Chlorogenic Acid and Geniposide Combination Prevents NASH in Rats Fed High-fat diet via Microbiota and TLR4-LPS Pathway

Objective: Chlorogenic acid and geniposide (CG) are derived from traditional Chinese medicine, Yinchenhao Recipe (QCHR), and can improve the clinical ecacy of NASH patients. This study investigated the effects of CG on NASH and expounded its Potential mechanism of action through the LPS-TLR4 pathway and microbiota. Methods: Rats were randomized into Control (C), Model (M), Chlorogenic Acid and Geniposide (CG), Pioglitazone (PH) and Bico (B) groups. After an 8-week high-fat diet (HFD), CG, PH and B oral treatment were initiated and carried out for a further 8 weeks. The stool samples were used in a16S rDNA V4 highly variable region measurement method in order to regulate the role of CG in gut microbiota. The concentrations of triglyceride (TG), cholesterol (CHO), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in LPS were detected by the corresponding methods. Results: Observations were made that CG signicantly improved the pathology of the liver and terminal ileum tissue. The accumulation of TG and the content of inammatory cytokines in the liver were signicantly decreased and the abundance of Proteobacteria was signicantly down-regulated. The expression of TLR4, AP-1, MyD88, and phosphorylated NF-κB p65 were signicantly decreased. All the ndings above indicated that CG was highly effective in improving the composition of gut microbiota, decreasing the production of endogenous LPS, and reducing the secretion of inammatory cytokines through the gut-liver axis. Conclusion: CG can regulate the abundance and diversity of the intestinal microbial community and improve liver inammation and steatosis in NASH rats by reducing LPS-TLR4-mediated inammation.


Introduction
Non-alcoholic fatty liver disease (NAFLD) is de ned as hepatic steatosis excluding the causes of signi cant alcohol consumption, steatogenic medication or hereditary disorders 1 -It's histologically characterized by steatosis 2 . About 25% of adults worldwide suffer from Nonalcoholic fatty liver disease (NAFLD) 3 and despite the known risk factors such as diabetes, obesity, age, gender and race, the prevalence of NAFLD is still increasing 4 . NAFLD will elevate the risk of all-cause mortality, liver-related deaths, malignancy, diabetes and coronary artery disease 5 . Histologically, NAFLD can be classi ed into non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) 1 . NASH, as an aggressive form of NAFLD, can develop into liver brosis, cirrhosis and hepatocellular carcinoma (HCC) in 5-15% of patients 6 . With the development of the brosis stage, the mortality risk of NAFLD patients increases exponentially 7 . Lifestyle changes are the basis of treatment for patients with NAFLD. The treatment of NASH can be divided into drugs that target disease pathogenesis (such as insulin resistance and de novo lipogenesis) and drugs that target downstream processes (such as cellular stress, apoptosis, in ammation and brosis). None of these are approved by the US Food and Drug Administration 6,8 .
Chlorogenic acid and geniposide are derived from the Yinchenhao Recipe, which can improve the clinical e cacy of NASH patients 9 . A profusion of studies have evidenced that chlorogenic acid and geniposide have antioxidant effects on different diseases [10][11][12][13][14] . Chlorogenic acid (CGA), one of the most common and abundant polyphenols, is present in a wide variety of beverages and foods. Chlorogenic acid has anti-in ammatory and antioxidant effects on the liver 15,16 . There is evidence that demonstrates how chlorogenic acid can improve obesity induced by a high-fat diet by improving intestinal dysbiosis 17 . Geniposide is an important active ingredient of Gardenia jasminoides Ellis fruit can increase SOD levels and cell viability, reduce ALT, AST and LDH levels and con rm the signi cant hepatic protection 18 .
Geniposide can also restore any impaired function of the intestinal barrier 19 . Our previous research evidenced that geniposide or chlorogenic acid can decrease fatty liver steatosis and serum ALT and AST level in rats with NASH, but the mechanism was unclear [20][21][22] .
The pathogenesis of NAFLD is highly complex, the "multiple hit" hypothesis considers that insulin resistance and hormones secreted from the adipose tissue, genetic and epigenetic factors, nutritional factors and gut microbiota are all involved in NAFLD 23 . The nutrients, bacterial products and metabolites from the gut will be ltered upon reaching the liver. This integral link between the gut and liver is de ned as the gut-liver axis. When the intestinal permeability increased, more bacterial products such as LPS crossed the epithelial barrier. LPS can be recognized by toll-like receptors (TLRs) (especially TLR4), inducing the synthesis of a variety of in ammatory cytokines, which induce in ammation, oxidative stress, and insulin resistance 24,25 .
Considering CG's antioxidant, blood fat reduction, cholesterol-lowering and prebiotic effects, the hypothesized that CG would ameliorate NAFLD through intestinal regulating was formulated. The experiment aim is to examine the regulatory effect of CG on intestinal microbiota,hepatic in ammation and steatosis in high-fat-diet-induced NASH rat model.

