Study On The Effect And Molecular Mechanism Of Paeoniflorin On Cholestatic Liver Injury

The incidence of cholestasis in chronic liver disease is about 35%,and there is an upward trend with age.Persistent cholestasis can develop into liver fibrosis,cirrhosis,and even liver cancer,bringing serious economic burden to patients.Clinical studies have found that reused Radix Paeoniae Rubra has a significant effect in the treatment of cholestatic hepatitis,significantly alleviate the abnormality of serum liver function indexes and the degree of liver tissue lesions,and improve the quality of life of patients.Previous studies of our group showed that paeoniflorin(PF),the main active component of Radix Paeoniae Rubra,could significantly improve serum biochemical indexes,hepatocyte injury and inflammatory cell infiltration in rats with cholestasis.However,the mechanism of PF in the treatment of cholestatic liver injury has not been fully revealed.Therefore,highthroughput bioinformatics methods such as network pharmacology and metabonomics combined with conventional molecular biology techniques were used to explore the mechanism of PF on cholestatic liver injury in our study.Rats were intragastrically administrated with α-naphthylisothiocyanate(ANIT)to establish cholestatic liver injury model.The therapeutic effects of PF on cholestatic liver injury were investigated by serum biochemical indexes including alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),total bile acid(TBA),alkaline phosphatase(ALP),γ-glutamyl transpeptidase(γ-GT),albumin(ALB)and the histopathology of the liver of rats;The effects of PF on SIRT1/FXR,Nrf2 and NF-κB/NLRP3 inflammatory body signaling pathways and the expression of inflammatory factors in rat liver tissue were detected by RT-PCR,western blotting and immunohistochemical techniques;Non-targeted metabonomics was used to identify the fecal and urinary biomarkers and metabolic pathways of PF in the treatment of cholestatic liver injury.Then,combined with the network pharmacology method,an interaction network of "biomarker-target-PF" was constructed to identify the upstream targets of PF regulating biomarkers and systematically reveal the mechanism of PF on cholestatic liver injury.The results showed that the different doses of PF significantly improved the levels of serum ALT,AST,ALP,TBIL,TBA,ALB,and γ-GT(P < 0.05,P < 0.01).Histopathology showed that different doses of PF alleviate cholestatic liver injury such as neutrophil infiltration and hepatocyte necrosis in different degrees.The results of molecular biological methods showed that PF significantly decreased the m RNA expression levels of NLRP3,Caspase-1,ASC,TNF-α,IL-1 β and IL-18 in liver tissue,In addition,PF significantly increased the expression levels of SIRT1,FXR and Nrf2 protein,and decreased the expression levels of NF-κB p65 and NLRP3 in liver tissue(P< 0.05).Non-targeted metabonomics showed that PF significantly changed the expression levels of 13 biomarkers such as sepiapterin,taurochenodesoxycholic acid,homovanillic acid,L-Phenylalanine,linoleic acid,and phosphocreatine.In addition,based on the pathway enrichment analysis of fecal biomarkers,16 metabolic pathways such as linoleic acid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis regulated by PF were obtained.Through the construction of "fecal biomarker-targetPF" network,it was found that PF regulated the metabolite linoleic acid,Ndesmethylcitalopram,3-methoxytyramine,PE [15:0/ 18:4(6Z,9Z,12 Z,15Z)]and sepiapterin by acting on CYP2C9,ABCB1,MAOB,MTOR and CDC25 B targets.Urine non-targeted metabonomics studies showed that PF significantly changed the expression levels of six urinary biomarkers,such as thiamine triphosphate,thiamine monophosphate and carboxyphosphoramide.Six metabolic pathways such as thiamine metabolism,sulfur metabolism and vitamin B6 metabolism regulated by PF were obtained by further enriching urine biomarkers.Through the construction of "urine biomarker-target-PF" network,it is found that PF regulated the change of urine biomarker sulfate through GSTP1 and CDK2.This study confirmed the therapeutic effect of PF on cholestatic liver injury in rats,found that PF improved cholestatic liver injury by activating SIRT1/FXR signaling pathway and inhibiting NF-κB/NLRP3 inflammasome signaling pathway,and clarified the metabolic characteristics of feces and urine in the treatment of cholestatic liver injury by PF.Finally,the mechanism of PF in the treatment of cholestatic liver injury was systematically revealed.