清肠温中方含药血清调控NLRP6途径对Caco-2/HT29-MTX细胞共培养模型MUC2表达的影响

Effect of Serum Containing Qingchang Wenzhong Decoction Regulating NLRP6 Pathway on MUC2 Expression in Caco-2/HT29-MTX Cell Co-Culture Model

目的:研究清肠温中方含药血清调控NLRP6途径影响Caco-2/HT29-MTX细胞共培养模型MUC2分泌的作用机制。方法:使用SD大鼠制备清肠温中方含药血清;Caco-2/HT29-MTX细胞共培养构建单层肠上皮细胞模型;IL-1β刺激共培养细胞模拟体外肠道炎症模型;si-NLRP6质粒转染干扰NLRP6,研究清肠温中方作用机制。qPCR检测NLRP6、IL-18的mRNA表达水平;Western blot检测NLRP6、IL-18蛋白表达水平;免疫荧光染色观察MUC2表达。结果:正常培养的Caco-2/HT29-MTX细胞中,NLRP6敲除后,MUC2表达明显减少。清肠温中方含药血清增加IL-1β诱导的细胞炎症模型中NLRP6和IL-18 mRNA及蛋白表达水平(P<0.05,P<0.01),且一定程度上抵消si-NLRP6的抑制作用。与模型组相比,清肠温中方含药血清干预后,促进MUC2蛋白表达恢复,si-NLRP6后一定程度上削弱了清肠温中方的作用。结论:清肠温中方含药血清通过调控NLRP6信号途径,进一步调节MUC2分泌,修复UC黏液屏障损伤。

Objective:To investigate the mechanism by which Qingchang Wenzhong Decoction(QCWZD)regulates the secretion of MUC2 in a co-culture model of Caco-2/HT29-MTX cells through the NLRP6 pathway.Methods:SD rats were used to prepare QCWZD-containing serum.Caco-2/HT29-MTX cells were co-cultured to create a monolayer intestinal epithelial cell model.IL-1 stimulated the co-cultured cells to mimic an in vitro intestinal inflammation model,and si-NLRP6 plasmid was transfected to interfere with NLRP6 to study the action mechanism of QCWZD.The mRNA expression of NLRP6 and IL-18 was detected using qPCR.Western blot was used to detect the protein expression of NLRP6 and IL-18,and immunofluorescence staining was used to evaluate MUC2 expression.Results:In normal cultured Caco-2/HT29-MTX cells,if NLRP6 is knocked down,MUC2 expression will decrease significantly.QCWZD-containing serum increased NLRP6 and IL-18 mRNA(P<0.01,P<0.001)and protein expression levels in IL-1β-induced cellular inflammation model(P<0.001,P<0.001),and to some extent counteracted the inhibitory effect of si-NLRP6.Compared with the model group,QCWZD intervention promoted the recovery of MUC2 protein expression,and si-NLRP6 could somewhat weaken the effect of QCWZD.Conclusion:QCWZD-containing serum regulates MUC2 secretion and repairs UC mucus barrier damage by modulating the NLRP6 signaling pathway.