2.1.CG attenuated HFD-induced obesity and liver injury
After the 16 week experiment, rats in the M group gained more epididymal fat weight than the control group, while the CG group gained less compared with the M group, but there was no signi cant difference between the PH and B groups (Fig 1A, B). The liver wet weight and hepatic index were signi cantly increased in the M group, respectively, compared with the C group. Post intervention at 8-weeks, the liver wet weight and hepatic index in the CG and PH group were signi cantly decreased compared to those in the M group, however, there was no signi cant difference between the B and M groups (Fig. 1C, D). The serum levels of ALT and AST were signi cantly increased in the M group compared with the control group and signi cantly decreased after the CG intervention at 8-weeks (Fig. 1E, F).

CG profoundly changed the composition of the intestinal microbiota in HFD-fed rats
To reveal the effects of HFD and the administration of CG on the microbiota structure, we sequenced the bacterial 16S rRNA at baseline and after 16 weeks, OTU levels by Shannon diversity index were signi cantly increased in the CG, PH and B groups compared with the M group ( Fig.2A). Unweighted PCoA, PCoA and NMDS analyses were conducted to provide an overview of the gut microbiota composition of the ve animal groups at baseline and the end of the trial. The plotted scores showed a substantial change in gut microbiota composition in rats fed an HFD. The results indicated that the HFDinduced NASH model had a considerable impact on the gut microbiota composition. CG intervention shifted the overall structure of the HFD-disrupted gut microbiota toward that of the control rats ( Fig. 2B-D). Venn diagrams also showed that 377 OTUs were shared between the C and CG groups, 277 OTUs were shared between the C and M groups (Fig. 2E). A hierarchical cluster analysis (HCA) was conducted, generating a heatmap that provided a comprehensive overview of which groups were clustered. The heatmap showed that the distance between the C and CG groups was closer than C and M's ( Fig. 2F). 16 weeks of HFD feeding induced extensive changes in the gut microbial community structure at the phylum level compared with the C group. There was an increase in the abundance of Firmicutes (95.0% vs. 82.4%) and decreases in the abundances of Actinobacteria (0.03% vs. 0.13%), Fusobacteria (0.007% vs. 0.002%) and Proteobacteria (0.1% vs. 0.3%). However, CG intervention mitigated the HFD-induced decrease in Actinobacteria and Proteobacteria, and the HFD-induced increase in Firmicutes. At the genus level, Streptococcus, Staphylococcus, Corynebacterium_1 were decreased in the M group compared to the C group, all of which were reversed by CG intervention. CG intervention decreased the abundances of Lactobacillus and Romboutsia compared with the HFD group (Fig. 2G, H). The plot showed that the abundance of Firmicutes, Actinobacteria and Fusobacteria had a signi cant difference in ve groups ( Fig.2I). In our experiment, the abundance of Firmicutes was much higher in the M group compared to the C group, and reversed by CG, PH and B intervention (Fig. 2J). However, the abundance of Bacteroidetes was similar among the ve groups (Fig.2K). The ratio of Firmicutes/Bacteroidetes (F/B) was also compared as a featured sign of obesity among the ve groups. As indicated in Figure 2L, HFD significantly increased the ratio of F/B, and its effect can be conversed by CG and B.

