Farnesoid X receptor contributes to oleanolic acid‐induced cholestatic liver injury in mice

Abstract Farnesoid X receptor (FXR) is a nuclear receptor involved in the metabolism of bile acid. However, the molecular signaling of FXR in bile acid homeostasis in cholestatic drug‐induced liver injury remains unclear. Oleanolic acid (OA), a natural triterpenoid, has been reported to produce evident cholestatic liver injury in mice after a long‐term use. The present study aimed to investigate the role of FXR in OA‐induced cholestatic liver injury in mice using C57BL/6J (WT) mice and FXR knockout (FXR−/−) mice. The results showed that a significant alleviation in OA‐induced cholestatic liver injury was observed in FXR−/− mice as evidenced by decreases in serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase as well as reduced hepatocyte necrosis. UPLC‐MS analysis of bile acids revealed that the contents of bile acids decreased significantly in liver and serum, while increased in the bile in FXR−/− mice compared with in WT mice. In addition, the mRNA expressions of hepatic transporter Bsep, bile acid synthesis enzymes Bacs and Baat, and bile acids detoxifying enzymes Cyp3a11, Cyp2b10, Ephx1, Ugt1a1, and Ugt2b5 were increased in liver tissues of FXR−/− mice treated with OA. Furthermore, the expression of membrane protein BSEP was significantly higher in livers of FXR−/− mice compared with WT mice treated with OA. These results demonstrate that knockout of FXR may alleviate OA‐induced cholestatic liver injury in mice by decreasing accumulation of bile acids both in the liver and serum, increasing the export of bile acids via the bile, and by upregulation of bile acids detoxification enzymes.


