were reliable in some extents. But more studies still need to be conducted in order to verify the outcome of the current meta-analysis. Overall, the results of this metaanalysis are credible and stable to a certain degree.
There are some limitations in the present study. Firstly, only three studies for FasL rs5030772 site were included in analysis and only one article was screened out for OA subgroup analysis due to shortage of original studies. Secondly, the heterogeneity was a little bit high (I 2 > 50%) in some models for overall analyses, leading to a cautious acceptance of the results. So we performed subgroup analyses stratified by diagnosis to make the result more credible. What's more, some of the included articles did not match the confounding factors such as age, sex and ethnicity between case group and control group. And different factors for matching might also increase the probability of residual confounding. Such confounding factors might influence the final results. However, this meta-analysis has some strength. For example, to our best knowledge, this is the most comprehensive meta-analysis focused on the association of Fas/ FasL gene polymorphism with the susceptibility of MSDD, including OA, IVDD and RA. Several strategies and rigid criteria were set to assess the methodological quality of each study; all of the included studies possessed high or moderate qualities.

Conclusions
In summary, the current meta-analysis suggested that Fas (rs1800682) and FasL (rs763110) polymorphism were associated with the susceptibility of IVDD. Fas (rs2234767) was correlated to the risk of OA and RA. Fas (rs1800682) and Fas (rs2234767) are more likely to be associated with MSDD for Chinese people. FasL (rs763110) is related to the progression of MSDD for both Caucasoid and Chinese race groups. However, FasL (rs5030772) might not be associated with MSDD.
Because of the above-mentioned limitations, large-scale studies, including larger populations and considering more confounding factors, are required to verify the outcomes of this meta-analysis.


Effect of exercise training intensity on mitochondrial dynamics and mitophagy in post myocardial infarction rats

: Myocardial infarction (MI) is the most common type of heart disease. According to recent studies, mitochondrial dysfunction has been suggested as a central player in cardiac disease and evidences point out the association of mitochondrial morphology with development of heart diseases. Exercise training plays a protective role against cardiovascular disease. However, the role of exercise training on proteins involved in mitochondrial dynamics and mitophagy system are not well understood. Therefore, the aim of the present study was to investigate these on cardiac mitochondrial dynamic and mitophagy proteins in rats with myocardial infarction. The present study was post-test design experiment with the control group. after MI with ligation of the left anterior descending coronary artery (LAD) and ensuring the creation of MI by echocardiography, male rats were subjected to high intensity interval training (HIIT), moderate (MIIT), low (LIIT), sedentary myocardial infarction (SED-MI) and healthy control groups. After six weeks exercise, the levels of MFN2, DRP1, Parkin, P62 and PGC-1α proteins were measured by ELISA method. Data analysis showed that proteins levels of MFN2, PGC-1α, Parkin and P62 decreased significantly in SED-MI group compared to healthy control while DRP1 protein levels increased significantly (P≤0.05). Also, MFN2 and PGC-1α proteins increased in MIIT group compared with SED-MI group and DRP1 protein levels were significantly decreased (P≤0.05). Moderate-intensity interval training (MIIT) resulted to improve mitochondrial fusion and fusion proteins in rats with myocardial infarction. While high and low intensity interval training (HIIT, LIIT), despite increasing MFN2 and PGC-1α and reducing DRP1, failed to improve fusion and mitochondrial fission .

INTRODUCTION
MMP, Ca2+ overloading, or generation of ROS1. Ca2+ overloading has been shown to induce mitochondrial fragmentation in both neonatal and adult rat cardiomyocytes [1]. The function of mitochondria as cellular power plants is essential to meet the eukaryotic cells energy demands.
Nevertheless, the role of mitochondria is not limited to energy consumption supply, but also includes, for example, calcium storage, intracellular signaling and cell death [2]. Because of its wide range of cellular functions, the mitochondria have a central role in cell homeostasis; consequently, disturbance on mitochondrial function is associated with various disorders. Major chronic diseases such as neurodegenerative disorders, cancer, aging, diabetes, and heart failure are well known to be associated with mitochondrial dysfunction [2]. For instance, in myocardium disease, mitochondrion dysfunction leads to various deleterious consequences, including increased oxidative stress, contractile dysfunction and apoptosis [2].
Mitochondria have a complex functional morphology with, at basis, a compartmented double-membrane system and a usual tubular shape. However, this organelle presents a great morphological variability, which could be associated with different cellular environments in physiological conditions or pathological stress. In this sense, mitochondria may assume different shapes in various cell types, and this morphological change associated with distribution is generally termed mitochondrial dynamics [3].
Mitochondria dynamic has been well studies in exercise training research, as the majority of the literature on mitochondrial dynamics focused in skeletal muscle [4][5][6][7][8][9]. Nevertheless, emerging attention has been directed toward the cardiac muscle and the role of mitochondrial dynamics on cardiovascular diseases. Indeed, cardiomyocytes mitochondria occupy more than one-third of the cell volume, and produces more than 30 kg of ATP daily [3], which is crucial for normal heart function. Interestingly, mitochondrial dysfunction has been suggested as a central player in cardiac disease and evidences point out the association of mitochondrial morphology with development of heart diseases [10]. For instance, Schaper et al. observed that myocardial tissue from patients with dilated cardiomyopathy presented mitochondria in large clusters in cytoplasm free of myofibrils and they fall within a wide range of size from very small to very large, as well as the variance in their shape [11]. Similarly, it was shown that mitochondria in the heart failure (HF) were small and fragmented, in human HF [12]. Moreover, this change in mitochondria morphology was associated with mitochondrial fusion protein, suggesting that mitochondria fusion is decreased in HF, impairing mitochondrial function and resulting in the cell loss and downward progression of the HF [12]. In this line, other studies reported the disturbance on mitochondrial size and number in cardiac hypertrophy [13] and cardiac hypoxia [1].

reactive oxygen species
In addition to the mitochondrial dynamic, a growing body of evidence suggests that mitochondrial autophagy plays an essential role in the regulation of optimal performance of mitochondria population, which is commonly referred as mitochondrial quality control. Autophagy is a mechanism of cell destruction by lysosomes and particularly important for cell survival during energy stress, degradation of misfolded proteins, dysfunctional cytoplasmic organelles, as well as dysfunctional mitochondria, which is known as mitophagy [14]. Therefore, mitophagy is also essential to the maintenance of cellular metabolism and homeostasis. Indeed, it has been shown that the mitophagy is involved in the pathogenesis of cardiovascular disease, diabetes, inflammatory disorders, and cancer [14]. Therefore, it seems that clarifying the mechanisms involved in the regulation of mitochondrial function and fusion process, fission and mitophagy with exercise training intensity can provide a critical understanding about the role of regular exercise on improving of myocardial infarction and could be of high importance in the context of clinical settings. Therefore the purpose of this study was to investigate of exercise training intensity effect on mitochondrial dynamics and mitophagy system in infarcted rats.

