清肠温中方对溃疡性结肠炎小鼠NLRP6/IL-18/MUC2轴及肠道黏液屏障的影响

Effects of Qingchang Wenzhong Decoction on NLRP6/IL-18/MUC2 Axis and Intestinal Mucus Barrier in Ulcerative Colitis Mice

目的研究清肠温中方对葡聚糖硫酸钠(DSS)诱导的溃疡性结肠炎(UC)小鼠NLRP6/IL-18/MUC2轴及肠道黏液屏障的影响及作用机制。方法24只SPF级雄性C57BL/6小鼠随机分为空白组、模型组、清肠温中方组和美沙拉嗪组,每组6只。空白组自由饮用去离子水,模型组、清肠温中方组和美沙拉嗪组自由饮用3%DSS溶液制备UC模型。造模7 d后,清肠温中方组、美沙拉嗪组予相应药液灌胃,空白组、模型组予去离子水灌胃,连续7 d。每日称量小鼠体质量,记录小鼠疾病活动指数(DAI)评分,HE染色观察结肠组织病理变化,AB-PAS染色观察结肠组织杯状细胞情况,Western blot检测结肠组织核苷酸结合寡聚化结构域样受体蛋白6(NLRP6)、白细胞介素(IL)-18蛋白表达,免疫荧光染色检测结肠组织黏蛋白2(MUC2)蛋白表达。结果与空白组比较,模型组小鼠体质量下降,DAI评分显著升高(P<0.01),结肠长度显著缩短(P<0.01),结肠组织黏膜屏障破坏,杯状细胞数量减少,黏液分泌减少;结肠组织NLRP6、MUC2蛋白表达显著降低(P<0.01),IL-18蛋白表达显著升高(P<0.01)。与模型组比较,清肠温中方组小鼠体质量增加,DAI评分显著降低(P<0.01),结肠长度显著增加(P<0.05);结肠黏膜损伤减轻,炎性细胞浸润减少,杯状细胞数量增多;结肠组织NLRP6、MUC2蛋白表达显著升高(P<0.01),IL-18蛋白表达显著降低(P<0.01)。结论清肠温中方能明显缓解UC小鼠疾病活动度,降低DAI评分,修复结肠组织病理损伤,增加杯状细胞数量,促进黏液分泌,其作用机制可能与调节NLRP6/IL-18/MUC2轴,修复肠道黏液屏障有关。

Objective To study the effects and mechanism of Qingchang Wenzhong Decoction(QCWZD)on NLRP6/IL-18/MUC2 axis and intestinal mucus barrier in DSS-induced ulcerative colitis(UC)mice.Methods Totally 24 SPF C57BL/6 male mice were randomly divided into blank group,model group,QCWZD group,and mesalazine group,with 6 mice in each group.The blank group was free to drink deionized water,the model group,QCWZD group and mesalazine group were free to drink 3% DSS solution to prepare the UC model.After 7 days of modeling,the QCWZD group and mesalazine group were given corresponding dose of drugs by gavage,the blank group and model group were given deionized water by gavage,for consecutive 7 d.The body mass was weighted everyday,DAI scores were recorded daily,HE staining was performed to detect the histopathological changes of colonic tissue,ABPAS staining was performed to observe the condition of colonic goblet cells,Western blot was performed to detect NLRP6 and IL-18 protein expression in colonic tissue,and immunofluorescence was performed to detected MUC2 protein expression in colonic tissue.Results Compared with the blank group,the body mass of the model group mice decreased,the DAI score significantly increased(P<0.01),and the colon length significantly shortened(P<0.01);the mucosal barrier of colonic tissue was destroyed,the number of goblet cells reduced,and the secretion of mucus was reduced;the expressions of NLRP6 and MUC2 protein in colonic tissue were significantly decreased(P<0.01),while the expression of IL-18 protein significantly increased(P<0.01).Compared with the model group,the QCWZD group showed an increase in body mass,a significant decrease in DAI score(P<0.01),and a significant increase in colon length(P<0.05);colonic mucosa damage was reduced,inflammatory cells infiltration was reduced,and the number of goblet cells increased;the expressions of NLRP6 and MUC2 protein in colonic tissue significantly increased(P<0.01),while the expression of IL-18 protein was significantly reduced(P<0.01).Conclusion QCWZD can significantly alleviate disease activity,decrease DAI score,repair of pathological damage to colon tissue,increase the number of goblet cells and promote mucus secretion in UC mice,and its mechanism of action may be related to the regulating of NLRP6/IL-18/MUC2 axis,repairing intestinal mucus barrier.