| INTRODUCTION
Drug-induced cholestatic liver injury (DIC) is caused by damaged bile acid homeostasis and is the primary pattern of drug-induced liver injury (Hoofnagle & Björnsson, 2019). DIC may further develop into serious consequences such as liver fibrosis, liver cirrhosis, liver cancer, and liver failure and has become the key factor of drug withdrawal (Li & Apte, 2015). A portion of Traditional Chinese Medicine (TCM) has been considered hepatotoxic based on modern medicine theory, although they are still widely prescribed in the clinic and have been used to treat various diseases for over 2000 years . The development of modern medicine and a wider application of Chinese herbal medicine have led to an increased incidence of TCM-associated liver toxicity, and especially DIC has increasingly been identified Oh et al., 2015). Oleanolic acid (OA) is a natural triterpenoid that is widely used as a complementary and alternative medicine in China and other countries (Liu et al., 2018). Several studies have indicated that OA treatment may lead to liver toxicity if given in small doses for an extended period or in a large dose over a short time (Liu et al., 2018). Previously, we found that OA induced cholestatic liver injury in Kunming mice administered with OA at a dose of 225 mg/kg once daily for 10 consecutive days  or in C57BL/6J male mice treated with OA at 457-685.5 mg/kg once a day for four consecutive days . Nonetheless, the mechanisms underlying OA-induced cholestatic liver injury have yet to be defined.
The free bile acids are conjugated to glycine (G) and taurine (T) through bile acid-CoA synthetase (Bacs) and bile acid-CoA amino acid N-acyltransferase (Baat). Many transporters are involved in the active transport of bile acids across the canalicular membrane to form bile, including bile salt export pump (Bsep), multidrug resistanceassociated protein 2 (Mrp2) and multidrug resistance protein 2 (Mdr2).
Among these transporters, Bsep plays a major role in efflux pathways (Song et al., 2015). Most of the bile acids are finally reabsorbed back to the liver through the Na+-taurocholate cotransport protein (Ntcp), and the organic anion transporter (Oatp), namely, bile acid enterohepatic circulation. During circulation, increased production of bile acids, excessive intake, and decreased excretion can contribute to cholestatic liver injury . Previously, we found that OA-induced liver injury occurred in mice when treated with OA at the dose of 90-135 mg/kg daily for 5 days mice developed liver injury with increased levels of serum ALT, AKP, and TBA. Serum unconjugated and conjugated bile acids in the OA-treated animals were increased, but the expression levels of Fxr, Bsep, Ntcp, Oatp1b2, Cyp7b1, Cyp8b1, and Cyp3a11 were decreased , suggesting that OA-induced liver injury was associated with bile acid homeostasis.
FXR plays a pivotal role in regulation of bile acid homeostasis. It is mainly expressed in hepatocytes, regulates hepatic bile acid biosynthesis and transport proteins, and participates in the formation, secretion, transport, uptake, and detoxification of bile acids (Shin & Wang, 2019). Activation of FXR can lead to suppression of bile acid synthesis enzymes such as Cyp7a1 and Cyp8b1 via small heterodimer chaperone (Shp) or fibroblast growth factor 15/19 (FGF15/19) pathways (Li & Chiang, 2013)
In contrast, studies have demonstrated that after inhibiting FXR or FXR gene knockout, the bile acid synthesis enzymes (Cyp7a1 and Cyp27a1) and uptake transporters (Ntcp and Oatp) were increased, while the bile acid efflux transporters (Bsep and Mdr2) and liver detoxification enzymes (Ugt1a1, Cyp3a11, and Cyp2b10) were decreased, resulting in bile acid overload and decreased bile acid detoxification ability, leading to increased toxicity (Cariello, Piccinin, Garcia-Irigoyen, Sabbà, & Moschetta, 2018;Yuan & Li, 2016). In addition, it has been demonstrated that knockout of FXR can ameliorate the increased liver injury in obstructive cholestasis by increasing the expression of the transporters Bsep and Mdr4, and by lowering serum and hepatic bile acid concentrations, suggesting a potential clinical role for FXR antagonists in the treatment of obstructive cholestatic liver disorders (Stedman et al., 2006). Based on these results, we propose that there may exist a beneficial effect of the downregulation of FXR in the homeostasis of bile acids in a pathological setting of OA-induced cholestatic liver injury.
Accordingly, in the present study, we aimed to investigate the regulatory role of FXR in OA-induced cholestasis using C57BL/6J mice and FXR gene knockout mice.  Lu et al., 2013); corn oil and OA were administered intragastrically once per day for 4 days, respectively. Body weights were controlled daily; at the end of the last dose administration, mice were treated with an intraperitoneal injection of pentobarbital anesthesia (60 mg/kg) (Shi et al., 2019). Mice were sterilized abdominally; following an approximately 2 cm longitudinal longitudinal transrectal incision, the abdomen was exposed to reveal the veins. Serum, liver, and gallbladder samples were collected. Blood was incubated at room temperature for 30 min, and serum fractions were obtained from blood by centrifugation for 15 min (1600 g, 4 C) for biochemical reagent detection and bile acid profile analysis.
After liver samples were weighed, they were divided into two parts: one part was immediately frozen in liquid nitrogen and then stored in À80 C for further research, and another part was soaked in 4% neutral formalin for histological evaluation. Gallbladders also were immediately frozen in liquid nitrogen and then stored in À80 C until use.

| Ethics statement
All procedures involving animals were in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and approved by Institutional Animal Use and Care Committee of Zunyi Medical University.

| Serum chemistry
Serum samples were analyzed using standard enzymatic assays using commercial kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bile acid (TBA) in accordance with the manufacturer's protocols (Jiang-Cheng Biological, China).

| Histopathology
Liver tissues were fixed in 4% neutral buffered formaldehyde solution for 24 h and then embedded in paraffin, and 3.5-μm-thick sections were obtained by cutting with a Leica RM2245 Biosystems microtome (Wetzlar, Germany). The slices were successively washed, dehydrated, and stained with hematoxylin and eosin (HE) for pathological examination (Olympus BX43, Tokyo, Japan). According to the area of liver tissue cell necrosis, hepatic necrosis was graded on a scale of 0 to 4+, in which score 0, represented no necrosis; score 1+, represented necrosis of 5-30% of hepatocytes; score 2+, represented necrosis of 30-50% of hepatocytes; score 3+, represented necrosis of 50-70% of hepatocytes; and score 4+, represented necrosis over 70% of hepatocytes (Oz et al., 2003). Each slide was evaluated blinded by two of our investigators (Hong Feng and Yan Hu) using light microscope; five fields were observed randomly at 200 times for each slice. achieved on a Thermo Hypersil Gold C 18 column (2.1 Â 150 mm, 1.9 μm), column temperature 40 C. The mobile phase consisted of 20% acetonitrile with 10 mM ammonium acetate (A) and 80% acetonitrile with 10 mM ammonium acetate (B), flow rate: 0.3 ml/min, injection volume: 2 μl. The initial composition of the mobile phase was 5% B, which was changed to 14% B in 6 min and then changed to 25% B in 6.3 min, hereafter changed to 50% B, maintaining the ratio for 10 min. It was then changed to 5% B, and after 3 min, it was changed to 100% B and held for 0.5 min, then changed to 5% B for 4 min.
Peak areas were used for comparison and statistical analysis. Bile acids were recognized and relatively quantified through the comparison with bile acid standards, including retention time, molecular weight, and mass spectra .