Study design and animals
A total of 60 adult male Wistar rats (320g ±20g ~16 weeks of age) were used with accessibility of food and water in ad libitum. To disinfect the surgical area betadine/alcohol wiping was utilized and the left anterior descending coronary artery (LAD) was ligated utilizing a 6-0 prolene (Ethicon Inc., USA) suture. [15]. At the end of operation, to recover the rats they were placed on the heating pad until anesthesia, warm sterile saline (0.5-1 mL, SC), Flunixin (1-2 mg/kg IM) and Cefazolin (25 mg/kg IM) were injected. By utilizing analgesia and hemodynamic monitoring for 24 h postoperative care was resumed [16,17]. to calculate the LV ejection fraction (EF) and fractional shortening (FS) the following formulas were applied [18,19] [%EF = (LVEDD2 -LVESD2)/ (LVEDD2) ×100] In this study the high degree myocardial damage rats which developed MI (FS ≤ 35%) based on echocardiographic result were included [20].

Training protocol
Thirty -five rats which met the inclusion criteria which survived a MI out of sixty MI-operated rats were randomly set to the low intensity training (low intensity training (LIT, n= 6), moderate intensity training (MIT, n = 6), and high intensity training (HIT, n=6), MI-Sedentary (MI-Sed, n = 6)) and Control (CNTRL, n = 6). By walking at a slow pace (5m/min) for 5 min/day, five days per week the MI-Trained groups familiarized with a motorized. Following this step, the rats rested for two days before maximal oxygen uptake (VO2 max). To measure VO2 max each maximal rat's running exercise test was utilized [21]. Control and MI-Sed animals did not received ET while LIT, MIT, and HIT animals were exercised according to the following table [22,23].
Running intensities increased to 0.02 m/s per week by considering previous studies [21,22]. During the exercise training, no treadmill slope were applied [23].

Myocardial mitochondria preparations
Mitochondria were isolated using the tissue around of the infarcted areas. In order to prepare the Myocardial mitochondria for extraction of proteins, the infarcted hearts were cut into pieces. The residual blood was The samples were then re-homogenized at 10.000×g for 10 min to attain the Mitochondria, contained in the final precipitation.
1 Enzyme-linked immunosorbent assay It should be noted that the whole aforementioned process were performed on ice and a BCA™ Protein Assay Kit (sc-202389, Santa Cruz Biotechnology, USA), with bovine serum albumin were employed at room temperature to determine the mitochondrial protein concentration [24].

ELISA1 analysis
Microtiter plates were coated with target protein diluted in 50 mM carbonate buffer at pH 9.0. Optimal concentrations determined by

STATISTICAL RESULTS
Weight changes, Ejection fraction (EF), and Fractional shortening (FS) among research groups at different stages of the study are presented in Table 1.   performance in response to the intensity and duration of exercise and more importantly is that these changes were not limited to a particular form of exercise [27][28][29]. It has been shown that exercise training improves mitochondrial dynamics and quality through enhanced mitophagy system. Evidence showed that mitophagy is regulated by AMPK, therefore, when mitochondria are disrupted, AMPK by increasing receptor p62/sequestosome triggers mitophagy system in two ways: First, by inhibiting mTOR3 through localizing to the lysosome, because mTOR through the phosphorylation inactivates the. If ULK1 is disabled, it will inhibits autophagy and mitophagy happens. Secondly, AMPK causing a progress in mitophagy through direct effect on ULK1.
Exercise may cause mitophagy through AMPK -ULK1 signaling pathways in skeletal muscle. But this hypothesis still is not agreed how   [30] . It has been suggested that the protective effects of exercise training due to improvement of glucose and fat metabolism is associated with increased mitochondrial biogenesis with activation of PGC-1α. Increased apoptosis and autophagy may be a sign of cardiomyocytes reduction that is caused by infarction. Accordingly, another way of protective activity of exercise training against myocardial infarction could be reduced apoptotic activity. Reduction in mitochondrial function and biogenesis after myocardial infarction is the main and dominant cause of heart failure development. It is postulated that PGC-1α could be used as a potential marker for heart recovery capacity after infarction. Finally, exercise with Beta-3 adrenergic receptor stimulation and activation of the signaling pathway of nitric oxide could also help protect against ischemia-reperfusion injury [30].
Generally, despite the lack of ROS measurements, it seems that interval is much more to be learned about the mechanisms responsible for exercise-induced cardioprotection against CAD.

CONFLICT OF INTERESTS STATEMENTS
The authors declare no conflict of interest.

ACKNOWLEDGEMENT
The writers would like to appreciate the faculty of sports sciences, University of Guilan.
3 transcription factor A 4 nuclear respiratory factor beta2


s2fieldsofstudy: Biology Medicine

text:
Effect of Neuronal Excitability in Hippocampal CA1 Area on Auditory Pathway in a Rat Model of Tinnitus

Background: Tinnitus is a common disorder that causes significant morbidity; however, the neurophysiological mechanism is not yet fully understood. A relationship between tinnitus and limbic system has been reported. As a significant component of the limbic system, the hippocampus plays an important role in various pathological processes, such as emotional disturbance, decreased learning ability, and deterioration of memory. This study was aimed to explore the role of the hippocampus in the generation of tinnitus by electrophysiological technology. Methods: A tinnitus model was established in rats through intraperitoneal injection of salicylate (SA). Subsequently, the spontaneous firing rate (SFR) of neurons in the hippocampal CA1 area was recorded with in vivo multichannel recording technology to assess changes in excitability induced by SA. To investigate the effect of excitability changes of hippocampus on the auditory pathway, the hippocampus was electrically stimulated and neural excitability in the auditory cortex (AC) was monitored. Results: Totally 65 neurons in the hippocampal CA1 area were recorded, 45 from the SA group (n = 5), and 20 from the saline group (n = 5). Two hours after treatment, mean SFR of neurons in the hippocampal CA1 area had significantly increased from 3.06 ± 0.36 Hz to 9.18 ± 1.30 Hz in the SA group (t = −4.521, P < 0.05), while no significant difference was observed in the saline group (2.66 ± 0.36 Hz vs. 2.16 ± 0.36 Hz, t = 0.902, P > 0.05). In the AC, 79.3% (157/198) of recorded neurons showed responses to electrical stimulation of the hippocampal CA1 area. Presumed pyramidal neurons were excited, while intermediate neurons were inhibited after electrical stimulation of the hippocampus. Conclusions: The study shows that the hippocampus is excited in SA-induced tinnitus, and stimulation of hippocampus could modulate neuronal excitability of the AC. The hippocampus is involved in tinnitus and may also have a regulatory effect on the neural center.

tinnitus signal that is generated in the thalamocortical system preventing habituation. [11] The CA1 area of the hippocampus is of significant importance for neurotransmission in and out of hippocampus, and the possibility of a role for this area in tinnitus is intriguing.
Neuronal excitability, recorded by direct insertion of an electrode into the targeted brain area, can provide a reliable understanding of neuronal activity. In this study, we recorded changes in the neuronal excitability in the hippocampus, to gain understanding of the possible regulatory effect of the hippocampus in tinnitus. We also electrically stimulated the hippocampus and recorded the neuronal excitability of the AC. The objective of our study was to investigate the potential role of the hippocampus, particularly the CA1 area, in tinnitus, to provide electrophysiological evidence for the pathogenesis of tinnitus.

Ethical approval
All procedures used in this study were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center (No. 2016-0010), and were conducted in accordance with these guidelines.

Animals
Adult male Sprague-Dawley rats (250-350 g) were obtained from the medical experimental animal center of Peking University Health Science Center. Animals were individually housed with free access to food and water, and were maintained under environmentally controlled conditions (12-h light-dark cycles, 22 ± 1°C, and 50-70% relative humidity).

