氯胺酮对心肌缺血再灌注损伤中Toll样受体-4和核因子-κB表达的影响

Effects of Ketamine on the Expression of Toll-like Receptor-4 and Nu-clear Factor-κB in Myocardial Ischemia-reperfusion Injury

目的研究氯胺酮对心肌细胞H9c2缺血再灌注损伤的保护作用及对Toll样受体-4(TLR-4)和核因子-κB(NF-κB)表达的影响.方法将处于对数生长期的大鼠心肌H9c2细胞分为3组:正常对照组(Con组)、缺氧复氧组(I/R组)、缺氧复氧+氯胺酮组(I/R+K组).各组细胞处理后采用细胞计数试剂盒(CCK-8)检测细胞增殖能力,试剂盒检测细胞内活性氧(ROS)水平及细胞外乳酸脱氢酶(LDH)含量,流式细胞仪检测细胞凋亡情况,实时荧光定量PCR(RT-PCR)检测TLR-4 mRNA水平.Western印迹检测TLR-4、NF-κB及凋亡相关蛋白活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)蛋白表达水平.结果与Con组相比,I/R组细胞增殖能力显著降低,细胞内ROS水平升高,细胞漏出LDH含量增多,细胞凋亡率显著增加,TLR-4和NF-κB表达水平明显上调,Caspase-3蛋白激活增多(P<0.05).与I/R组相比,I/R+K组细胞增殖能力明显升高,细胞内ROS水平降低,细胞漏出LDH含量减少,细胞凋亡率显著降低,TLR-4和NF-κB表达水平下调,Caspase-3蛋白激活减少(P<0.05).结论氯胺酮能够通过抑制TLR-4和NF-κB的表达,减轻细胞损伤,减少细胞凋亡,对心肌细胞缺血再灌注损伤起保护作用.

Objective To study the protective effect of ketamine on myocardial H9c2 ischemi-a-reperfusion injury and the expression of Toll-like receptor-4(TLR-4)and nuclear factor-κB(NF-κB).Methods Rat myocardial H9c2 cells in logarithmic growth phase were divided into three groups:normal control group(Con group),hypoxia reoxygenation group(I/R group),hypoxia reoxygenation+ketamine group(I/R+Group K).After treatment with each group of cells,CCK-8 was used to detect the cell proliferation ability.The kit was used to detect the levels of reac-tive oxygen species(ROS)and extracellular lactate dehydrogenase(LDH),and the apoptosis was detected by flow cytometry.RT-PCR TLR-4 mRNA levels were detected.Western blot was used to detect the expression of cysteine-containing Caspase-3 protein activated by TLR-4,NF-κB and apoptosis-related proteins.Results Compared with the Con group,the cell proliferation ability of the I/R group was significantly decreased,the intracellular ROS level was increased,the LDH content of the cells was increased,the apoptosis rate was significantly increased,and the expres-sion levels of TLR-4 and NF-κB were significantly up-regulated.Caspase-3 protein activation in-creased,the difference was statistically significant(P<0.05).Compared with the I/R group,the cell proliferation ability of the I/R+K group was significantly increased,the intracellular ROS level was decreased,the LDH content of the cells was decreased,the apoptosis rate was significant-ly decreased,and the expression levels of TLR-4 and NF-κB were down-regulated.Caspase-3 pro-tein activation was decreased,and the difference was statistically significant(P<0.05).Conclu-sion Ketamine can protect against myocardial cell ischemia-reperfusion injury by inhibiting the ex-pression of TLR-4 and NF-κB,reducing cell damage,reducing cell apoptosis.