Mechanism Of Metformin Alleviates CCl₄-induced Acute Liver Injury In Mice By Regulating Stress Response

Acute liver injury（ALI）refers to liver cell damage induced in a short period of time by triggering factors such as infection,improper drug use,and ischemia.Due to the complex mechanism of occurrence,there are still many challenges in prevention and treatment,and it is urgent to reveal the mechanism of acute drug-induced liver injury.CCl₄ has hepatotropic biological properties,and CCl₄ is closer to human acute drug-induced liver injury in mechanism and symptoms by inducing the inflammatory response of acute liver injury.Therefore,animal models of CCl₄-induced acute liver injury are often used to explain the effect of human ALI.Pathogenesis research.So far,it has been found that oxidative stress,inflammatory response,necrosis and apoptosis are closely related to the occurrence of ALI.At present,experimental results show that mild inflammation and stress response may be related to the protection of cellular homeostasis,but the relationship between them is not very clear.It is currently believed that when an external stimulus induces a stress response,an inflammatory response is induced if the stress response is insufficient to defend against homeostasis.The nucleus,endoplasmic reticulum,and mitochondria are key regulatory organelles of inflammation.Transcriptomics and other experiments have found that stress responses such as endoplasmic reticulum and mitochondria play their role through selective translation,that is,upregulating the expression of pro-repair proteins and/or inhibiting the expression of pro-damage proteins.Anti-damage effect.IL-33 acts as an"alarm"for the immune system,rapidly triggering the immune system to respond to viral invasion and liver toxic damage.It has been found that IL-33 can alleviate high-fat diet（HFD-）-induced hepatic steatosis and insulin resistance.When the liver is chronically injured,IL-33 promotes the development of liver fibrosis by activating the Th2 response and hepatic stellate cells.It is believed that depending on the immune environment of the body,the IL-33/ST2 axis activates cellular stress responses through different pathways,thereby playing a protective role.IL-33 overexpression mice also showed mild inflammation,liver fibrosis and other changes,indicating that IL-33 overexpression mice are a good animal model to elucidate the effect of inflammatory state on liver injury.Metformin,as a first-line drug for diabetes,has the function of inhibiting mitochondrial respiratory chain complex I,and it has been reported that inhibiting mitochondrial respiratory chain complex I with rotenone can effectively alleviate CCl₄-induced ALI.Recent studies have found that metformin plays a role in preventing or treating a variety of diseases through anti-inflammatory,anti-stress and immune regulation and other basic pathologies.Effects and mechanisms in animal models of injury.Therefore,in this experiment,wild-type and IL-33 gene overexpression mouse models of CCl₄ acute liver injury were used as the research objects.Combined with serum biochemical indicators,morphological observation,and transcriptomic analysis,the stress of mitochondria,endoplasmic reticulum and other organelles was analyzed.The response is an entry point to further elucidate the mechanism of metformin antagonizing acute inflammatory response-induced liver injury.Method:1.The mouse ALI model was constructed by intraperitoneal injection of CCl₄into wild-type and IL-33 gene-overexpressing mice.The specific groups were as follows:Wild-type mice were grouped into control group（WT-Na Cl）and olive oil group（WT-Oil）,model group（WT-CCl₄）,metformin group（WT-Met）,metformin pretreatment group（WT-CCl₄+Met）;IL-33 gene overexpression mice were grouped into control group（IL33-Na Cl）,olive oil group（IL33-Oil）,model group（IL33-CCl₄）,metformin group（IL33-Met）,metformin pretreatment group（IL33-CCl₄+Met）.2.The AST/ALT enzyme activity detection kit and HE staining were used to evaluate whether the acute liver injury model of wild-type and IL-33 gene overexpression mice was successfully constructed,and the effect of metformin pretreatment on wild-type and IL-33 gene overexpression model groups effects of liver injury in mice.3.The liver tissues of the wild-type and IL-33 gene overexpression mice in each experimental group were taken,and RNA was extracted after Trizol lysis,and the gene