| RNA extraction and real-time polymerase chain reaction analysis
Total RNA of liver tissues was extracted using the TRIzol reagent (Takara Bio Inc., Japan) according to the manufacturer's instructions.  Next, the membrane was incubated with the HRP-conjugated Affinipure Goat Anti-Rabbit lgG (1:5000, Proteintech, China) secondary antibody at room temperature for 40 min. All antibodies were dissolved in TBST (with 1% BSA). The signals were detected using an electrochemiluminescence kit (7 Sea Biotech, China) with a chemiluminescence detection system (Bio-Rad, USA), and the density of the immunoreactive bands was then analyzed.

| STATISTICAL ANALYSIS
All experimental data were expressed as mean ± standard error of the mean (SEM). and were evaluated using SPSS 18.0. Data were assessed using one-way analysis of variance (ANOVA) and followed by the LSD (equal variance assumed) or Dunnett' T3 (equal variance not assumed) test. The graphs were generated using GraphPad Prism version 7 (GraphPad Software). A P value of <0.05 was considered statistically significant.

| RESULTS
4.1 | Cholestatic liver injury is reduced in FXR À/À mice after OA administration The changes of body weight, liver index and serum biochemical indicators were evaluated following continuous OA administration for 4 days to determine the liver injury. As shown in Figure 1A, after OA was administered for 4 days, the body weight of WT mice in the OA-H group decreased significantly compared with the control group.
There was no significant difference in the body weight of FXR À/À mice between the control group and the OA-H group. Compared with the control group, the gallbladder weight and liver index of the WT mice increased markedly in the high dose OA-treated ( Figure 1B,C).
These results showed that liver injury occurred in the WT mice, while significantly less injury was found in the FXR À/À mice when treated with high dose of OA.
The levels of serum ALT, AST, ALP, and TBA were increased in both the OA-L and OA-H groups compared with the control group among WT mice (Figure 2), while the increases in the OA-H group were more profound, and particularly, the ALP level of the OA-H group was 2.37 times the upper level of normal (ULN), suggesting that cholestatic liver injury occurred in the WT mice of the OA-H group (Trauner et al., 2019). In the FXR À/À mice, the serum ALT, AST, and ALP levels also increased in the OA-H group after OA administration compared with the control group of FXR À/À mice, but the ALP level of the OA-H group was 1.45 times the ULN, which suggested that cholestatic liver injury had not occurred in FXR À/À mice (Trauner et al., 2019). Notably, in the OA-H group mice, all these serum biochemical indicators of FXR À/À mice were significantly lower than those of WT mice. More importantly, the serum TBA level was not elevated in the FXR À/À mice while a dramatic increase was observed in WT mice ( Figure 2D). The liver histopathology was used to further evaluate the liver injuries (Figure 3), which was agreed with the serum biochemical results. In WT mice, no lesions appeared in the control group, but necrosis of hepatocytes (black arrows) was observed in both OA-L and OA-H groups, and the extent of hepatocyte necrosis was dose-dependent, suggesting that the hepatocyte necrosis occurred in WT mice was caused by OA rather than spontaneous lesion in mice (Kawashita et al., 2019). While the hepatocyte necrosis did not occur in FXR À/À mice, some swelling hepatocytes (black arrows) were observed in the liver tissues of FXR À/À mice. These findings demonstrated that knockdown of FXR À/À attenuated OAinduced cholestatic liver injury in mice.
