Effect And Mechanism Of P2Y6 Receptor In Superior Cervical Ganglion On Cardiac Sympathetic Excitation Induced By Myocardial Ischemia Injury

Background:The main cause of death in patients with cardiovascular disease is myocardial ischemia.Cardiac activity is regulated by autonomic nerves.Superior cervical ganglion（SCG）and stellate ganglion send out cardiac sympathetic nerve to innervate the heart.The cardiac sympathetic nerve terminals release norepinephrine（NE）,which exerts a regulatory effect on cardiac function.When myocardial ischemia occurs,cardiac sensory afferent information is enhanced,affecting the function of sympathetic ganglion neurons in the neck,which can further strengthen cardiac sympathetic efferent nerve impulses and increase blood pressure and heart rate,aggravating myocardial ischemic injury.In recent years,the role of P2Y6receptor in the development of cardiovascular diseases has attracted much attention.P2Y6 receptors have an important role in vascular injury,vascular calcification,atherosclerotic vascular injury,and enhanced myocardial tone.This suggests that P2Y6 receptors may be a potential therapeutic target for cardiovascular system diseases.P2Y6 receptors were found to be involved in nociceptive transmission in sensory ganglia and expressed on SCG neurons,but their role and mechanism in myocardial ischemic injury are not clear.Objective:To explore the role of P2Y6 receptor in cervical sympathetic ganglia neuropathy induced by myocardial ischemia and its molecular mechanism,providing a new experimental basis for drug targets for the prevention and treatment of ischemic heart disease.Methods:Animal experimental methods:This study established a myocardial ischemia model by ligating the coronary artery of the rat heart.Fifty SD rats were randomly divided into normal control group（Ctrl）,sham operation group（Sham）,myocardial ischemia model group（MI）,myocardial ischemia model group plus negative control group（MI+NC shRNA）,and myocardial ischemia model plus P2Y6shRNA group（MI+P2Y6 shRNA）.Ten animals in each group.On the 7th day of myocardial ischemia modeling,P2Y6 shRNA and NC shRNA were injected by sublingual veins.（1）ECG and myocardial HE staining were observed to determine whether the myocardial ischemia model was successfully constructed;（2）Measure the blood pressure and heart rate of rats using animal blood pressure apparatus,and observe the differences in blood pressure and heart rate between myocardial ischemia model group and control group rats;（3）Double immunofluorescence labeling was used to detect whether P2Y6 receptor and Neu N were co-expressed in cervical sympathetic ganglion neurons;（4）To detect the cervical sympathetic ganglion P2Y6receptor by real-time quantitative PCR（Real-time PCR）;（5）Western blotting was used to detect the protein expressions of P2Y6 receptor in cervical sympathetic ganglia,inflammatory factor interleukin 1β（IL-1β）,tumor necrosis factor-α（TNF-α）,and pp65 and p65 in NF-κB signaling pathway.（6）Enzyme linked immunosorbent assay（Elisa）was used to detect the release of norepinephrine in the serum of model rats.Cellular experiment methods:PC12 cells were cultured by chemical oxygen-glucose deprivation（OGD）.After the OGD model was prepared successfully,PC12 cells were randomly divided into four groups as follows:normal control group（Ctrl）,oxygen-glucose deprivation model group（OGD）,oxygen-glucose deprivation model plus P2Y6 agonist UDP（OGD+UDP）,oxyglucose deprivation model plus P2Y6 inhibitor MRS2578（OGD+MRS2578）.Experiments:（1）Na₂O₄S₂concentration and time screening:the effect of different concentration of Na₂O₄S₂（1,5,10,30,100 mmo/L）and different time（0,4,12,24h）on cell survival was detected by CCK-8 method,and the most suitable modeling conditions for oxygen-glucose deprivation were selected by the cell morphology under different conditions;（2）The m RNA level of P2Y6 receptor in PC12 cells of each group was detected by real-time quantitative PCR;（3）The protein expression levels of P2Y6 receptor,IL-1β,TNF-α,pp65 and p65 in PC12 cells of each group were detected by western blotting.（4）Enzyme linked immunosorbent assay（Elisa）was used to detect the release quantity of inflammatory factor IL-1βin the supernatant of PC12 cell culture.Results:The results of animal experiments:（1）ECG and HE staining results showed that the ST segment of ECG in MI group was significantly elevated,and cardiomyocyte arrangement were disordered,while there was no change in control group,suggesting that the model of myocardial ischemia in rats was successfully built.