A UPLC-MS/MS-based metabolomics analysis of the pharmacological mechanisms of rabdosia serra against cholestasis
Graphical abstract
Introduction
Cholestasis is a clinical syndrome characterized by impaired bile acid formation and circulation. This condition is caused by injury to the hepatocytes and cholangiocytes, ultimately leading to intrahepatic retention of toxic bile acids followed by various hepatobiliary diseases, such as cholestatic hepatitis, primary biliary cirrhosis, cholecystitis, liver fibrosis, and liver failure (Yang et al., 2018). To date, the pathogenesis of cholestasis remains unclear. However, several factors have been suggested as crucial inducers of cholestasis, including accumulation of bile acids (Lee et al., 2018), dysregulation of transporters (Li et al., 2017b; Wu et al., 2018), oxidative stress (Zhao et al., 2017; Zhang et al., 2019), and inflammation (Li et al., 2017a; Goldstein and Levy, 2018).
The internal circulation of bile acid, including its synthesis, transport, metabolism, and elimination, is tightly related to the metabolic enzymes and transporters that are regulated by a complex network of nuclear receptor-mediated pathways, such as farnesoid X receptor (FXR), pregnane X receptor, and vitamin D receptor (Fan et al., 2019). These nuclear receptors, especially FXR, are considered vital therapeutic targets for cholestasis (Arab et al., 2017). In clinical practice, ursodeoxycholic acid (UDCA), obeticholic acid (OCA), and glucocorticoids are considered effective medicines for cholestatic hepatitis; however, their efficacy and cost are unsatisfactory. Owing to the continuous development of Traditional Chinese Medicine (TCM), single drug and combination preparations are often administered to treat chronic metabolic diseases owing to concepts, such as multi-component and multi-targeting. Further, their low adverse reactions and lack of drug dependence are appealing (Zhao et al., 2020). The use of herbal medicine and extracts has thus evolved as a trend for the prevention and treatment of cholestasis.
Rabdosia serra (RS), which belongs to the Labiatae family, has been administered for many years to treat jaundice, hepatitis, arthritis, enteritis, lepromatous leprosy, and acute cholecystitis in China (Zhang et al., 2020). RS is abundant in diterpenoids and flavones, which have diverse bioactivities, including antibacterial, anti-inflammatory, anti-oxidation, and antitumor activities. A study has proven that RS exerts pharmacodynamic effects in liver diseases, such as hepatitis, acute liver injury, and intrahepatic cholestasis (Min et al., 2018). However, the mechanism employed by RS to alleviate cholestatic liver injury is poorly understood.
As a robust and comprehensive approach, metabolomics is widely performed to probe the pathogenesis of diseases and the mechanism of drugs. Metabolomics offers significant advantages when comprehensively exploring the mechanisms of TCM (Wang et al., 2011) and provides a holistic insight into the relationship between metabolites and metabolic pathways in pathological and physiological states; this is because it enables the analysis of specific biomarkers during disease treatment (Sun et al., 2012). Currently, metabolomics is utilized as a popular approach for examining various liver diseases (Fu et al., 2019; Zhang et al., 2020).
According to previous studies, abnormal changes in anti-oxidative, cytoprotective metabolites and bile acids occur in intrahepatic cholestasis (Aoki et al., 2011; Yu et al., 2018). Bile acids are vital metabolites and signaling molecules involved in the process of lipid metabolism, immune response, and cholesterol homeostasis (Thomas et al., 2008; Dai et al., 2018). Here, we derived a serum and bile acid-targeted metabolomics method using UPLC coupled with Q-TOF mass spectrometry and characterized the metabolic profiles obtained upon RS administration as a treatment for cholestasis. A quantifiable LC-MS system was also used to determine bile acid levels in rat serum, bile, and liver. Tissue histopathology and biochemical evaluations were carried out to determine the therapeutic effect of RS. RT-qPCR was also performed to determine the gene expression of some transporters associated with bile acids to verify the metabolomics results.
Section snippets
Chemicals and reagents
α-Naphthylisothiocyanate (ANIT, Lot No. F1707046) and medicinal grade soybean oil (Lot No. F1613059) were purchased from Aladdin Company (Shanghai, China). Ursodeoxycholic Acid (UDCA, Lot No. 15C09094L) was supplied by Dr. Falk Pharma GmbH. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acid (TBA), total bilirubin (T-BIL), and lactate dehydrogenase (LDH) kits were provided by Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Cholic acid (CA),
Therapeutic effects of RS on cholestatic rats
The bile flow rates were significantly reduced in rats given ANIT (Fig. 1A); this is one of the most critical physiological signs of cholestasis. However, treatment with RS significantly regularized the bile flow rates at various times. Further, the liver index was significantly higher in the model group than in the control group. Both RS and UDCA treatment reduced the liver index levels relative to levels in the model group (Fig. 1B).
Serum ALT and AST levels were measured as indicators of
Discussion
Cholestasis is a pathological condition that involves intrahepatic accumulation of bile acids owing to a decrease in bile excretion or disruptions caused by tumors or inflammation (Ghonem et al., 2015). FXR agonists were previously demonstrated to act against inflammation in many hepatobiliary diseases, including cholestasis, cirrhosis, and IBD. In clinic trials, FXR agonists were administered to treat nonalcoholic fatty liver disease (NAFLD) and primary biliary cirrhosis (Gadaleta et al., 2011
Conclusion
In the present study, RS was found to protect the liver by regulating multiple metabolic pathways and promoting the excretion of bile acids. Meanwhile, RS played an essential role in reversing the imbalance of bile acids and protected against cholestasis by regulating the expression of transporters associated with bile acids. These findings lay a foundation for the development of RS as a therapeutic agent for cholestasis and provide a reference for the fabrication of extracts that can be used
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
CRediT authorship contribution statement
Kaihui Zhang: Writing – original draft, Writing – review & editing, Formal analysis. Yufeng Yao: Writing – original draft, Formal analysis. Meiqi Wang: Validation, Data curation. Fangle Liu: Validation, Data curation. Qian Wang: Investigation. Huanhuan Ma: Investigation. Yuanyuan Xie: Investigation. Yunxia Ma: Investigation. Pengyu Dai: Investigation. Chenchen Zhu: Conceptualization, Supervision. Chaozhan Lin: Conceptualization, Supervision.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
The work was supported by the National Natural Science Foundation of China (grant number 81673872, 81173535, and 81974520).
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Kaihui Zhang and Yufeng Yao contributed equally to this work.