The Effects And Mechanisms Of ALDH2 In The Senescence Of Fibroblasts Induced By Ischemia

Stress-induced cellular senescence is crucial in causing acute and chronic dysfunction in multiple organs due to ischemic injury.Studies have shown that fibroblasts are more resistant to ischemic injury compared to cardiomyocytes.Senescent fibroblasts have been identified in the infarct and marginal areas of mouse myocardial tissue after MI.It is suggested that myocardial fibroblasts are the main type of senescent cells in ischemic myocardial tissue.Therefore,regulating ischemiainduced fibroblast senescence may play a key role in the occurrence and development of myocardial injury and its repair.However,the molecular mechanism of fibroblast senescence induced by myocardial ischemia has not been fully understood.Ischemic injury decreased the activity of mitochondrial aldehyde dehydrogenase 2family member(ALDH2).ALDH2 is a glucose metabolic enzyme and aldehyde dehydrogenase located in mitochondria,which is expressed in large quantities in fibroblasts of various tissues.ALDH2 has been found to play a role in regulating the occurrence and development of ischemic tissue injury by decreasing the accumulation of 4-hydroxynonenal(4-HNE)in the tissues.4-HNE is the main product of lipid peroxidation caused by reactive oxygen species(ROS).Currently,4-HNE is considered to be a toxic substance and plays a significant role in cell signaling and fate regulation.It has been confirmed that ALDH2 is involved in regulating the senescence of endothelial cells and vascular smooth muscle cells.Activating ALDH2 can inhibit stress-induced cell senescence.However,it is not known whether ALDH2 can regulate fibroblast senescence after ischemia through 4-HNE.In this study,we used the mouse myocardial infarction model and the Oxygenglucose deprivation(OGD)cell model to induce cell senescence under stress conditions.We observed the dynamic changes of fibroblast senescence induced by ischemia.The aim was to investigate how ALDH2 regulates ischemia-induced fibroblast senescence based on 4-HNE,and how to activate ALDH2 to inhibit fibroblast senescence and improve myocardial ischemic injury.This research provides new ideas and a theoretical basis for identifying drug targets to intervene in ischemic diseases.Experimental ObjectiveThe aim is to observe the effect of ischemia on stress senescence of fibroblasts.To explore the role and mechanism of ALDH2 in ischemia-induced aging of fibroblasts and to provide a theoretical and experimental basis for finding drug targets to interfere with the aging of myocardial fibroblasts after ischemia and new strategies to improve ischemic myocardial injury..Experimental methods1.The MI model was established by ligating the left anterior descending branch of the mouse coronary artery.The expression of senescence marker protein in myocardial tissue was detected using the Western Blot method.Co-localization of senescent cells and fibroblasts was observed using the immunofluorescence double staining method to verify the effect of MI on the senescence of myocardial fibroblasts.The OGD model of mouse NIH3T3 fibroblasts was established,and the markers of cell senescence were detected using Western Blot,immunofluorescence,β-Galactosidase(β-gal)staining,activity detection,and RT-q PCR to investigate the effect of OGD on mouse fibroblast senescence.2.The key genes of fibroblast senescence induced by MI and OGD were screened,and the screening results were verified.The specific inhibitor CVT-10216 of the key gene ALDH2 and the specific activator Alda-1 were used to determine the regulatory effect of ALDH2 on fibroblast senescence after OGD.Confocal microscopy was used to observe mitochondrial morphology,and the Western Blot method was used to detect mitochondrial fusion and fission protein expression.JC-1 staining was used to detect mitochondrial membrane potential,while ATP content and ROS content were detected by a kit to clarify the effect of ALDH2 on mitochondrial damage in fibroblasts after OGD.The regulatory effect of ALDH2 based on 4-HNE on fibroblast senescence after OGD was determined by exogenous 4-HNE supplementation.3.Protein interaction omics were used to screen the interacting proteins of ALDH2 after OGD.Co-IP,molecular docking,Western Blot,and immunofluorescence were used to verify the results of the protein interaction omics analysis.After si RNA interference with interacting proteins,DSS cross-linking and other