The Role Of STEAP4 In Metformin Attenuating Myocardial Ischemia Reperfusion Injury

Objective:Acute myocardial infarction?AMI?has become one of the most serious and fatal diseases in the world.With the change of people's lifestyle,the incidence of AMI is still on the rise every year.So far,the most effective treatment is through percutaneous coronary intervention?PCI?as soon as possible to restore the blood perfusion of the heart.However,perfusion itself also brings many side effects,mainly due to the myocardial ultrastructure,energy metabolism function and electrophysiological changes caused by ischemia,which are more prominent after recanalization of blood vessels,and may even lead to severe arrhythmia and eventually sudden cardiac death.The phenomenon is known as myocardial ischemia reperfusion injury?MIRI?.The occurrence of MIRI is complex,involving a variety of mechanisms,such as the massive production of intracellular oxygen free radicals,calcium overload,the inflammatory effect of white blood cells,the lack of high-energy phosphate compounds,and cell apoptosis.In the heart after reperfusion,the number of myocardial cell survival directly affects the subsequent cardiac function and quality of life,while the apoptosis induced by MIRI seriously reduces the activity of myocardial cells,which can lead to further deterioration of cardiac function after reperfusion.Therefore,it is inevitable to focus on how to reduce the apoptosis induced by MIRI.Studies have found that cardiac ischemic preconditioning and post-conditioning can effectively improve MIRI,but the clinical operation involves ethical and moral issues,so the feasibility is not good.To explore effective drug intervention to prevent apoptosis of myocardial cells has become an important breakthrough in the prevention of MIRI.Metformin,as a derivative of metformin,is a traditional hypoglycemic drug.However,in recent years,more and more pleiotropic properties of this drug have been discovered,including anti-inflammatory,antioxidant,endothelial function protection,thrombosis reduction and myocardial apoptosis.Its anti-apoptotic effect on myocardial cells in diabetic patients with MIRI has been reported,such as the reduction of myocardial cell apoptosis by activating AMPK pathway.However,the protective effect of MIRI alone is still controversial clinically,so metformin,as a cheap,safe and widely used hypoglycemic drug,deserves further discussion on its anti-apoptosis mechanism.six-transmembrane epithelial antigen of prostate 4?STEAP4?,initially found to induce fat cell differentiation,its and promote fat cells to absorb glucose,affect the cell energy metabolism and resistance to apoptosis.Metformin has been shown to increase the expression of STEAP4 in human CD34⁺cells under hypoxia.Recently,STEAP4was found to be significantly reduced in patients with cardiac dysfunction,with a significant correlation.Therefore,we hypothesized that STEAP4 may play a role in the process of metformin protecting MIRI from apoptosis,ultimately alleviating the apoptosis of MIRI and protecting heart function.In conclusion,the purpose of this study was to investigate the effect of metformin on myocardial ischemia-reperfusion injury.SD rats and H9c2 cell lines were selected as subjects for the cost experiment,and the in vivo ischemia reperfusion injury model and hypoxia re-oxygenation model of H9c2 myocardial cells were established in SD rats to explore the following two aspects:?1?observe and analyze whether metformin preconditioning group has a protective effect on MIRI after ischemia reperfusion injury in rats;?2?observe the protective effect of metformin on hypoxia and re-oxygenation of H9c2 cells by applying RNA interference technology to low expression of STEAP4,and to explore the possible mechanism of metformin in myocardial protection.Methods:1.60 healthy male SD rats were randomly divided into 3 groups?A?Control group;2.?B?ischemia reperfusion group?I/R?;?C?ischemia/reperfusion+metformin group?I/R+metformin?.The rats in group C were treated with intraperitoneal injection of metformin into 100ul normal saline once a day 14 days before ischemia reperfusion according to the standard of 250mg/kg body weight.Group A and group B were injected with the same volume of normal saline for 14 consecutive days.Group B and group C established the ischemia reperfusion model by thoracotomy ligation of the left anterior descending coronary artery for half an hour and reperfusion for 2 hours.Rats in group A also needed thoracotomy and puncture of the left anterior descending coronary artery,but no ligation and follow-up operations were performed.2.The normal state?37?,5%CO₂?under cultivation H9c2 cells randomly divided into 6 groups:?A?the Control group?Control?;?B?hypoxia re-oxygenation group?hypoxia re-oxygenation,H/R?,with Earle's to replace normal culture medium,and cultivation in the anoxic incubator?37?,5%CO₂,94%N₂?,6 hours,then Earle's culture with normal culture medium in the normal conditions?37?and 5%CO₂?train4 hours.?C?H/R+metformin?10uM?was dissolved in the medium and added to the cell culture plate.Cells were pretreated with metformin?10uM?for 12 hours before re-oxygenation.?D?H/R+metformin?20uM?,same as the