4.2 | Bile acids levels are lower in FXR À/À mice than in wild-type mice following OA administration 4.3 | mRNA expression levels of genes involved bile acids homeostasis are altered in FXR À/À mice compared with those in wild-type mice FXR is known to regulate transporters and enzymes involved in bile acids homeostasis, and lower concentration bile acids were observed in FXR À/À mice in our experiment. Hence, we next investigated F I G U R E 4 Levels of liver bile acids at 24 h after OA continuous administration for 4 days. The detection of the bile acids was performed as described in Section 2, and samples were collected at 24 h after the last dose (OA-L: oleanolic acid low dose group; OA-H: oleanolic acid high dose group). Data are presented as the mean ± SEM (n = 3-5). *P < 0.05, as compared with the control group without OA treatment; # P < 0.05, comparison between WT mice and FXR À/À mice under the same dose of OA treatment mRNA expression levels in the liver of genes involved in regulation of bile acid homeostasis at 24 h after the last dose following 4 days of continuous OA administration. Following OA treatment, Fxr mRNA was dose-dependently suppressed in WT mice (Figure 7). Similarly, in WT mice, the bile acid efflux transporters (Bsep, Mdr2, and Mrp2) and uptake transporters (Ntcp, Oatp1b2) were also significantly F I G U R E 5 Levels of serum bile acids at 24 h after OA continuous administration for 4 days. The detection of the bile acids was performed as described in the section of materials and methods, and samples were collected at 24 h after the last dose (OA-L: oleanolic acid low dose group; OA-H: oleanolic acid high dose group); mean ± SEM (n = 3-5). *P < 0.05, as compared with the control group without OA treatment; # P < 0.05, comparison between WT mice and FXR À/À mice under the same dose of OA treatment F I G U R E 6 Levels of bile acids in the bile at 24 h after OA continuous administration for 4 days. The detection of the bile acids was performed as described in Section 2, and samples were collected at 24 h after the last dose (OA-L: oleanolic acid low dose group; OA-H: oleanolic acid high dose group). Data are presented as the mean ± SEM (n = 3-5). *P < 0.05, as compared with the control group without OA treatment; # P < 0.05, comparison between WT mice and FXR À/À mice under the same dose of OA treatment suppressed in the OA-H group compared with the control group (Figure 7), while the gene expression of the transporter Ostβ that is responsible for the efflux of bile acids back into the blood was increased. In addition, the expression of BSEP membrane protein in the liver was down-regulated after OA treatment as determined by Western blotting (Figure 7). On the contrary, there were no noticeable changes in the mRNA expression levels of these genes in FXR À/À mice between the control group and OA treated groups. Interestingly, in the OA-H group, the mRNA expression of Bsep, Mdr2, Mrp2, Ntcp, and Oatp1b2 was significantly higher in FXR À/À mice compared with WT mice, which was also observed for BSEP membrane protein expression. These results indicated that increased mRNA expression of bile acid transporters might contribute to mild liver damage in FXR À/À mice relative to WT mice when treated with OA.
We next determined the mRNA expression levels of bile acid synthesis enzymes (Shp, Cyp7a1, Cyp8b1, Cyp27a1, and Cyp7b1) and bile acid conjugated enzymes (Baat and Bacs) in the liver. As shown in To further explore the causes of less liver injury in FXR À/À mice, we analyzed the mRNA expression of the liver bile acids detoxifying enzymes Cyp3a11, Cyp2b10, Ephx1, Ugt1a1, Ugt2b5, and Sult2a1 ( Figure 9). We found that the mRNA expressions of Cyp3a11, Ephx1, Ugt1a1, and Ugt2b5 were suppressed in WT OA-H mice compared with the WT mice controls, while the mRNA expressions of Cyp2b10, Ephx1, and Ugt1a1 were significantly increased in FXR À/À OA-H mice compared with the FXR À/À control. Obviously, the mRNA expression levels of the detoxifying enzymes Cyp3a11, Cyp2b10, Ephx1, Ugt1a1, and Ugt2b5 were much higher in FXR À/À mice compared with WT mice in the OA-H treated group. Overall, these results indicated that FXR knockdown might reduce the OA-induced cholestatic liver injury by up-regulating the mRNA expression of liver bile acid detoxification enzymes.