Experiment 1
Rats were randomly divided into a saline group (n = 5) and a salicylate (SA) group (n = 5). Rats in the SA group were injected with SA (350 mg/kg, i.p.), which has previously been shown to induce tinnitus. [12][13][14][15] This tinnitus model has been widely used in experimental studies and its reliability has been verified by both audiology and behavioral testing. [16][17][18] The saline group rats received an injection of an equal volume of saline.
Rats were anesthetized with isoflurane (3.0-5.0% during induction for 3 min and then 1.5-2.5% for maintenance) through a gas pump (RWD R520IP, Shenzhen, China). [19] When full-depth anesthesia was reached, as assessed by foot withdrawal in response to a foot pinch, the rats were placed in a stereotaxic apparatus (RWD 68026R, Shenzhen, China) for surgery. The body temperature of the rats was maintained at 37.0°C by means of an animal heating pad. The CA1 area (AP = −4.56 mm, ML = 1.90 mm, DV = −2.80 mm) was confirmed according to the rat brain stereotaxic coordinates (Paxinos and Watson, 2007). The hippocampal CA1 area of the left hemisphere was exposed as mentioned above. The dura of the brain was removed with a surgical microscope, and a 16-channel silicon electrode was implanted in the dorsal hippocampus to record the neuronal activity in the CA1 area.
The basal spontaneous firing rate (SFR) of the CA1 area was recorded before treatment with saline or SA. The SFR was also recorded at six time points after the treatment: 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h.

Experiment 2
Twelve rats were anesthetized and placed in stereotaxic head frame on a heating pad. The hippocampal CA1 area of the left hemisphere was exposed as in experiment 1 and a stimulating electrode was inserted into the CA1 area for delivering electrical stimulation. The AC (AP = −3.96 mm, ML = −6.50 mm, DV = − [4.40-5.40] mm) of the right hemisphere was exposed as previously described. [20] The dura mater was removed and the recording electrode was then advanced along the dorsoventral axis into the AC.
Electrical stimulation was performed using a custom-made bipolar tungsten electrode. Electrical stimuli were delivered as shock trains (pulse duration: 0.1 ms, train duration: 1 min, rate 50 Hz). The SFR of the AC was recorded before and immediately after delivering electrical stimulation to the CA1 area. For each time point, the recording duration was more than 3 min.

Spike train analysis
The electrophysiological techniques used in this study are similar to those described in previous publications. [21,22] The iridium oxide electrode was composed of two shanks, each of which had eight recording sites (100 µm each site, 0.2-0.4 MΩ impedance), with an intershank distance of 200 µm. Recordings sites were staggered to provide a two-dimensional arrangement. Three stainless steel screws, threaded into the cranium, were used as ground electrodes during recordings. The electrode was moved slowly to the target region using stereotaxic coordinates.
The output of the electrode was connected to a 16-channel preamplifier (Plexon Inc, Dallas, TX, USA) using a flexible low-noise cable. [23] Signals were sent to a multichannel acquisition processor and recorded. Signals were analyzed using Offline Sorter (Plexon Inc, Dallas, TX, USA) and NeuroExplorer (Nex Technologies, Madison, AL, USA) for spike sorting. The valley-seeking scan algorithm was used for principal component analysis and K-mean scan was selected for automatic neuron clustering. Then, manual verification of automatic clusters was performed. Abnormal waveforms such as interference waveforms and waveforms occurring within the refractory period (1 ms) were removed. [24] Timestamps of neuronal spiking were imported to NeuroExplorer for comparison of crosscorrelations to prevent cell data duplication.

Histological studies
At the end of recordings, rats were deeply anesthetized and perfused with 4% paraformaldehyde solution. Rat brains were removed for histological analysis. The locations of the electrodes were confirmed by microscope observation. Only rats with correctly implanted electrodes were included in the data analysis.

Statistical analysis
Statistical analysis was performed with SPSS 24.0 (SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± standard error. The Shapiro-Wilk test was used to verify whether the data conformed to a Gaussian distribution. Mean values of normally distributed continuous variables were compared using the independent sample t-test, while those with a nonnormal distribution were compared with the Mann-Whitney U-test. Paired sample t-tests were used to identify the significance of pairwise comparisons. A P < 0.05 was considered as significantly different.

Effect of salicylate on neuronal firing rate of hippocampal CA1 area
We were able to record the firing activity of 65 neurons in the hippocampal CA1 area, 45 from the SA group (n = 5) and 20 from the saline group (n = 5) [ Figure 1a]. The mean SFR was 3.06 ± 0.36 Hz before SA injection and 3.60 ± 0.37 Hz at 3.0 h after SA administration, while the mean SFR of the saline group was 2.66 ± 0.36 Hz and 2.06 ± 0.41 Hz before and 3.0 h postsaline treatment, respectively. However, t-tests indicated no statistical difference at the time point of 0 h and 3.0 h in both the saline group (t = 1.009, P > 0.05) and SA group (t = −1.023, P > 0.05).
Considering that the onset time of tinnitus is usually about 2.0 h after SA treatment, we also evaluated changes in the SFR at 2.0 h after SA injection. As shown in Figure 1b, 2.0 h after SA injection, the SFR of 29 neurons increased and that of 16 neurons decreased. However, the SFR of nine neurons increased and that of 11 neurons decreased in the saline group. More specifically, 2.0 h after treatment, the mean SFR of neurons in the hippocampal CA1 area had significantly increased from 3.06 ± 0.36 Hz to 9.18 ± 1.30 Hz in the SA group (t = −4.521, P < 0.05), while no significant difference was noticed in the saline group (2.66 ± 0.36 Hz vs. 2.16 ± 0.36 Hz, t = 0.902, P > 0.05). The Mann-Whitney U-test revealed significant differences between the SA group and the saline group at the time points of 0.5 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h, indicating an elevation of the overall SFR of the CA1 area by SA treatment, as shown in Figure 2. As compared with basal SFR, neuronal discharges changed significantly at the time point of 0.5 h, 2.0 h, and 2.5 h in the SA group (t-test, P < 0.05) as shown in Figure 3.