previous group.?E?H/R+metformin?40uM?,same as the previous group.?F?H/R+metformin?80uM?,same as the previous group.The cell activity of each group was detected by cck-8 assay,and bcl-2/Bax ratio of each group was detected by western blot assay.The apoptosis of each group was analyzed to determine the optimal concentration of metformin for subsequent experiments.3.The normal state?37?and 5%CO₂?under cultivation H9c2 cells randomly divided into 5 groups:?A?the Control group?Control?,without any processing;?B??hypoxia re-oxygenation,H/R?,with Earle's culture to replace normal culture medium,and cultivation in the anoxic incubator?37?and 5%CO₂,94%N₂?,6 hours,then Earle's culture with normal culture medium in the normal conditions?37?and 5%CO₂?train 4 hours.?C?H/R+metformin,metformin was dissolved in the medium and added to the cell culture plate,and cells were pretreated for 12 hours before hypoxic re-oxygenation.?D?H/R+siNC+metformin,H9c2 cells were transiently transfected with siRNA NC and transfected for treatment in group C for 24 hours.?E?H/R+siSTEAP4+metformin,H9c2 cells were transiently transfected with STEAP4siRNA and treated with group C 24 hours later.4.After the ligation of the left anterior descending coronary artery in rats,the myocardial ischemia model was successfully established by observing and analyzing the changes in ST segment of the II,so as to prepare for the subsequent cardiac reperfusion.5.After cardiac reperfusion,serum of rats in each group was collected and the levels of troponin and creatine kinase isoenzyme?CK-MB?were detected by ELISA to identify the degree of cardiac injury.6.Myocardial infarction area of SD rats after ischemia reperfusion was determined by TTC staining.7.The degree of apoptosis of cardiac myocytes is measured by terminal deoxynucleotidyl transferase-mediated dUTP Nick end labeling?TUNEL?staining.8.Hematoxylin and eosin?HE?staining was used to evaluate the myocardial pathological changes and injuries.9.Trypan blue staining was used to determine the survival rate of H9c2 cells.10.Flow cytometry was used to measure the apoptosis rate of H9c2 cells.11.Mitochondrial membrane potential of H9c2 cells was determined by rhodamine 123?Rh123?staining.12.Levels of STEAP4 mRNA expression and apoptosis-related molecules?Bcl-2,Bax?were detected by Real Time PCR.13.Western Blot was used to detect the expression of apoptosis-related molecules?Bcl2,Bax?,mitochondrial apoptosis-related molecules?Cytochrome C,Cleaved Caspase9,Cleaved Caspase3?,STEAP4 and PPAR-?in rat cardiac myocytes and H9c2 cells.Results:1.After ligation,the ST segment of lead II electrocardiogram of myocardial ischemia reperfusion rats in each group was significantly higher than that in the control group;after reperfusion,the myocardial infarction-related marker proteins CK-MB and troponin in rats were significantly higher than those in the control group.However,after the pretreatment with metformin,these indicators all decreased significantly.2.The myocardial infarction area and apoptosis rate of myocardial ischemia reperfusion rats in each group were significantly higher than those in the control group,while the myocardial infarction area and apoptosis rate of the metformin preconditioning group were significantly decreased.3.Myocardium of ischemia reperfusion rats showed pathological changes such as fibrosis,intercellular edema and inflammatory cell immersion.Metformin can improve myocardial injury and alleviate myocardial pathological changes.4.In ischemia reperfusion rats,the expression of STEAP4 was decreased compared with the normal group,while the expression of pro-apoptotic protein?Bax?was increased and the expression of anti-apoptotic protein?Bcl-2?was decreased,which was reversed after metformin pretreatment.5.In H9c2 cells,metformin was found to improve cell status at 40uM concentration.We also found that in H9c2 cells and hypoxia re-oxygenation group contrast,metformin pretreatment has improve hypoxia re-oxygenation injury situation,including improve cell survival rate,reduce the apoptosis rate,increase the Bcl-2 expression,decrease Bax levels,increase STEAP4,PPAR-?expression quantity,reduce mitochondrial apoptotic molecules?Cytochrome C,Cleaved Caspase9,Cleaved Caspase3?expression level,increase the mitochondrial membrane potential.6.when knocked at low STEAP4 protein expression after partial reversal of metformin in H9c2 cells under hypoxia re-oxygenation of protection,performance of anti-apoptotic proteins?Bcl-2?,promoting apoptosis protein expression?Bax?increased,PPAR-?levels drop,falling mitochondrial membrane potential and mitochondrial apoptosis of related molecules?Cytochrome C,Cleaved Caspase9,Cleaved Caspase3?activation.Conclusion:1.Metformin can effectively reduce the apoptosis of rat cardiac myocytes and improve MIRI.2.Metformin can improve the hypoxic re-oxygenation injury of H9c2 cells,inhibit mitochondrial apoptosis,and activate the expression of STEAP4 and PPAR-?.Therefore,the STEAP4/PPAR-?related signaling pathway may be a new explanation for metformin's protective effect on the cardiovascular system.The signaling pathway including STEAP4 may be a new target for maintaining mitochondrial function and inhibiting apoptosis.