基于Nrf2/HO-1/GPX4通路介导的铁死亡探讨白鲜皮致肝损伤的作用机制OA

Objective To investigate the mechanism of liver injury induced by Cortex Dictamni based on ferroptosis mediated by the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/glutathione peroxidase 4(GPX4)pathway.Methods(1)In vivo experiment:Forty male ICR mice were randomly divided into 4 groups:control group,high-dose Cortex Dictamni group(9.180 g·kg-1),medium-dose Cortex Dictamni group(6.885 g·kg-1),and low-dose Cortex Dictamni group(5.508 g·kg-1).Drugs were administered by gavage(20 mL·kg-1)once daily for 7 consecutive days.Serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST),as well as hepatic ferrous ion(Fe2+)and malondialdehyde(MDA)contents,were measured by ELISA.Pathological changes of liver tissue were observed by HE and Prussian blue staining.mRNA and protein expression levels of Nrf2,HO-1,GPX4,GCLM and FPN1 in liver tissue were detected by RT-PCR and Western Blot.(2)In vitro experiment:HepaRG cells were treated with Cortex Dictamni aqueous extract at concentrations of 1,2,4,6,8 and 10 mg·mL-1 for 24 hours,and cell viability was measured by CCK-8 assay to screen the optimal intervention concentrations.HepaRG cells were then divided into control group,high-dose Cortex Dictamni group(8 mg·mL-1),medium-dose group(6 mg·mL-1)and low-dose group(4 mg·mL-1).After 24 hours of intervention,mRNA and protein expression levels of Nrf2,HO-1,GPX4,GCLM and FPN1 were detected by RT-PCR and Western Blot.Results(1)Compared with the control group,serum ALT levels were significantly increased in the medium-and high-dose Cortex Dictamni groups(P＜0.05,P＜0.001),and serum AST levels were significantly elevated in the high-dose group(P＜0.01).Compared with the control group,mice in all Cortex Dictamni groups exhibited marked hepatocyte injury,including cell swelling and rupture,nuclear deformity,severe disruption of cellular structure,disordered hepatic cord arrangement,massive inflammatory cell infiltration,and significantly increased brown iron deposits(P＜0.001).Hepatic Fe2+and MDA levels were significantly elevated(P＜0.05,P＜0.01,P＜0.001),while mRNA and protein expression levels of Nrf2,HO-1,GPX4,GCLM and FPN1 were significantly decreased in liver tissue(P＜0.001).(2)Compared with the control group(0 mg·mL-1),HepaRG cell viability was significantly reduced in all Cortex Dictamni treatment groups(2,4,6,8,10 mg·mL-1)in a dose-dependent manner(P＜0.05,P＜0.001).mRNA and protein expression levels of Nrf2,HO-1,GPX4,GCLM and FPN1 were significantly decreased in all Cortex Dictamni dose groups compared with the control group(P＜0.001).Conclusion Cortex Dictamni aqueous extract can induce liver injury.The underlying mechanism may be related to the inhibition of the Nrf2/HO-1/GPX4 pathway and its downstream target proteins GCLM and FPN1,leading to intracellular iron accumulation and lipid peroxidation,thereby triggering ferroptosis.