Ribes diacanthum Pall modulates bile acid homeostasis and oxidative stress in cholestatic mice by activating the SIRT1/FXR and Keap1/Nrf2 signaling pathways.

Cholestatic liver injury (CLI) is a pathophysiological syndrome characterized by the accumulation of bile acids (BAs), which leads to significant hepatic dysfunction. This condition is frequently associated with disturbances in BAs homeostasis and the induction of oxidative stress. Ribes diacanthum Pall (RDP), a conventional folk medicinal plant, has been employed in Mongolia, the Inner Mongolia region of China, and other areas for the remediation of hepatic disorders. However, the specific mechanism and chemical composition by which RDP exerts its effects remain unknown. The aim of this research was to assess the protective impact of RDP on CLI and probe into the underlying mechanism and pinpoint the active constituents of RDP. For this study, a CLI mouse model induced via bile duct ligation (BDL) was used to investigate the hepatoprotective effect of RDP. Mice were administered low, medium, or high doses of RDP for 6 consecutive days, beginning 3 days prior to BDL induction. Subsequently, serum biochemical parameters, hepatic histopathology, and cholestatic markers were analyzed. An HPLC-QTOF-MS/MS analysis was also conducted to identify the prototype constituents in RDP. Furthermore, component-directed network pharmacology was utilized to identify the active constituents, central targets, and signaling cascades of RDP. Eventually, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were adopted to confirm the associated antioxidant enzymes, BAs transporters, and metabolic enzymes. Molecular docking was applied to forecast the binding affinity between the components and core targets. RDP effectively ameliorated the pathological liver damage and cholestasis in BDL-induced CLI mice. Moreover, 43 components within RDP were identified through HPLC-QTOF-MS/MS analysis. Altogether 106 potential targets were detected, and the high-affinity targets, namely Keap1 and SIRT1, were located through the PPI network. The results of GO and KEGG analysis indicated that the reaction to oxidative stress and BAs homeostasis are significantly associated with the RDP treatment of CLI. In the in vivo experimental study, the findings revealed that RDP alleviated the BDL-induced oxidative damage. Simultaneously, RDP augmented the expressions of BAs efflux transporters and the metabolic enzymes in liver tissues, thus promoting BAs excretion and metabolism in cholestatic rodents. Mechanically, RDP attenuated hepatic oxidative stress and the accumulation of BAs, protecting the liver from BDL-induced cholestasis via the Keap1/Nrf2 and SIRT1/FXR signaling axis. The molecular docking result indicated that bolusanthol C and 3,6,3',4'-tetrahydroxyflavone possess a superior binding affinity to the two core targets (Keap1, SIRT1). These results suggest that RDP ameliorate CLI by regulating BAs homeostasis and alleviating oxidative stress through the SIRT1/FXR and Keap1/Nrf2 signaling pathways, presenting a novel therapeutic strategy for cholestasis. Additionally, bolusanthol C and 3,6,3',4'-tetrahydroxyflavone may function as key pharmacological agents in RDP, responsible for its protective effects against CLI.

Bayoude A ，Zhang J ，Shen Y ，Tilyek A ，Chai C ... -  《-》

Caffeoylquinic acids from Silphium perfoliatum L. show hepatoprotective effects on cholestatic mice by regulating enterohepatic circulation of bile acids.

The incidence of cholestatic liver disease (CLD), which is primarily marked by abnormal bile acids (BAs) metabolism and can result in significant hepatic injury, is rising. Nevertheless, there remains a lack of effective treatments and drugs in clinical practice. Silphium perfoliatum L. (SP) is rich in various structural types of caffeoylquinic acid (CQA) compounds, and it is a traditional herb of North American Indians with hepatobiliary therapy effects. However, its therapeutic effect and mechanism of action on CLD have never been studied. To determine if SP-8, an extract rich in CQAs from SP, protects against cholestatic liver injury induced by alpha-naphthylisothiocyanate (ANIT) and to clarify its mechanism based on the farnesoid x receptor (FXR) signaling pathway and enterohepatic circulation of BAs. The therapeutic efficacy of SP-8 was evaluated by assessing the serum biochemical indices, inflammatory factors, and liver histopathology. Targeted metabolomics of the BAs was studied in the feces, liver, serum, and bile using UPLC-MS/MS. Additionally, a Western blot analysis was used to examine the expression levels of the peroxisome proliferator-activated receptor γ (PPARγ), the FXR, and proteins related to the synthesis and transport of BAs. 16S rRNA gene sequencing was performed to evaluate the gut microbiota (GM). Finally, molecular docking simulations were conducted to assess the interaction between seven types of CQAs from SP-8 with FXR and PPARγ. SP-8 significantly enhanced the health status of cholestatic mice induced by ANIT as evidenced by a notable reduction in the liver function indices and pro-inflammatory factors, restoration of liver pathological damage, and acceleration of BAs excretion through the feces. In addition, the levels of harmful secondary BAs in the liver and blood were significantly reduced by SP-8. Furthermore, the results of the study on the mechanism of action confirmed that SP-8 not only regulated FXR and PPARγ but also significantly ameliorated the GM structure, thereby promoting the enterohepatic circulation of BAs and achieving the homeostasis of the BAs in the blood and liver. In addition, SP-8 successfully reduced the inflammatory response by strongly suppressing the nuclear translocation of NF-κBp65. According to the molecular docking results, the extract's primary active ingredients could be the seven CQAs in SP-8, as they exhibited a strong affinity for both FXR and PPARγ. Finally, the Mantel test analysis revealed a significant correlation among cholestatic-associated parameters, the GM, and BAs. It was confirmed for the first time that the SP-8 extract of Silphium perfoliatum L. that is rich in seven CQAs had a strong therapeutic effect on ANIT-induced CLD. Its mechanism may involve the regulation of the FXR signaling pathway and the amelioration of the GM structure to promote the homeostasis of BAs enterohepatic circulation. This study provides a potential candidate medicinal herb and its components for the development of CLD therapeutic drugs.

