Elsevier

Phytomedicine

Volume 91, October 2021, 153683
Phytomedicine

A UPLC-MS/MS-based metabolomics analysis of the pharmacological mechanisms of rabdosia serra against cholestasis

https://doi.org/10.1016/j.phymed.2021.153683Get rights and content

Highlights

  • A rapid, sensitive metabolite detection technology method based on UPLC-MS/MS for metabolomics research.

  • RS was found to protect the liver by regulating multiple metabolic pathways and promoting the excretion of bile acids.

  • 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.

  • The present study clarifies the correlation between molecular mechanisms and metabolites, which is crucial for understanding the pharmacological mechanism of extracts.

Abstract

Background

Rabdosia Serra, the dried aerial parts of Rabdosia serra (Maxim.) Hara (RS) from the Labiatae family, is a traditional Chinese herbal medicine called Xihuangcao. Although RS has been found to exert a therapeutic effect on cholestasis, the underlying molecular mechanism remains unclear.

Purpose

This study was designed to investigate the pharmacological effect and mechanism of RS on cholestatic rats using metabolomics platform.

Methods

Histopathology and biochemical evaluations were performed to determine the therapeutic effect of RS and developed a rapid metabolite detection technology method based on UPLC-MS/MS to perform metabolomics research. Further, quantitative real-time polymerase chain reaction (RT-qPCR) was used to study the effect of RS on the bile acid metabolism pathway at the transcriptional level.

Results

RS significantly reduced the bile flow rates in cholestatic rats and decreased the levels of ALT, AST, TBA, T-BIL, and LDH, which were increased in the model group. Histological analysis showed that RS alleviated the liver injury induced by ANIT. Serum metabolomics results revealed 33 of the 37 biomarkers were found to be significantly altered by ANIT, and 26 were considerably changed following treatment with RS. Metabolic pathway analysis revealed four pathways such as primary bile acid biosynthesis, biosynthesis of unsaturated fatty acids, and arachidonic acid and tryptophan metabolism. The bile acid secretion process and the inflammation and oxidative stress processes are the major biochemical reactions following treatment with ANIT and RS. Bile acid-targeted metabolomics study showed that TCA, GCA, GCDCA, and GDCA might be sensitive biomarkers that induced liver injury. we found that treatment with RS regulated the levels of bile acid in the serum and liver and restored the proportion of bile acids, especially CA and conjugated bile acids, such as TCA and GCA, in the bile duct. RS increased the mRNA expression levels of FXR, SHP, BSEP, and MRP2 in livers, and IBABP, OST-α, and OST-β in the ileum.

Conclusion

: In this study, RS was found to protect the liver by regulating multiple metabolic pathways and promoting the excretion of bile acids. Simultaneously, 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. We demonstrated the correlation between molecular mechanisms and metabolites, provide a reference for the fabrication of extracts that can be used to treat cholestasis.

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).

References (45)

  • L. Ding et al.

    Bile acid nuclear receptor FXR and digestive system diseases

    Acta Pharm. Sin. B.

    (2015)
  • J.J. Eloranta et al.

    The role of FXR in disorders of bile acid homeostasis

    Physiology

    (2008)
  • K. Fu et al.

    Metabolomics and lipidomics reveal the effect of hepatic Vps33b deficiency on bile acids and lipids metabolism

    Front. Pharmacol.

    (2019)
  • R.M. Gadaleta et al.

    Farnesoid X receptor activation inhibits inflammation and preserves the intestinal barrier in inflammatory bowel disease

    Gut

    (2011)
  • N.S. Ghonem et al.

    Fibrates and cholestasis

    Hepatology

    (2015)
  • J. Goldstein et al.

    Novel and emerging therapies for cholestatic liver diseases

    Liver Int.

    (2018)
  • R. Heidari et al.

    Mitochondria protection as a mechanism underlying the hepatoprotective effects of glycine in cholestatic mice

    Biomed. Pharmacother.

    (2018)
  • G.M. Hirschfield et al.

    Pathogenesis of cholestatic liver disease and therapeutic approaches

    Gastroenterology

    (2010)
  • H.R. Kast et al.

    Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor

    J. Biol. Chem.

    (2002)
  • Y.C. Lee et al.

    Manual acupuncture relieves bile acid-induced itch in mice: the role of microglia and TNF-α

    Int. J. Med. Sci.

    (2018)
  • P. Lefebvre et al.

    SREBF2-Embedded mir33 links the nuclear bile acid receptor FXR to cholesterol and lipoprotein metabolism

    Arterioscler. Thromb. Vasc. Biol.

    (2015)
  • M. Li et al.

    Mechanisms of bile acid mediated inflammation in the liver

    Mol. Aspects Med.

    (2017)
  • Cited by (0)

    1

    Kaihui Zhang and Yufeng Yao contributed equally to this work.

    View full text