The Protective Effect And Mechanism Of PNaKtide On Myocardial Cell Ischemia/Reperfusion Injury

Coronary heart disease(CHD)is currently one of the leading causes of human death.Every year,millions of people die from CHD.With the development of medicine,considerable progress has been made in the treatment of CHD,especially acute myocardial infarction(AMI).At present,reperfusion therapy at the myocardial level,including thrombolysis,PCI,and CABG,is very mature.However,in the course of reperfusion therapy,infarct size often does not decrease along with the restoration of blood flow,and even leading to enlarged infarct size,reperfusion arrhythmia,and myocardial stunning.That is the ischemia-reperfusion injury we usually talk about.The reason of myocardial ischemia-reperfusion injury is not known yet.It is generally believed that in the process of ischemia-reperfusion,oxidative stress caused by the production of a large number of free radicals and reactive oxygen species is a major factor in the occurrence of ischemia-reperfusion injury.Preventing and alleviating the myocardial ischemia-reperfusion injury can improve the clinical efficacy of myocardial infarction.Many experiments have been conducted in the prevention and treatment of myocardial ischemia and reperfusion injury,but these are often unable to be used clinically due to various reasons.Until now,there is no therapeutic drug for ischemic reperfusion injury.In this paper,a series of studies using antioxidant p Na Ktide were carried out,hoping to provide new ideas for clinical prevention and treatment of myocardial ischemia-reperfusion injury.ObjectivesOxidative stress plays an important role in myocardial ischemia-reperfusion(I/R)injury.At present,studies have confirmed that p Na Ktide,known as an antioxidants,can inhibit Na/K-ATPase/Src/ reactive oxygen species(ROS)signaling,and has been proved to have a good therapeutic effect in cell and animal models in diseases such as obesity,fatty liver,atherosclerosis and tumors.Now,a large number of studies have shown that abnormal accumulation of ROS plays an important role in the process of myocardial ischemia-reperfusion injury,the elimination of ROS provides an effective relief of myocardial ischemia-reperfusion injury.Therefore,p Na Ktide,a peptide inhibits the generation ROS,whether also has the effect of alleviating myocardial ischemia-reperfusion injury,became our research target.This study aimed to explore the role and possible mechanism of p Na Ktide in myocardial ischemia-reperfusion injury,and to provided a new clue of the treatment of ischemic heart disease.Methods1.We established models separately for H9C2 cell physical hypoxia model,cobalt chloride-induced chemical oxidative stress injury model and rat myocardial infarction model.2.To explore the concentration gradient of p Na Ktide in the physical hypoxia model of H9C2 cells.Experiment grouping: a: control group;b: control + p Na Ktide 50 u M;c: hypoxia group;d: hypoxia group + p Na Ktide 10 u M;e: hypoxia group + p Na Ktide 20 u M;f: hypoxia group + p Na Ktide 50 u M.In the p Na Ktide experimental group,p Na Ktide was added 2 h before the experiment,and then hypoxia was performed for 12 h.After the end of hypoxia,2 h reoxygenation was performed.The optimal concentration of p Na Ktide for cytoprotection was determined by the difference of LDH release concentration in the culture supernatant and the cell death rate after trypan blue staining of H9C2 cells.3.We screened the results according to the concentration gradient and used the optimal concentration of p Na Ktide for hypoxia/reoxygenation experiments.The experiments were divided into: a: control group;b: optimal concentration of control group + p Na Ktide;c: hypoxia group;d: hypoxia group + optimal concentration of p Na Ktide.After the completion of hypoxia/reoxygenation,ROS detection was performed and TUNEL staining was used to evaluate the apoptosis.4.Proteins of H9C2 cells in each experimental group were extracted,and protein concentration was measured by BCA method.Western Blot assay was used to detect the levels of Caspase 3 and PARP in each experimental group and test the relationship with p Na Ktide concentration.5.Cobalt chloride was used to induce H9C2 cell chemical injury model for research, experimental grouping: a: control group;b: control + p Na Ktide 50 u M;c: Cobalt chloride group;d: Cobalt chloride group + p Na Ktide 10 u M;e : Cobalt chloride group + p Na Ktide 20 u M;f: Cobalt chloride group + p Na Ktide 50 u M.In the p Na Ktide experimental group,p Na Ktide was added 2h before the