Liver Protective Effects And Molecular Pharmacological Mechanisms Of Auraptene

Objective:To investigate the liver protective effects and molecular pharmacological mechanisms of auraptene?AUR?.Methods:?1?The protective effect of AUR on lithotolic acid?LCA?-induced cholestatic liver injury via farnesoid X receptor?FXR?activation in mice was investigated.Two dimensional virtual screening of a natural product library for FXR agonists was performed.Molecular docking and dual-luciferase reporter gene assays were carried out to investigate the FXR activation effect of AUR.Cholestatic mice model was induced by intraperitoneal injection of LCA.The levels of serum aminotransferase and bile acids as well as hematoxylin-eosin?H&E?staining were used to evaluate the protective effect of AUR on LCA-induced cholestatic liver injury.The levels of total bile acids in serum,liver and intestine of mice were measured using liquid chromatography-tandem mass spectrometry to observe the regulation of AUR on bile acid transporting on tissue level.The expression levels of hepatic uptake transporter Na⁺/taurocholate cotransporting polypeptide?Ntcp?,export transporters bile salt export pump?Bsep?,multidrug resistance-related protein 2?Mrp2?,bile acid synthetic enzymes cholesterol 7?-hydroxylase?Cyp7a1?,sterol 12?-hydroxylase?Cyp8b1?,and bile acid metabolic enzymes hydroxysteroid sulfotransferase 2a1?Sult2a1?were examined to clarify the regulation mechanism of AUR on the transporting,synthasis,and metabolism of bile acids.The liver sections of 5-bromodeoxyuridine?BrdU?staining and expression of forkhead box M1b?FoxM1b?,Cyclin D1,Cyclin B1 were were used as indexes to elucidate the mechanism of AUR promoting hepatocyte proliferation.The anti-inflammatory effects of AUR were investigated by the expression of inflammatory-related genes nuclear factor-?B p65?NF-?B p65?,tumor necrosis factor-??TNF-??,interleukin-1??IL-1??and interleukin-6?IL-6?.FXR antagonist guggulsterone?GS?and FXR gene silencing test was performed in vivo and in vitro to determine whether the liver protective effects of AUR were dependent on FXR.?2?Anti-fibrotic effects of AUR on thioacetamide?TAA?-induced liver fibrosis in mice.Mice were injected intraperitoneally with TAA on Monday,Wednesday and Friday to induce liver fibrosis model.At the same time,AUR was administered intragastrically on Tuesday,Thursday and Saturday for total 8 weeks.The severity of liver damage induced by TAA and the protective effect of AUR were evaluated by the body weight gain,liver weight and liver/body weight ratio.Serum ALT?alanine aminotransferase?,AST?aspartate transaminase?,total bilirubin?TBIL?,total bile acids?TB?,total cholesterol?TC?,triglycerides?TG?and H&E staining were determined as indicators of protective effects of AUR on liver injury induced by TAA in mice.The expression levels of bile acid transporters and synthesis enzymes were determined to evaluate the mechanism of AUR on the regulation of intrahepatic bile acid.Masson's Trichrome staining of liver sections and the expression of transforming growth factor-?1?TGF-?1?,?-smooth muscle actin??-SMA?,tissue inhibitor of metalloproteinase-1?TIMP-1?and collagen I-?1?COL1-?1?were determined to evaluate the anti-fibrosis effects of AUR.The expression of TNF-?,IL-1?,IL-6 and FXR target gene NF-?B were determined to investigate the mechanism of AUR on TAA-induced inflammatory response.FXR antagonist GS and FXR siRNA were performed in vivo and in vitro to verify the role of FXR in anti-fibrotic effects of AUR on TAA-induced liver fibrosis.?3?The preventive effect of AUR on CCl₄ induced chemical liver injury.Mice were injected intraperitoneally with CCl₄ to induce chemical liver injury.The protective effects of AUR on chemical liver injury induced by CCl₄ in mice were evaluated by H&E staining and serum biochemical indicators.The expression levels of bile acid transporters Ntcp,Bsep and Mrp2 were determined to evaluate the effect of AUR on the regulation of intrahepatic bile acids.The expression of synthetic enzymes Cyp7a1,Cyp8b1 and metabolic enzyme Sult2a1 were determined to elucidate the effect of AUR on the synthesis and metabolism of bile acids.The number of BrdU-positive hepatocytes was used as an index to reveal the effect of AUR on hepatic repair and the expression levels of FoxM1b,Cyclin D1,Cyclin B1 were detected to further evaluate the molecular mechanism of AUR on hepatocyte proliferation.The expression of inflammatory factors NF-?B,TNF-?,IL-1?and monocyte chemotactic protein 1?MCP-1?were determined to investigate in the anti-inflammatory mechanism of AUR on CCl₄ induced inflammatory response.In vitro FXR gene silencing experiments using mouse primary cultured hepatocytes was performed to investigate the effect of AUR on FXR and FXR related target genes.Results:?1?In the study of LCA-induced cholestatic liver injury,AUR,an agonist of FXR,reduced the uptake and promoted the efflux of intrahepatic bile acids by down-regulating the expression of Ntcp and up-regulating of Bsep and Mrp2.AUR reduced synthesis of bile acid and promoted its metabolism by repressing the expression of Cyp7a1 and Cyp8b1 and increasing the expression of Sult2a1.AUR also promoted hepatocyte proliferation via inducing the expression of Cyclin D1,Cyclin B1 and their up-stream regulatory protein Fox M1b.AUR alleviated liver inflammation by inhibiting the expression of inflammation-related factors.In vivo and in vitro studies further demonstrated that the anti-cholestasis effect of AUR on cholestatic liver injury induced by LCA was due to the FXR-mediated regulation of above genes after activation of FXR.?2?In the study of TAA-induced liver fibrosis in mice,AUR reduced the level of toxic bile acids by decreasing the uptake and synthesis of bile acids and promoting its efflux.AUR reduced collagen deposition and inhibited activation of hepatic stellate cells?HSCs?by down-regulating the expression of fibrosis related genes TGF-?1,?-SMA,TIMP-1 and COL1-?1.The anti-inflammatory effect of AUR was mediated through the downregulation of NF-?B,TNF-?,IL-1?and IL-6.The regulatory effect of AUR on these factors was eliminated by FXR antagonist GS.In vitro gene silencing experiments directly demonstrate that FXR was involved in the anti-fibrosis effect of AUR in TAA-induced fibrosis mice.?3?In the experiment of CCl₄-induced chemical liver injury,prophylactic administration of AUR reduced the uptake and synthesis of toxic bile acids by down-regulating the expression of Ntcp,Cyp7a1 and Cyp8b1.AUR increased the efflux and metabolism of bile acid by up-regulating the expression of Bsep,Mrp2 and Sult2a1.AUR also promoted hepatocyte proliferation and regeneration by inducing the expression of Fox M1b,cyclin D1 and Cyclin B1.AUR relieved inflammatory response induced by CCl₄ via a down-regulating effect on the expression of inflammatory cytokines NF-?B,TNF-?,IL-1?and MCP-1.In vitro FXR gene silence test further proved that the mechanism of liver protection by AUR is closely related to the gene regulation which is mediated by FXR.Conclusion:AUR was proved as a novel agonist of FXR.Through the activation of FXR,AUR was involved in the regulation of bile acid related transporters and enzymes,effectively reducing the level of bile acids in liver.AUR could promote the proliferation of hepatocytes and reduced the inflammatory reaction of liver,as well as inhibite the activation of HSCs to protect against LCA-induced cholestatic liver injury,TAA-induced liver fibrosis and CCl₄-induced chemical liver injury in mouse model.