大黄素甲醚对脂多糖诱导肠上皮细胞损伤自噬与缝隙连接蛋白的作用影响

Effect of physcion on autophagy and gap junction protein in LPS-induced intestinal epithelial cell injury model

目的:研究大黄素甲醚(physcion,PHY)对脂多糖(LPS)诱导人小肠上皮细胞(HIEC-6)损伤模型炎症反应、自噬以及缝隙连接蛋白的作用影响,探讨PHY对肠道损伤的保护作用。方法:体外培养肠上皮细胞作为研究对象,将细胞分为对照组,模型组(LPS 1.0μg·mL^(-1)),PHY低(2.5μg·mL^(-1))、中(5.0μg·mL^(-1))、高(10.0μg·mL^(-1))剂量组,CCK-8法检测各组细胞活力情况,ELISA法检测各组细胞分泌炎症因子TNF-α、IL-1β的水平,Western blot法检测各组细胞TLR4信号通路蛋白、自噬蛋白Beclin-1、LC3及缝隙连接蛋白Cx43的表达情况。结果:LPS与各实验浓度的PHY对肠上皮细胞活力均无明显影响。与模型组比较,PHY中、高剂量对肠上皮细胞损伤炎症因子TNF-α、IL-1β的分泌具有显著的抑制作用(P<0.01),对TLR4蛋白表达亦具有明显的抑制作用(P<0.01);自噬蛋白表达方面,与模型组比较,PHY可上调Beclin-1、LC3的蛋白表达,其中PHY高剂量组Beclin-1蛋白表达明显增加(P<0.01),PHY各剂量对LC3蛋白表达均有显著的促进作用(P<0.01);而PHY各剂量对缝隙连接蛋白Cx43表达呈明显抑制作用(P<0.01)。结论:PHY可有效抑制肠上皮细胞损伤时释放炎症因子的水平,与TLR4信号通路表达呈正相关,其减轻LPS诱导的肠上皮细胞损伤作用与上调自噬蛋白Beclin-1、LC3及抑制缝隙连接蛋白Cx43的表达具有一定的相关性。

OBJECTIVE To investigate the effect of physcion on inflammatory factor、autophagy and gap junction protein in lipopolysaccharide(LPS)-induced human intestinal epithelial cell(HIEC-6)injury model,and to explore the protective mechanism of physcion on intestinal injury.METHODS HIEC-6 cells were cultured in vitro and divided into control group,model group(LPS 1.0μg·mL^(-1)),physcion low(2.5μg·mL^(-1)),medium(5.0μg·mL^(-1))and high(10.0μg·mL^(-1))dose groups.CCK-8 was used to detect the cell vitality of each group.ELISA was used to detect the levels of inflammatory cytokines TNF-αand IL-1β.Western blot was used to detect the expression of TLR4 signaling pathway protein,autophagy protein Beclin-1、LC3 and gap junction protein Cx43 of each group.RESULTS LPS and physcion of various experimental concentrations had no significant effect on the viability of HIEC-6 cell.Compared with model group,medium and high dose physcion could significantly inhibit the level of inflammatory cytokines TNF-αand IL-1β(P<0.01).It also significantly inhibited the expression of TLR4 protein(P<0.01).Compared with model group,physcion could up-regulate the protein expression of Beclin-1 and LC3.The protein expression of Beclin-1 in the physcion high-dose group was significantly increased(P<0.01),and the protein expression of LC3 was significantly promoted by each dose of physcion(P<0.01).In addition,each dose of physcion significantly inhibited the expression of Cx43 protein(P<0.01).CONCLUSION Physcion could effectively inhibit the level of inflammatory cytokines released by intestinal epithelial cell injury,and it was positively correlated with the expression of TLR4 signaling pathway.The effect of physcion on LPS-induced intestinal epithelial cell injury was correlated with up-regulation of autophagy protein Beclin-1、LC3 and inhibition of the expression of gap junction protein Cx43.