Cholestatic liver disease is a clinically pathological condition characterized by abnormal bile flow, always accompanied by the accumulation of intrahepatic and extrahepatic bile acids (Fernandez-Murga et al., 2018). Many factors can evoke cholestasis, including hepatitis, gene mutations, metabolic disorders, and drugs (Gijbels et al., 2019). As a drug-induced cholestasis, estrogen-induced cholestasis mainly occurs in vulnerable women taking oral contraceptives or receiving postmenopausal hormone replacement therapy (Bach et al., 2020). 17α-ethinylestradiol (EE), one of the synthetic estrogen derivatives, is extensively employed in research on estrogen-induced cholestasis (Marrone et al., 2016). Ursodeoxycholic acid (UDCA) is the most widely prescribed medicine for cholestasis, gallstones, and fatty liver disease (Li et al., 2016). However, about 40% of patients with cholestasis show poor responses to UDCA treatment (Hirschfield et al., 2015). Consequently, it is essential to develop novel therapeutic medicine for cholestasis.

During cholestasis development, bile excretion disorder is the most intuitive pathological manifestation, along with the variation of endogenous bile acids composition and proportion (Hirschfield et al., 2010). In hepatocytes, primary bile acids are synthesized from cholesterol through enzymatic reactions (Šarenac and Mikov, 2018) and subsequently excreted by transporters on the membranes of hepatocytes (Phelps et al., 2019). After being transported to the capillary duct between hepatocytes and combined with amino acids (Hua et al., 2021), the primary bile acids enter the small intestine through the bile duct with bile flow (Bogatyrev et al., 2020). Primary bile acids are converted to secondary bile acids by the action of intestinal flora (Jiao et al., 2018). Most of the bile acids in the intestine are absorbed into the portal circulation through bile acid transporters distributed on intestinal epithelial cells (Tiratterra et al., 2018), and then enter the hepatocytes through uptake transporters on the hepatocyte membrane (Bowman et al., 2019), a process also known as the enterohepatic circulation of bile acids (Sun et al., 2021b). Normally, the composition and proportion of bile acids are in a stable state (Setchell et al., 1997), but when homeostasis is disrupted, various pathological disorders in the hepatoenteric system may occur, including cholestasis (Zhang et al., 2008). Bile discharge disorders also lead to the accumulation of toxic bile acids (Oizumi et al., 2017). Therefore, targeting endogenous bile acids contributes to understanding the influence of cholestasis and medicine treatment on bile acid homeostasis.

Bile acids can act as ligands to activate various nuclear receptors, including the farnesoid X receptor (FXR, NR1H4), which belongs to a subclass of metabolic receptors within the nuclear receptor superfamily (Keitel et al., 2019). FXR is mainly located in the liver and intestine (Sun et al., 2021a) and plays a vital role in regulating bile acids homeostasis (Trauner et al., 2017). Activation of FXR has been shown to inhibit cholesterol 7α-hydroxylase (CYP7A1), a key rate-limiting enzyme in the classical bile acid synthesis pathway (Chiang and Ferrell, 2020). FXR could induce transporters, such as multidrug resistance-associated protein 2 (MRP2) and bile salt export pump (BSEP), to reinforce bile acids efflux while inhibiting Na⁺-dependent taurocholate cotransporter (NTCP) from reducing bile acids uptake by hepatocytes (Stofan and Guo, 2020). Targeting FXR activity has emerged as a novel strategy to treat cholestasis and other hepatic diseases (Kowdley et al., 2018; Hirschfield et al., 2019; Trauner et al., 2019). Some FXR agonists, such as obeticholic acid, have been approved by the FDA for the treatment of primary biliary cirrhosis in patients who are intolerant to or non-responsive to first-line therapy (van Golen et al., 2018).

Paeoniflorin (Supplementary Figure S1A) is the most abundant bioactive constituent in Paeonia lactiflora Pall., Paeonia suffruticosa Andr., or Paeonia veitchii Lynch, which is one of the most widely used herbal medicine for hepatic disease over 2,000 years in China (Ma et al., 2020). It has been well established that paeoniflorin has numerous pharmacological effects on hepatic diseases, such as hepatic ischemia/reperfusion alleviation (Xie et al., 2018), cholestasis alleviation (Wei et al., 2020), hepatic fibrosis attenuation (Wang et al., 2021), nonalcoholic fatty hepatic disease prevention (Ma et al., 2016), and so on. Crucially, paeoniflorin has been shown to remarkably alleviate alpha-naphthylisothiocyanate (ANIT)-induced cholestatic hepatitis (Zhao et al., 2017; Zhou et al., 2017; Chen et al., 2021). However, the pharmacology effect of paeoniflorin on EE-induced cholestasis has not been thoroughly evaluated.

