Effects Of Expression Inhibition Of RAGE Gene By SiRNA On Inflammatory Response After Acute Myocardial Infarction Of Rats

Objective To study the effects of expression inhibition of RAGE gene by siRNA on the inflammatory response after acute myocardial infarction of rats.Methods Primary cardiocytes were cultured and infected with AD-Ager-RNAi in different concentrations,and the best multiplicity of infection(MOI)was calculated.Then,all cells were divided into 3 groups:(1)Blank control group(Con)in which the cells were cultured in normal condition without any intervention;(2)Control siRNA group in which the cells were infected with control siRNA called CON098 and then cultured in hypoxia condition for 4h;(3)RAGE siRNA group in which the cells were infected with RAGE targeting siRNA called AD-Ager-RNAi and then cultured in hypoxia condition for 4h.Expression of the RAGE-gene was analyzed by qPCR,and Western Blot was used to detect protein expression level of RAGE and phosphorylation of ERX1/2.Experimental animals were divided into 4 groups:(1)12 SD rats belong to Sham group in which the hearts of rats were exposed without left anterior descending(LAD)artery ligation;(2)12 SD rats belong to acute myocardial infarction(AMI)group in which the hearts of rats were ligated in the LAD artery;(3)12 SD rats belong to control siRNA(control siRNA)group in which the hearts of rats were ligated in the LAD artery and injected with CON098 around the infarcted area after 30min of ligation.(4)12 SD rats belong to RAGE siRNA group(RAGE siRNA)in which the hearts of rats were ligated in LAD artery and injected with AD-Ager-RNAi around the infarcted area after 30min of ligation.Cardiac ultrasound was performed to test the heart function at one week and three weeks after operation,respectively.3 weeks after operation,rats were executed and cardiac tissues from different regions were harvested.The mRNA levels of RAGE in different regions of hearts were detected by qPCR,the protein expression levels were detected by Western Blot analysis and immunohistochemistry analysis.Results In the cells exeperiments,the expression level of RAGE mRNA of RAGE siRNA group was significantly lower than that of the control siRNA group(p=0.009),and slightly higher than that of the Con gourp with no significant difference(p=0.051);The expression level of RAGE protein of RAGE siRNA group was significantly lower than that of the control siRNA group(p=0.029)while there were no significant difference between RAGE siRNA group and Con group(p=0.064);Also,the expression level of pERKl/2 protein of RAGE siRNA group was significantly lower than that of the control siRNA group(p=0.011)while there were significant difference between RAGE siRNA group and Con group(p=0.067).However,the RAGE mRNA level,RAGE protein expression level and pERK1/2 protein expression level of control siRNA group were significantly higher than that of the Con group(p<0.001).In animal exeperiments,the RAGE mRNA level in infarcted regions was significantly higher than that in non-infarcted regions(p<0.001);The protein expression levels of RAGE and pERKl/2 in infarcted regions was significantly elevated compared with that in non-infarcted regions(p<0.001).There were no significant difference in RAGE mRNA level,RAGE protein expression level and pERKl/2 expression level between cardiac tissue of Sham group and non-infarcted regions of AMI group(p>0.05).The mean EF of RAGE siRNA group injected with AD-Ager-RNAi was significantly higher than that of control siRNA group injected with control siRNA at three weeks after injection(p=0.011),but not at one week after injection(p=0.409).The RAGE mRNA level and protein expression level of RAGE siRNA group were significantly lower than that in control siRNA group(p<0.001,p=0.034,respectively)according to Western Blot analysis.Immunohistochemistry analysis also indicated that the RAGE and pERKl/2 protein expression levels in RAGE siRNA group were significantly decreased compared with that in control siRNA group(p>0.05).There were no difference of RAGE mRNA level,RAGE protein and pERKl/2 pretein expression levels between RAGE siRNA group and Sham group(p>0.05).Conclusions RAGE targeting siRNA could efficiently inhibit RAGE gene expression in cardiocytes cultured in hypoxia condition.Injection of RAGE targeting siRNA(AD-Ager-RNAi)around the infracted regions could efficiently inhibit the expression of RAGE protein and pERK1/2 protein expression,subsequently attenuate the overwhelming inflammatory responses induced by MAPK/ERK1/2 pathway and improve the heart function after acute myocardiac infarction.