Toll样受体4在大鼠心肌缺血再灌注损伤中表达的实验研究

目的 观察心肌缺血再灌注早期Toll样受体4 mRNA(TLR4 mRNA)及蛋白表达,探讨TLR4在心肌缺血再灌注损伤中的作用.方法 雄性SD大鼠随机分为2组:假手术组(Sham组)、缺血再灌注组(IR组),每组36只,建立大鼠心肌缺血再灌注模型,按照不同的再灌注时间(0、0.5、1、24和8 h)处死动物.光镜和电镜观察心肌组织形态及超微结构改变.免疫组化检测心肌TLR4蛋白表达情况.实时定量逆转录聚合酶链反应定量心肌TLR4 mRNA表达水平.酶联免疫吸附试验测定心肌中肿瘤坏死因子-α(TNF-α)含量.结果 (1)Sham组心肌组织形态及超微结构改变不明显;IR组心肌损伤较重,缺血心肌恢复血液灌注8 h内,其病理学变化未见显著改善.(2)Sham组与IR组TLR4蛋白都有阳性表达,IR组TLR4表达增加,且以再灌注1 h最为明显(19.62±3.84,P＜0.01).(3)与Sham组比较,IR组心肌,TLR4 mRNA表达水平均出现不同程度上调,以再灌注1 h达到峰值(4.03±0.85)×10-2,P＜0.01],而Sham组各时间点未见明显改变.(4)IR组心肌TNF-α水平高于各对应时间点Sham组(P均＜0.05),且心肌TLR4 mRNA表达与TNF-α呈正相关(r=0.728,P＜0.01).结论 心肌缺血再灌注早期,心肌TLR4表达迅速上调,TLR4的激活可能通过促进TNF-α等炎性因子的产生分泌增多来介导心肌缺血再灌注损伤.

Myocardial Toll like receptor 4 expression in a rat model of myocardial ischemia reperfusion injury

Objective To explore the role of TLR4 in myocardial ischemia reperfusion injury(MI/RI) by observing the dynamic TLR4 expression changes at mRNA and protein levels early after myocardial ischemia reperfusion.Methods Male SD rats were randomly divided into Sham and IR group and the rats were killed according to different reperfusion time(0,0.5,1,2,4 and 8 hours).Myocardial changes under light microscope and transmission electronic microscope were observed.TLR4 expressions at protein and mRNA levels were detected by immunohistochemistry and realtime RT-PCR respectively.Myocardial TNF-α was determined by ELISA.Results (1)Myocardial injury was observed in IR but not in Sham group and histopathological and uhrastructual changes in IR group remained unchanged up to 8 hours after reperfusion.(2)Positive TLR4 protein staining was visualized in both Sham and IR groups and significantly increased and peaked at 1 hour of reperfusion in IR group.(3)Compared to Sham group,TLR4 mRNA level was upregulated in myocardium in IR group and peaked at 1 hour of reperfusion.(4)Concentration of TNF-α in IR group was significantly higher than that of Sham group at corresponding time points(all P＜0.05),and myocardial TLR4 mRNA level correlated positively with myocardial TNF-α (r=0.728,P＜0.01).Conclusion Expression of TLR4 in myocardium during early after myocardial ischemia repeffusion was upregulated and actived TLR4 might play an important role in MI/RI through promoting myocardial TNF-α excretion.