低蛋氨酸通过调节自噬基因ATG16L1和miR130a改善肠上皮细胞炎症反应和细胞紧密连接

Mechanism of autophagy genes ATG16L1 and miR130a in inhibiting intestinal inflammatory response in low intestinal methionine

目的探究低蛋氨酸通过调节自噬基因ATG16L1和miR130a在改善肠上皮细胞炎症反应和细胞紧密连接的机制。方法将人肠上皮细胞Caco-2分为4组:对照组、低蛋氨酸组、肠上皮细胞炎性模型组(模型组)和模型+低蛋氨酸组。其中模型组和模型+低蛋氨酸组使用具核梭杆菌感染6 h建立感染模型;对照组和模型组使用常规培养基,低蛋氨酸组中蛋氨酸水平为正常培养基的10%。酶联免疫吸附法用于检测炎性因子水平。在培养后12 h、24 h和48 h使用流式细胞术检测凋亡。Western blot和qPCR法检测LC3Ⅱ/LC3Ⅰ、caspase、ATG16L1蛋白、ATG16L1 mRNA和miR-130a的水平。结果模型组细胞凋亡率显著低于对照组(P <0. 01),低蛋氨酸组与对照组细胞凋亡无显著差异(P> 0. 05),模型+低蛋氨酸组的细胞凋亡率显著低于模型组(P <0. 01)。低蛋氨酸组与对照组炎性因子水平无显著差异(P> 0. 05),感染24 h后模型组各项炎性因子水平均显著升高(P <0. 01),模型+低蛋氨酸组的TNF-α、IL-1、IL-8均显著低于模型组(P <0. 01)。低蛋氨酸组与对照组Caspase和LC3Ⅱ/LC3Ⅰ无显著差异(P> 0. 05),建模后Caspase显著升高,而LC3Ⅱ/LC3Ⅰ显著降低(P <0. 01),而模型+低蛋氨酸组的Caspase显著低于模型组,而LC3Ⅱ/LC3Ⅰ显著高于模型组(P <0. 01)。低蛋氨酸组与对照组ATG16L1 mRNA和miR-130a的表达无显著差异(P> 0. 05),建模后24 h ATG16L1 mRNA显著降低而miR-130a显著升高(P <0. 01),模型+低蛋氨酸组的ATG16L1 mRNA显著高于模型组而miR-130a显著低于模型组(P <0. 01)。结论低蛋氨酸可以抑制肠上皮细胞炎症反应损伤并抑制细胞凋亡,这可能由于低蛋氨酸通过促进ATG16L1和抑制miR130a,上调炎症模型中肠细胞的自噬水平,保护细胞。

Objective To explore the mechanism of low methionine improving the intestinal inflammatory response and cell tight junctions by regulating the autophagy genes ATG16L1 and miR130a. Methods Human intestinal epithelial cells of Caco-2 were divided into 4 groups:control group,low methionine group,intestinal epithelial inflammatory model group( model group) and model + low methionine group. The model group and the model + low methionine group were infected with Fusobacterium nucleus for 6 h to establish an infection model. The control medium and the model group used conventional medium,and the methionine level in the low methionine group was 10% of the normal medium.ELISA was used to detect inflammatory factor levels. Apoptosis was detected by flow cytometry at 12 h,24 h and 48 h after culture. Western blot and qPCR were used to detect the levels of LC3Ⅱ/LC3Ⅰ,caspase,ATG16L1 protein,ATG16L1 mRNA and miR-130 a. Results The apoptosis rate of the model group was significantly lower than that of the control group( P < 0. 01). Low methionine had no significant effect on apoptosis( P > 0. 05). The apoptotic rate of the model + low methionine group was significantly lower than that of the model group( P < 0. 01). Low methionine had no significant effect on inflammatory factor levels( P > 0. 05). After 24 hours of infection,the levels of inflammatory factors in the model group were significantly increased( P < 0. 01). TNF-α,IL-1 and IL-8 in the model + low methionine group were significantly lower than the model group( P < 0. 01). Low methionine had no significant effect on Caspase and LC3Ⅱ/LC3Ⅰ. Caspase were significantly increased and LC3Ⅱ/LC3Ⅰ were significantly decreased after modeling( P < 0. 01). Caspase in the model + low methionine group were significantly lower than those in the model group( P < 0. 01),and LC3Ⅱ/LC3Ⅰ were significantly higher than those in the model group( P < 0. 01). Low methionine had no significant effect on the expression of ATG16L1 mRNA and miR-130 a. At 24 h after modeling,ATG16L1 mRNA was significantly decreased and miR-130 a was significantly increased( P < 0. 01). The ATG16L1 mRNA in the model + low methionine group was significantly higher than that in the model group and miR-130 a was significantly lower than the model group( P < 0. 01). Conclusion Low methionine can inhibit the intestinal inflammatory response and inhibit apoptosis,which may be due to the low methionine can protect cells by up-regulating the autophagy level of intestinal cells in the inflammatory model by promoting ATG16L1 and inhibiting miR130a.