expression of each experimental group was analyzed by transcriptomics;the WT-Na Cl group was used as the control,and the WT-Na Cl group was used as the control.Changes of gene expression in liver tissue of mice in-CCl₄ group and WT-CCl₄+Met group;with IL33-Na Cl group as control,the changes in liver tissue gene expression of mice in IL33-CCl₄ group and IL33-CCl₄+Met group were analyzed by GO enrichment.Analysis and KEGG signaling pathway enrichment analysis clarified the biological functions of differentially expressed genes.4.The effect of metformin pretreatment on the gene expression of wild-type and IL-33 gene overexpression mice CCl₄ acute liver injury model was analyzed by Venn diagram,and WT-CCl₄+Met was analyzed by GO enrichment analysis and KEGG signaling pathway enrichment analysis.The biological function of hepatic tissue expression restoration gene of mice in WT-CCl₄+Met group and IL33-CCl₄+Met group.5.Take the liver tissues of wild-type and IL-33 gene overexpression mice from each experimental group,extract RNA after Trizol cleavage,reverse transcribed into c DNA,and detect the expression changes of mitochondrial respiratory chain complex subunits and antioxidant genes by q PCR.6.The liver tissues of wild-type and IL-33 gene overexpression mice were taken from each experimental group,and the protein was extracted after RIPA cleavage,and the mitochondrial respiratory chain complex subunits,antioxidant proteins and endoplasmic reticulum stress-related proteins were detected by Western Blot.expression changes.Result:1.From the changes of HE staining and liver function enzymes,we found that 1)the serum AST/ALT levels of the mice in the WT-CCl₄ group and the IL33-CCl₄group increased sharply.The structure of the hepatic cord is destroyed,hepatic lobule boundary blurred,hepatic cord structure destruction and a large number of inflammatory cells are infiltrated.Compared with the WT-CCl₄ group,there was no change in the main indicators of acute liver injury in the IL33-CCl₄ group.2)The serum AST/ALT levels of mice in the WT-CCl₄+Met group and IL33-CCl₄+Met group were significantly decreased,the hepatocytes were arranged regularly,the boundaries of the hepatic lobules were clearly defined,the damage to the hepatic cord structure was reduced,and the number of inflammatory cell infiltration decreased.3)Compared with the WT-CCl₄+Met group,the IL33-CCl₄+Met group had a significantly lower serum AST/ALT level and a significantly lower degree of liver histological damage.2.Through transcriptomic analysis of differentially expressed genes,we found:1)In wild-type mice:compared with WT-Na Cl group,a total of 935 genes were up-regulated and 914 genes were down-regulated in WT-CCl₄ group.Compared with WT-Na Cl group,344 genes were up-regulated and 509 genes were down-regulated in WT-CCl₄+Met group.2)In IL-33 gene overexpression mice:compared with IL33-Na Cl group,1025 genes were up-regulated and 706 genes were down-regulated in IL33-CCl₄ group.Compared with IL33-Na Cl group,320 genes were up-regulated and310 genes were down-regulated in IL33-CCl₄+Met group.It is suggested that metformin alleviates CCl₄-induced acute liver injury by stabilizing gene expression.3.Through the KEGG signal pathway enrichment analysis,we found that:1)Compared with the WT-Na Cl group and the IL33-Na Cl group,the differentially expressed genes in the WT-CCl₄ group and the IL33-CCl₄ group had similar functions,and the down-regulated genes were mainly enriched in steroid hormone synthesis,cytochrome P450 to foreign substances,cytochrome P450 to drug metabolism and amino acid synthesis and other related pathways;while the up-regulated genes are mainly enriched in cytokine receptor interaction,IL-17 signaling pathway and other inflammatory pathways and other related signaling pathways.4.Through Venn diagram analysis,it was found that the expression of 1428differentially expressed genes in the liver tissue of the mice in the WT-CCl₄+Met group was restored,and the expression of 1415 differentially expressed genes in the liver tissue of the mice in the IL33-CCl₄+Met group was restored;further through the KEGG signal Pathway analysis of the functions of the above expression restoration genes:WT-CCl₄+Met group and IL33-CCl₄+Met group metabolism-related genes are mainly enriched in steroid hormone biosynthesis,P450 enzyme metabolism of foreign substances,P450 enzyme metabolism of drugs and amino acids