Study On Anti-MIRI Of Salvianolic Acid B And Ginsenoside Re With Their Compatibility Mechanism Based On HiPSC-CMs

Myocardial ischemia-reperfusion injury(MIRI)refers to the pathological process of tissue damage aggravation after the ischemic heart recovers blood perfusion within a certain period of time.It involves a variety of mechanisms that will lead to abnormal mechanical activity of the heart,such as oxidative stress,autophagy,inflammatory response,mitochondrial dysfunction and calcium overload.Currently,there is still a lack of effective drugs for clinical treatment.Human cardiomyocytes(hiPSC-CMs)with the same genetic background and biological characteristics as those of human heart are cardiomyocytes directionally differentiated after reprogramming from human cells,which refer to cardiomyocytes derived from human induced pluripotent stem cells in full.They are excellent biological cell models for studying the myocardial activity of drugs.The real time xCELLigence analysis system(RTCA)with realtime,continuous,unlabeled and injury-free monitoring cells and recording and analyzing the characteristics of cell physiological activity is used to measure the contraction-relaxation and field potential signals of myocardial cells,so as to obtain the physiological activity changes of myocardial cells and reflect the mechanical activity of the heart.Guanxin Danshen Formulation is an effective compound for the treatment of coronary heart disease with Salviae miltiorrhizae and Notoginseng as the sovereign and ministerial drugs,respectively.Studies have shown that it has a myocardial protection effect,and can treat left ventricular remodeling caused by MIRI.Besides,one of the symptoms of coronary heart disease is myocardial ischemia caused by coronary artery stenosis,suggesting that Guanxin Danshen Formulation may have certain efficacy in the treatment of MIRI.In addition,our research group has confirmed in the previous studies that the combination of salvianolic acid B in Salviae miltiorrhizae and ginsenoside Re in Notoginseng can effectively prevent the apoptosis of endothelial cells induced by oxidized low density lipoprotein through the anti-oxidative and anti-inflammatory mechanisms,and that salvianolic acid B at 60μg/mL and ginsenoside Re at 120μg/mL have the optimal synergistic protection.Therefore,in this study,salvianolic acid B,an active ingredient of Salviae miltiorrhizae in Guanxin Danshen Formulation,and ginsenoside Re,an active ingredient of Notoginseng were used alone or in combination to improve the MIRI effect,using the research strategy of optimizing Chinese herbal compound to provide a theoretical basis for the research and development of the role of Guanxin Danshen Formulation in the clinical treatment of MIRI.HiPSC-CMs was used as the model for in vitro experiments.The myocardial injury model of hiPSC-CMs was established by hypoxia for 6 hours and reoxygenation for 24 hours.Five dosage groups of salvianolic acid B(7.5,15,30,60 and 120μg/mL)and five dosage groups of ginsenoside Re(7.5,15,30,60 and 120μ/mL)were selected to study the effects of the two on the impedance and field potential of hiPSC-CMs and its myocardial injury model.Based on the results of this part of the study and the previous research results of the research group,salvianolic acid B(15,30,and 60μg/mL)and ginsenoside Re(30,60,and 120μg/mL)were combined in three different doses into nine groups to explore the effects of the combination of salvianolic acid B and ginsenoside Re on the impedance and field potential of hiPSC-CMs and its myocardial injury model.Then SD rats were selected for animal experiments and divided into sham operation group.Myocardial ischemia reperfusion model group;Salvianolic acid B low dose group(15mg/kg),medium dose group(30mg/kg),and high dose group(60mg/kg);Ginsenoside Re low dose group(15mg/kg),medium dose group(30mg/kg),and high dose group(60mg/kg);The MIRI models of rats in the salvianolic acid B(60mg/kg)plus ginsenoside Re(60mg/kg)group and the positive control propranolol(2.5mg/kg)group were established by ligation of the left anterior descending coronary artery(ischemia for 30 minutes and reperfusion for 24 hours)in the model group and the rats in the administration groups after four days of intragastric administration.The TTC staining method was used to observe the ischemic area of the rat heart,and HE staining method was used to observe the pathological morphology of the myocardial structure.The activity of serum myocardial enzymes was determined by automatic biochemical analyzer.The effects of salvianolic acid B,ginsenoside Re and their combination on the protection of MIRI were explored.The kit was used to detect the possible antioxidant mechanism during the protection of MIRI by salvianolic acid B and salvianolic acid B combined with ginsenoside Re.The L-type channel agonist bay-k-864420nM was added into hiPSC-CMs to establish an intracellular calcium overload model,to study the effect of ginsenoside Re on protecting hiPSC-CMs against calcium overload,and the Langendorff isolated heart perfusion technique was used to study the effects of ginsenoside Re on the contraction function of primary myocardial cells and calcium transient in rats with hypoxic-reperfusion injury,and to explore the possible mechanism of ginsenoside Re in regulating calcium channels in myocardial cells in order to alleviate the protective effect of MIRI on the heart.In order to verify the experimental results,the expressions of calcium channel related proteins(SERCA,RyR2,and α1C)in the gene were detected by PCR.The results showed that salvianolic acid B,ginsenoside Re and their combination could enhance the contractility of hiPSC-CMs,alleviate myocardial damage and improve field potential function.Salvianolic acid B,ginsenoside Re and their combination could effectively reduce the myocardial ischemic area of the rat MIRI model,improve myocardial structural damage,reduce the activity of myocardial enzymes,and protect the heart.Salvianolic acid B and salvianolic acid B together with ginsenoside Re could reduce the malondialdehyde(MDA)content in serum of MIRI rats,and enhance the superoxide dismutase(SOD)activity and reduced glutathione(GSH)activity,which played an antioxidant role in the process of improving myocardial injury.Ginsenoside Re has the function of improving the damaged cell contraction and calcium transient of primary myocardial cells in rats with hypoxia-reperfusion injury.By increasing SERCA and reducing the expression of RyR2 and α1C proteins,it plays a role in antagonizing calcium overload in myocardial cells and regulating calcium channel in myocardial cells to reduce MIRI.In summary,the mechanism of salvianolic acid B reducing MIRI may be that it reduces MDA level,improves SOD activity and GSH activity,and enhances the myocardial antioxidant capacity to protect the heart.The mechanism of ginsenoside Re in reducing the protection of the heart by MIRI may be related to its efficacy in regulating the calcium channel in myocardial cells,increasing the expression of SERCA protein,reducing the expression of RyR2 and α1C proteins,antagonizing intracellular calcium overload,and maintaining cellular calcium homeostasis.The mechanism of cardioprotection by the combination of salvianolic acid B and ginsenoside Re may be that MIRI protects the heart by reducing the antioxidant mechanism of myocardial oxidative stress,such as MDA level,improving SOD activity and GSH activity.