| Objective: This research established by coronary artery ligation method rat myocardial ischemia-reperfusion injury(Myocardial ischemia reperfusion injury,MIRI)model,Shuxinshengmai Dan on Myocardial ischemia reperfusion injury in the rat cardiac function change,at the same time,from the aspect of organization pathology and molecular biology to detect and analyze the rats of myocardial tissue pathology change and myocardial ultrastructure change,TLR4/NF-κB signaling pathway related factor of the protein and gene expression to explore Shuxinshengmai Dan on the protective effect and mechanism of rat MIRI,so as to provide theoretical basis for the research and clinical application of new drugs.Methods: 1.The SPF SD rats,male and female each half,the rats were randomly divided into blank group and control group,model group,salviae miltiorrhiza injection group(salviae miltiorrhiza group,positive control drug),Shuxinshengmai Dan group(low dose group,hereinafter referred to as low group;Medium dose group,hereinafter referred to as medium group;High-dose group,hereinafter referred to as the high group).2.Rat myocardial ischemia-reperfusion injury model was established by ligation of anterior descending coronary artery.3.Electrocardiogram(ECG)was used to detect the changes of ECG in rats.4.Hemodynamic changes were detected by right carotid artery cannula.5.Enzyme linked immunosorbent assay(ELISA)was used to detect IL-6 and TNF-αin the serum of rats.6.HE staining and transmission electron microscopy were used to observe the myocardial tissue of rats with myocardial ischemia-reperfusion injury,and the effects of Shuxinshengmai Dan on the myocardial tissue and its ultrastructure were analyzed.7.The protein and gene expression levels of TLR4,NF-κB,MyD88,HMGB1,IL-6 and TNF-α were detected by real-time fluorescence quantitative PCR(Rt-PCR),and western blot assay,and the regulatory effect of Shuxinshengmai Dan on TLR4/NF-κB signaling pathway and the protective mechanism of myocardium were analyzed.Results:(1)Hemodynamics: compared with the model group,the hemodynamics indexes of each treatment group were improved.Compared with the salvia miltiorrhiza group,the improvement was more obvious in the Shuxinshengmai Dan group.(2)The levels of IL-6 and TNF-α in the serum of rats were determined by ELISA showed that compared with the model group,the levels of IL-6 and TNF-α in each treatment group were decreased,and the declines were more significant in the middle group.(3)The changes of myocardial tissue and its ultrastructure were observed by HE staining and transmission electron microscopy.Compared with the model group,the results of HE staining showed that the degree of myocardial cell irregularity was reduced,inflammatory infiltration was reduced,and muscle fiber fracture and dissolution were reduced in each treatment group.Compared with the model group,the results of transmission electron microscopy showed that the ultrastructural changes of myocardial tissue were observed by transmission electron microscopy showed that compared with the model group,the muscle fibers and mitochondria in each treatment group were more orderly,and mitochondrial swelling and vacuity were reduced.(4)Rt-PCR was used to detect the expression levels of TLR4,NF-κB,MyD88,HMGB1,IL-6,and TNF-α genes in rat cardiac tissue.Compared with the model group,the expression levels of the genes to be measured in each drug group were improved,and the expression levels of TLR4 genes in the low group were significantly decreased compared with the salvia miltiorrhiza group(P < 0.05).The expression of NF-κB gene in each treatment group was significantly decreased,and there was no significant difference between the treatment groups(P > 0.05).Compared with the model group,the expression level of MyD88 in each group decreased significantly in each treatment group,but there was no significant difference between each treatment group(P > 0.05).Compared with the model group,the expression of HMGB1 decreased in all the treatment groups,and the decrease was more obvious in the low group compared with the salvia miltiorrhiza group,with statistically significant difference(P < 0.05).Compared with the model group,the expression of IL-6 gene decreased in each group,and the decrease was more obvious in the group of Shuxinshengmai Dan than the group of salvia miltiorrhiza.Compared with the model group,the expression of TNF-α decreased in all treatment groups,especially in the low group.(5)Western blot was used to detect the expression levels of TLR4,NF-κB,MyD88 and HMGB1 proteins in rat myocardial tissue.Compared with the model group,the expression levels of TLR4,NF-κB,MyD88 and HMGB1 proteins were all decreased in each treatment group,and the expression levels of proteins in the low group of Shuminsenmaekan were more significantly decreased than those in the other treatment groups.Conclusion:(1)Shuxinshengmai Dan may protect the myocardium by improving the cardiac function,pathological changes and ultrastructure of the myocardial tissue of MIRI in rats.(2)Shuxinshengmai Dan inhibited the contents of IL-6 and TNF-α in serum,indicating that the protective effect of Shuxinshengmai Dan on MIRI in rats maybe related to the inhibition of inflammatory response.(3)Shuxinshengmai Dan down-regulated the expression TLR4,NF-κB,MyD88,HMGB1,IL-6 and TNF-α genes in myocardial tissue,and down-regulated the protein expression of TLR4,NF-κB,MyD88 and HMGB1 in myocardial tissue,indicating that shuxinshengmai Dan can inhibit the inflammatory response and protect the MIRI of rats maybe related to regulating the TLR4/NF-κB signaling pathway. |
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