Zhang G ，Jia W ，Liu L ，Wang L ，Xu J ，Tao J ，Xu M ，Yue M ，Luo H ，Hai P ，Yue H ，Zhang D ，Zhao X ... -  《-》

Wedelolactone alleviates cholestatic liver injury by regulating FXR-bile acid-NF-κB/NRF2 axis to reduce bile acid accumulation and its subsequent inflammation and oxidative stress.

Cholestatic liver diseases (CLD) comprise a variety of disorders of bile formation, which causes chronic exposure to bile acid (BA) in the liver generally and results in hepatotoxicity and progressive hepatobiliary injury. Wedelolactone (7-methoxy-5, 11, 12-trihydroxy-coumestan, WED), the natural active compound derived from Ecliptae Herba, has been reported with valuable bioactivity for liver protection. Nevertheless, the effect of WED on cholestatic liver injury (CLI) remains unexplored. The present study aims to elucidate the protective effect of WED on Alpha-naphthylisothiocyanate (ANIT)-induced CLI mice, and to investigate its potential pharmacological mechanism. The anit-cholestatic and hepatoprotective effects of WED were evaluated in ANIT-induced CLI mice. Non-targeted metabolomics study combined with ingenuity pathway analysis (IPA) was used to explore the key mechanism of WED. The BA metabolic profile in enterohepatic circulation was analyzed to evaluate the effect of WED in regulating BA metabolism. Furthermore, molecular dynamics (MD) simulation and cellular thermal shift assay (CETSA) were used to simulate and verify the targeting activation of WED on the Farnesoid X receptor (FXR). The core role of FXR in WED promoting BA transportation, and alleviating BA accumulation-induced hepatotoxicity was further evaluated in WT and FXR knockout mice or hepatocytes. WED dose-dependently alleviated ANIT-induced cholestasis and liver injury in mice, and simultaneously suppressed the signaling pathway of nuclear factor-kappa B/nuclear factor-erythroid 2-related factor 2 (NF-κB/NRF2) to relieve inflammation and oxidative stress. At the metabolite level, WED improved the metabolic disorder in CLI mice focusing on the metabolism of BA, arachidonic acid, and glycerophospholipid, that closely related to the process of BA regulation, inflammation, and oxidative damage. WED targeting activated FXR, which then transcribed its target genes, including the bile salt export pump (BSEP) and the BA transporter, and subsequently increased BA transportation to restore the damaged enterohepatic circulation of BA. Meanwhile, WED alleviated hepatic BA accumulation and protected the liver from BA-induced damage via NF-κB/NRF2 signaling pathway. Furthermore, FXR deficiency suppressed the protective effect of WED in vitro and in vivo. WED regulated BA metabolism and alleviated hepatic damage in cholestasis. It protected the liver according to adjusted BA transportation and relieved BA accumulation-related hepatotoxicity via FXR-bile acid-NF-κB/NRF2 axis. Our study provides novel insights that WED might be a promising strategy for cholestatic liver disease.

Wang MQ ，Zhang KH ，Liu FL ，Zhou R ，Zeng Y ，Chen AL ，Yu Y ，Xia Q ，Zhu CC ，Lin CZ ... -  《-》

Oleanolic acid alleviates ANIT-induced cholestatic liver injury by activating Fxr and Nrf2 pathways to ameliorate disordered bile acids homeostasis.