（2）The results of blood pressure and heart rate showed that the blood pressure and heart rate of MI rats were higher than those of Ctrl group（P<0.01）,while the application of P2Y6 shRNA could inhibit the increase of blood pressure and heart rate induced by MI（P<0.01）,while NC shRNA had no inhibitory effect（P>0.05）.（3）The results of cardiac sympathetic nerve discharge showed that the amplitude of cardiac sympathetic nerve discharge was significantly enhanced（P<0.001）and the discharge frequency was accelerated in the MI group compared with the Ctrl group,while P2Y6shRNA reduced the amplitude and frequency of cardiac sympathetic nerve discharge in the MI group（P<0.001）,and there was no significant difference between the MI+NC shRNA group and the MI group（P>0.05）.（4）NE level in serum of rats was measured by enzyme immunosorbent assay.The results showed that the NE level in MI group was significantly higher than that in control group（P<0.05）,and P2Y6shRNA significantly decreased the NE level in MI rats（P<0.05）.（5）Immunofluorescence results showed that the co-expression levels of P2Y6 receptor and Neu N in SCG were significantly enhanced in the MI group compared with the Ctrl group（P<0.01）;the co-expression levels were reduced in the MI+P2Y6 shRNA group compared with the MI group（P<0.01）;there was no significant difference in the MI+NC shRNA group compared with the MI group（P>0.05）.（6）Real-time quantitative PCR results showed that the P2Y6 m RNA level in the SCG in the MI group was significantly higher than that in the Ctrl group（P<0.001）,and P2Y6shRNA significantly reduced the level of P2Y6 m RNA in the MI group（P<0.001）,while NC shRNA did not have this reduction effect（P>0.05）.（7）The protein blotting results showed that the expression levels of P2Y6 receptor,IL-1β,TNF-α,pp65 and p65 proteins were significantly increased in the SCG of rats in the MI group compared with the Ctrl group（P<0.01）,and P2Y6 shRNA significantly decreased the expression levels of the above proteins in the MI group（P<0.01）,while NC shRNA did not have such a decreasing effect（P>0.05）.Results of cell experiments:（1）CCK-8 results showed that the cell viability of PC12 cells in the 6 h treatment group decreased to below 50%compared with the 0 h treatment group（P<0.001）,and the cell viability of PC12 cells in the 12 and 24 h treatment groups did not differ from the 6 h treatment group;compared with the Ctrl group,the cell viability of 1 mmol/L Na₂O₄S₂ treatment was around 80%（P<0.05）,the cell viability of the 5 mmol/L concentration treatment group decreased to less than50%（P<0.001）,and the cell viability of PC12 cells treated with 10,30 and 100mmol/L Na₂O₄S₂ concentrations did not change significantly from the 5 mmol/L concentration treatment group（P>0.05）;Under the light inverted microscope,we observed the morphology of the cells treated with 5 mmol/L concentration at different times.The OGD 24 h cells basically showed wrinkling,complete disappearance of synapses and rounding of cytosol.Therefore,we selected 5 mmol/L Na₂O₄S₂treatment of PC12 cells for 24 h as the most suitable modeling condition.（2）Real-time PCR results showed that P2Y6 m RNA levels were significantly higher in the OGD group compared with the Ctrl group（P<0.01）,significantly lower in the OGD+MRS2578 group compared with the MI group（P<0.01）,while significantly higher in the MI+UDP group compared with the OGD group（P<0.01）.（3）The western blotting results showed that the proteins expression levels of P2Y6 receptor,IL-1β,TNF-α,pp65 and p65 were significantly increased in the OGD group compared with the Ctrl group,and the above proteins levels were significantly lower in the OGD+MRS2578 group compared with the OGD group,while significantly increased in the OGD+UDP group compared with the OGD group.（4）Elisa assay results showed that the IL-1βcontent in the supernatant of PC12 cells in the OGD+UDP group increased compared with the OGD group,and the difference was statistically significant（P<0.001）;while the IL-1βcontent in the supernatant of cells in the OGD+UDP+P65 inhibitor group decreased significantly compared with the OGD+UDP group（P<0.01）.Conclusion:P2Y6 receptor in the superior cervical ganglion neurons are involved in cardiac sympathetic neuropathy due to myocardial ischemia,and the molecular mechanisms involving the NF-κB pathway and the release of inflammatory factors IL-1βand TNF-α.