Genipin Alleviates CCl₄-induced Acute Liver Injury By Inhibiting Ferroptosis

Objective Genipin is the main active metabolite of Gardenia jasminoides.Previous studies have shown that it can protect liver through multiple mechanisms such as anti-oxidative stress,protection of mitochondria,anti-inflammation and so on.Ferroptosis is a new form of regulated cell death which is different from apoptosis、pyroptosis、autophagy and necroptosis.The cell death resulted from accumulation of intracellular iron ion level,imbalance of redox and accumulation of lipid peroxides.The main purpose of this study is to explore whether ferroptosis is involved in CCl₄-induced acute liver injury in mice,and to further explain the molecular mechanism of the hepatoprotective effect of genipin from the pathway of system Xc-/GPx4,and provide a theoretical basis for highconnotation drug screening and new development based on traditional Chinese medicine.Methods（1）The acute liver injury model of C57BL/6 mice treated with CCl₄ was established.The general morphology of liver,histopathological（Knodell）score after H&E staining,TUNEL staining to evaluate the degree of cell necrosis,iron level,MDA（malondialdehyde）and ROS（reactive oxygen species）level,C11BODIPY581/591 to evaluate lipid peroxidation was compared to the control group.The changes of mitochondrial volume,morphology,membrane integrity and crest number were observed by Transmission electron microscope.The expression of ferroptosis core module protein x CT-GPx4 was assesed by Western Blot.（2）C57BL/6 mice were randomly divided into six groups: control group,CCl₄ model group,CCl₄+ genipin treatment group,genipin group,CCl₄+Fer-1 control group and Fer-1 group.The liver morphology,Knodell score,TUNEL staining degree,iron、MDA and ROS level and C11BODIPY581/591 evaluation of lipid peroxidation were observed.The changes of mitochondrial volume,morphology,membrane integrity and crest number were observed by Transmission electron microscope.The expression of ferroptosis core module protein x CT-GPx4 was assessed by Westernblot,and the expression of p53 protein in the possible upstream signal pathway was also detected.（3）Normal hepatocyte line L-O2 was divided into control group,Erastin（iron death inducer）group,Erastin+ genipin group,genipin group,Erastin+Fer-1 group and Fer-1n group.The ability of cell proliferation,the level of MDA and ROS were detected.and the changes of mitochondrial volume,morphology,membrane integrity and crest number were observed by electron microscope.Results（1）Compared with the control group,the levels of serum ALT and AST in CCl₄-induced acute liver injury group were significantly increased,hepatic structure disorder,hepatocyte necrosis and inflammatory cell infiltration were significantly increased,and the level of iron,MDA,ROS and lipid peroxidation in liver tissue were significantly increased.Electron microscopic observation showed that decreased or vanished mitochondria cristae,a ruptured outer mitochondrial membrane,and a condensed mitochondrial membrane.The expression of x CT and GPx4 protein decreased significantly.（2）Compared with CCl₄ group,genipin treatment group and ferroptosis inhibitor Fer-1 group could significantly alleviate the pathological injury of liver tissue,decrease the levels of ALT and AST,iron、ROS and MDA level,decrease the level of lipid peroxidation,alleviate the changes of mitochondria and hepatocyte necrosis under transmission electron microscope.The expression of ferroptosis core module protein x CT-GPx4 was up-regulated and the expression of p53 was decreased by Western blot assessment.（3）The cell activity detected by CCK-8 showed that the number of cells decreased significantly in erastin group,genipin and Fer-1 could significantly increase the cell survival rate,and the level of LDH,ROS and MDA in erastin group increased significantly,while genipin and Fer-1 treatment could alleviate the cell injury caused by erastin and decrease the content of ROS and MDA.Transmission electron microscope showed that the volume of mitochondria decreased,the membrane density increased and the mitochondrial crest decreased or disappeared in erastin group.Genipin and Fer-1 treatment can alleviate it.Conclusions Ferroptosis is involved in CCl₄-induced acute liver injury in mice.Genipin inhibits ferroptosis by up-regulating the expression of system Xc-and GPx4,thus alleviates acute liver injury.