Effect of electrical stimulation of the CA1 area on firing rate of auditory cortex
A total of 198 neurons from 12 rats in the AC were recorded; their basal SFR was 8.04 ± 0.19 Hz. As shown in Figure 4, 157 (79.3%) neurons in the AC showed responses to electrical stimulation of the hippocampal CA1 area, and the remaining 41 neurons (20.7%) showed no obvious changes (N-neurons). The SFR of 90 neurons significantly increased after hippocampal stimulation (E-neurons), while 67 neurons showed a reduction in SFR, as compared to the basal level (I-neurons). The SFR of E-neurons was significantly lower than that of I-neurons. The SFR of E-neurons increased significantly, while that of I-neurons decreased markedly after electrical stimulation, as shown in Figure 5.  Tinnitus is a common clinical symptom; however, the underlying mechanism is not well understood. To gain a better understanding of the pathophysiology of tinnitus, we used the SA-induced tinnitus animal model, developed by Jastreboff et al., [25] which has a tinnitus pattern akin to a 70 dB pure tone at 10 kHz. Many tinnitus patients also experience negative emotions, such as anxiety, nervousness, and insomnia which implicate a role for the limbic system in this condition. According to Jastreboff's model of tinnitus, the limbic system is involved in the development of tinnitus and gives rise to negative cognition and emotion, intensifying the patient's attention to tinnitus, and resulting in a mutual enhancement between tinnitus and negative emotion. Rauschecker et al. [26] proposed that the limbic system acts as a switch for cognition of tinnitus. In this study, we focused on the hippocampus as a target for intervention, if limbic-auditory interactions could be shown in tinnitus, and found that SA significantly increased the SFR of the hippocampus, indicating involvement of the hippocampus in tinnitus.
In the first part of our study, we observed that the excitability of the hippocampus in the SA group was significantly     according to previous studies, which indicates that excitability of the hippocampus is consistent with changes in SA concentration. [27] This suggests that the hippocampus may be involved in SA-induced tinnitus. However, the excitability of hippocampal neurons also increased significantly at 0.5 h after SA administration. Considering the complicated functions of hippocampus, particularly as a key regulator of the hypothalamic-pituitary-adrenal axis, the increase might be the consequence of stress response. [28] As the crux of the auditory system, the AC has been extensively studied in animals and humans with tinnitus.
A functional imaging study showed that neural activity was increased in regions involved in the neural pathway of the auditory system, such as AC, inferior colliculus, and medial geniculate body. [29] However, resting state fMRI showed increased functional connection between the AC and hippocampus. [30,31] We delivered electrical stimulation to the hippocampus and recorded the neuronal excitability of the AC, to gain insight into the possible regulatory effect of the hippocampus in tinnitus. In the second part of our study, we found that 79.3% ( In summary, neuronal excitability in the hippocampus increased after SA treatment, indicating the potential involvement of the hippocampus in tinnitus. Electrical stimulation of the hippocampus could induce neuronal excitability changes in the AC, implying possible regulation of the central auditory system by the limbic system during tinnitus. Our study provides useful electrophysiological evidence related to the pathogenesis of tinnitus. The hippocampus may be a target region for treatment of emotion, learning and memory disorders in tinnitus. However, the neuronal activity of rats was evaluated under anesthesia, that might affect the electrophysiological results, and similar studies should be performed in awake animals.


extfieldsofstudy: Medicine Biology

text:
A new species of Kurixalus from western Yunnan, China (Anura, Rhacophoridae)

Abstract A new species of the genus Kurixalus (Anura: Rhacophoridae) is described from western Yunnan, China. Genetically the new species, Kurixalus yangi sp. n., is closer to Kurixalus naso than to other known congeners. Morphologically the new species is distinguished from all other known congeners by a combination of the following characters: smaller ratios of head, snout, limbs, IND, and UEW to body size; male body size larger than 30 mm; curved canthus rostralis; weak nuptial pad; brown dorsal color; absence of large dark spots on surface of upper-middle abdomen; presence of vomerine teeth; gold brown iris; single internal vocal sac; serrated dermal fringes along outer edge of limbs; granular throat and chest; rudimentary web between fingers; and presence of supernumerary tubercles and outer metacarpal tubercle.

Introduction
The genus Kurixalus Ye, Fei, & Dubois in Fei (1999) distributes widely in eastern India, Indochina, Sunda Islands, Philippine archipelago, montane forests of southern China, and adjacent continental islands, and cur rently contains 15 species (Frost 2018). Owing to its morphological conservativeness, the taxonomy and systematics of Kurixalus were once very confusing (Yu et al. 2017a). For instance, Kurixalus hainanus (Zhao, Wang, & Shi in Zhao et al. 2005) was once thought to be a synonym of Kurixalus odontotarsus (Ye & Fei in Ye et al. 1993) by some authors (e.g., Fei et al. 2010) or a synonym of Kurixalus bisacculus (Taylor, 1962) by Yu et al. (2010). On the basis of broad sampling, recently Yu et al. (2017a) suggested that K. hainanus is valid and revealed six lineages that might represent undescribed species in the genus Kurixalus, one of which occurs in western Yunnan, China and northern Myanmar and is genetically closer to Kurixalus naso (Annandale, 1912) than to other known congeners with a divergence of 6.18% estimated from COI sequences (clade C, Fig. 1).
Here we further describe the lineage consisting of specimens from western Yunnan, China as a new species. Morphological comparisons demonstrate that the new species is distinctive from K. naso and other known congeners and therefore warrants taxonomic recognition.

Materials and methods
Sampling. Specimens were collected during fieldwork in Dehong Autonomous Prefecture, western Yunnan, China in June and July, 2014 (Fig. 2). They were euthanized with diethyl ether anesthesia and fixed by 90% ethanol before being stored in 70% ethanol. Liver tissues were preserved in 99% ethanol. Specimens were deposited at Kunming Institute of Zoology, Chinese Academy of Sciences.
Morphology. Morphometric data were taken using digital calipers to the nearest 0.1 mm. Morphological terminology follows Fei (1999). Measurements include:

SVL
snout-vent length (from tip of snout to vent); HL head length (from tip of snout to rear of jaws); HW head width (width of head at its widest point); SL snout length (from tip of snout to anterior border of eye); IND internarial distance (distance between nares); IOD interorbital distance (minimum distance between upper eyelids); UEW upper eyelid width (maximum width of upper eyelid); ED eye diameter (diameter of exposed portion of eyeball); TD tympanum diameter (the greater of vertical or horizontal diameter of tympanum); DNE distance from nostril to eye (from posterior border of nostril to anterior border of eye); FLL forelimb length (distance from elbow to tip of third finger); THL thigh length (distance from vent to knee); TL tibia length (distance from knee to heel); FL foot length (distance from proximal end of inner metatarsal tubercle to tip of fourth toe); TFL length of foot and tarsus (distance from tibiotarsal joint to tip of fourth toe).  A multivariate principal component analysis (PCA) was conducted using SPSS 17.0 (SPSS Inc.) based on a correlation matrix of size-standardized measurements (all measurements divided by SVL). Scatter plots of the scores of the first two factors of the PCA were used to examine the differences between the new species and K. naso. Additionally, the differences between the new species and its two congeners known from Yunnan, China (K. odontotarsus and K. hainanus) were also similarly examined based on morphometric data.