Cholestasis is a clinical syndrome with high incidence and few effective treatments. Oleanolic acid (OA) is a triterpenoid compound with anti-cholestatic effects. Studies using bile duct ligation or lithocholic acid modeling have shown that the alleviating effect of OA on cholerosis is related to the regulation of nuclear factor erythroid 2 related factor (Nrf2) or farnesoid X receptor (Fxr). This study aims to investigate the underlying mechanism of OA against alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury based on Nrf2 and Fxr dual signaling pathways. The ANIT-induced rats model was used with or without OA treatment. Serum biochemical indexes, liver histopathological changes and glutathione level were examined. Bile acids (BAs) targeted metabolomics based on UHPLC-MS/MS were performed. siRNA, RT-qPCR and western blot analysis were used to prove the role of Fxr and Nrf2 pathway in OA's anti-cholestatic liver injury in vivo and in vitro. OA significantly alleviated ANIT-induced liver injury in rats, reduced primary bile acids, accelerated metabolism of BAs and reduced the intrahepatic accumulation of BAs. The expressions of bile salt export pump (Bsep), Na+-taurocholic cotransport polypeptide (Ntcp), UDP-glucuronyl transferase 1a1 (Ugt1a1) and Fxr in rat liver were markedly up-regulated, the activation of Nrf2 was promoted, and the expression of cholesterol 7α-hydroxylase (Cyp7a1) was decreased after OA treatment. Moreover, Fxr or Nrf2 silencing attenuated the regulation of OA on BAs homeostasis related transporters and enzymes in rat primary hepatocytes. OA may regulate BAs-related transporters and metabolic enzymes by activating Fxr and Nrf2 pathways, thus alleviating the cholestatic liver injury induced by ANIT.

Liu J ，Liu J ，Meng C ，Huang C ，Liu F ，Xia C ... -  《-》

Desmodium styracifolium (Osb.) Merr. Extracts alleviate cholestatic liver disease by FXR pathway.

Cholestatic liver disease (CLD) is a disease characterized by cholestasis. Farnesoid X receptor (FXR) is a nuclear receptor that maintains homeostasis in bile acid metabolism. Studies have shown that gut microbiota interfered with the FXR pathway. Modulation of FXR to inhibit cholestasis has become a key measure in the treatment of CLD. In traditional folk medicine, Desmodium styracifolium (Osb.) Merr. was used as a primary treatment for gallstones, gonorrhea, jaundice, cholecystitis and other diseases. Modern pharmacological studies had also found that the herb has anti-calculus, anti-inflammatory, antioxidant, diuretic and liver damage. Therefore, we speculated that Desmodium styracifolium (Osb.) Merr. extracts (DME) could alleviate CLD through the FXR pathway and might be associated with the gut microbiota. However, studies of DME alleviating CLD through the FXR pathway have not been reported. To study the effect and mechanism of DME in relieving CLD through in vivo and in vitro experiments. First, mice were administrated with alpha-naphthyl isothiocyanate (ANIT) to establish a CLD model in vivo. Meanwhile, HepG2 cells were induced by lithocholic acid (LCA) to establish the CLD model in vitro. To evaluate the therapeutic effect of DME on CLD mice, hematoxylin-eosin (HE) staining, and biochemical indicators were performed. The prototype of the blood components in mice serum was detected by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). 16S rDNA sequencing was used to analyze the gut microbiota. Finally, the protein and mRNA expression of the FXR pathway in mice liver tissues or HepG2 cells were detected by Western blot, qRT-PCR, or immunofluorescence. Pathological testing and biochemical indexes showed that DME significantly reduced serum ALT, AST, ALP, TBIL, DBIL, TBA and liver TBA levels, and attenuated liver tissue injury, necrosis and jaundice in CLD mice. In addition, MetagenomeSeq analysis of gut microbiota showed that DME significantly up-regulated the abundance of Parvibacter, down-regulated the abundance of Paenalcaligenes, and regulated bile acid homeostasis. In terms of mRNA expression, DME significantly upregulated the mRNA levels of Nr1h4, Abcb11, Cyp7a1 and Slc10a1. Meanwhile, in terms of protein expression, DME significantly up-regulated the protein expression levels of FXR, BSEP, CYP7A1 and NTCP, which regulated bile acid homeostasis. Finally, the molecular docking results showed that the components of DME, such as Lumichrome, Daidzein and Folic acid, all had good binding ability with FXR, and the surface plasmon resonance (SPR) results also showed that both Lumichrome and Daidzein had a relatively high affinity with FXR. DME alleviated CLD through the FXR pathway, and the mechanisms might be associated with the gut microbiota.

Zhang Z ，Guan G ，Tang Z ，Wan W ，Huang Z ，Wang Y ，Wu J ，Li B ，Zhong M ，Zhang K ，Nong L ，Gao Y ，Cao H ... -  《-》