Terminal ileum epithelial barrier damage is diminished by administration of GC in rats fed an HFD, leading to the inhibition of LPS leakage
The HE staining demonstrated that the HFD-induced terminal ileum injury was alleviated by CG intervention (Fig.3A). The terminal ileum tissue of the rats in the C group was structurally intact, and the intestinal villi were neat and had an orderly arrangement. The terminal ileum tissue of rats in the M group presented damage to the intestinal mucosal barrier, the intestinal epithelial cells were detached, and the intestinal mucosal mechanical barrier was impaired. After treatment with CG, the ileal mucosal structure fully recovered, and the propria intestinal gland was abundant with goblet cells. After treatment with PH, the intestinal villi were orderly arranged, and there was a small amount of intestinal epithelial cells detached. After treatment with B, an intact terminal ileum structure, rich intestinal gland, and abundance of goblet cells can be observed. The expression levels of tight junction proteins were also examined, such as Occludin and zonula occluden-1 (ZO-1). The protein levels of Occludin and ZO-1were reduced in the M group, compared to the C group, and CG and B treatment rescued the protein expression of Occludin ( Figure 3B,D) and ZO-1( Figure 3C,E) through Immunohistochemistry. The plasma level of endotoxin was determined, which is an LPS and one of the main components of the cell wall of gram-negative bacteria.. With the increase of gram-negative intestinal bacteria in the M group, the LPS content in the plasma of M group was signi cantly increased compared with that in the C group. After treatment, the LPS content in the plasma was signi cantly decreased in the CG group, but not in the PH group and B group (Fig. 3F). From these results it can be observed that CG can prevent leaking of endotoxin from the gut into the bloodstream by upregulating the expression of TJ proteins.

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Hepatic steatosis is a predominant feature of NAFLD. For this reason, examined the effect of CG on NASH induced by the HFD diet was examined. As shown in Fig. 4A, in contrast to the C group, there was extensive steatosis and ballooning of hepatocytes and partial hepatocyte necrosis with a large area of nuclear fragmentation or dissolution with in ammation in the M group by H&E. Notably, HFD-induced steatosis and ballooning in the liver were decreased by CG treatment (Fig. 4A). The oil red O staining also demonstrated that HFD-induced lipid droplets in the liver were reduced by CG treatment (Fig. 4B,C). The TG content was signi cantly increased in the liver tissue of the M group compared with that of the C group. After treatment, the TG content in the liver tissue was signi cantly decreased in the CG group, PH group and B group (Fig. 4D). Similarly, HFD feeding markedly enhanced cholesterol in the liver of rats and was further reversed to normal levels by CG and PH treatment (Fig. 4E). Many cells/prominent ballooning 2 2.5. CG inhibited the activation of nuclear factor κB signaling and decreased the content of in ammatory cytokines (IL-1β, IL-6, TNF-α) Changes in gut microbiota control metabolic LPS-induced inflammation in high-fat diet-induced insulin resistance and in ammation in mice. LPS and TLR4 signaling control the production of proinflammatory cytokines, leading to chronic inflammation and insulin resistance in HFD-fed mice 26, 27 . The expression levels of TLR4, AP-1, MyD88 and phosphorylated NF-κB p65 were increased signi cantly in the liver tissue of the M group compared with that of the C group. After treatment, the expression levels of TLR4, AP-1, MyD88, and phosphorylated NF-κB p65 in the liver tissue were signi cantly decreased in the CG, PH, and B group ( Fig. 5A-F). These results show that HFD-fed rats produce higher levels of pro-inflammatory cytokines in hepatic and adipose tissue, including tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL1β). The concentration of these cytokines after 14 weeks of HFD feeding was measured. IL-1β, IL-6 and TNF-α content in the liver tissue of the M group were signi cantly increased compared with that of the C group. After treatment, the IL-1β, IL-6, TNF-α content in the liver tissue were signi cantly decreased in the CG, PH and B group (Fig. 5G-I). The content of IL-1β, IL-6, TNF-α in plasma was also detected. Notably, a similar conclusion was drawn in regards to both plasma and liver tissue (Supplement Fig 2).
Administration of CG improves microbiota dysbiosis, increases tight junction proteins in the gut, and decreases the production of endotoxin. CG also attenuates NASH through inhibiting activation of TLR4-LPS signaling and the production of proinflammatory cytokines.

