| Objective:The purpose of this study was to explore the protective effects of electroacupuncture on myocardial ischemia model rats and the effects of electroacupuncture serum on H9C2 cells,by investigating the effects of the expression of hyperpolarization activated cyclicnucleotide 2(HCN2)protein and m RNA in H9C2 cells after electroacupuncture at Shenmen point at different time periods in rats with myocardial ischemia,so as to provide experimental support for the further study of the effective substances of electroacupuncture serum.Methods:90 healthy male SD rats were reared under the same conditions,and the electrocardiogram of the rats was recorded and analyzed by the Powerlab 8-lead physiological recording system.The rat myocardial ischemia model was replicated by ligation of the left anterior descending branch of coronary artery.The rats with abnormal ECG and the failure of model replication were eliminated.The rats,that were successfully replicated the rat model,were randomly divided into normal group,model group,electroacupuncture 1d group,electroacupuncture 3d group,electroacupuncture 5d group and electroacupuncture 7d group.Each group finally included 6 rats.From the next day,all electroacupuncture group rats was treated with electroacupuncture on both sides of the "Shenmen" acupoints,with a frequency of 2HZ,and the current intensity is measured by the slight shaking of rat paws,15 minutes at a time,once a day.Setting the number of consecutive electroacupuncture days according to the different electroacupuncture treatment groups.After the last electroacupuncture treatment,rat myocardium and hippocampus tissues and abdominal aorticblood were collected,centrifuged at 4 ℃ and3500 rpm for 20 minutes,and the supernatant was packed at 100 μl,and the rest of the serum was inactivated 30 min in a 56℃ water bath,use.22 μm microporous membrane to remove bacteria in the ultra-clean worktable.Enzyme-linked immunosorbent assay(ELISA)was used to detect serum cTn-I and cTn-T levels,as well as the concentration of cAMP and cGMP in serum and myocardium;Rat cardiomyocyte line H9C2(2-1)cells were cultured in microbial incubator,at the condition of saturated humidity,5%CO2 and 37℃,with Medium 199/EBSS medium containing 10% fetal bovine serum(FBS)and 100U/ml penicillin and 100ug/ml streptomycin.The growing H9C2 cells were inoculated at a certain density,abandoned the original growth medium,and cultured in the medium containing 10%electroacupuncture serum for 24 hours.Total protein and total RNA were collected.Western blotting(WB)was used to detect the expression of HCN2 protein in H9C2 cells,and Reverse Transcriptase Polymerase Chain Reaction(RT-PCR)was used to detect the expression of HCN2 m RNA in H9C2 cells.Results:1 The effects of electroacupuncture in different periods:1.1 ECG results show1)HR of rats with different duration of electroacupuncture showed different trends before and after the replication of AMI model.Compared with that before model replication,the HR of rats in each group decreased after model replication,but the difference was not statistically significant(p>0.05),and the difference was not statistically significant,although the HR has increased after electroacupuncture treatment(p>0.05).2)Compared with before model replication,the ST Height in electroacupuncture groups at different time stages increased significantly after model replication,with statistical significance(p<0.01);Compared with after model replication,the ST Height decreased significantly after electroacupuncture treatment at different time intervals,and the difference was statistically significant(p <0.01).3)The T amplitude of rats with different time course of electroacupuncture showed different changes before and after MI model replication and after electroacupuncture treatment.After model replication,T amplitude of rats were elevated or inverted,indicating that the model was replicated successfully.After electroacupuncture treatment of different time periods,the elevated or inverted T amplitude tended to be normal,indicating that different time periods of electroacupuncture treatment had different therapeuticeffects.1.2 ELISA results show1)Compared with the normal group,the contents of cTn-I and cTn-T in serum of the model group were significantly increased,and the difference was statistically significant(p<0.01).Compared with the model group,the serum cTn-T content in electroacupuncture at different time courses groups was significantly reduced,and the difference was statistically significant(p<0.01 or p<0.05).The content of cTn-I in the electroacupuncture 1d group was significantly reduced,and the difference was statistically significant(p<0.05).Compared