methods were used to explore the effect of ALDH2 interacting proteins on their enzyme activity and its specific mechanism.Western Blot,β-gal staining,activity detection,RT-q PCR,and other methods were used to clarify the regulatory role of ALDH2 interacting proteins on aging fibroblasts after OGD.4.Neonatal mouse cardiac fibroblasts were isolated in vitro.The study examined the effects of ALDH2 activation on senescence and 4-HNE content of cardiac fibroblasts after OGD using Western Blot,β-gal staining,activity detection,and RT-q PCR.A coculture system for mouse cardiac fibroblasts and cardiomyocytes was established to assess the effect of senescence of cardiac fibroblasts after ALDH2 activation and OGD inhibition on co-cultured cardiomyocytes using CCK-8 cell viability assay and TUNEL staining.MI mice were administered the ALDH2 activator Alda-1,and myocardial tissue was collected 3 days after MI to determine the effect of activating ALDH2 on the aging of myocardial fibroblasts using immunofluorescence double staining.Echocardiography,TTC staining,and H&E staining were used to assess the effects of ALDH2 activation on cardiac function,myocardial infarction size,and myocardial tissue injury in mice.Experimental results1.Influence of ischemia on fibroblast senescenceThe results of differential expressed genes(DEGs)enrichment analysis in a mouse myocardial fibroblast dataset(GSE111059)showed that DEGs were enriched in the cell senescence pathway after myocardial infarction(MI)for 3 days(p<0.05).The detection results of cell senescence markers showed that the expression of cell senescence marker was significantly increased after 3 days of MI and 4 hours of OGD(p<0.05),which was the critical time point for inducing fibroblast senescence.2.The mechanism of ALDH2 in fibroblast senescence induced by ischemiaThe integrated analysis of differential protein data of myocardial fibroblast DEGs 3days after MI and NIH3T3 4 hours after OGD showed that ALDH2 might be the key target of ischemic-induced fibroblast senescence.The OGD model of NIH3T3 cells showed that the protein expression level of ALDH2 was significantly increased after OGD(p<0.05),and the enzyme activity was significantly decreased(p<0.05).Activation of ALDH2 could significantly inhibit the expression of cell senescence markers(p < 0.05).ALDH2 had no significant effect on the rate of glycolysis after OGD4 h(p>0.05)but could significantly reduce the content of 4-HNE in cells after OGD 4h(p<0.05).In vitro supplementation of different concentrations of 4-HNE showed that1-10 μM of 4-HNE could significantly induce senescence of NIH3T3 cells(p<0.05).These results indicate that ALDH2 regulates fibroblast senescence after OGD based on4-HNE.3.The interaction between HSPA8 and ALDH2 induced fibroblast senescence under ischemic conditionThe OGD model of NIH3T3 cells revealed that OGD can lead to an interaction between HSPA8 and ALDH2.Treatment with OGD resulted in the partial translocation of HSPA8 protein to the mitochondria(p<0.05).Inhibiting the expression of HSPA8 using si RNA increased the expression of ALDH2 tetramer(p<0.05)and the activity of ALDH2 enzyme(p<0.05)after OGD.Additionally,inhibiting HSPA8 significantly prevented senescence of NIH3T3 cells after OGD(p<0.05).4.Mechanism by which ALDH2 regulates myocardial fibroblast senescence and influences ischemic myocardiumThe OGD model of neonatal cardiac primary fibroblasts showed that activating ALDH2 could significantly inhibit the senescence of cardiac fibroblasts 4 hours after OGD(p<0.05),and the content of 4-HNE in the cells was significantly decreased(p<0.05).It was found that activating ALDH2 in myocytes significantly improved the survival rate of myocytes after OGD(p<0.05)and reduced the apoptosis of myocytes(p<0.01).In the mouse MI model,it was found that the administration of ALDH2 activator Alda-1 could significantly inhibit the aging of fibroblasts in myocardial tissue after 3 days of MI(p<0.01),improve the cardiac function and myocardial tissue damage in mice after MI,and reduce the myocardial infarction size(p<0.001).Experiment conclusion1.ALDH2 can reduce the content of 4-HNE in fibroblasts and improve the cellular senescence induced by ischemia.2.HSPA8 interacts with ALDH2,inhibits ALDH2 enzyme activity,and induces fibroblast senescence after OGD.3.Activation of ALDH2 can inhibit the senescence of myocardial fibroblasts induced by ischemia and improve the cardiac function of myocardial infarction mice.