Discussion
There is an increasing amount of evidence suggesting that alterations in the gut microbiota correlate with liver disease or immunological disease 28, 29 . As high-throughput sequencing technology has progressively matured, many studies have recorded the changes in the gut microbiota of NASH individuals. Using probiotics and prebiotics to regulate gut microbiota has become a new approach to prevent and treat NAFLD [30][31][32] . In this study, the community structure of the rat fecal microbiome induced by a high-fat diet through 16s rRNA gene sequencing was demonstrated. Observations were made that intestinal microbiota and in ammatory cytokines such as TNF-α, IL-1β and IL-6 had signi cantly interfered with the 16-week HFD in rats. However, the CG intervention at 8-weeks corrected the disturbance of intestinal microbiota to a certain extent and reduced the production of LPS in plasma, while inhibiting the TLR4-LPS pathway. The imbalance of in ammation cytokines and the reversal of steatohepatitis in high-fat rats was also corrected. These results suggest that CG may have major signi cance in the treatment of NASH.
The pathogenesis of NAFLD is highly complex, including insulin resistance, hormones secreted from the adipose tissue, genetic and epigenetic factors, nutritional factors and gut microbiota 23 . Recent research into the effects of chlorogenic acid and geniposide on the gut and liver has indicated that geniposide can administer cholesterol metabolism by regulating FXR-mediated gut-liver crosstalk of bile acids 33 .
Research also indicated that geniposide can ameliorate barrier dysfunction via AMPK-mediated inhibition of the MLCK pathway 19 . Previous studies demonstrated that geniposide has bene cial effects on liver diseases both in vivo and vitro 34,35 . The present study concludes that the action mechanism of the CG combination on NAFLD is multi-targeting. For this reason, it is necessary to further investigate the effects of the CG combination on regulation of microbiota in NAFLD.
CG, derived from the Yinchenhao Recipe (QCHR), is a traditional Chinese medicine formula composed of capillaris (Artemisia capillaris), gardenia (Gardenia jasminoides) and rhubarb (Rheum rhabarbarum). The main active compounds of YCHR are chlorogenic acid and geniposide. YCHR is comprised of 12 g capillaris (Artemisia capillaris), 9 g gardenia (Gardenia jasminoides), and 9 g rhubarb (Rheum rhabarbarum) 36 In the present study, the dose given to rats equates to approximately 4 g/day for humans. 37  To determine the underlying mechanism of how CG improves in ammation and obesity in rats, the gut microbiota in each group at the 16th week was detected. In the present study, observations were made that the OTU levels were signi cantly increased in the CG, PH and B groups compared with the M group according to the Shannon diversity index. Venn diagrams depicted that there were far more OTUs in the CG group than those in the C and M groups, and the lowest in the M group. According to PCoA, NMDS and hierarchical clustering analysis, CG reversed the overall change in the intestinal ora structure induced by HFD, and CG was allocated between the C and M groups.
A recent study revealed that the administration of S. liforme can improve physiological parameters of the heart and liver through regulating gut microbiota in HFD-induced rats, physiological parameters of the liver being closely correlated to special bacteria 41 . Similarly, in the present study, at the phylum level, the intestine microbiota of M was characterized by an increase in the abundance of Firmicutes and a decrease in the abundance of Bacteroides, Proteobacteria and Actinobacteria, which was similar to the previously reported gut microbiota in the intestines of obese people 42,43 . Prior research has suggested that Firmicutes are bene cial for obese people to obtain energy from food, and eventually gain weight 44 .
The claim that CG can reduce the weight gain of HFD rats can be reasonably believed. This is because CG decreases the abundance of Firmicutes. I.N. Abdallah, et al showed that the ratio of Firmicutes / Bacteroidetes is elevated in the intestines of obese people 45 , and it was also decreased in the CG group compared to the M group. At the genus level, Romboutsia were decreased in the M group compared to the C group, all of which was reversed by CG intervention. Romboutsia belongs to Bacteria-Firmicutes-ClostridiaClostridiales-Peptostreptococcaceae, which is related to body energy metabolism 46 . The above indicates that the direct modulating effects of CG on gut bacteria may play a pivotal role in the control of obesity. Observations were made that the abundance of Streptococcus, Staphylococcus, Corynebacterium_1 was increased in the CG group. Streptococcus thermophilus, as a probiotic cocktail, can improve liver in ammation in NASH patients 47 . Thus, the results above demonstrate that the administration of CG remodels the structure of the gut environment and improves the gut microbiota dysbiosis induced by HFD feeding. This nding may partially explain the bene cial role of CG in microbiota in HFD-induced NASH.
Notably, the present study demonstrated that the reduction of Proteobacteria can lower the production of endogenous LPS. The immunohistochemical results of the small intestine also showed that CG can restore the intestinal barrier by up-regulating tight junction proteins and inhibit the leakage of intestinal endotoxin into the blood. The present study further demonstrated that the LPS-mediated TLR4/NF-κB pathway is integral to the pathogenesis of NASH 48 . CG directly inhibits endotoxin-induced activation of the TLR4-LPS pathway and production of proin ammatory cytokines including IL-1β, IL-6, and TNF-α (Fig. 5). TNF-α induces hepatocyte cell death, causes insulin resistance, which results in hepatocyte steatosis, and regulates KCs' activation through an autocrine mechanism. IL-6 is a potential mediator of insulin resistance 49 . IL-1β induces steatosis, hepatocyte injury, and brosis 50 . The in ammation cytokines like TNF-α, IL-1β, and IL-6 are critical to the pathogenesis of NASH. The reduction of cytokine such as TNF-α, IL-1β and IL-6 and the level of LPS in plasma through CG, PH and B was established.
From the above, the conclusion was drawn that CG attenuated HFD-induced obesity and liver injury in rats by regulation of gut microbiota and inhibition of the TLR4-LPS pathway.
There are several limitations to the present study. Firstly, although CG can improve major indicators of NASH in the livers, its effect on metabolic parameters remains unclear due to the limitation of the highfat-diet-induced NASH rat model. Body weight does not decrease signi cantly compared with the M group after CG treatment (data not shown). Secondly, better grouping strategies can be set, such as adding a control group administrated with natural compounds. This can better clarify the impact of a high-fat diet on the intestinal ora. Chlorogenic acid and geniposide could also be added separately in individual groups to evaluate the advantages of their combination. Finally, the correlations of gut microbiome changes with physiological parameters and LPS-TLR4 pathway in rats fed a high-fat diet require further investigation.
In general, CG can reduce the abundance of bacteria producing LPS by changing the composition of the intestinal ora. CG can also increase the expression of Tj protein and reduce the entry of LPS into the blood through the intestine. This contributes to preventing liver injury through the inhibiting activation of the LPS-TLR4 pathway, decreasing the translocation of NF-κB and AP-1 and decreasing the production of in ammatory cytokines including IL-1β, IL-6, and TNF-α. CG can provide a therapeutic effect on NASH by targeting intestinal microbiota then manipulating intestinal microecological imbalance. The results of the present study highlight the therapeutic potential of CG in NASH management.

