The Effect Of Verbenalin In Regulating MDMX/PPARα-mediated Ferroptosis In Alleviating Alcoholic Steatohepatitis

Alcohol is one of the leading causes of morbidity and mortality from liver-related diseases worldwide.Alcoholic steatohepatitis（ASH）is a serious danger to human health and,if left untreated,can cause more serious liver damage,liver fibrosis,cirrhosis and even liver cancer.However,there is still a lack of effective treatment for alcoholic steatohepatitis.Notably,there is growing evidence that damage in alcoholic steatohepatitis is associated with iron death.Ferroptosis is a process of cell death caused by cell metabolism and lipid peroxidation overloaded with iron,which has been linked to cancer and several liver diseases.Several studies have shown that murine bimicroprotein X（MDMX）affects ferroptosis in relation to lipid peroxidation in cells,and it is a protein isolated from mouse c DNA expression libraries that can interact with p53,which can significantly inhibit ferroptosis in various cancer models.Verbenalin is the main compound L（Verbenaceae）in verbena and is generally considered a safe food by the U.S.Food and Drug Administration.Verbena,first recorded in the"Supplementary Record of Famous Doctors".Verbenalin（VE）,its main active ingredient,has been found to have many biological effects,including anti-obesity,anti-inflammatory and antioxidant activity,removal of jaundice and treatment of malaria.It can treat lump buildup,dysmenorrhea,blocked throat,edema,jaundice,and malaria.Studies have shown that verbenalin has a certain therapeutic effect on liver damage caused by palmitic acid（PA）,oleic acid（OA）,ethanol and acetaminophen.To further explore the mechanistic role of verbenin in alcoholic steatohepatitis.We have designed the following five parts to investigate this topic:1.Effects of VE in the alcoholic steatohepatitis mouseWe purchased 70 C57BL/6J male mice（body weight 18g-22g）,and then divided each group of 10 into normal group,model group,model group plus 12.5mg/kg,25mg/kg,50mg/kg verbenalin group,normal plus 50mg/kg verbenalin group,and silybin group.The model group mice were fed alcoholic feed to establish a model of alcoholic fatty liver injury,and the drug group began to give daily gavage of low,medium and high doses of VE on the first day of modeling.The normal group was fed control feed.After molding,fast for 24h.Anesthetize the mouse to sacrifice it,followed by the collection of plasma and liver tissue.In order to detect the damage of liver tissues in each group,HE staining,serum ALT,AST and TG biochemical indexes were used to detect liver tissue damage,and the experimental results showed that we successfully established a model of alcoholic fatty liver injury induced by alcoholic feed,and the experimental data showed that VE had an improving effect on injury.2.VE inhibits oxidative stress-induced iron death in alcoholic fatty liver injuryFirstly,through the detection of a series of indicators such as ROS,4-HNE,MDA,GSH,SOD and other indexes in serum and tissues,the results show that VE has a certain effect on oxidative stress in alcoholic fatty liver injury.The optimal concentration of VE for AML-12 cells was screened by CCK8.At the same time,we detected a series of oxidative stress indicators such as ROS,4-HNE,MDA,GSH,SOD in cells,and the results showed that VE had a certain protective effect on alcohol-induced damage of mouse liver cells.In addition,the results of projection electron microscopy showed that VE had a certain mitigating effect on mitochondrial dysfunction.At the same time,the detection of Fe²⁺content showed that VE reduced the release of Fe2+in alcoholic steatohepatitis.The above experimental results show that VE inhibits oxidative stress-induced ferroptosis in alcoholic steatohepatitis。3.VE affects ferroptosis by targeting MDMXNetwork pharmacology results show that VE can target MDMX.The effect of VE on its activity was determined using the MDMX Activity Kit.The experimental results showed that VE could bind to MDMX and reduce the activity of MDMX in liver injury.At the same time,the thermal stability experiment was carried out,and the results showed that VE improved the heat resistance of MDMX.Then,we inhibited MDMX with MDMX inhibitor（NCS207895）on AML-12 cells and Ethanol（200m M）for 24hours,and the detection of ROS,4-HNE,MDA,GSH,SOD and other indicators showed the effect of VE on oxidative stress in AML-12 cells.In addition,transmission electron microscopy and Fe²⁺content were used to detect the effects of mitochondria and iron death.The experimental results showed that VE could inhibit the expression of MDMX,and when MDMX was inhibited and given VE treatment,it was found that VE could inhibit ferroptosis by affecting MDMX.Then,MDMX was overexpressed by MDMX overexpression plasmid on AML-12 cells,and then treated with Ethanol（200m M）and VE（100μM）for 24 h,and oxidative stress indicators were detected by ELISA.Transmission electron microscopy to detect the effect of overexpression of MDMX on mitochondria in AML-12 cells;At the same time,Fe2+experiments were used to explore the effect of overexpression of MDMX on VE’s inhibition of ferroptosis.The results showed that after overexpression of MDMX in AML-12 cells,VE’s ability to inhibit AML-12 ferroptosis was weakened.4.MDMX affects lipid peroxidation and iron death by affecting PPARαactivityTo determine the molecular mechanism of action of MDMX,co-immunoprecipitation（Co-IP）experiments are performed in AML-12 cells.The results showed that MDMX affects PPARα.The effect of MDMX on its activity was determined using the PPARαActivity Kit.PPAR has been shown to be a transcription factor involved in the regulation of lipid homeostasis.We hypothesize that MDMX may influence ferroptosis by mediating PPARα.To verify this,we treated AML-12cells stimulated with the PPARα-specific agonists pirinixic acid（10μM）and Ethanol（200 m M）for 24 h.ELISA to detect indicators of oxidative stress;Transmission electron microscopy to detect the effect of PPARα-specific agonists on mitochondria in AML-12 cells;At the same time,Fe²⁺experiments were used to explore the effect of PPARα-specific agonists on ferroptosis.Next,we determined the protective effect of the MDMX inhibitor NCS207895（5μM）against oxidative stress-induced iron death in the presence or absence of the PPARαantagonist GW6471（10μM）in AML-12 cells.Indicators of oxidative stress are detected by ELISA.Furthermore,levels of Fe²⁺indicate that the protective effect of NCS207895 against ferroptosis is inhibited in GW6471-treated AML-12 cells.This observation was further confirmed using transmission electron microscopy.These results suggest that reducing PPARαactivity is a key pathway for MDMX to inhibit cellular antioxidant defenses.5.VE by affecting MDMX/PPARα-mediated lipid peroxidation and iron death in AML-12 cellsTo study the effect of verbenalin on PPARαactivity,PPARαactivity in serum and liver tissues was detected by ELISA.In addition,we analyzed the protective effect of VE（100μM）on oxidative stress-induced iron death with the presence or absence of the PPARαantagonist GW6471（10μM）in AML-12 cells,to detect oxidative stress indicators by ELISA.Fe²⁺levels indicated that the protective effect of verbenalin on iron death was inhibited in GW6471-treated AML-12 cells.This observation was further confirmed using transmission electron microscopy.These results suggest that verbenalin regulates MDMX/PPARα-mediated antioxidant defense and ferroptosis in AML-12 cells.