Succinate Receptor GPR91 As A Target For Prevention And Treatment Of Myocardial Ischemia-and Hypoxia-induced Injury

In recent years,the incidence of cardiovascular and cerebrovascular diseases is on the rise.According to the statistics of the world health organization(WHO)in 2012,the annual number of deaths due to cardiovascular diseases is as high as 17.4 million,becoming the first killer threatening human health.Myocardial ischemia and hypoxia are more common in coronary blood insufficiency or plateau hypoxia environment.The above two conditions often induce the injury of myocardial tissue.Therefore,the research on the prevention and treatment of myocardial ischemia and hypoxic heart disease has become an important part in the field of cardiovascular disease.G protein coupled receptor(GPCR)is a kind of protein located on the cell membrane with seven transmembrane domains,and is one of the most important drug targets in the body.There are many kinds of GPCR ligands.However,it was surprised that the intermediate products of glucose metabolism were identified as the ligands of GPCR.Succinic acid is an intermediate product of the tricarboxylic acid cycle.Studies have shown that it is a specific ligand of G protein-coupled receptor 91,which was often called as an orphan receptor before its ligand was pared.It has important physiological functions and pathological significance,which also implies succinic acid more important roles.It has been suggested that the increase of blood pressure in hepatic blood insufficiency is related to the increase of blood concentration of succinic acid,which is also involved in the regulation of renin release and leads to the increase of blood pressure.The latest results of the study also found a large number of GPR91 expression in heart tissue,suggesting that GPR91 is probably closely related to heart function.Based on the above research background,the following two parts of experiments have been carried out in this research.1)In the first part,using the rat myocardial ischemia model caused by coronary artery ligation and the rat myocardial hypoxia model induced by simulation of the plateau environment,the changes of GPR91 mRNA and protein expression and the pathological changes of rat myocardial tissue were detected.2)In the second part,with the primary culture of new borned-rat myocardial cells under hypoxia condition,the cells survival state and the protein or phosphorylated protein expressions of some key molecules,including BNP,PI3 K,and Akt were detected with or without intracellular GPR91 siRNA interference,in order to verify whether GPR91 involved in the hypoxia-induced pathological processes of myocardial cells.Part 1: Rat myocardial ischemia and hypoxia models were prepared according to the literatures.The former was induced by coronary artery ligation and the latter was induced by low air pression equipment.In details,myocardial ischemia model of rats were prepared by ligattion with the anterior descending branch of the left coronary artery,while those in the sham operation group were only ligated with the anterior descending branch of the left coronary artery.Hypoxia model of rats were prepared by letting rats live in the condition of low air pression for 1,3,5 and 7 days.GPR91 mRNA and protein expression levels in rat cardiac myocytes were detected by PCR and Western blotting.The results showed that after the scheduled time of ischemia,compared with the sham operation group,the mRNA and protein levels in the myocardium of rats in the 12 h and 24 h groups were up-regulated(P<0.01,P<0.05).The results showed that compared with the normal pressure group,the mRNA expression level of GPR91 in rat cardiac myocytes at 1d,3d,5d and 7d of the depression group was significantly increased(P<0.05),and the protein expression level was also significantly increased after 7d(P<0.05).By analyzing the pathological sections of myocardial tissue,the myocardial cells in the low-pressure 1d group showed degeneration and swelling,inflammatory cell infiltration could be seen on 3d and 5d,and myocardial degeneration,swelling and fracture could be seen after 7d,with unclear nuclei.The results indicated that GPR91 was involved in the pathological process of myocardial ischemic injury.Part 2: Primary culture of rat myocardial cells was obtained using the established procedure.In order to observe the influence of hypoxia and the roles of GPR91 on the primary culture of rat myocardial cells,the cells were treated with the low oxygen environment,and then interferenced with GPR91 siRNA technology.In our experiment,compared with that of the control group,GPR91 reduction in mRNA and protein expressions in myocardial cells,the increasement of myocardial cells survival rate(P<0.05),the down-regulation of BNP protein and PI3 K and Akt phosphorylated protein were detected(vs normal pressure group,P<0.05).These results suggest that GPR91 may be involved in the pathological process of myocardial ischemic injury by regulating the PI3K/Akt pathway.In summary,it has been proved that GPR91 was involved in the pathological processes of ischemic and hypoxic myocardial injury based on investigations in the animal and the cells models,and might be realized by regulating the phosphorylation of PI3 K and Akt proteins in its signal transduction pathway.This study is not only significant both in theory and practice for the prevention and treatment of myocardial diseases,especially caused under the conditions of insufficient blood supply or hypoxia,but also provides a basis for the research and development of drugs targeted to GPR91.