The present study explored whether paeoniflorin had a valuably alleviated effect on EE-induced cholestasis in rats and further clarified whether this effect is related to the regulation of bile acid transporters via FXR, which affected endogenous bile acid homeostasis. The findings prove that paeoniflorin might become a potential candidate for cholestasis.

Endogenous and exogenous estrogens, including their metabolites, would result in intrahepatic cholestatic liver injury in susceptive women who exhibited abnormal bile flow and hepatitis (Padda et al., 2011). As a hepatotoxic substance, EE has been widely applied in rodents to explore underlining mechanisms of estrogen-induced cholestatic hepatic diseases, which belongs to drug-induced cholestatic hepatic disorders (Yang et al., 2019; Ming et al., 2021). Previous reports have proven that EE injection reduced bile flow and led to cholestasis via the FXR-meditated Extracellular regulated protein kinases (ERK)-Liver kinase B1 (LKB1)-Adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway (Li et al., 2017). To illuminate the pharmacological activity of paeoniflorin on EE-induced cholestasis and explore whether the effect is relevant to FXR, the current study adopted a rodent model in Sprague-Dawley rats injected with EE once daily for five successive days. In this animal model, EE-induced cholestasis with a defect of pivotal canalicular solute transporters of bile acids, including BSEP, MRP2, and NTCP (Marrone et al., 2016). Currently, UDCA is the first-line clinical medicine for cholestasis, so it is applied as the positive control in the current study. Previous reports have fully demonstrated the improved effect of UDCA administration on EE-induced cholestatic liver injury (Li et al., 2016; Di Guida et al., 2018).

Paeoniflorin is the primary active component in Paeonia lactiflora pall, which is extracted from the dried root of the plants. Previous studies have demonstrated that paeoniflorin exerted effectively protective effects on various hepatic diseases (Tu et al., 2019), including ANIT-induced cholestasis (Zhao et al., 2013; Chen et al., 2015; Chen et al., 2021) and bile duct ligation-induced cholestatic liver injury (Wei et al., 2020). This study fully elucidated that paeoniflorin showed an alleviative effect on EE-induced hepatotoxicity for the first time, as characterized by significant reduction of hepatotoxicity biochemical serum markers, including ALT, AST, ALP, and γ-GT, which were elevated by EE induction. Moreover, ameliorative hepatic and ileal histopathology was observed in paeoniflorin-treated group.

Cholestasis is mainly manifested as decelerated bile flow. In this study, injection of EE significantly decreased bile flow and elevated cholestatic biochemical indicators, including TBIL, DBIL, and TBA. Administration of paeoniflorin or UDCA could significantly improve these trends. In the development of cholestasis, oxidative stress is an important driving force that cannot be ignored. The results indicated that the MDA level was elevated while the SOD level was declined in the liver of EE-treated rats, indicating that oxidative stress was activated. The present study showed that paeoniflorin could restore indicators of oxidative stress. Overall, paeoniflorin alleviated EE-induced cholestasis, which was characterized by increasing bile flow, decreasing serum biomarkers, ameliorating hepatic and ileal histopathology, and improving oxidative stress.

Bile acid is one of the major ingredients in bile. In the enterohepatic system, bile acids can facilitate the assimilation of lipids, fat-soluble vitamins, and cholesterol in the intestine (Lee et al., 2020). Destabilization of bile acid homeostasis may trigger various pathological changes, including cholestasis (Fickert and Wagner, 2017). Moreover, when cholestasis occurs, the body accumulates a large quantity of bile, leading to hepatocellular necrosis with the turbulence of phospholipids, unsaturated fatty acids, and sphingolipids metabolism, which further aggravates hepatic diseases (Lin et al., 2019). Compared to the control group, the levels of key bile acids in serum were markedly elevated after EE induction, indicating that the uptake function of bile acid by hepatocytes was damaged. Meanwhile, the levels of CA, TCA, β-MCA, and DCA were significantly increased in the liver while considerably decreased in the bile, indicating that the function of the bile acid efflux transporter on the hepatocyte membrane is disturbed, leading to the reduction of bile acids efflux into bile and accumulation in the liver. Among bile acids with abnormal levels, LCA and DCA are considered hepatotoxic, and their accumulation exacerbates hepatocyte injury caused by cholestasis (Chen et al., 2014; Li et al., 2016). Administration of paeoniflorin in cholestatic rats could maintain sensitive endogenous bile acids in the serum, bile, and liver at relatively stable levels, indicating that the bile acid homeostasis was maintained when bile excretion was promoted by paeoniflorin. Since bile acid homeostasis is mainly regulated by bile acid transporters on the hepatocyte membrane, it can be speculated that paeoniflorin has a regulatory effect on bile acid transporters, which is inseparable from the influence of upstream nuclear receptors of the transporters.

