P38γ Regulates CCl4-Induced Acute Liver Injury Through The Nrf2/HO-1 Signaling Pathway

Objective:Acute liver injury（ALI）,also known as acute liver failure（ALF）,refers to a group of serious liver injury diseases caused by various causes with poor prognosis.Mild acute liver injury can gradually heal on its own,but it can also turn into chronic liver injury,which can lead to liver fibrosis,cirrhosis or liver cancer,while acute injury can develop into acute liver failure with high mortality when it is extremely severe.Under the long-term effect of exogenous factors,short-term exposure of the organism to large doses of exogenous substances can cause acute liver injury.Dysfunction of the liver will lead to dysregulation of several systems such as digestion,coagulation and endocrine,reducing the quality of life and in severe cases endangering life.The treatment of acute liver injury is still a long-standing challenge in clinical medicine.The extracellular signal-regulated kinase P38γ（also known as SAPK3 and MAPK12）,one of the four isoforms of the P38 MAPK family,has been shown to have an important role in cancer,cardiac and neurodegeneration.P38γhas a specific distribution in tissues,is mainly expressed in skeletal muscle,and affects apoptosis and metastasis of tumor cells.It has also been shown that P38γis highly expressed in liver tissue of patients with NAFLD and regulates the pathogenesis by controlling the infiltration of regulatory neutrophils.Therefore,it is highly likely that P38γhas an important regulatory role in acute liver injury.In the present study,we focused on a model of acute liver injury induced by carbon tetrachloride and investigated the effects of P38γon it,as well as the effects of lipid metabolism-related genes and oxidative stress levels in H₂O₂-induced AML-12 cells.In addition,the specific pathway mechanisms by which P38γregulates acute liver injury were specifically investigated.Methods:this experiment was divided into in vivo and in vitro experiments,respectively.In vivo experiments:Male C57BL/6J mice were selected at 8-10 weeks of age and randomly grouped into four groups:normal group,CCl₄-induced model group,AAV9control group and AAV9-P38γgroup,with 6-12 mice in each group.Except for the normal group which was injected with the same amount of olive oil,the mice in the other three groups were injected intraperitoneally with 10%CCl₄ diluted in olive oil at a dose of 0.5 m L/kg,and the mice were anesthetized and executed after acute liver injury was induced within 24 h.The blood and liver were collected.The model was successfully constructed by small animal imaging,immunofluorescence and RT-q PCR,and then the liver tissues of each group of mice were examined by H&E staining and immunohistochemistry to examine the degree of liver injury.Finally,the expression levels of genes related to oxidative stress（i NOS and NOX4）,inflammatory factors（TNF-α,IL-1βand IL-6）and lipid metabolism（FASN,ACOX-1,SREBP-1c and PPAR-α）were detected by Western blotting and RT-q PCR.In vitro experiments:mouse normal hepatocytes（AML-12 cells）were used,and the expression of P38γwas firstly examined after stimulating AML-12 cells with different concentrations of H₂O₂ for different times to determine the optimal concentration and time of stimulation.Finally,P38γoverexpression plasmid and P38γ-si RNA were transfected into the cells,and the changes of Nrf2/HO-1 signaling pathway and the expression levels of the above mentioned genes were detected by Western blotting and RT-q PCR,so as to observe the specific regulatory mechanism of P38γon acute liver injury.Results:In vivo results:In the CCl₄-induced acute liver injury model in mice,significant pathological changes in inflammation,lipid metabolism and oxidative stress-related protein expression occurred,and the expression of P38γwas also significantly increased.And in vivo silencing of P38γwas observed to effectively ameliorate the pathological changes and the expression of inflammation,lipid metabolism and oxidative stress-related proteins in mouse liver.In vitro results:The expression of P38γwas significantly increased in H₂O₂-stimulated AML-12 cells.Subsequent transfection of P38γ-si RNA attenuated stimulus-induced inflammation,lipid metabolism and oxidative stress-related protein expression,whereas transfection of P38γoverexpression plasmid exacerbated stimulus-induced inflammation,lipid metabolism and oxidative stress.On the other hand,both overexpression and silencing of P38γin AML-12 cells affected the Nrf2/HO-1 signaling pathway,then suggesting that P38γmay ultimately contribute to oxidative stress,lipid metabolism and inflammatory responses in acute liver injury by regulating the Nrf2/HO-1 signaling pathway.Conclusion:the above experimental results suggest that P38γregulates CCl₄-induced acute liver injury through the Nrf2/HO-1 signaling pathway.