Results
Morphometric data of the new species and K. naso are summarized in Table 1. We retained the first two principal components which accounted for 63.03% of the total variance and had eigenvalues above 2.0 ( Table 2). Loadings for PC 1, which accounted for 48.69% of the total variance, were all positive except for TD and were most heavily loaded on HL, SL, IND, UEW, FLL, THL, TL, and TFL (Table 2). Differentiation was found along the PC 1 axis between K. naso and the new species (Fig. 3). This result indicates that the new species differs from K. naso by a series of characters associated with the head and limbs such as shorter HL, shorter SL, narrower IND, narrower UEW, shorter FLL, shorter THL, shorter TL, and shorter TFL. The second principal component (PC 2) accounted for 14.34% of the total variance and loaded heavily and positively on IOD and negatively on HW (Table 2), but no clear separation was observed along this axis between the new species and K. naso (Fig. 3). In addition, the new species can be separated from K. odontotarsus and K. hainanus by having smaller ratio of head length to body size (Fig. 4).  Diagnosis. The new tree frog species is assigned to the genus Kurixalus based on a combination of the following characters: tips of digits enlarged to discs, bearing circum-marginal grooves; small body size (SVL range of 31.6-34.7 mm in adult males; Table 1); finger webbing poorly developed and toe webbing moderately developed; ser rated dermal fringes along outer edge of forearm and tarsus; an inverted triangularshaped dark brown mark between eyes; dorsal brown ") (" saddle-shaped marking; and coarse dorsal and lateral surfaces with small, irregular tubercles (Nguyen et al. 2014a, Nguyen et al. 2014b, Yu et al. 2017b). Our previous molecular study placed the new species in Kurixalus with other known congeners (Yu et al. 2017a).
Kurixalus yangi sp. n. can be distinguished from its congeners by a combina tion of the following characters: male body size larger than 30 mm; smaller ratio of head length to body size; curved canthus rostralis; weak nuptial pads; brown dorsal color; absence of large dark spots on upper-middle abdomen; presence of vomerine teeth; gold brown iris; single internal vocal sac; serrated dermal fringes along outer edge of limbs; granular throat and chest; interorbital space longer than upper eyelid; rudimentary web between fingers; and presence of supernumerary tubercles and thenar tubercle.
Description of holotype. A small rhacophorid; HL shorter than HW; snout pointed, no dermal prominence on tip, projecting beyond margin of lower jaw in ventral view; canthus rostralis blunt and curved; lore region oblique, slightly concave; nostril oval, slightly protuberant, closer to tip of snout than eye; IND slightly narrower than IOD; pineal spot absent; pupil oval, horizontal; tympanum distinct, rounded, slightly less than half ED; supratympanic fold distinct, curving from posterior edge of eye to insertion of arm; vomerine teeth in two oblique patches, touching inner front edges of oval choanae; tongue notched posteriorly; single internal vocal sac.
Relative length of fingers is I < II < IV < III. Tips of all four fin gers expanded into discs with circum-marginal and transverse ventral grooves; relative width of discs is I < II < IV < III; nuptial pad present on first finger; fingers weakly webbed at base; lateral fringes on free edges of all fingers; subarticular tubercles prominent and rounded, formula 1, 2, 2, 1; supranumerary tubercles present; two metacarpal tuber cles present; series of white tubercles forming serrated fringe along outer edge of forearm.
Heels overlapping when legs at right angle to body; relative length of toes is I < II < III < V < IV; tips of toes expanded into discs with circum-marginal and transverse ventral grooves; toe discs smaller than finger discs; relative size of discs is I < II < III < V < IV; webbing moderate on all toes, webbing formula is I1.5-2II1-2III1-2IV2-1V following Myers and Duellman (1982); subarticular tubercles prominent and rounded, formula 1, 1, 2, 3, 2; supernumerary tubercles present; inner metatarsal tubercle distinct, oval; outer metatarsal tubercle absent; series of tubercles forming serrated dermal fringe along outer edge of tarsus and fifth toe.
Numerous small to large tubercles scattered on top of head, upper eyelids, dorsum, and flanks; patch of white tubercles below vent; white tubercles on tibiotarsal articulation; throat and chest finely granulated and abdomen coarsely granulated; dor sal surface of limbs smooth with tuberculs and ventral surface of thighs granulated.
Color of holotype in life. Iris golden brown; dorsal surface brown, mottled with green patches and a dark brown saddle-shaped mark on dorsum behind eye; a dark   brown inverted triangular-shaped mark between eyes, posterior of which extends to and touches the saddle-shaped mark; lateral head and tympanic region brown, mottled with green patches below canthus and dark brown spots on edge of upper jaw; flank light yellow, mottled with green and brown patches; limbs dorsally brown with three clear dark brown bands, mottled with green; palm of hand light red; rear, anterior, and venter of thigh red; inner side of tarsus and foot red; chest and abdomen white, fringed with yellow and mottled with small brown spots; chin clouded with dark brown and mottled with yellow patches.
Color of holotype in preservative. In preservative, green, yellow, and red faded. Dorsal ground color brown, pattern same as in life. Flank white with brown patches; margin of lower jaw clouded with dark brown; chin, chest, and abdomen white with scattered brown spots; palm of hand dirty white; anterior, posterior, and venter of thigh dirty white, with many fine brown speckling scattered on venter of thigh; inner side of tarsus and foot dirty white.
Variations. Because the holotype and paratypes of the new species are all male, sexual dimorphism could not be determined. IND is smaller than IOD in holotype and most paratypes, but IND is larger than IOD in paratype KIZ 14102913 (Table 1). In addition, IOD is larger than UEW in holotype and most paratypes, but IOD is smaller than UEW in paratypes KIZ 14102912 and KIZ 14102913 (Table 1). Additionally, color pattern of paratype KIZ 14102912 also differs from other specimens in that its chin has much less spotting.
Distribution and natural history. The new species is known from border region with northern Myanmar in western Yunnan, China (Fig. 2) and northern Myanmar according to Yu et al. (2017a). At the type locality, the new species was found calling on leaves of bushes adjacent to a road at night (Fig. 8). Specimens from the other two sites were found calling on broad leaves at the edge of an evergreen forest. Tadpoles, eggs and females were not found.
Comparisons. The new species, Kurixalus yangi sp. n., is genetically closer to K. naso than to other known members of Kurixalus according to our previous work (Yu et al. 2017a), but morphologically it can be separated from K. naso by having smaller ratios of head, snout, IND, UEW, and limbs divided by SVL (Table 2 and Fig. 3). The smaller IND and UEW ratios in the new species can be observed when comparing these distances with the IOD, which is generally larger in the new species but smaller in K. naso (Table 1).
Currently, three Kurixalus species (K. odontotarsus, K. hainanus, and K. lenquanensis Yu, Wang, Hou, Rao, & Yang, 2017) are recognized in Yunnan, China (Yu et al. 2017a, Yu et al. 2017b). The new species differs from K. odontotarsus and K. hainanus by having smaller ratio of head length to body size and no large dark spots on abdomen (versus larger ratio of head length to body size and large dark spots on entire abdomen; Figs 4, 9) and from K. lenquanensis by larger body size (SVL of 31.6-34.7 mm in adult males), more pointed snout, and presence of green coloration on dorsal surface and lateral side of head and body (versus smaller body size [SVL of adult males less than 30 mm], somewhat rounded snout, and absence of green coloration on dorsum; Fig. 9, Yu et al. 2017b).
The new species is distinguished from Kurixalus idiootocus (Kuramoto & Wang, 1987) by larger body size, absence of a pair of symmetrical large dark patches on chest, and single internal vocal sac (versus smaller body size [SVL of adult males less than 30 mm], presence of a pair of symmetrical large dark patches on chest, and single external vocal sac; Yu et al. 2017a); from Kurixalus berylliniris Wu, Huang, Tsai, Li, Jhang, & Wu, 2016 by gold brown irises, weak nuptial pads, and coarsely granular abdomen (versus emerald to light green irises, greatly expanded nuptial pads, and smooth abdomen; Wu et al. 2016); from Kurixalus wangi Wu, Huang, Tsai, Li, Jhang, & Wu, 2016 by larger body size, weak nuptial pads, and presence of supernumerary tubercles on foot (versus smaller body size [SVL of 28.6-31.6 mm in adult males], greatly expanded nuptial pads, and absence of supernumerary tubercles on foot; Wu et al. 2016); and from Kurixalus eiffingeri (Boettger, 1895) by weak nuptial pads, oblique loreal region, and curved canthus rostralis (versus greatly expanded nuptial pads, vertical loreal region, and straight canthus rostralis; Wu et al. 2016).

