基于VIP信号改变的清热化湿调枢方对预防结肠炎小鼠肠黏膜屏障损伤的作用

The effect of Qingrehuashitiaoshu decoction granules based on VIP signal on preventing colonic mucosal barriers injury in DSS-induced colitis in mice

目的基于VIP及其受体变化,观察清热化湿调枢方对于预防DSS诱导的结肠炎小鼠模型肠黏膜屏障的损伤作用及其机制。方法选用24只C57BL/6雄性小鼠,正常组6只予纯水自由饮用,其余18只分为模型组、美沙拉秦对照组、清热化湿调枢方组,予2.5%葡聚糖硫酸钠(DSS)水溶液自由饮用,并分别予灌胃等量纯水、美沙拉秦混悬液(0.52g·kg^(-1)·d^(-1))、清热化湿调枢方(1.69g·kg^(-1)·d^(-1)),连续干预7d。观察各组小鼠的体质量、疾病活动指数(DAI)评分、结肠长度;结肠组织HE染色病理切片;ELISA法检测血清IL-2、IL-4水平;q-PCR法检测结肠组织IL-10 mRNA水平;Western Blot法检测sIgA、Claudin2、MUC2、VIP、VPAC1、VPAC2水平。结果与正常组小鼠比较,模型组小鼠体重下降,DAI分数升高,结肠长度缩短,差异有统计学意义(P<0.05),病理切片显示肠黏膜上皮结构损伤,炎症浸润;血清IL-2、IL-4差异不显著;结肠组织IL-10 mRNA、sIgA、Claudin2、MUC2、VIP、VPAC2水平降低,差异有统计学意义(P<0.05),结肠组织VPAC1水平升高,差异有统计学意义(P<0.05)。与模型组比较,清热化湿调枢方和美沙拉秦对照组都能降低小鼠DAI分数、恢复结肠长度,差异有统计学意义,另外,清热化湿调枢方比美沙拉秦更能恢复小鼠的体质量;清热化湿调枢方和美沙拉秦组小鼠有更紧致的肠黏膜上皮结构及更少的炎性细胞浸润;血清IL-2差异不显著,仅有清热化湿调枢方能提高小鼠血清IL-4水平;清热化湿调枢方和美沙拉秦对照组能恢复结肠组织中IL-10 mRNA、SIgA、Claudin2、MUC2、VIP、VPAC2水平,降低VPAC1水平,差异均有统计学意义(P<0.05)。相关性分析显示,VIP的改变与体重、Cladudin2和VPAC2存在相关性;VPAC1与SIgA存在相关性;VPAC2与DAI分数、IL-10 mRNA、MUC2、VIP存在相关性。结论清热化湿调枢方在一定程度上对于DSS诱导的结肠炎具有预防作用,并能恢复损伤的肠黏膜机械、化学、免疫屏障,且能调节免疫。其中,机械屏障的修复作用可能由VIP-VPAC2驱动;化学屏障的修复与VPAC2相关;免疫屏障的修复与VPAC1相关。

Objective Based on the changes of vasoactive intestinal peptide(VIP)signal and its receptors,to observe the protective effect and mechanism of Qingrehuashitiaoshu decoction(QRHSTSD)on colonic mucosal barriers injury in DSS-induced colitis mice model.Methods Twenty-four C57BL/6 male mice were selected.Six mice in the normal group were given pure water for free drinking.The remaining 18 mice were divided into model group,5-ASA group and QRHSTSD group.The mice were given 2.5%dextran sodium sulfate(DSS)aqueous solution for free drinking,and were given the same amount of pure water or 5-ASA suspension(0.52g/kg/d),QRHSTSD granules(1.69g/kg/d)for 7 days.The body weight,disease activity index(DAI)scores,colonic length and HE staining pathological section of the distal colon tissue of mice in each group were observed.Serum levels of IL-2 and IL-4 were detected by ELISA.The level of IL-10 mRNA in colon tissue was detected by q-PCR.The levels of sIgA,Claudin2,MUC2,VIP,VPAC1 and VPAC2 were detected by Western Blot.Results Compared with the normal group,the body weight of the mice in the model group decreased,the DAI score increased,and the colonic length was shortened.There was no significant difference in serum IL-2 and IL-4.The levels of IL-10 mRNA,sIgA,Claudin2,MUC2,VIP and VPAC2 in colon tissue were decreased,and the level of VPAC1 in colon tissue was increased.Compared with the model group,QRHSTSD and 5-ASA group could reduce the DAI scores and restore the colonic length of mice,and the difference was statistically significant.In addition,QRHSTSD could restore the body weight of mice more than 5-ASA.The mice in the QRHSTSD and 5-ASA group had tighter colonic mucosal epithelial structure and less inflammation.There was no significant difference in serum IL-2,and only QRHSTSD could increase serum IL-4 level in mice.The levels of IL-10 mRNA,SIgA,Claudin2,MUC2,VIP and VPAC2 in colon tissue were restored and the level of VPAC1 was decreased in QRHSTSD group and 5-ASA group.Conclusion QRHSTSD has a preventive effect on DSS-induced colitis to some degree,and can restore the damaged colonic mucosal mechanical,chemical,and immune barriers,and can regulate immunity.The regulation of mechanical barrier may be driven by VIP-VPAC2.The regulation of chemical barrier is related to VPAC2.And the regulation of immune barrier is related to VPAC1.