| Objective: The purpose of the present study was to investigate liver protective effects and molecular pharmacological mechanisms of alisol B 23-acetate(AB23A).Methods:(1) Promotive effect of AB23 A on liver regeneration via farnesoid X receptor(FXR) activation in mice following partial hepatectomy was investigated. AB23A(12.5, 25 or 50 mg/kg) was treated to mice by oral gavage once a day for 7 days. On the 4th, 5th, 6th or 7th day, partial hepatectomy or sham surgeries were performed. Liver weight to body weight ratio, hepatocyte proliferation ratio and mitotic index were used to evaluate the promotive effect of AB23 A on liver regeneration. The gene and protein expression of cell cycle protein Cyclin D1, Cyclin B1 and their upstream gene forkhead box M1b(FoxM1b) was determined to elucidate the mechanism that AB23 A promoted hepatocyte proliferation. The levels of serum ALT, AST and hepatic bile acids were used to evaluate the hepatoprotective effect of AB23 A. The expression of bile acid synthetic enzyme Cyp7a1 and efflux transporter Bsep was determined to clarify the mechanism underlying the decreased bile acid in liver by AB23 A. FXR antagonist guggulsterone(GS) was used to block FXR signaling to determine whether the promotive and protective effects of AB23 A were related with FXR activation. In vitro experiment using mice primary hepatocytes was used to determine the effect of AB23 A on FXR target genes. Furthermore, gene silence experiment was used to determine the effect of FXR gene silence on the expression of FXR target genes modulated by AB23 A. In addition, dual-luciferase reporter assay and molecular docking were used to evaluate the effect of AB23 A on FXR activation.(2) Protective effect of alisol B 23-acetate against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis was investigated. AB23A(10, 20 or 40 mg/kg) was treated to mice by oral gavage once daily for 7 days. On the 5th or 6th day 4 h after AB23 A treatment, mice were orally administered 75 mg/kg dose of ANIT. The levels of serum ALT, AST, ALP, γ-GTP, total bilirubin, total bile acids, hepatic total bile acids and biliary total bile acids, as well as histopathological examination were used to evaluate the protective effect of AB23 A on ANIT-induced hepatotoxity and cholestasis. The expression levels of hepatic uptake transporters Ntcp, Oatp1b2 and efflux transporters Bsep, Mrp2, Mdr2, Mrp3, Mrp4 were determined to elucidate the mechanism of the effect of AB23 A on bile acid transport. The expression of bile acid synthetic enzymes Cyp7a1, Cyp8b1, Cyp27a1 and bile acid conjugating enzymes Bal, Baat, was examined to clarify the mechanism of the effect of AB23 A on bile acid synthesis and conjugation. The bile acid metabolic enzymes Cyp3a11, Cyp2b10, Sult2a1 and Ugt1a1 expression was determined to explore the mechanism of the effect of AB23 A on bile acid metabolism. FXR antagonist GS was used to block FXR signaling to evaluate whether the lowing- hepatic bile acids and hepatoprotective effects of AB23 A were related with FXR activation. In addition, dual-luciferase reporter assay was used to evaluate the effect of AB23 A on FXR activation in vitro.(3) Protective effect of alisol B 23-acetate against carbon tetrachloride-induced hepatotoxicity via activation of FXR and STAT3 in mice was investigated. Chenodeoxycholic acid(CDCA) which is a FXR agonist, was used as a positive control drug. AB23A(10, 20 or 40 mg/kg) was treated to mice by oral gavage once daily for 7 days. On the 5th or 6th day 4 h after AB23 A treatment, mice were injected intraperitoneally with 750 μl/kg of CCl4. The levels of serum ALT, AST, total bile acids, histopathological examination and TUNEL assay were used to evaluate the protective effect of AB23 A on CCl4-induced hepatotoxicity. BrdU-positive hepatocytes and mitotic index were used to evaluate the effect of AB23 A on hepatocyte proliferation. The expression of FoxM1 b, Cyclin D1 and Cyclin B1 was determined to elucidate the mechanism of the effect of AB23 A on hepatocyte proliferation. The expression levels of bile acid transporters Ntcp, Bsep, Mrp2 and bile acid synthetic enzymes Cyp7a1, Cyp8b1 were determined to elucidate the mechanism underlying decreased bile acid in liver in AB23A-treated mice. In addition, the expression of p-STAT3 and STAT3 target genes Bcl-xl, SOCS3 was examined to clarify the mechanism that AB23 A reduced hepatocyte apoptosis.