experiment,and Cobalt chloride 800 u M was added for 20 h to detect.We conducted the cell death after trypan blue staining of H9C2 cells.6.To investigate the effect of p Na Ktide on H9C2 cell cycle and cell proliferation.The experimental groups were: a: control group;b: p Na Ktide 10 u M group;c: p Na Ktide 20 u M group;d: p Na Ktide 50 u M group.Flow cytometry was used to detect cell cycle after PI staining.H9C2 cells were cultured for 24 h,48h and 72 h respectively,and cell proliferation was detected using CCK8 kit.7.Vivo experiments in rat for p Na Ktide,Experimental grouping: a.Sham group: After thoracotomy,the silk thread passes through the left anterior descending coronary artery,but no ligation;b.Myocardial infarction group;c.Myocardial infarction + scramble peptide group;d.Myocardial infarction + p Na Ktide group.In the peptidomimetic experimental group,the polypeptide was injected from the tail vein of the rat before being ligated for 30 minutes before the ligation of the anterior descending artery.After ligation for 40 minutes,the ligature was released for 2 hours and then the reperfusion was performed.The whole blood was then taken for LDH measurement and heart tissue H&E staining and pathological scoring;8.Mechanism of p Na Ktide on cytoprotection mechanism for H9C2.Experimental grouping: a: control group,b: hypoxia group,c: hypoxia + p Na Ktide 10 u M group,d: hypoxia + p Na Ktide 20 u M group,e: hypoxia + p Na Ktide 50 u M group.Western Blot was used to detect the relationship between Src,Src p Y418,ERK1/2 and P-ERK1/2 levels and p Na Ktide concentration in each group of cells,and explore the role of p Na Ktide in Src and ERK1/2 signaling pathways.Result:1.Before hypoxia,H9C2 cells were treated with p Na Ktide 10 u M,20 u M,and 50 u M,the experimental results showed that with the increase of p Na Ktide concentration,LDH release concentration and trypan blue staining detected H9C2 cell death rate gradually decreased.The concentration-dependent relationship with p Na Ktide was most obvious when p Na Ktide concentration was 50 u M.2.Physical hypoxia experiment was performed with p Na Ktide concentration of 50 u M.Under hypoxia,active oxygen production increased significantly and fluorescence intensity increased significantly.After p Na Ktide was added,the fluorescence intensity was significantly reduced.That is,active oxygen production was inhibited.TUNEL staining showed that apoptotic cells increased significantly after hypoxia,and the number of apoptotic cells decreased significantly after adding p Na Ktide.3.Western Blot experiments found that as the concentration of p Na Ktide increased,the levels of caspase 3 and PARP gradually decreased.p Na Ktide inhibited the activation of apoptosis protein in a concentration-dependent manner.4.The experiment of chemical injury of H9C2 cells induced by cobalt chloride showed that after the p Na Ktide 10 u M,20 u M and 50 u M were added before chemical injury,the death rate of H9C2 cells gradually decreased with the increase of p Na Ktide concentration,in a concentration-dependent manner with p Na Ktide.5.H9C2 cells were pretreated with p Na Ktide concentrations of 10 u M,20 u M,and 50 u M.The data showed that p Na Ktide had no effect on the proliferation and cycle of H9C2 cells.6.The rat model of myocardial infarction found that serum LDH levels increased significantly under hypoxic conditions.H&E staining of cardiac tissue revealed myocardial fibrosis and neutrophil infiltration in the infarcted area,whereas serum LDH levels decreased significantly after p Na Ktide pretreatment.At the same time,myocardial fibrosis and neutrophil infiltration were weakened,suggesting that p Na Ktide has the effect of attenuating myocardial ischemia-reperfusion injury.7.The study of myocardial protection mechanism of p Na Ktide found that the expression of Src and ERK1/2 in H9C2 cells was significantly increased under hypoxic conditions,and that after pretreatment with p Na Ktide,the level of Src and ERK1/2 were decreased with the increase of p Na Ktide concentration,showing a concentration-dependent relationship.The results suggest that p Na Ktide can inhibit the activity of Src/ERK1/2/ROS signaling pathway,reduce the production of ROS and inhibit the apoptosis of cardiomyocytes,thereby weakening myocardial cell ischemia-reperfusion injury,showing significant myocardial protection.Conclusions:In vitro and in vivo studies,we have shown that p Na Ktide can significantly inhibit hypoxia-induced cardiomyocyte injury and is a potential therapeutic drug for myocardial ischemia-reperfusion injury.