| Backround and Objective:Coronary heart disease was a common clinical disease caused by the myocardial ischemia and hypoxia.Studies had found that P2X7 receptor was involved in the transmission of nociceptive information.Purine receptors were related to the cardiovascular function and disease processes.Long noncoding RNAs(LncRNAs)are involved in the basal regulation of protein-coding genes,by affecting mRNA transcription,splicing,export,stability and translation.LncRNAs are involved in the pathogenesis of neurous system diseases.LncRNAs are abnormally expressed in some complex disease process.This study was to explore the role of P2X7 receptor in the pathophysiological function and possible mechanism of abnormal sympathetic nerve function and morphology mediated by myocardial ischemia,and the possible function of the LncRNA existed specific in peripheral ganglion.It may provide new therapeutic targets for the prevention and treatment of coronary heart disease.Methods:We used the ligation of the rat heart left anterior descending coronary artery as the myocardial ischemic model.Rats were randomly divided into the control group(Con),myocardial ischemic group(MI),sham operation group(Sham),MI rats treated with BBG group(MI+BBG),MI rats treated with P2X7 siRNA group(MI+P2X7 si),MI rats treated with mlncRNA2 siRNA group(MI+ mlncRNA2 si)and MI rats treated with Scramble siRNA group(MI+SC si).The studies were made as follows:(1)After the preparation of myocardial ischemia model,HE stain of cardiac tissue was used to observe cardiac tissue in myocardial ischemic rats.(2)Each experimental group's blood pressure and heart rate were monitored by non-invasive blood pressure determinator.(3)Detection of electrocardiogram(ECG)in the time of pre-operation and post-operation were made to analyse the heart rate variability(HRV)and record the changes of the cardiac autonomic nerve function were recorded(4)Creatine kinase(CK),creatine kinase isoform MB(CK-MB),lactate dehydrogenase(LDH)and aspartate amino transferase(AST)were measured in each group.(5)The distribution and density of nerve fibers around the ischemic myocardium were observed by immunohistochemistry.(6)The expression levels of mlncRNA2 and the P2X7 receptor mRNA in rat SCG and SG were analyzed by Real-time PCR.(7)The expression levels of P2X7 receptor immunoactivity and protein in rat SCG and SG neurons were analyzed by immunohistochemistry and western blot in each group.and the phosphorylation of P38 and ERK1/2 was tested by western blotting.(8)The immunoactivity of TNF-a and IL-6 in rat SCG and SG were detected by immunohistochemistry.(9)The concentration of TNF-a,IL-6,EPI and NA in rat serum of each group were tested by ELISA.Results:(1)The expression levels of mlncRNA2 in SCG and SG were significantly higher in MI group than those in Con group by Real-time PCR(n=3,p<0.01).(2)After the 30 days of myocardial ischemia,HE stain of cardiac tissue in myocardial ischemic rats showed the myocardial fibers appeared coagulation necrosis and the nuclear fragmentated or disappeard,the cytoplasm broke into irregular granular and the interstitial appeared edema.The normal myocardial fibers dyed pink,cytoplasm and nucleus showed clear,cell membrane was integrity,cell arrangement were rule and the intercellular space was moderate.(3)Compared with Con,Sham,MI+BBG,MI+P2X7 si,MI+mIncRNA2 si groups,the systolic blood pressure(SBP),diastolic blood pressure(DBP)and heart rate(HR)in the myocardial ischemic group were increased(n=5)(p<0.01).There were no significant difference about SBP,DBP and HR among Con,Sham,MI+BBG,MI+P2X7 si,MI+mlncRNA2 si groups(n=5)(p>0.05).It was the same results between MI group and MI+SCsi groups(n=5)(p>0.05).(4)ECG showed the elevated ST segment and abnormal Q wave in myocardial ischemic rats.The analysis of HRV showed the values of LF,HF,SDANN,RMSSD in MI group were obviously lower than those in Con,Sham,MI+BBG,MI+P2X7 si,MI+mlncRNA2si groups(n=10)(p<0.01).While the value of LF/HF in MI group was higher than that in the other five groups(n=10)(p<0.01).(5)Compared with data in Con,Sham,MI+BBG,MI+P2X7 si,MI+mlncRNA2 si groups,the concentration of CK,CK-MB,LDH and AST in blood serum of the myocardial ischemic group rats were increased(n=8)(p<0.01).(6)The results of immunohistochemistry showed that in myocardial ischemic rats the expression levels of GAP43 and TH in the cardiac nerve fibers around the ischemic myocardium were higher than those in Con group and Sham groups(n=6)(p<0.01).The values of TH/GAP43 in myocardial ischemic rats were significantly increased in comparison with those in Con group,Sham group(n=6)(p<0.01).It indicated the newborn nerve fibers were mainly the sympathetic nerves.(7)The results in immunohistochemistry and Western blot showed that there were apparent significance on the expressions of P2X7 receptor in SCG/SG neurons among MI group and Con,Sham,MI+BBG,MI+P2X7 si,MI+mlncRNA2 si groups(n=6)(p<0.01).There was no visible difference in the expression of P2X7 receptor in Con,Sham,MI+BBG,MI+P2X7 si,MI+mlncRNA2 si groups n=6)(p>0.05).The expression of P2X7 receptor in MI group was the same level as MI+SC si group(n=6)(p>0.05).(8)The TNF-a and IL-6 immunoactivity levels of SCG/SG neurons in MI group were much stronger than those in Con,Sham,MI+BBG,MI+P2X7 Si,MI+mlncRNA2 si groups(n=10)(p<0.01).There was no difference among Con,Sham,MI+BBG,MI+P2X7 si,MI+mlncRNA2 si groups(n=10)(p>0.05).It was the same results between MI and MI+SC si groups(n=10)(p>0.05).(9)The results in Western blot showed that the phosphorylation of P38 and ERK1/2 in MI group were significantly higher than those in Con,Sham,MI+BBG,MI+P2X7 si,MI+mIncRNA2 si groups(n=6)(p<0.01).No obvious difference was existed in Con,Sham,MI+BBG,MI+P2X7 si,MI+mlncRNA2 si groups(n=6)(p>0.05).It was the same results between MI and MI+SC si groups(n=6)(p>0.05).(10)The results in ELISA showed that the concentration levels of TNF-?,IL-6,EPI,NA in MI group were much greater than those in Con,Sham,MI+BBG,MI+P2X7 si,MI+mlncRNA2 si groups(n=6)(p<0.01).No difference was found among Con,Sham,MI+BBG,MI+P2X7 si,MI+mlncRNA2 si groups(n=6)(p>0.05).It was the same results between MI and MI+SC si groups(n=6)(p>0.05).Conclusions:Expression levels of P2X7 receptor in the SCG/SG and cardiac sympathetic nerves of myocardial ischemic rats were increased.Up-expression levels of P2X7 receptor in SCG/SG and cardiac sympathetic nerves in myocardial ischemic rats were decreased after treated by P2X7 receptor antagonist BBG,P2X7 siRNA,mlncRNA2 siRNA.The dominant phenomenon of myocardial sympathetic nerve can be suppressed after treated by P2X7 receptor antagonist BBG,P2X7 siRNA,mlncRNA2 siRNA.BBG,P2X7 siRNA or mlncRNA2 siRNA may inhibite the excitation transmission of cervical sympat-hetic nerves to improve the myocardial ischemic injury.P2X7 receptors in SCG/SG and cardiac sympathetic nerves was involved in the sympathoexcitatory reflex process mediated by myocardial ischemic nociceptive signal. |
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