Inflammation-related genes are mainly enriched in cytokine receptor interactions;and down-regulated genes in the liver tissue of IL33-CCl₄+Met mice are also enriched in chemokine signaling pathways,tumor necrosis factor signaling pathway and NF-k B signaling pathway and other inflammation-related signaling pathways,the anti-inflammatory effect of metformin in IL-33 gene-overexpressing mice was significantly better than that in wild-type mice.5.By detecting the changes of mitochondrial respiratory chain complex m RNA and related protein levels,we found:1)Compared with the WT-Na Cl group and the IL33-Na Cl group,the mitochondrial respiratory chain complex MT-ND1,NDUFV1,MT-ATP6 and ATP5A1 m RNA were significantly decreased in the WT-CCl₄ group and the IL33-CCl₄ group,while the mitochondrial respiratory chain complex subunit MT-ND1 and NDUFA9 protein expression decreased.2)Compared with WT-CCl₄group and IL33-CCl₄ group,the m RNA expression of mitochondrial respiratory chain complex in liver tissue of mice in WT-CCl₄+Met group and IL33-CCl₄+Met group was increased,and the subunits of mitochondrial respiratory chain complex were increased.The trend of protein expression was consistent with that of m RNA.3)Compared with wild-type mice,in IL-33 gene overexpressing mice,the expression of mitochondrial respiratory chain complex m RNA and the expression of respiratory chain subunit proteins in the IL33-CCl₄+Met group increased more significantly,and the expression of mitochondrial respiratory chain complex subunits tends to be more normal.6.By detecting the changes in the m RNA expression and protein expression level of the antioxidant protein SOD2,we found that:1)Compared with the WT-Na Cl group and the IL33-Na Cl group,the liver tissue of the WT-CCl₄ group and the IL33-CCl₄ group had antioxidant activity.The m RNA and protein expression of protein SOD2 decreased.2)Compared with the WT-CCl₄ group and the IL33-CCl₄group,the m RNA and protein expressions of the antioxidant protein SOD2 in the liver tissue of the mice in the WT-CCl₄+Met group and the IL33-CCl₄+Met group were increased.3)Compared with wild-type mice,in IL-33 overexpressing mice,the m RNA and protein expression of antioxidant protein SOD2 in IL33-CCl₄+Met group increased more significantly,making hepatocytes more prone to redox stabilization.7.By detecting the changes in the expression of endoplasmic reticulum stress-related proteins p-e IF2αand Bip,we found that:1)Compared with the WT-Na Cl group and the IL33-Na Cl group,the expressions of p-e IF2αand Bip protein in tissues were significantly increased in WT-CCl₄ group and IL33-CCl₄ group.2)Compared with the WT-CCl₄ group,the expression of p-e IF2αand Bip protein in the liver tissue of WT-CCl₄+Met group did not change significantly;compared with the IL33-CCl₄group,the expression levels of p-e IF2αand Bip protein were significantly decreased in IL33-CCl₄+Met group.3)Compared with wild-type mice,the expression of p-e IF2αand Bip proteins in IL33-CCl₄+Met group decreased more significantly,which could more significantly inhibit CCl₄-induced endoplasmic reticulum response.In conclusion:1.Metformin pre-treatment of CCl₄ acute liver injury model mice,especially IL-33 gene overexpression mice,significantly improved liver pathological changes and biochemical indicators,indicating that the anti-CCl₄-induced liver injury effect of metformin may be related to IL-33 gene overexpression mice.related inflammatory response and other changes.2.Metformin can increase the expression of mitochondrial respiratory chain complex in wild-type mice and IL-33 overexpressing mice,indicating that metformin can alleviate CCl₄-induced liver injury by inhibiting mitochondrial dysfunction.3.Metformin can reduce the expression of endoplasmic reticulum stress-related proteins in liver tissue of IL-33 gene-overexpressing mouse models of acute liver injury,indicating that metformin inhibits the endoplasmic reticulum stress injury response of hepatocytes,which is one of the mechanisms of reducing liver injury.4.Comprehensive analysis of the transcriptomic experimental results of chemokines,tumor necrosis factor,oxidative stress and other signaling pathways related to inflammation and stress response,indicating that metformin may improve cellular stress resistance,reduced expression of inflammatory factors attenuated CCl₄-induced liver injury at the nucleus and mitochondria/endoplasmic reticulum level.