(4) Protective effect of alisol B 23-acetate via FXR-mediated regulation of transporters and enzymes in estrogen-induced cholestatic liver injury was investigated. CDCA which is a FXR agonist was used as a positive control drug. AB23A(7.5, 15 or 30 mg/kg) was treated to mice by oral gavage once daily for 7 days. Since the 3th day 4 h after AB23 A treatment, mice received subcutaneous injections of EE(10 mg/kg) once daily for 5 successive days. The levels of serum ALP, total bile acids, bile flow and biliary bile acids, as well as histopathological examination were used to evaluate the protective effect of AB23 A on EE-induced cholestatic liver injury. The expression levels of bile acid efflux transporters Bsep、Mrp2 and uptake transporter Ntcp were determined to elucidate the mechanism of the effect of AB23 A on bile acid transport. The expression levels of bile acid synthetic enzymes Cyp7a1, Cyp8b1 and upstream transcription factors Shp, Fgf15 were examined to clarify the mechanism of the effect of AB23 A on bile acid synthesis. The bile acid metabolic enzymes Cyp3a11, Cyp2b10, Sult2a1 and Ugt1a1 expression was determined to explore the mechanism of the effect of AB23 A on bile acid metabolism. FXR antagonist GS was used to block FXR signaling to determine whether the protective effect of AB23 A was related with FXR activation.Results:(1) In the study of mice following partial hepatectomy, AB23 A dose-dependently promoted hepatocyte proliferation via upregulating hepatocyte proliferation-related protein FoxM1 b, Cyclin D1 and Cyclin B1 expression, and attenuated liver injury via an inhibition in Cyp7a1 and an induction in efflux transporter Bsep expression resulting in reduced hepatic bile acid levels. These changes in the above genes, as well as accelerated liver regeneration and hepatoprotection in AB23A-treated mice were abrogated by FXR antagonist GS in vivo. In vitro evidences also directly showed the regulation of these genes by AB23 A was abrogated when FXR was silenced. Dual-luciferase reporter assay and molecular docking further demonstrated the effect of AB23 A on FXR activation in vitro.(2) In the study of ANIT-induced hepatotoxity and cholestasis, AB23 A decreased hepatic uptake and increased efflux of bile acid through down-regulation of hepatic uptake transporter Ntcp and up-regulation of efflux transporters Bsep, Mrp2 and Mdr2 expression. AB23 A also reduced bile acid synthesis in liver through repressing bile acid synthetic enzymes including Cyp7a1 and Cyp8b1, and increased bile acid conjugation through increasing bile acid conjugating enzymes Bal and Baat. Furthermore, AB23 A increased bile acid metabolism through an induction in gene expression of Sult2a1 which is a phase II enzyme. Further in vivo and in vitro studies indicated that the hepatoprotective effect of AB23 A against ANIT-induced liver injury and intrahepatic cholestasis was due to FXR-mediated regulation of above genes.(3) In the study of CCl4-induced hepatotoxicity, AB23 A promoted hepatocyte proliferation via an induction in hepatic levels of FoxM1 b, Cyclin D1 and Cyclin B1 which are involved in hepatocyte DNA replication and mitosis. AB23 A also reduced hepatic bile acids through decreasing hepatic uptake transporter Ntcp, bile acid synthetic enzymes Cyp7a1, Cyp8b1, and increasing efflux transporter Bsep, Mrp2 expression. The effects of AB23 A were related with FXR activation. In addition, AB23 A induced STAT3 phosphorylation, STAT3 target genes Bcl-xl and SOCS3, resulting in decreased hepatocyte apoptosis.(4) In the study of EE-induced cholestatic liver injury, AB23 A increased hepatic efflux and decreased uptake of bile acid through an induction in efflux transporters Bsep, Mrp2, and an inhibition in hepatic uptake transporter Ntcp expression. AB23 A also reduced hepatic bile acid synthesis through repressing bile acids synthetic enzymes Cyp7a1 and Cyp8b1. Furthermore, AB23 A increased bile acid metabolism through an induction in gene expression of Sult2a1. Further studies indicated that the hepatoprotective effect of AB23 A against EE-induced cholestatic liver injury was due to FXR-mediated regulation of above genes.Conclusion: AB23 A promotes liver regeneration via FXR activation in mice following partial hepatectomy. AB23 A also protects against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis. Furthermore, AB23 A protects against CCl4-induced hepatotoxicity via activation of FXR and STAT3 in mice. In addition, AB23 A produces protective effect against EE-induced cholestatic liver injury via FXR-mediated regulation of transporters and enzymes. |
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