F I G U R E 8
The mRNA expression levels of liver bile acid synthesis enzymes and conjugated enzymes after OA continuous administration for 4 days. Samples were collected at 24 h after the last dose (OA-L: oleanolic acid low dose group; OA-H: oleanolic acid high dose group). Data are presented as the mean ± SEM (n = 5-8). *P < 0.05, as compared with the control group without OA treatment; # P < 0.05, comparison between WT mice and FXR À/À mice under the same dose of OA treatment F I G U R E 9 The mRNA expression levels of liver bile acid detoxifying enzymes after OA continuous administration for 4 days. Samples were collected at 24 h after the last dose (OA-L: oleanolic acid low dose group; OA-H: oleanolic acid high dose group). Data are presented as the mean ± SEM (n = 5-8). *P < 0.05, as compared with the control group without OA treatment; # P < 0.05, comparison between WT mice and FXR À/À mice under the same dose of OA treatment

| DISCUSSION
In the present study we demonstrated that cholestatic liver injury was observed 24 h after the final dose of a 4-day continuous administration of high dose OA. FXR À/À mice were protected from OA-induced cholestatic liver injury, which might be attributed to not only the increased mRNA expression of Bsep, Cyp3a11, Cyp2b10, Ephx1, Ugt1a1, Ugt2b5, Bacs, and Baat, but also to the decreased bile acid overload.
At present, ALP > 2 times ULN is used as the basis for determining the diagnostic criteria of cholestatic liver injury based on the "Practical Guidelines for the Diagnosis and Early Treatment of Drug-induced Liver Injury" (Tajiri & Shimizu, 2008). In the study of cholestatic liver injury, animal models often combine evidence of cholestasis, intrahepatic bile duct dilatation, hepatocyte feathery degeneration and other typical pathologies (Khamphaya et al., 2016). In the present study, we showed that the ALP was >2 times ULN in WT mice treated with OA-H for 4 days, along with hepatocyte necrosis and increased ALT, AST, and TBA levels. However, a significant lower ALP level (1.45 times the ULN) was observed in FXR À/À mice treated with OA-H. Histopathological examination of liver tissues also revealed less hepatocytes necrosis in FXR À/À mice treated with OA-H. Accordingly, our results clearly indicated that high dose OA induced cholestatic liver injury in WT mice, while liver damage in FXR À/À mice was reduced.
Bile acids are the main components of bile and are responsible for promoting the absorption of lipids and fat-soluble vitamins, which play an important role in maintaining cholesterol metabolism. Dysfunction in the formation, secretion, and excretion of bile acids may lead to the accumulation of a large amount of bile acids in the liver, which in turn leads to liver damage (Kong et al., 2013;Liu et al., 2019). It has been reported that the decrease in bile acid content in the serum and liver can be used as a prognostic indicator of cholestatic liver injury, while the increased bile acid content in the bile can reduce the liver toxicity induced by bile acid Wang et al., 2014Wang et al., , 2017Wang et al., , 2020.
Our results were consistent with the observations in a study showing that the Xiaoyan Lidan Formula ameliorates ANIT-induced intrahepatic cholestatic liver injury in rats by reversing the increase of hepatic bile acids and decreasing serum bile acids levels induced by ANIT (Zhang et al., 2020). In this work, the elevated serum bile acids and increased bile acids in the bile were also observed in FXR À/À mice, which may in part explain why FXR À/À mice may have a reduction in the OA-induced cholestatic liver injury.
Unexpectedly, modest liver injury was observed in FXR À/À mice at 24 h after the last dose following treatment with OA for 4 days, but the levels of liver bile acids increased, which was inconsistent with findings from previous studies (Anakk et al., 2011;Kong et al., 2019).
It has been reported that changes in bile acids are related to the duration of drug exposure, doses administered, and compensatory mechanisms involved in the process of cholestatic liver injury (Tian et al., 2017). To further understand changes in liver bile acids caused by OA in FXR À/À mice, a similar analysis was made for liver bile acids at 3 h after the last dose of 4-day OA administration. In this context, T-αMCA levels were decreased in FXR À/À mice compared with WT mice after OA treatment. Thus, we postulated that FXR À/À mice exhibited reduced hepatotoxicity due to low content bile acids in the early stages of OA administration. This hypothesis was further supported by the measurement of liver bile acids at 3 h following a single administration of OA, which showed a significant reduction of TCA, GCA, TUDCA, TLCA, THDCA, TCDCA, and T-α/βMCA in FXR À/À mice compared with WT mice. These results implied that FXR À/À mice attenuated the OA-induced cholestatic hepatotoxicity by reducing the level of liver bile acid in the early stage, while the lower content of intrahepatic bile acids in the WT mice might be the self-protection function of WT mice after OA-induced cholestatic liver injury.
