基于NF-κB p65信号通路探讨金汁修复受损的小鼠肠黏膜上皮细胞的机制

Mechanism of purified feces in repairing damaged mouse intestinal epithelial cells through NF-κB p65 signaling pathway

目的观察金汁含药血清对脂多糖(LPS)诱导的小鼠肠黏膜上皮细胞自噬相关蛋白及NF-κB p65信号通路的影响,探讨其对肠黏膜上皮细胞的保护作用机制。方法选择雄性BALB/c小鼠制备空白血清和不同浓度(10%,15%,20%)的金汁含药血清。将LPS诱导的体外培养的小鼠肠黏膜上皮细胞随机分成空白组、模型组(LPS+空白血清)及金汁低含药血清组(LPS+10%金汁含药血清)、金汁中含药血清组(LPS+15%金汁含药血清)、金汁高含药血清组(LPS+20%金汁含药血清),各组干预24 h后,用透射电镜观察肠黏膜上皮细胞自噬情况,q-PCR法检测细胞中LC3A、LC3B mRNA表达情况,Western blot法检测细胞中LC3Ⅰ/LC3Ⅱ、p65、p-p65表达情况,ELISA法检测细胞上清液中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量。结果与空白组比较,模型组细胞内的双层膜结构明显减少,细胞中LC3A、LC3B mRNA相对表达量及LC3Ⅰ/LC3Ⅱ均明显降低(P均<0.05),p65、p-p65相对表达量及IL-1β、IL-6、TNF-α含量均明显升高(P均<0.05)。与模型组比较,金汁高含药血清组自噬体明显增多;金汁高含药血清组LC3A mRNA相对表达量和金汁低、中、高含药血清组LC3B mRNA相对表达量及LC3Ⅰ/LC3Ⅱ均明显升高(P均<0.05),金汁低、中、高含药血清组细胞中p65、p-p65相对表达量和金汁中、高含药血清组IL-1β、IL-6、TNF-α含量均明显降低(P均<0.05)。结论金汁含药血清可通过抑制NF-κB p65信号通路,促进肠黏膜上皮细胞自噬而修复受损的小鼠肠黏膜上皮细胞。

Objective It is to observe the effect of purified feces drug serum on autophagy related proteins and NF-κB p65 signal pathways of lipopolysaccharide(LPS)-induced mouse intestinal epithelial cells(IECs),and to explore its protective mechanism on intestinal epithelial cells.Methods Male BALB/c mice were selected to prepare blank serum and purified feces containing serum at different concentrations(10%,15%,20%).The LPS-induced mouse intestinal mucosal epithelial cells cultured in vitro were randomly divided into blank group,model group(LPS+blank serum)and purified feces low drug-containing serum group(LPS+10%purified feces drug-containing serum),purified feces medium drug-containing serum group(LPS+15%purified feces drug-containing serum)and purified feces high drug-containing serum group(LPS+20%purified feces drug-containing serum).After 24 hours of drug intervention,the autophagy of cells in each group was observed by transmission electron microscope,the expression of LC3A,LC3B mRNA was detected by Q-PCR,the expression of LC3Ⅰ/LC3Ⅱ,p65 and p-p65 was detected by Western blot,the contents of interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)in cell supernatant were detected by ELISA.Results Compared with the blank group,the bilayer membrane structure in the cells of the model group was significantly reduced,the relative expression of LC3A and LC3B mRNA and LC3Ⅰ/LC3Ⅱin the cells were significantly decreased(all P<0.05),and the relative expression of p65 and p-p65 and the content of IL-1β,IL-6 and TNF-αwere significantly increased(all P<0.05).Compared with the model group,the autophagosomes were significantly increased in the purified feces high drug-containing serum group;the relative expression of LC3A mRNA in the purified feces high drug-containing serum group and the relative expression of LC3B mRNA and LC3Ⅰ/LC3Ⅱin the purified feces low,medium and high drug-containing serum groups were significantly increased(all P<0.05),the relative expression of p65 and p-p65 in the cells of the purified feces low,medium and high drug-containing serum groups and the content of IL-1β,IL-6 and TNF-αin the purified feces medium and high drug-containing serum groups were significantly decreased(all P<0.05).Conclusion Purified feces containing serum could promote autophagy of mouse intestinal mucosal epithelial cells,thus to repair damaged intestinal mucosal epithelial cells though inhibiting NF-κB p65 signaling pathway.