Discussion
Species diversity of the genus Kurixalus seems to be underestimated, with at least five unnamed lineages in the K. odontotarsus species group, with the exception of the new species described here, remaining to be described according to our earlier work (Yu et al. 2017a; Fig. 1). Taxonomic confusion in the K. odontotarsus species group mainly involved K. bisacculus. Of the remaining five clades that might represent unnamed species, four (clades F, G, H, and K; Fig. 1) were placed in K. bisacculus (Stuart and Emmett 2006, Thy et al. 2010, Yu et al. 2010, Nguyen et al. 2014a, Nguyen et al. 2014b). Even K. hainanus (clade J) was considered a synonym of K. bisacculus (Yu et al. 2010). A reason for this situation is the relatively low divergence of 16S rRNA sequences between K. bisacculus and these clades, which resulted in these lineages being considered conspecific though morphological differences exist between them (e.g., Yu et al. 2010). Another source of taxonomic confusion in the K. odontotarsus species group involves K. verrucosus, as specimens from northern Myanmar (Kurixalus sp5; Fig. 1) and Kurixalus naso from southern Tibet (clade A, Fig. 1) had been wrongly treated as K. verrucosus in previous molecular studies (Yu et al. 2010, Yu et al. 2013, Li et al. 2013, Nguyen et al. 2014a, Nguyen et al. 2014b) according to Yu et al. (2017a). Additionally, with the exceptions of those unnamed lineages revealed by Yu et al. (2017a), cryptic species likely also exist in Philippine populations of K. appendiculatus according to Gonzalez et al. (2014). In short, combination of the two recent molecular studies based on broad sampling (Gonzalez et al. 2014, Yu et al. 2017a has provided a relatively clear genetic framework for the taxonomy of Kurixalus and more morphological studies will be necessary to verify the specific status of those lineages. Phylogenetically, the K. odontotarsus species group is comprised of two clades; one contains K. yangi sp. n., K. naso, and K. sp5 and one contains other species from Indochina and southern China (Fig. 1). Kurixalus yangi sp. n. is known from western Yunnan, China and northern Myanmar, K. naso is known from southern Tibet and northeastern India, and K. sp5 is known from northern Myanmar. This pattern suggests that frogs of Kurixalus might have colonized the Indian subcontinent from northern Indochina.


extfieldsofstudy: Chemistry Medicine

text:
Yinchenhao Decoction Alleviates Liver Fibrosis by Regulating Bile Acid Metabolism and TGF-β/Smad/ERK Signalling Pathway

Yinchenhao decoction (YCHD), comprising Yinchenhao (Artemisiae Scopariae Herba), Zhizi (Gardeniae Fructus) and Dahuang (Radix Rhei et Rhizoma), is widely used for treating various diseases. We aimed to investigate the bile acid metabolic mechanism of YCHD in dimethylnitrosamine (DMN)-induced liver fibrosis model. Rats received DMN (10 mg/kg, intraperitoneally) for four successive weeks for liver fibrosis induction and were treated with YCHD for the last 2 weeks. Histopathological analysis showed that YCHD prevented DMN-induced histopathological changes in liver tissues. Serum liver function in YCHD group improved. Ultraperformance liquid chromatography-mass spectrometry analysis showed that YCHD significantly restored both free and conjugated bile acid levels increased by DMN, to normal levels. RT-qPCR results showed that YCHD treatment upregulated the expression of genes related to bile acid synthesis, reabsorption, and excretion. Western blotting analysis showed that YCHD downregulated α-SMA, TGF-β1, p-Smad3, and p-ERK1/2 expression in chenodeoxycholic acid (CDCA)-activated hepatic stellate cells (HSCs). The viability of CDCA-activated HSCs significantly increased after treatment with YCHD and PD98059 (an ERK inhibitor) compared to YCHD treatment alone. Our findings suggest that YCHD alleviated DMN-induced liver fibrosis by regulating enzymes responsible for bile acid metabolism. Additionally, it inhibits CDCA-induced HSC proliferation and activation via TGF-β1/Smad/ERK signalling pathway.

YCHD Attenuates DMN-induced Hepatic Histopathological Changes. Haematoxylin and eosin
(H&E) staining showed that hepatocytes in rats receiving DMN alone exhibited increased swelling, degeneration, and lymphocyte infiltration in intralobular, periportal, and bridging areas. Treatment with YCHD clearly improved DMN-induced pathological changes. No remarkable hepatocyte degeneration was found in the liver tissues of YCHD-treated rats (Fig. 1A).
In the control group, Sirius red staining showed a normal distribution of collagen with a thin rim around the terminal hepatic vein. Liver specimens obtained from DMN-treated rats showed extensive perilobular fibrosis as indicated by increased hepatic collagen in comparison with control rats. YCHD-treated rats showed decreased collagen deposition (Fig. 1B). Significantly higher fibrosis scores were observed in DMN-treated rats than in normal control rats; YCHD significantly improved these scores compared with DMN (Table 1).

YCHD Reduces Hepatic hydroxyproline (Hyp) Content and Improves Serum Liver Function.
To evaluate the anti-fibrosis effects of YCHD further, Hyp content in liver tissues was measured. Compared with the DMN group, the YCHD group showed a significant decrease in Hyp content (Table 1). Rats with DMN-induced liver damage exhibited significantly increased serum alanine aminotransferase (ALT), aspartate aminotransferase  (AST), and alkaline phosphatase (ALP) activities (P < 0.01), and serum contents of total bile acid (TBA) and total bilirubin (TBIL) (P < 0.01), along with reduced serum albumin (Alb) level when compared with control rats. YCHD treatment completely reversed these indices of DMN-induced damage in liver function (Fig. 2).

YCHD Regulates Bile Acid Metabolism.
For the metabolic profiling of bile acids after YCHD treatment, 16 individual bile acids in the serum and hepatic tissues (see Supplementary Figs S1 and S2) of rats were quantified using ultra performance liquid chromatography-mass spectrometry (UPLC-MS). Six free bile acids including cholic acid (CA), ursodeoxycholic acid (UDCA), CDCA, hyodeoxycholic acid (HDCA), and lithocholic acid (LCA), six taurine-conjugated bile acids including taurocholate (TCA), taurodeoxycholate (TDCA), tauroursodeoxycholate (TUDCA), taurochenodeoxycholate (TCDCA), taurohyodeoxycholic acid (THDCA), and taurolithocholicacid (TLCA), and four glycine-conjugated bile acids including glycocholate (GCA), glycodeoxycholate (GDCA), glycoursodeoxycholate (GUDCA), and glycochenodeoxycholate (GCDCA) were quantified. The changes in bile acid composition after YCHD treatment were identified by comparing the relative bile acid contents in the serum and hepatic tissues of rats (Fig. 3). UDCA and LCA contents in the liver were lower than the limit of quantitation. The levels of all other free, taurine-conjugated, and glycine-conjugated bile acids were significantly upregulated in the DMN-treated rat samples compared with control samples; however, these levels recovered to normal values upon YCHD treatment.