Animals and treatment
Sprague-Dawley (SD) rats (body weight 200 ± 20 g) were purchased from the Shanghai Sippe-Bk Lab Animal Co., Ltd. All rats were housed and kept under controlled conditions (23 ± 2°C), humidity (60 ± 10%) and a 12-hour light-dark cycle. The rats adapted to the conditions for a week and were then randomly allocated into a high-fat-diet (HFD) model group and normal diet control group (C group; n = 6). Rats were fed according to a high-fat diet (77.5% common feed + 0.5% sodium cholate + 2% cholesterol + 5% soya bean + 5% sucrose + 10% lard from) for 16 weeks in order to produce a NAFLD rat model 51 . At the beginning of the 9th week, the rats in the HFD-fed model group were randomly divided into 4 groups (n = 6 in each group): Model group (M group), Chlorogenic acid and Geniposide group (CG group), Pioglitazone hydrochloride group (PH group) and Bi d triple viable capsule (Bi co) group (B group) and a single rat as the experimental unit. The groups mentioned above were fed a HFD for another 8 weeks. The rats in the C group were fed with common feed for 16 weeks. From the 9th week, rats in the M and C groups were fed with double distilled water in the dose of 10 ml/kg-day absorbed intact in the stomach. Rats in the CG group were fed with chlorogenic acid and geniposide aborbed intact in the stomach. Rats in the CG group were also fed a HFD for 16 weeks, followed by a daily gavage of a mixture of a dosage of 60 mg/kg geniposide and a dose of 60 mg/kg-day chlorogenic acid (weight ratio = 66.7:1) in addition to a HFD for an additional 8 weeks. Rats in the PH group were fed with pioglitazone hydrochloride in the dose of 10 mg/kg-day absorbed intact in the stomach. Rats in the B group were fed with bi co in the dose of 210 mg/kg-day absorbed intact in the stomach.

