Shikonin Attenuates Acetaminophen-induced Hepatotoxicity By Upregulation Of Nrf2 Through Akt/GSK3? Signaling

Object: Acute liver failure caused by acetaminophen(APAP)overdose is a common and major cause of global drug-induced liver injury(DILI).Shikonin(SHK)is a natural crystalline powder extracted from the roots of shikonin.Recent studies have shown that SHK can reduce acute liver injury in mice with concanavalin A.However,the role of SHK in APAP-induced liver toxicity has not been determined.This experiment aims to explore the potential protective effects and mechanisms of SHK in APAP-induced in vitro liver toxicity,and to study the protective effects of SHK on APAP-induced acute liver injury in vivo,in order to promote the development of effective treatment strategies for APAP liver toxicity.Methods:(1)Hematoxylin-eosin staining(HE staining)was used to detect pathological conditions of mouse liver tissue in APAP-induced liver injury model by SHK pretreatment;meanwhile,mouse serum was detected by AST and ALT kits.(2)L-02 cells or mouse liver tissue were treated with SHK,APAP and/or various pathway inhibitors,then GSH,ROS and MPO levels were detected by kits.(3)MTT assay: after using SHK,APAP and/or various pathway inhibitors,test the cell viability of L-02 cells.(4)Western blotting: After using SHK,APAP,and/or various pathway inhibitors,the expression levels of p-Akt,p-GSK3?,and Nrf2 in L-02 cells or mouse liver tissue were detected.(5)Real-time quantitative gene amplification fluorescence(RT-qPCR): After using SHK,APAP,and/or various pathway inhibitors,RT-qPCR was used to detect the mRNA expression changes of Nrf2 and Nrf2 targeted genes.Results:(1)HE staining,AST,ALT,GSH,ROS and MPO results in a mouse model of acute liver injury induced by APAP suggest that SHK can prevent APAP induced liver damage.(2)MTT assay,and ROS and GSH to detect cell viability and oxidative stress of L-02 cells after SHK and/or APAP treatment,the results suggest that SHK can inhibit Hepatic toxicity and oxidative stress in L-02 cells.(3)SHK can increase the expression of Nrf2 and its downstream target genes HO-1,GCLC and GCLM in L-02 cells;after addition of the Nrf2 inhibitor brutasol Reversible increase of L-02 cell viability,decrease of ROS content and increase of GSH content caused by SHK.(4)Application of database analysis and experimental techniques suggest that SHK may induce Nrf2 expression in L-02 cells through the PI3K/Akt/GSK3? pathway.(5)The results of western blotting and HE staining suggest that SHK prevents APAP-induced liver toxicity through Akt/GSK3?/Nrf2 in vivo.Conclusion: The present study indicates that SHK protects APAP-induced liver injury via inhibition of oxidative stress through AKT/GSK3 pathway-dependent Nrf2 upregulation in hepatocytes.Hence,SHK may be a promising candidate for treatment of APAP hepatotoxicity.