There are many enzymes and transporters that participate in bile acid homeostasis, and essentially, all are regulated by FXR (Schadt et al., 2016). Inhibition of FXR may upregulate the expression of bile acid synthases (Cyp7a1, Cyp8b1, Cyp27a1, and Cyp7b1) and reuptake transporters (Ntcp, Oatp), resulting in the accumulation of liver bile acids and inducing liver injury (Zhong et al., 2018). The mRNA expression of bile acid synthases and bile acid uptake transporters decreased in WT mice following OA-H treatment; however, a similar decrease in the gene expression of these bile acid synthases and bile acid uptake transporters was not observed in FXR À/À mice after OA-induced cholestatic liver injury. These results could in part explain why the intrahepatic bile acid content of FXR À/À mice was higher than that of WT mice at 24 h after the final dose of a 4-day continuous administration of high dose OA, suggesting that FXR À/À mice might reduce OA-induced liver damage through other ways, such as bile acid efflux.
However, the regulation of these bile acid synthases and uptake transporters in the scenario of OA treatment and FXR knockdown warrants further investigation. Other studies have noted that inhibition of FXR downregulates the expression of the bile acid efflux transporters Bsep, Mdr2, and Mrp2, leading to the accumulation liver bile acids and subsequent liver damage (Guo et al., 2016). Given that BSEP is the main bile acid efflux transporter and is responsible for transporting conjugated bile acids (Song et al., 2015), we evaluated the expression of BSEP protein. The expression of Bsep mRNA and BSEP protein was markedly higher in FXR À/À mice than in WT mice when treated with OA, suggesting that knockdown of FXR promoted liver bile acids efflux, and thus, reduced the toxicity caused by OA ( Figure 10). Our findings demonstrated that the disruption of Bsepmediated efflux was the dominant factor involved in OA-induced cholestatic liver injury. Previous studies (Sayin et al., 2013;Song et al., 2015) have shown that the expression of Bsep was suppressed in FXR gene knockout mice. It is worthy to note that in the present study, we observed a similar downregulation of Bsep in the liver of FXR À/À mice compared with the WT control ( Figure 7). However, our results showed that the expression of Bsep mRNA and BSEP protein was significantly higher in the liver of FXR À/À mice compared with WT mice when treated with OA. The increase in Bsep expression does not appear to be solely mediated by FXR, other mechanisms, such as Nrf2, glucocorticoid receptor, and other nuclear (hormone) receptors in cholestasis (Martin et al., 2010;Weerachayaphorn et al., 2010), might also be involved, which need to be further explored. It is generally believed that the increased expression of bile acid detoxifying enzymes and conjugated enzymes is also related to the reduction of cholestatic liver injury. Compared with WT mice after OA treatment, the expressions of conjugating enzymes Baat and Bacs in FXR À/À mice were significantly higher. The increase in the expression of these conjugating enzymes may enhance the conjugation of bile acids and thus reduce their toxicity. In addition, we observed significantly higher expressions of bile acid detoxification enzymes Cyp3a11, Cyp2b10, Ugt1a1, Ugt2b5, and Ephx1 in the FXR À/À mice compared with the WT mice when treated with OA. In the cases of Cyp2b10, Ugt1a1, and Ephx1, an induction of gene expression caused by OA treatment was also present in the FXR À/À mice. The increased expression of these detoxification enzymes may be involved in the polyhydroxylation, sulfation, or glycosylation of toxic bile acids, converting them into more hydrophilic substances for excretion ( Figure 10), thereby reducing bile acid toxicity (Cho et al., 2010).
Although the exact mechanism has yet to be defined, these adaptive changes in the FXR-null mice may play an important role in alleviating OA-induced cholestatic liver injury.

| CONCLUSION
In this study, we demonstrated that OA-induced cholestatic liver injury in mice was significantly reduced when FXR was knocked out.
This phenomenon may be mediated through the modulation of the expressions of genes involved in the export and synthesis of bile acids, leading to a decrease in the accumulation of bile acids both in the liver and serum, as well as the upregulation of bile acids detoxification enzymes.

ACKNOWLEDGMENTS
We would like to express our gratitude to Prof. Hua Yang from the Department of Pathology of Affiliated Hospital of Zunyi Medical University for the kind assistance in histopathology evaluation. A special thanks also goes to Yanliu Lu for the technical guidance.

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.