YCHD Regulates the Expression of Bile Acid Metabolism-Associated Genes.
To clarify the regulatory mechanisms of YCHD on bile acid metabolism, we further quantified the expression of bile acid metabolism-associated genes in rat liver tissues. The expression of nuclear receptor genes, including farnesoid X receptor (FXR), small heterodimer partner (SHP), liver receptor homolog-1 (LRH-1), and hepatocyte nuclear factor-4α (HNF4α), was markedly downregulated in the DMN group; however, FXR and SHP expression was significantly upregulated upon YCHD treatment (Fig. 4A). The expression of bile acid synthesis-associated genes, including cholesterol 7-alpha-hydroxylase (CYP7A1), sterol 12-alpha-hydroxylase (CYP8B1), and sterol 27-hydroxylase (CYP27A1), was downregulated in the DMN group compared with the control group, and YCHD treatment restored their expression to normal levels (Fig. 4B). Although the expression of bile acid reabsorption-associated genes, including organic anion-transporting polypeptides (OATP) 2, 3, and 4, and Na+-taurocholate co-transporting protein (NTCP), was downregulated in the DMN group compared with the control group, the expression of OATP4 and NTCP was significantly upregulated in the YCHD group compared with the DMN group (Fig. 4C). Moreover, the expression of bile acid excretion-associated genes, including multidrug-resistance-associated protein 3 (MRP3) and bile salt export pump (BSEP), was studied. MRP3 expression was significantly upregulated in the DMN group and significantly downregulated in the YCHD group. The results for BSEP expression were the opposite of those seen for MRP3 (Fig. 4D).
YCHD Inhibits the Expression of TGF-β1, p-Smad3, and p-ERK1/2 in Hepatic Tissues of Rats. Western blot analysis revealed that TGFβ1, α-SMA, p-Smad3, and p-ERK1/2 protein expression levels in DMN-induced liver fibrosis group were significantly higher than those in the control group (Fig. 5). Furthermore, YCHD treatment significantly reduced these protein expression levels. However, there was no significant difference among the DMN, control, and YCHD groups in the protein expression levels of p-Smad2.

YCHD Suppresses CDCA-induced HSC-T6 Cell Proliferation and Activation by Inhibiting TGF-β1/
Smad/ERK Signalling Pathways. Excessive CDCA may represent an endogenous danger signal to initiate liver inflammation 8 ; therefore, we used CDCA to induce proliferation in HSCs. As shown in Fig. 6A, cell viability was determined after exposing HSCs to CDCA at different concentrations. Because CDCA significantly increased the viability of HSC-T6 cells, subsequent experiments were performed with 100 μM CDCA. YCHD exposure significantly decreased the viability of HSC-T6 cells in a dose-dependent manner (Fig. 6B). CDCA significantly increased α-SMA, TGFβ1, p-Smad3, and p-ERK1/2 expression; however, they were decreased by YCHD treatment ( Fig. 6C,D). Consequently, YCHD may inhibit cell proliferation and HSC activation by negatively regulating TGF-β1/Smad/ERK signalling pathways.
To further clarify whether YCHD inhibits HSC proliferation and activation via TGF-β1/Smad/ERK signalling pathways, we measured the viability of HSCs and YCHD-induced expression of p-ERK1/2 protein in the presence of PD98059, an ERK inhibitor. Treatment with PD98059 and YCHD significantly enhanced the viability of CDCA-induced HSC-T6 cells compared with YCHD treatment (Fig. 7A). Additionally, the expression of α-SMA and p-ERK1/2 was significantly higher after YCHD and PD98059 treatment than after YCHD treatment alone ( Fig. 7B-D). These findings indicate that YCHD could inhibit p-ERK1/2 to inhibit the proliferation and activation of HSCs.

Discussion
Fibrosis is a common and serious complication of various diseases and occurs frequently. It is characterised by abnormal or excessive accumulation of ECM components, particularly fibrillar collagens, leading to disrupted tissue function in the affected organs 3,20-24 . Fibrosis is a key driver of progressive organ dysfunction in many inflammatory and metabolic diseases, including liver disease, pulmonary fibrosis, kidney disease, and heart failure [25][26][27][28][29][30] . TCM has been used since ancient times and was widely considered as an alternative or complementary therapy for the treatment of various diseases [31][32][33][34][35] . Although increasing evidence has shown that liver fibrosis can regress in many cases in the past decades 36 , there is a lack of clinically effective drugs for the treatment of cirrhosis. YCHD is a clinically effective treatment for liver and gallbladder dampness-heat syndrome in cholestatic liver diseases 18 . The intraperitoneal administration of DMN for 4 weeks could induce liver fibrosis and cause specific histological features of fibrotic liver tissue in animal model 37 . In the present study, we demonstrated that YCHD protects against pathological changes of liver fibrosis, and regulates hepatic Hyp content and the serum levels of ALT, AST, ALP, TBA, and TBIL. These data indicated that YCHD effectively reduced liver injury and fibrosis induced by DMN. Furthermore, we investigated the mechanisms of action of YCHD against liver fibrosis by analysing the metabolic profiles of bile acids.
Smit et al. hypothesised that toxic bile may cause hepatic injury 38 , and since then, the relationship between toxic bile acids and chronic liver injuries has been studied extensively. It has been reported that serum GCA, TCA, and TCDCA levels are higher in patients with obstructive cholestasis than in healthy individuals 39 . Patients with advanced cirrhosis also have increased levels of GCDCA, a toxic bile acid, compared with normal individuals 40 . The serum levels of CA, GCA, and TCA are significantly increased in animals with histopathological signs of hepatocellular necrosis, which were in accordance with the levels of standard biomarkers of liver injury 41 . In our study, the levels of free bile acids such as CA, DCA, CDCA, and HDCA; conjugated bile acids, especially TCDCA; and four glycine-conjugated bile acids were higher in the serum/hepatic tissues of DMN-induced liver fibrosis rats than in normal rats. This indicates that DMN increases the release of these bile acids from the liver into the blood. However, their levels were decreased in rats treated with YCHD compared with DMN-treated rats. This finding is consistent with the YCHD-induced improvement in liver function as shown by the levels of biochemical markers of liver injury, such as ALT, AST, ALP, TBA, and TBIL, indicating that YCHD decreases the release of toxic bile acids, including CA, DCA, CDCA, and GCDCA, into the blood stream in response to liver injury.
Bile acids are synthesised via two pathways. The initial step in the classical pathway of hepatic synthesis of bile acids is the enzymatic addition of a 7α hydroxyl group by CYP7A1 to form 7α-hydroxycholesterol, which is further hydroxylated by CYP8B1 42 . Alternatively, bile acid synthesis is initiated by CYP27A1. It has been reported that FXR activation in rats with established cirrhosis leads to the accelerated resolution of liver fibrosis 43 , and Bile acid excretion associated genes. All data were determined by RT-qPCR. ACTB was as the internal standard. Values are expressed as mean ± SD. Significant differences were analyzed by one-way ANOVA. # P < 0.05, ## P < 0.01, ### P < 0.0001 (vs. Control group).
FXR expression in the rat liver significantly decreases in α-naphthylisothiocyanate (ANIT)-induced cholestasis model 44 . FXR induces the expression of SHP, which then binds to HNF4α and LRH-1 to inhibit the transcription of CYP7A1 gene. In this study, FXR, SHP, CYP7A1, CYP8B1, and CYP27A1 levels were upregulated in YCHD-treated rats, whereas they were downregulated in DMN-treated rats. This indicates that YCHD inhibits the synthesis of bile acids by regulating the negative feedback pathway.
The re-uptake of conjugated bile acids into hepatocytes occur through NTCP (SLC10A1) or OATP transporters 45 . NTCP (SLC10A1) mutation significantly elevates plasma levels of conjugated bile acids; however, it does not translate into liver injury, indicating that enterohepatic circulation of bile acids may be caused by other transporters, such as OATP, when NTCP is absent 46 . Bile acid excretion occurs across canaliculus and basolateral membranes into the duodenum or back into the systemic circulation via BSEP and MRP3 47,48 . In our study, MRP3 was activated in DMN-induced liver fibrosis rats, and the expression of NTCP, OATP2/3/4, and BSEP was significantly upregulated in YCHD-treated rats compared with that in DMN-treated rats. YCHD may increase bile acid reabsorption and restore the balance in bile acid excretion, thereby reducing the impact of toxic bile acids on liver injury and fibrosis in rats.
CDCA, which is considered to be potentially toxic, has been reported to induce mitochondrial injury and death of bile duct epithelial cells 49 and to induce HSC proliferation and activation 19 . Hence, we explored the effect of YCHD on CDCA-induced HSC activation in this study. α-SMA is commonly used as an important marker of myofibroblast formation 50,51 , which reflects the activation of HSCs 52 . Therefore, we investigated α-SMA expression in HSC-T6 cells further. The results showed that CDCA could significantly upregulate α-SMA expression; however, the upregulated expression of α-SMA was decreased by YCHD treatment, indicating that toxic bile acids, such as CDCA, can induce HSC activation.
An interaction between the TGF-β1/Smad pathway and the ERK pathway has been established 53,54 , and TGF-β1/Smad/ERK signalling contributes to the progression of liver fibrosis 55 . In this study, we found that YCHD suppresses the protein expression of TGF-β1, p-Smad3 and p-ERK1/2, whereas DMN activates TGF-β1/Smad/ERK signalling to induce liver fibrosis in rats. To confirm these effects, we further quantified the expression of TGF-β1, p-Smad2, and p-ERK1/2 signalling in CDCA-activated HSCs. The results indicated that YCHD suppresses CDCA-induced HSC proliferation and activation by inhibiting TGF-β1/Smad/ERK signalling pathway.
Overall, this study demonstrated that YCHD prevents histopathological changes of hepatic dysfunction in DMN-induced liver fibrosis rats by regulating bile acid metabolism enzymes, which are associated with the increase in bile acid synthesis, reabsorption, and excretion. Furthermore, YCHD inhibits CDCA-induced HSC proliferation and activation by regulating TGF-β1/Smad/ERK signalling.