Nuclear receptors have been demonstrated to regulate target gene expression in numerous metabolic processes, including bile acid synthesis and conjugation, through ligand-activated transcription (Beuers et al., 2015). FXR is a crucial regulator in the maintenance of bile acid homeostasis (Gomez-Ospina et al., 2016), as FXR activation restores the levels and metabolic abnormalities of bile acids pool and exerts therapeutic effects on cholestasis in rodents (Dong et al., 2019). In contrast, specific FXR deficiency would lead to the reduction of bile flow. Nowadays, several FXR agonists are undergoing clinical trials for various types of cholestasis treatment (Kowdley et al., 2018; Trauner et al., 2019). The present experiment explored whether FXR was involved in the alleviation of paeoniflorin against EE-induced cholestasis. The results illustrated that EE induced a significant decrease in FXR expression, and paeoniflorin administration could restore the mRNA and protein expression of FXR in a dose dependent manner. As a weak FXR agonist, UDCA administration upregulated EE-suppressed FXR expression at both mRNA and protein levels.

LXRα is distributed in the liver, kidney, and intestine, known as metabolically active receptor. Both FXR and LXRα can form heterodimers with the retinoid X receptors and heterodimers in the active state bound to the response element on the DNA in the basal state (Hiebl et al., 2018). According to the analysis of mammalian one-hybrid and transient transfection reporters, paeoniflorin has been found to activate LXRα in HepG2 cells (Lin, 2013). In this study, it was observed that EE injection down-regulated LXRα expression while over 200 mg/kg paeoniflorin could markedly increase LXRα expression at the protein level. In contrast, UDCA intervention showed no significant influence on LXRα.

Bile acid transporters and metabolic enzymes are essential for maintaining bile acid homeostasis. As two crucial bile acid transporters on the hepatocyte membrane, BSEP and MRP2 regulate the transport of free and conjugated bile acids into bile. EE treatment significantly decreased the mRNA and protein expression of BSEP and MRP2, leading to bile flow obstruction and bile acids accumulation. BSEP and MRP2 expression were significantly increased by paeoniflorin administration, promoting hepatic bile acid efflux. NTCP regulates the transition of conjugated bile acid uptake into hepatocytes from the portal venous blood. The expression of NTCP was reduced in EE-induced cholestasis, which contributed to the relief of cholestasis. Previous studies have shown that estrogen-induced cholestasis reduced basolateral organic anion-transporting polypeptides, such as NTCP and OATPs (Geier et al., 2003). Nonetheless, the decreases of BSEP and MRP2, which are mainly responsible for canalicular secretion of bile acids, played dominant roles in EE-induced cholestasis. Paeoniflorin and UDCA administration further reduced NTCP expression, contributing to the improvement of cholestatic liver injury.

As a bile acid synthetic enzyme, CYP7A1 plays a crucial role in bile acid synthesis. By activating the hepatic small heterodimer partner, FXR can repress CYP7A1 to reduce bile acid synthesis in hepatocytes (Chiang et al., 2000). The present study revealed that paeoniflorin down-regulated the protein level of CYP7A1 in a dose-dependent manner. Nonetheless, decreased bile acid synthesis enzyme in cholestatic rats might be an adaptive response to reduce drug-induced hepatotoxicity.

In summary, the present study demonstrated that paeoniflorin dose-dependently alleviated EE-induced cholestasis and maintained bile acid homeostasis in rats. Moreover, the results also indicated a correlation between nuclear receptors, bile acid transporters, synthetic enzyme, and bile acid homeostasis with the alleviation effect of paeoniflorin on EE-induced cholestasis. The impact of paeoniflorin against cholestasis may account for the activation of the FXR-mediated bile acid homeostasis signaling pathway, which contributes to upregulation of bile acids efflux and downregulation of bile acids uptake and synthesis (Figure 8). The present study suggested that paeoniflorin may become a potential therapeutic agent for estrogen-induced cholestasis.