with the 1d acupuncture group,the cTnT content of the 5d electroacupuncture group and the cTn-I content of the 3d electroacupuncture group were higher,and the difference was statistically significant(p<0.01 or p<0.05).Compared with the 3d electroacupuncture group,the content of cTn-T was higher in the 5d and 7d electroacupuncture groups,and the content of cTnI was lower in the 7d electroacupuncture group,the difference was statistically significant(p<0.01 or p<0.05).2)Compared with the normal group,the content of cAMP in serum was significantly decreased,while the content of cAMP in the myocardial tissue of the model group was significantly increased,and the difference was statistically significant(p< 0.01).Compared with the model group,cAMP content of electroacupuncture 1d group in serum and myocardium was decreased,but the difference was not statistically significant(p>0.05);the content of serum cAMP was increased in electroacupuncture3 d group while decreased in myocardium,and the difference was statistically significant(p<0.01),but there was no statistical significance in the changes of cAMP content in serum and myocardium(p>0.05);serum cAMP level in 7d electroacupuncture group was increased,and the difference was statistically significant(p<0.01),but the change of cAMP content in myocardium was not statistically significant(p>0.05).Compared with the electroacupuncture 1d group,the serum cAMP content of electroacupuncture 3d and 7d groups was higher,the difference was statistically significant(p<0.01).Compared with electroacupuncture3 d group,the serum cAMP content of electroacupuncture 5d group is lower,and the difference is statistically significant(p<0.01).Compared with electroacupuncture 5d group,the cAMP level in serum were higher in electroacupuncture 7d group(p<0.01).3)Compared with the normal group,the cGMP content in serum and myocardial tissue of the model group increased,and the difference was statistically significant(p<0.05).Compared with the model group,the serum cGMP content in the electroacupuncture1 d and 5d groups decreased,and the content increased in electroacupuncture 3d group,and the difference was statistically significant(p<0.05);the serum cGMP content of rats in the electroacupuncture 7d group decreased,but the difference was not statistically significant(p>0.05);the cGMP content of myocardial tissue decreased in electroacupuncture1 d group,but the difference was not statistically significant(p>0.05);the content of cGMP in the myocardial tissue of the electroacupuncture 3d group decreased,and the difference was statistically significant(p<0.05),but the changes of the content in myocardial tissue of the 5d and 7d electroacupuncture groups was not statistically significant(p>0.05).Compared with the electroacupuncture 1d group,the serum cGMP content of the electroacupuncture 3d group was significantly increased,and the difference was statistically significant(p<0.01);the change of the level in serum of electroacupuncture 5d and 7d groups was not statistically significant(p>0.05).Compared with the electroacupuncture 3d group,the serum cGMP content decreased and the myocardial tissue content increased in the electroacupuncture 5d and 7d groups,and the difference was statistically significant(p<0.01 or p<0.05).2 Effect of electroacupuncture serum on the expression of HCN2 in H9C2 cells:2.1 Western-blot revealedCompared with the normal serogroup,the HCN2 protein expression of the model serogroup was significantly increased after intervention,and the difference was statistically significant(p<0.01).Compared with the model serogroup,the expression of HCN2 protein in electroacupuncture serogroups after intervention was significantly reduced,and the difference was statistically significant(p<0.01).The expression of HCN2 protein was relatively lower at electroacupuncture 1d,3d,and 5d serogroup,and the expression was increased in electroacupuncture 7d serogroup,but the difference was not statistically significant(p>0.05).2.2 RT-PCR revealed:Compared with the normal serogroup,the HCN2 m RNA expression of H9C2 cells in the model serogroup decreased after intervention,and the difference was statistically significant(p<0.01).Compared with the model serogroup after intervention,the difference in electroacupuncture 1d,3d and 5d serogroups was not statistically significant(p>0.05).After the intervention of the 7d electroacupuncture serum group,the expression of HCN2 m RNA increased,and the difference was statistically significant(p<0.01).Conclusion:Electroacupuncture serum at different time courses significantly regulates the expression of HCN2 protein and m RNA in H9C2 cells. |
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