YCHD Preparation.
Granules of herbal concentrates comprising Yinchenhao (90 g), Zhizi (54 g), and Dahuang (36 g) were purchased from Shuguang Hospital. The granules were immersed in 1.2 L of cold water (6 mL/g of crude drug granules) for 30 min. This mixture was then boiled, slowly fried for 40 min, and filtered when hot under gravity. The remaining pellets were re-immersed in 0.8 L of water (4 mL/g of crude drug granules), boiled, slowly fried for 40 min, and filtered when hot under gravity. The filtrates were combined and concentrated to 400 mL at a final concentration of 630 mg/mL in a water bath at 95 °C. The fingerprint of YCHD was determined for quality control by HPLC 56 .
Preparation of YCHD-medicated Serum. Sprague-Dawley rats were randomly divided into YCHD (n = 8) and vehicle control (n = 8) groups. Rats in the YCHD group received YCHD (3.15 g/kg, p.o.) twice a day for three days, whereas the control group received physiological saline (p.o.) twice a day for three days. One hour after the last administration, the rats were intraperitoneally anesthetised with amobarbital, and blood was sampled from the abdominal aorta and centrifuged. Aliquots of the separated serum were collected into 10-mL ampoules, and preserved at −80 °C until analysis.
This study was carried out in accordance with the recommendations of the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996)   Animal Experiments. Twenty-four 7-week-old male Wistar rats (200-250 g) were housed under standard conditions of temperature (17-25 °C) and light (12-h photoperiod). The rats were randomised into control (n = 9) and DMN-treated (n = 15) groups. In the DMN-treated group, 10 mg/kg DMN was administered intraperitoneally for three consecutive days each week for 4 weeks, and the rats in the control group received an equal volume of physiological saline. At the end of the second week, six DMN-treated rats were co-treated daily with YCHD (3.15 g/kg, p.o.), and the remaining nine rats received an equal volume of water. At the end of the fourth week, all animals were killed, and the liver and serum samples were collected for subsequent analysis. The study protocol was approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine.
Histological Analysis. Formalin-fixed liver tissues were processed, and 4-μm thick slices were stained with H&E and Sirius red. The specimens were observed for histopathological changes by light microscopy. Fibrosis was graded by three blinded pathologists according to the description by Scheuer 57 . Fibrosis scores were obtained after thorough examinations of three different areas of the tissue slide from each rat.
Hepatic Hydroxyproline Content and Serum Biochemistry Analysis. Liver specimens (100 mg) were prepared for Hyp determination according to a modified method described by Jamall. The Hyp content served as an indirect measure of tissue collagen content and was weighed wet (μg/g). The serum levels of ALT, AST, ALP, Alb, and TBIL were measured according to the instructions provided by the manufacturers of corresponding analytical kits. TBA was measured at a clinical laboratory of Shuguang Hospital.
Metabolic Profiling Analysis. Chromatographic analyses were performed with a Waters ACQUITY UPLC system using a ZQ2000 mass spectrometer. UPLC/MS-based metabolic profiling analysis of bile acids was carried out on serum samples according to established methods 58 .
RT-qPCR. The expression of genes associated with bile acid metabolism was determined by RT-qPCR. Total RNA was extracted from liver tissues using the RNAsimple Total RNA Kit (TIANGEN, Beijing, China) and reverse-transcribed using the ReverTra Ace ® RT-qPCR kit (TOYOBO, OSAKA, Japan). qPCR was performed on an ABI 7500 PCR System (Applied Biosystems, Foster City, CA, USA) under the following conditions: 95 °C for 30 s, 95 °C for 5 s (40 cycles); 60 °C for 30 s, and 72 °C for 15 s. The primer pairs used in this study are shown in Table S1. Each sample was run three times. The relative expression level of genes was calculated using beta-actin as the internal control.

MTS Assay.
HSCs were seeded in a 96-well plate at a density of 2.0 × 10 3 /well in triplicate to monitor cell viability. The cells were cultured for 1, 2, and 3 days at 37 °C. CellTiter 96 AQueous One solution (20 μL) containing MTS was added to each well. After 4 h of incubation at 37 °C, the absorbance at 490 nm was measured. Cell viability was calculated with respect to control samples. Western Blot Analysis. HSC cell lysates were collected, and the protein concentrations were determined by the BCA protein assay. Equal amounts of proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes as previously described 59 . Then, the membranes were incubated overnight at 4 °C with the primary antibodies, and GAPDH as an internal control. Subsequently, the membranes were incubated with a secondary antibody (Li-Cor Biosciences) for 1 h at room temperature. Finally, the target protein bands were visualised using the Li-Cor Odyssey scanner and software (Li-Cor Biosciences). Data Analysis. Statistical analysis was performed using the SPSS software (Chicago, IL, USA) and the GraphPad Prism software (San Diego, CA, USA). All values are expressed as the mean ± standard deviation (SD) and statistically analysed using one-way ANOVA. P < 0.05 was considered statistically significant.