| Objective: To observe the expression of micro RNA(mi R)-30 c in myocardial tissue of rats with myocardial ischemia-reperfusion injury(MI/RI)after cardiopulmonary bypass(CPB);To observe whether overexpression of mi R-30 c could reduce myocardial apoptosis and MI/RI in MI/RI rats under CPB by inhibiting the activation of Nuclear factor(NF)-κB,and thus play a role in myocardial protection.Methods:Part 1 To detect the differential expression of mi R-30c12 rats were divided into two groups(n=6),which were named sham group(S)and ischemia-reperfusion group(I/R).CPB model was established.Group S continued cardiopulmonary bypass for 150 minutes.In I/R group,myocardial ischemia lasted for30 min and reperfusion lasted for 120 min.During the surgery,Heart rate(HR)and Mean arterial pressure(MAP)were recorded,Hematocrit(HCT)was monitored,and plasma cardiac troponin I(c Tn I),Creatine kinase-MB(CK-MB)levels and the relative expression level of m RNA of mi R-30 c in cardiac tissue were measured at the end of the experiment.Part 2 To investigate the role of overexpression of mi R-30c(1)mi R-30 c was overexpressed by viral transfection18 rats were randomly divided into 3 groups(n=6),which were classified as normal(Nor)group,control adeno-associated virus(AAV-NC)group,and mi R-30 c overexpression adenoassociated virus(AAV-mi R-30c)group.The AAV-NC group and the AAV-mi R-30 c group were respectively injected with control virus and mi R-30 c overexpressing virus through the tail vein pump.Twenty-one days after the transfection,the myocardial tissue was taken to observe the virus fluorescence under a fluorescence microscope,and the relative expression level of mi R-30 c m RNA in the myocardial tissue was detected.(2)To evaluate the role of mi R-30c72 rats were divided into 6 groups(n=12),named normal rats sham(NOR +S)group and ischemia/reperfusion(NOR +I/R)group,control virus rats sham operation(NC+S)group and ischemia/reperfusion(NC+I/R)group,mi R-30 c overexpression rats sham operation(mi R-30c+S)and ischemia/reperfusion(mi R-30c+I/R)group.CPB models were established,respectively.Each S group continued to bypass for 150 min,each I/R group was ischemia for30 min,and reperfusion for 120 min.In the end,arterial blood and myocardial tissue samples were taken to detect the following indexes : the contents of myocardial enzymes(CK-MB and c Tn I);Infarct size(IS);Ultrastructure of myocardium and mitochondrial Flameng score of myocardium;apoptosis rate of myocardial cells;The relative expression level of m RNA of mi R-30 c in myocardial tissue;The relative expression levels of NF-κB P65,phosphorylated NF-κB P65(p-p65),Bax,Bcl-2 in myocardial tissue.Part 3 To explore the mechanism of action of mi R-30c(1)Dosage selection of Betulinic acid(BA)30 rats were randomly divided into 5 groups(n=6),which were named sham(S)group,BA5 group,BA10 group,BA15 group and BA20 group according to different doses of BA(5,10,15,20mg/kg),respectively.CPB model was established.Each BA group was slowly injected with the corresponding dose of BA after the CPB flow was stable,and then BA was continuously pumped at a dose of 10mg/kg/h until the end of the experiment.The same amount of solvent(0.5% twain-80 normal saline)was injected into the S group.At the end of the experiment,the protein expression levels of p-65 and p-p65 were detected in myocardial tissue.(2)To observe whether BA affects the effect of mi R-30c36 rats overexpressed with mi R-30 c were randomly divided into 3 groups(n=12),which were identified as mi R-30 c group,mi R-30c+TW group,and mi R-30c+BA group.MI/RI models under CPB were established in each group.10 min before ischemia,15mg/kg BA was slowly injected in the mi R-30c+BA group through the tail vein,and then BA was continuously pumped at a dose of 10mg/kg/h until the end of the experiment.The mi R-30c+TW group was injected with the same amount of solvent(0.5% twean-80 normal saline),while the mi R-30 c group was only treated with ischemia/reperfusion.Arterial blood and heart samples were collected at the end of the experiment,and the following indexes were detected :(1)the content of myocardial enzymes(CK-MB and c Tn I);(2)IS;(3)Ultrastructure of myocardium and mitochondrial Flameng score of myocardium;(4)apoptosis rate of myocardial cells;(5)Relative protein expression levels of P65,p-p65,Bax and Bcl-2 in myocardial tissue.Results:Part 1MAP ≥ 60 mm Hg and HCT ≥ 20% during CPB in the two groups of rats.Rats in I/R group could achieve cardiac arrest and resuscitation.Plasma concentrations of CK-MB and c Tn I in I/R group were increased comparing the S group(P<0.05);The expression of mi R-30 c in myocardial tissue was significantly decreased in group I/R(P<0.05).Part 2(1)Green virus fluorescence was observed in the AAV-NC group and AAV-mi R-30 c group under the fluorescence microscope,but not in the Nor group;There was no difference in mi R-30 c expression between NOR group and AAV-NC group(P>0.05),while the relative expression level of mi R-30 c in AAV-mi R-30 c group was significantly increased compared with that in AAV-NC group(P<0.05).(2)Compared with the S groups(NOR +S,NC+S,and mi R-30c+S),the corresponding I/R groups(NOR +I/R,NC+I/R,and mi R-30c+I/R)had increased CK-MB content,c Tn I content,IS,myocardial mitochondrial Flameng score,myocardial apoptosis rate,p-p65 expression level,and Bax expression level(P<0.05),while the m RNA expression of mi R-30 c and the protein expression of Bcl-2 were decreased(P<0.05);Compared with NC+S group,the relative expression of mi R-30 c was increased in mi R-30c+S group(P<0.05);Compared with NC+I/R group,the content of CK-MB and c Tn I,IS,myocardial mitochondrial Flameng score,myocardial apoptosis rate,p-p65 expression and Bax expression were decreased in mi R-30c+I/R group,while the m RNA expression of mi R-30 c and Bcl-2 protein expression were increased(P<0.05).Part 3(1)Compared with the S group,the expression of P-65 in BA20 group was increased(P<0.05),the expression of p-p65 in BA15 and BA20 groups was increased(P<0.05).(2)Compared with mi R-30c+TW group,the content of CK-MB and c Tn I,IS,myocardial mitochondrial Flameng score,myocardial apoptosis rate,p-p65 expression and Bax expression in mi R-30c+BA group were increased to varying extent,while the expression of Bcl-2 was decreased(P<0.05).Conclusion:(1)The expression of mi R-30 c was decreased in myocardial tissue of MI/RI rats under CPB.(2)Overexpression of mi R-30 c can inhibit the activation of NF-κB in the myocardium of M/RI rats under CPB,reduce the apoptosis of cardiomyocytes,and reduce MI/RI in rats,and it has the effect of myocardial protection.(3)NF-κB activator BA attenuates the myocardial protective effect of mi R-30 c,NF-κB/Bcl-2 pathway is involved in the myocardial protection process of mi R-30c... |
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