Sulfiredoxin-1 Alleviates Myocardial Hypertrophy And Fibrosis Interacting With PRDX2 Through Regulating AKT/GSK3 β

Objectives:End-stage heart failure is the main cause of human death worldwide,caused by various diseases such as obesity,thyroid hormone dysfunction,diabetes,hyperlipidemia,heart valve disease,hypertension,myocardial infarction and various types of cardiomyopathy.Cardiac hypertrophy is an important driving factor for end-stage heart disease.There are currently no effective pre-measures.Studies have found that oxidative stress is a key factor in pathological changes such as myocardial hypertrophy under various pathological conditions.At the same time,there are also evidences shown that inhibiting oxidative stress in the myocardium can alleviate pathological myocardial hypertrophy and fibrosis.Sulfiredoxin-1(SRXN1)is an endogenous antioxidant protein belonging to the sulfiredoxin family,which catalyzes oxidized peroxidase to its reduced form,thereby restoring its redox activity.It plays an important role in various physiological processes such as apoptosis,cell proliferation,invasion,and redox balance.SRXN1 is part of a thiol-based antioxidant system that preferentially oxidizes and reduces ROS levels under conditions of oxidative stress.At the same time,some studies suggest that SRXN1 can inhibit mitochondrial apoptosis through the PI3K/AKT signaling pathway and alleviate myocardial injury.Studies have also shown that in the early stage of hyperglycemic disease,SRXN1 can alleviate the oxidative stress response of cardiomyocytes,thereby reducing the inflammatory response and related damage of cardiomyocytes caused by high-glucose environment.Peroxidases(PRDXs)are a broad and abundant family of enzymatically active antioxidant enzymes.It can reduce reactive oxygen species,such as H2O2,produced by cells under various stimulation conditions through redox reactions.Thereby promoting the normal physiological function of cells and preventing cell oxidative stress damage.Existing studies have found that overexpression of SRXN1 can increase the content of PRDX family protein PRDX1 in myocardial tissue,while interference with SRXN1 can reduce the content of PRDX family protein PRDX1,2,3,and 4.Among them,PRDX1 has been shown to reduce myocardial stress caused by stress load.Cellular inflammation and oxidative stress and fibrosis,PRDX2 expression is reduced in cardiomyocyte hypertrophic cells,and is involved in the inhibition of oxidative stress response,which can activate the activity of AKT/GSK3β signaling pathway after being inhibited in cells.The AKT/GSK3β signaling pathway has been confirmed to be one of the important signaling pathways involved in cardiac hypertrophy and fibrosis.However,the mechanism by which SRXN1 regulates AKT/GSK3β through PRDXs to alleviate cardiac hypertrophy has not been reported yet.Therefore,we intend to study the interaction between the antioxidant protein SRXN1 and PRDXs and regulate the activity of AKT/GSK3β signaling protein to alleviate myocardial hypertrophy and fibrosis caused by excessive activation of adrenergic receptors by reducing myocardial oxidative stress injury and inflammatory response.Process-related biological functions and mechanisms involved.Methods:Part Ⅰ:Isoprenaline(ISO)was used to establish rat myocardial hypertrophy models in vitro and in vivo,and the degree of myocardial hypertrophy was detected by echocardiography and QRT-PCR.The content of SRXN1 protein in ISO cardiomyocytes and myocardial tissue was determined by immunofluorescence IF and Western Blot WB.Part Ⅱ:Lentivirus overexpression vector was used to overexpress SRXN1 protein in H9c2 rat cardiomyocytes.The overexpression effect of SRXN1 gene was detected by WB,QRT-PCR,fluorescence staining,DCFH-DA staining,MDA kit and SOD kit respectively.After ISO induction,cells with overexpression of SRXN1 became hypertrophic.Inflammatory response,cellular oxidative stress response and fibrotic changes,and the contents of PRDX1 and PRDX2 downstream of SRXN1 were detected.Part Ⅲ:Lentiviral vector was used to knock down PRDX2 in SRXN1 overexpressed H9c2 cells,and then WB,QRT-PCR,fluorescence staining,DCFH-DA staining,MDA kit and SOD kit were used to detect the hypertrophic and inflammatory reactions of PRDX2 gene knockdown cells induced by ISO.Cellular oxidative stress response and fibrotic changes.Part Ⅳ:SRXN1 protein was overexpressed in the myocardium of SD rats by adenovirus overexpression vector and intracardial injection under direct vision.In vivo fluorescence imaging system,echocardiography,Hematoxylin-Eosin HE staining,Masson staining,WB,QRT-PCR,MDA kit and SOD kit were used to detect the effect of adenovirus transfection respectively.Myocardial hypertrophy,inflammatory response,myocardial oxidative stress response and fibrosis changes were observed after ISO induction in rats in each experimental group.Results:Part Ⅰ:ISO-induced echocardiography results in rats showed that cardiac function had corresponding changes in cardiac hypertrophy.Iso-induced H9c2 cells and rat myocardium significantly upregulated the expression of ANP and BNP,and significantly decreased the protein content of SRXN1.Part Ⅱ:The expression levels of ANP,BNP,TNF-α,IL-1β and IL-6 genes,the activity of AKT/GSK3β signaling pathway,and the oxidative stress level of H9c2 cells with SRXN1 overexpression were significantly decreased compared with those of H9c2 cells without SRXN1 overexpression,while the antioxidant capacity was significantly increased.Part Ⅲ:Knockdown of PRDX2 gene in H9c2 cells restored ISO-induced up-regulation of AKT/GSK3β signaling pathway activity,increased cell surface area,up-regulated expression of genes related to cardiac hypertrophic and inflammatory factors,increased oxidative stress response and increased content of collagen,inflammatory reactive protein and myocardial fibrosis protein.And there was a trend of aggravation of related lesions.Part Ⅳ:The degree of cardiac hypertrophy,the expression levels of ANP,BNP,TNF-α,IL-1β and IL-6 genes,the activity of AKT/GSK3β signaling pathway,the oxidative stress level and antioxidant capacity of SRXN1 overexpressed rats were significantly lower than those of the unoverexpressed rats after ISO induction.There was no significant difference between the ISO-induced control adenovirus injection group and the ISO-induced normal saline group.Conclusions:SRXN1 decreased in ISO-induced myocardial hypertrophy in vitro and in vivo due to oxidative reactions.Overexpression of SRXN1 protein in rat cardiomyocytes in vitro and in vivo alleviates ISO-induced myocardial hypertrophy,myocardial inflammatory response,and myocardial fibrosis by reducing myocardial oxidative stress response and regulating AKT/GSK3β signaling pathway.