Ethics statement
Experiments were conducted following established animal protocols and the guidelines approved by the animal experimental ethics committee of Xiamen University (approval No. XMULAC20200055).
All methods with respect to animals were carried out in accordance with relevant ARRIVE guidelines.

Sample collection
At the end of the 16th week, blood was extracted from the abdominal aorta in all the rats and the body weight was weighed. Rats were intraperitoneally injected with 10% chloral hydrate for euthanasia and then liver and epididymal adipose tissue samples were weighed and collected. Fecal samples were collected immediately for gut microbiota analysis. The liver and terminal ileum samples were stored in the 10% formaldehyde for histopathological analysis. The rest of the liver samples were stored at -80°C condition for western blotting and biochemical analysis.

Determination of AST, ALT in serum
Blood samples were collected from the abdominal aorta and stored at room temperature (RT) for 1 h. After centrifugation at 3500 rpm for 10 min, serum samples were collected for determination of ALT and AST levels using commercial kits (Nanjing Jiancheng Bioengineering Institute).

Histopathological evaluation of liver and the terminal ileum tissue
Formalin-xed and para n-embedded the liver and ileum tissue, stained with hematoxylin-eosin (HE), Immunohistochemistry of ileum sections were incubated with antibodies against zonula occludens 1 (ZO-1) (1:100; GB111402, Servicebio, Wuhan, China) and Occludin (1:100; GB111401, Servicebio, Wuhan, China). Livers embedded in an optimum cutting temperature compound were used for oil red O staining for assessment of hepatic steatosis. The procedure was performed as previously detailed and observed under an optical microscope (magni cation 400×). All tissues were evaluated by two experienced and 'blinded' pathologists and the histological scoring system for Nonalcoholic steatohepatitis (NASH) was conducted according to the NAS score system:0 = basically no in ammation, 1 ≤ 400-fold eld of view, 2 = 400-200-fold eld of view, 3⩾200-100-fold eld of view, 4 = up to the 40-fold eld of view. Color thresholding and measurement of area fraction with ImageJ was also utilized (National Institutes of Health, Bethesda, MD).

Determination of triglyceride (TG) and cholesterol (CHO) in liver
100mg of liver samples were weighed and then 0.9mL of absolute ethanol was added in to the centrifuge tube. Afterwards, the liver samples were homogenized with an electric homogenizer upon ice water and centrifuged at 4°C 2500 r/min for 10 min. 2.5 µl of supernatant for TG determination was acquired. The TG content was determined by using a commercial kit (Nanjing Jiancheng Bioengineering Institute) and the cholesterol was measured by a cholesterol assay kit (Nanjing Jiancheng Bioengineering Institute).
The nal concentrations of triglycerides and cholesterol were corrected for protein content.

Determination of LPS in the plasma
Blood samples were collected from the portal vein and then centrifuged at 4°C 2500 r/min for 5min.
Plasma was transferred into LPS-free cups and stored at -20°C for analysis. The content of LPS in plasma was detected by an endpoint chromogenic method utilizing a test kit.

Western Blotting
The liver tissue was lysed in RIPA with 1% PMSF and the protein was then fractionated by electrophoresis on 10% SDS-PAGE. Then, the protein was transferred to the PVDF membrane. Western blotting was used

Statistical analysis
Data were analyzed by using SPSS 21.0 software and data that conformed to the normal distribution were expressed as the mean ± SD. T-test was used for comparison between two groups, and one-way analysis of variance was utilized for comparison among multiple groups. P-value < 0.05 was considered to be signi cantly different. Figure 1 Articulates the effect of CG on body weight, obesity and serum transaminases. A. Fat weight of rats in epididymal tissue at the 16th week. B. Fat weight/body weight of Rats at the 16th week. C. The liver wet weight of rats at week 16. D. Hepatic index (hepatic index = liver weight/body weight × 100) of rats. The effect of CG on the liver injury was determined by detecting the serum levels of ALT(E) and AST(F). Statistical analyses were conducted with one-way ANOVA. #P < 0.05, ##P<0.01, ###P < 0.001 versus C; *P < 0.05, **P < 0.01, ***P < 0.001 versus M.