标题：抑制Toll样受体4表达减轻缺氧/复氧心肌细胞损伤

      作者：万为国，叶天新，陈修寰，杨波，张翠    武汉大学人民医院心内科，湖北 武汉 430060

      卷次： 2019年30卷24期

      【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。
      【关键词】 Toll样受体4；缺氧/复氧；凋亡；心肌细胞；RNA干扰
      【中图分类号】 R542.2 【文献标识码】 A 【文章编号】 1003—6350（2019）24—3129—04

Suppression of TLR4 alleviates H9C2 cardiomyocytes injury induced by anoxia/reoxygenation.
WAN Wei-guo, YETian-xin, CHEN Xiu-huan, YANG Bo, ZHANG Cui. Department of Cardiology, Renmin Hospital of Wuhan University,Wuhan 430060, Hubei, CHINA
【Abstract】 Objective To demonstrate whether the injury in H9C2 cardiomyocytes following anoxia/reoxygen-ation (A/R) would be alleviated via inhibiting Toll like receptor 4 (TLR4). Methods According to random number tablemethod, 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, trans-fection of negative control TLR4 shRNA plasmid) and TLR4-shRNA plasmid-treated group (A/R+shRNA group, cardio-myocytes experienced the same time of A/R after transfection of negative control TLR4-shRNA plasmid). TLR4 mRNAwas 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 apopto-sis were analyzed by the CCK-8 assay and flow cytometry, respectively. The levels of interleukin 6 (IL-6) and tumor ne-crosis factor α (TNF-α) were assessed by enzyme-linked immunosorbent assay (ELISA). Whether inhibition of TLR4 ex-pression ameliorate A/R-induced cardiomyocyte injury was investigated by reducing apoptosis and secretion of inflam-matory factors. Results Compared with the Con group, the expression of TLR4 and NF-κBp65, the level of IL-6 andTNF-α, apoptotic H9C2 cardiomyocytes and Bax/Bcl-2 ratio were significantly increased, and the cell survival rate andI-κBα were significantly decreased after A/R (all P<0.05). Compared with the A/R group, the expression of TLR4 andNF-κBp65, IL-6 and TNF-α, apoptosis and Bax/Bcl-2 ratio in the shRNA+A/R group significantly decreased, whilethe cell survival rate and I-κBα expression increased significantly (all P<0.05). Conclusion Suppression on TLR4 alle-viated cardiomyocyte injury after A/R by inhibiting the cell apoptosis and inflammation.
      【Key words】 Toll like receptor 4 (TLR4); Anoxia/reoxygenation (A/R); Apoptosis; Cardiomyocytes; RNAi