Protective Effect And Mechanism Of Puerarin On Acute Liver Injury Induced By Acetaminophen

Acetaminophen(APAP)is a common antipyretic and analgesic drug,but excessive use can cause serious liver damage.Puerarin is an isoflavone compound extracted from pueraria root,which has significant antioxidant activity and liver protection.Oxidative stress is the key cause of ap AP-induced liver injury,and Nrf2,as a key coordinating factor of antioxidant stress,plays an indispensable role in improving and alleviating acute liver injury.This study was intended to investigate the effect of puerarin on APAP-induced liver injury from both organism and cellular levels,and to explore whether its potential protective mechanism is related to the activation of Keap1/Nrf2 signaling pathway,so as to clarify the protective mechanism of puerarin on APAP-induced liver injury.The main research contents and results are as follows:1 Protective effect of puerarin on ap AP-induced acute liver injury in miceMice were given puerarin 200,400 and 800 mg/kg intragastric administration for7 days,once a day,respectively.After the last intragastric administration,300 mg/kg APAP was injected intraperitoneally to establish acute liver injury model,and samples were taken after fasting for 24 hours.The effects of puerarin on liver tissue structure,liver function,oxidative damage and mitochondrial membrane potential of liver injury mice were studied by histopathological observation,blood biochemical examination,spectrophotometry and JC-1 fluorescence probe.Results:(1)APAP results in increased liver organ index,necrosis of liver cells around central vein and loss of cord-like structure.Pretreatment with medium and high doses of puerarin can significantly reduce the liver organ index and improve the pathological changes of liver tissue.(2)Puerarin can reduce the contents of ALT,AST,AKP,γ-GT and TBIL in serum of mice with acute liver injury,and alleviate liver function injury.(3)Puerarin can increase the contents of GSH,SOD and CAT in ap AP-induced acute liver injury of mice,and reduce the accumulation of MDA and ROS in the liver,thus reducing the oxidative stress in the liver.(4)Puerarin can significantly inhibit the decrease of mitochondrial membrane potential and alleviate mitochondrial dysfunction in mice with acute liver injury induced by APAP.Conclusion: Puerarin can protect mice from acute liver injury induced by APAP mainly through its antioxidant activity.2 Protective effect of puerarin on oxidative damage of Hep G2 cells induced by APAPCCK-8 method was used to detect the cell activity under different concentrations of APAP and puerarin,and to determine the modeling concentration of APAP and puerarin concentration.After that,the cells were divided into groups,and the effects of puerarin on the function,degree of oxidative damage and mitochondrial membrane potential of Oxidative damaged Hep G2 cells were studied by spectrophotometry,DCFH-DA fluorescence probe method and JC-1 fluorescence probe method.Results:(1)When the concentration of APAP was 10 m M,the cell survival rate was 77%,which could be used to construct oxidative damage cell model.When puerarin concentration was less than or equal to 60 μM,there was no significant effect on cell viability.Therefore,puerarin concentration should be controlled within 60 μM,and 15,30,60 μM could be used as the dosage.(2)After cell grouping treatment,puerarin can significantly down-regulate the content of ALT and AST in the culture medium of oxidative damaged Hep G2 cells,increase the content of GSH,SOD and CAT in cells and decrease the content of MDA and ROS,thus alleviating the oxidative damage of cells.(3)Puerarin can significantly restore the decrease of mitochondrial membrane potential and alleviate mitochondrial dysfunction in oxidative damaged Hep G2 cells.Conclusion: Puerarin can protect APAP-induced oxidative damage Hep G2 cells mainly by alleviating oxidative stress.3 Effects of puerarin on Keap1/Nrf2 signaling pathway3.1 Effects of puerarin on HEPATIC Keap1/Nrf2 signaling pathway in APAP-induced acute liver injury mice: Nrf2 signaling pathway is the regulatory center of oxidative stress response in the body.In this study,APAP was used to construct acute liver injury model in mice,and q PCR,Western Blot and other experimental techniques were used.The effects of pretreatment with different concentrations of puerarin on the transcription and expression of liver Keap1/Nrf2 signaling pathway related proteins Keap1,Nrf2,GCLC,GCLM,HO-1 and NQO1 were detected,and the protective effect of puerarin on liver by activating Nrf2 signaling pathway in mice was preliminatively explored from the perspective of signaling pathway.Results: Puerarin down-regulated the expression of Keap1 in mouse liver from both transcription and translation levels,and up-regulated the expression of GCLC,GCLM,HO-1 and NQO1 downstream of Nrf2 signaling pathway.Conclusion: Puerarin can activate the Nrf2 signaling pathway in mouse liver and promote the expression of antioxidant proteins downstream of the pathway.3.2 Effects of puerarin on Keap1/Nrf2 signaling pathway in Ap AP-induced oxidative damage Hep G2 cells: Oxidative damage model of Hep G2 cells was constructed by APAP,and the effects of pretreatment with different concentrations of puerrin on the transcription and expression of Keap1,Nrf2,GCLC,GCLM,HO-1 and NQO1 related proteins in the Keap1/Nrf2 signaling pathway were detected by q PCR and Western Blot.Afterwards,the expression levels of Nrf2 protein in cytoplasm and nucleus were determined respectively to explore whether puerarin can activate Nrf2 signaling pathway by promoting Nrf2 nuclear migration,thus playing a protective role on liver cells.Results: Puerarin down-regulated the expression of Keap1 in Hep G2 cells at both transcriptional and translational levels,promoted the nuclear transfer of Nrf2,and promoted the expression of downstream antioxidant related proteins GCLC,GCLM,HO-1 and NQO1.Conclusion: Puerarin can promote Nrf2 nuclear transfer and activate Nrf2 signaling pathway by inhibiting Keap1 expression.In conclusion,puerarin plays a protective role in ap AP-induced acute liver injury by affecting liver organ index,hepatocyte pathological changes,serum biochemical indices,liver antioxidant content and mitochondrial membrane potential.In cell experiments,puerarin plays a protective role on APAP-induced oxidative damage Hep G2 cells by affecting cell activity,biochemical indices of cell culture medium,intracellular antioxidant content and mitochondrial membrane potential.The mechanism is that puerarin exerts antioxidant activity by inhibiting the transcription and translation of Keap1 and promoting the nuclear transfer of Nrf2,thereby activating the expression of GCLC,GCLM,NQO1 and HO-1 genes downstream of the antioxidant response element.