KLF4在LPS诱导的心肌细胞损伤中的作用研究

The role of KLF4 in LPS induced cardiomyocyte injury

背景KLF4作为一种转录因子在保持血管内皮功能中发挥重要作用,然而其是否能够保护心肌细胞免受脂多糖诱导的损伤尚不清楚。目的探讨KLF4在脂多糖诱导的心肌细胞损伤中的作用。方法分离培养原代大鼠乳鼠心肌细胞,将其随机(随机数字法)分为5组:空白组,阴性对照组(NC组),NC+脂多糖刺激组(NC+LPS组),KLF4过表达组,KLF4过表达+LPS组。采用MTT法检测细胞活性,采用试剂盒检测细胞活性氧(ROS),超氧化物歧化酶(SOD2),谷胱甘肽过氧化物酶(Gpx)和丙二醛(MDA)的水平;采用酶联免疫吸附法(Elisa)检测细胞中肿瘤坏死因子(TNFα),白细胞介素-1β(IL-1β)和IL-6的水平。采用Tunel染色检测细胞凋亡。采用免疫印迹检测TLR4和核因子E2相关因子2(NRF2)的蛋白水平。结果心肌细胞转染KLF4过表达腺病毒后,细胞中KLF4的表达明显高于NC组(P<0.05)。NC+LPS组细胞活性明显低于NC组(P<0.05),KLF4过表达+LPS组细胞活性高于NC+LPS组(P<0.05)。与对照组相比,NC+LPS组中的心肌细胞TNFα、IL-1β、IL-6蛋白表达水平明显升高(P<0.0001),KLF4过表达+LPS组心肌细胞TNFα、IL-1β、IL-6蛋白表达水平则显著降低(P<0.0001)。NC+LPS组心肌细胞ROS和MDA水平明显高于NC组,而SOD2和Gpx的活性低于NC组(P<0.0001);KLF4过表达+LPS组心肌细胞ROS和MDA水平的水平明显降低,而SOD2和Gpx的活性明显升高(P<0.0001)。LPS组心肌细胞凋亡数量明显高于NC组,KLF4过表达+LPS组心肌细胞凋亡数量明显降低(P<0.001)。LPS组心肌细胞TLR4总蛋白水平高于NC组,NRF2的核蛋白水平低于NC组;KLF4过表达+LPS组心肌细胞TLR4的总蛋白水平降低,NRF2的核蛋白水平显著增高(P<0.001)。结论:KLF4可抑制LPS诱导的心肌细胞损伤,其作用机制可能是通过抑制LPS诱导的心肌细胞中TLR4的表达,促进NRF2向细胞核转移来抑制炎症因子释放,减轻氧化应激损伤及抑制细胞凋亡。

Objective To investigate the role of KLF4 in lipopolysaccharide induced cardiomyocyte injury.Methods Primary rat cardiomyocytes were isolated and cultured,and randomly divided into 5 groups:control group,negative control(NC),LPS group,KLF4 overexpression group,KLF4 overexpression+LPS group.MTT method was used to detect cell activity,ROS,SOD 2,GPX and MDA were detected by kit,TNFα,IL-1β and IL-6 were detected by ELISA.TUNEL staining was used to detect apoptosis.The protein levels of TLR4 and Nrf2 were detected by Western blot.Results The expression of KLF4 in cardiomyocytes was significantly higher than that in the NC group(P<0.001).The cell activity of LPS group was significantly lower than that of NC group(P<0.001),and that of KLF4 overexpression+LPS group was higher than that of LPS group(P<0.001).The levels of TNFα,IL-1β and IL-6 in LPS group were significantly higher than those in the NC group(P<0.0001),and the levels of TNFα,IL-1β and IL-6 in KLF4 overexpression+LPS group were lower than those in LPS group(P<0.0001).The levels of ROS and MDA in LPS group were significantly higher than those in the control group,while the activities of SOD2 and GPX were lower than those in the NC group(P<O.OOOl).The levels of ROS and MDA in KLF4 overexpression+LPS group were lower than those in LPS group,while the activities of SOD2 and GPX were higher than those in LPS group(P<0.0001).The number of apoptosis in LPS group was significantly higher than that in the NC group,and that in KLF4 overexpression+LPS group was lower than that in LPS group(P<0.001).The level of TLR4 wan higher and Nrf2 protein in the nucleus of LPS group was lower than that of the NC group.The level of TLR4 was lower and Nrf2 protein in the nucleus of KJLF4 overexpression+LPS group was significantly higher than that of LPS group(P<0.001).Conclusions KLF4 can alleviate LPS induced cardiomyocyte injury by regulating TLR4 and NRF2 signals.