抑制Toll样受体4表达减轻缺氧/复氧心肌细胞损伤  

Suppression of TLR4 alleviates H9C2 cardiomyocytes injury induced by anoxia/reoxygenation

作　　者：万为国[1] 叶天新 陈修寰 杨波[1] 张翠[1] WAN Wei-guo;YETian-xin;CHEN Xiu-huan;YANG Bo;ZHANG Cui(Department of Cardiology,Renmin Hospital of Wuhan University,Wuhan 430060,Hubei,CHINA)

机构地区：[1]武汉大学人民医院心内科

出　　处：《海南医学》2019年第24期3129-3132,共4页Hainan Medical Journal

基　　金：国家自然科学基金(青年)资助项目(编号：81500278)

摘　　要：目的研究抑制H9C2心肌细胞Toll样受体4(TLR4)基因表达对缺氧/复氧(A/R)诱导细胞损伤的影响及其机制。方法H9C2心肌细胞按随机数表法分为正常对照组(Con组):正常培养的H9C2心肌细胞、A/R组:按常规的方法进行缺氧/复氧处理、阴性对照组(NC组)和shRNA+A/R组:分别转染阴性对照TLR4-shRNA质粒和TLR4-shRNA质粒后缺氧/复氧相同的时间。Weston-blot法和RT-PCR法检测RNA干扰(RNAi)的效果;CCK-8法检测心肌细胞的存活率;流式细胞仪检测细胞凋亡;Weston-blot法测定B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关蛋白(Bax)、核因子-κBp65(NF-κBp65)和NF-κB抑制剂I-κBα)蛋白水平;ELISA法测量培养液中白细胞介素6(IL-6)和肿瘤坏死因子α(TN-α)的浓度。观察抑制TLR4表达后是否能通过减轻细胞凋亡和炎症因子的分泌改善A/R诱导的心肌细胞损伤。结果与Con组比较,A/R组H9C2心肌细胞TLR4与NF-κBp65表达、IL-6和TNF-α水平、细胞凋亡及Bax/Bcl-2比值增加,而细胞存活率和I-κBα表达明显降低,差异均有统计学意义(P<0.05);与A/R组比较,shRNA+A/R组TLR4与NF-κBp65表达、IL-6和TNF-α水平、细胞凋亡及Bax/Bcl-2比值下降,而细胞存活率和I-κBα表达明显升高,差异均有统计学意义(P<0.05)。结论抑制TLR4能通过抑制炎症反应和细胞凋亡减轻A/R诱导的H9C2心肌细胞损伤。Objective To demonstrate whether the injury in H9C2 cardiomyocytes following anoxia/reoxygenation(A/R)would be alleviated via inhibiting Toll like receptor 4(TLR4).Methods According to random number table method,H9C2 cardiomyocytes were divided into the control group(Con group,normal cultured H9C2 cardiomyocytes),A/R group(cardiomyocytes were treated according to conventional A/R methods),negative control group(NC group,transfection of negative control TLR4 shRNA plasmid)and TLR4-shRNA plasmid-treated group(A/R+shRNA group,cardiomyocytes experienced the same time of A/R after transfection of negative control TLR4-shRNA plasmid).TLR4 mRNA was detected by RT-PCR.TLR4,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(BAX),nuclear factor-κBp65(NF-κBp65)and inhibitor of kappa B(I-κBα)protein were examined by Western blot analysis.Cell viability and apoptosis were analyzed by the CCK-8 assay and flow cytometry,respectively.The levels of interleukin 6(IL-6)and tumor necrosis factorα(TNF-α)were assessed by enzyme-linked immunosorbent assay(ELISA).Whether inhibition of TLR4 expression ameliorate A/R-induced cardiomyocyte injury was investigated by reducing apoptosis and secretion of inflammatory factors.Results Compared with the Con group,the expression of TLR4 and NF-κBp65,the level of IL-6 and TNF-α,apoptotic H9C2 cardiomyocytes and Bax/Bcl-2 ratio were significantly increased,and the cell survival rate and I-κBαwere significantly decreased after A/R(all P<0.05).Compared with the A/R group,the expression of TLR4 and NF-κBp65,IL-6 and TNF-α,apoptosis and Bax/Bcl-2 ratio in the shRNA+A/R group significantly decreased,while the cell survival rate and I-κBαexpression increased significantly(all P<0.05).Conclusion Suppression on TLR4 alleviated cardiomyocyte injury after A/R by inhibiting the cell apoptosis and inflammation.

关 键 词：TOLL样受体4 缺氧/复氧 凋亡 心肌细胞 RNA干扰 

分 类 号：R542.2[医药卫生—心血管疾病]