The Effect Of A Chinese 4-herb Formula,yiqi-huoxue Granule Via Mi R-1 Targeting Mapk3 In Myocardial Ischemia/reperfusion Injury Yaqi Fan(internal Medicine Of Traditional Chinese Medicine)

Objective:The model of cardiomyocyte hypoxia-reoxygenation was used to stimulate ischemia-reperfusion in vivo,observe the intervention effect of Yiqi-Huoxue granule on myocardial ischemia-reperfusion injury,detect the expression levels of microRNA-1,MAPK3,Bax,Bcl-2,etc.Explore the mechanism of Yiqi-Huoxue granule.Method:Experiment 1:Adult rat cardiomyocytes were divided into 3 groups by random number table method:control group,model group,and Yiqi-Huoxue granule group.Place the model group（M）and Yiqi-Huoxue granule group（YH）into the hypoxic chamber for 3h,then open the hypoxic chamber,transfer the cells to the cell thermostat incubator which is keeping 37℃,5%CO₂,and perform various tests after 0.5 h of normal culture.1.Detection of the degree of myocardial injury:lactate dehydrogenase（LDH）release amount was detected by the colorimetric method in cell culture medium;the content of creatine kinase（CK）activity was detected by ultraviolet spectrophotometry in the cardiomyocytes;2.Detection of apoptosis indicators:q RT-PCR was used to detect changes in Bax and Bcl-2 m RNA expression;Western Blot was used to detect the protein changes in Bax and Bcl2 expression;3.Signal pathway detection:q RT-PCR was used to detect the changes of microRNA-1expression,and Western Blot method was used to detect the phosphorylation changes of MAPK3 protein expression.Experiment 2:Use H9c2 cardiomyocytes to verify the conclusion of experiment one.H9c2 cell was be transfected by microRNA-1 mimics,and was divided into 4 groups by random number table method:model group（M）,model+Transfection group（T）,Model+Yiqi-Huoxue granule group（YH）,Model+Transfection+Yiqi-Huoxue granule group（T+YH）.Put the 4 groups of cells into the hypoxic chamber for 3 h,then open the hypoxic chamber,transfer the cells to the cell thermostat incubator which is keeping37℃,5%CO₂,and perform various tests after 0.5 h of normal culture.1.Detection of the degree of myocardial injury:LDH release amount was detected by the colorimetric method in cell culture medium;2.Detection of apoptosis indicators:q RT-PCR was used to detect changes in Bax and Bcl-2 m RNA expression;Western Blot was used to detect the protein changes in Bax and Bcl2 expression;3.Signal pathway detection:q RT-PCR was used to detect the changes of microRNA-1expression,and Western Blot method was used to detect the phosphorylation changes of MAPK3 protein expression.Result:1.In the experiment of adult rat cardiomyocytes damage induced by H/R,compared with group C,the LDH release and CK activity of group M was increased,the m RNA express ratio of Bax/Bcl-2 and the protein express ratio of Bax/Bcl-2 was increased significantly,the difference was statistically significant（P<0.01）.Compared with the M group,the LDH release and CK activity of group M was decreased,the m RNA express ratio of Bax/Bcl-2 and the protein express ratio of Bax/Bcl-2 was decreased,the difference was statistically significant（P<0.01 or P<0.05）.2.In the experiment of adult rat cardiomyocytes damage induced by H/R,compared with group C,the expression of microRNA-1 in group M was significantly was increased,and the phosphorylation changes of MAPK3 protein was significantly increased,the difference was statistically significant（P<0.01）.Compared with the YH group,the expression of microRNA-1 was reduced and the phosphorylation changes of MAPK3 protein was significantly decreased,the difference was statistically significant（P<0.01 or P<0.05）.3.In the experiment of H9c2 cells damage induced by H/R,compared with group C,the LDH release and CK activity of group M was increased,the m RNA express ratio of Bax/Bcl-2 and the protein express ratio of Bax/Bcl-2 was increased significantly,the difference was statistically significant（P<0.01）.Compared with the M group,the LDH release and CK activity of group M was decreased,the m RNA express ratio of Bax/Bcl-2 and the protein express ratio of Bax/Bcl-2 was decreased,the difference was statistically significant（P<0.01）.4.In the experiment of H9c2 cells damage induced by H/R,compared with group C,the expression of mi RNA-1 in group M was increased significantly,and the phosphorylation changes of MAPK3 protein was increased significantly,the difference was statistically significant（P<0.01）.Compared with group M,the mi RNA-1expression in the YH group was significantly reduced,and the phosphorylation changes of MAPK3 protein was significantly reduced,the difference was statistically significant（P<0.01）.5.In the experiment of H9c2 cells transfected with microRNA-1 mimics,compared with group M,the ratio of Bax/Bcl-2 m RNA and protein in the YH group was significantly reduced,the ratio of Bax/Bcl-2 m RNA and protein in the T group was increased significantly,and the difference was statistically significant（P<0.01）.Compared with the YH group,the ratio of Bax/Bcl-2 m RNA and protein in the T group was significantly increased,the ratio of Bax/Bcl-2 m RNA and protein in the YH+T group was significantly increased,the difference Statistical significance（P<0.01）.Compared with the T group,the ratio of Bax/Bcl-2 m RNA and protein in the YH+T group was significantly reduced,and the difference was statistically significant（P<0.01）.6.In the experiment of H9c2 cells transfected with microRNA-1 mimics,compared with group M,the phosphorylation changes of MAPK3 protein in the YH group was significantly reduced,and the phosphorylation changes of MAPK3 protein in the T group was increased,the difference was statistically significant（P<0.01 or P<0.05）.Compared with the YH group,the phosphorylation changes of MAPK3 protein in the T group increased significantly,and the phosphorylation changes of MAPK3 protein in the YH+T group increased,and the difference was statistically significant（P<0.01 or P<0.05）.Compared with the T group,the phosphorylation changes of MAPK3 protein in the YH+T group was significantly reduced,and the difference was statistically significant（P<0.01）.Conclusion:1.Yiqi-Huoxue granule has a protective effect on myocardial ischemia-reperfusion injury;2.Micro RNA-1 can regulate myocardial ischemia-reperfusion injury by targeting MAPK3;3.Yiqi-Huoxue granule can regulate MAPK3 through microRNA-1 to play an anti-myocardial ischemia-reperfusion injury role.