补脾益肠丸干预Notch信号重塑Th17/Treg细胞平衡治疗溃疡性结肠炎的作用机制

Action mechanism of Bupi Yichang Pills interfering with Notch signaling to reshape Th17/Treg cell homeostasis in the treatment of ulcerative colitis

目的:基于Th17/Treg细胞平衡探究补脾益肠丸(BPYCP)干预Notch信号通路治疗溃疡性结肠炎(UC)的作用机制。方法:葡聚糖硫酸钠诱导小鼠UC,BPYCP和5-ASA同步给药;实验期内观察小鼠临床症状及称重,实验结束时采集结肠样品、测量其长度并称重,病理组织技术评价结肠损伤;流式细胞术检测肠系膜淋巴结中Th17/Treg细胞比例,qPCR和ELISA检测相关核转录因子及分泌的细胞因子IL-17A/IL-10的表达水平;Western Blot和qPCR检测结肠组织中Notch信号分子DLL1、DLL3、DLL4、Jagged1、Jagged2、Notch1、Notch2、Notch3、Hes1、Hes2和Hes5的表达水平;相关性分析Notch1基因与Th17/Treg细胞的相关性,进一步流式检测Th17/Treg细胞的Notch1表达水平。结果:BPYCP可有效缓解DSS诱导的小鼠实验结肠炎,并呈典型的剂量依赖相关性,体质量损失减小、疾病活动指数下降、生存率上升,结肠显著延长,而结肠重量、结肠重量/结肠长度、结肠重量指数显著下降,结肠组织溃疡及炎性细胞浸润明显改善;与DSS组比较,中、高剂量BPYCP给药处理的结肠炎小鼠CD4^(+)CCR6^(+)、CD4^(+)IL-17A+Th17细胞及其转录调控因子(AHR,BATF,RORɑ和RORγt)、细胞因子IL-17A表达水平显著降低(P<0.05,P<0.01);与此同时,中、高剂量BPYCP给药处理的结肠炎小鼠CD4^(+)CD25^(+)、CD4^(+)CD25^(+)Foxp3^(+)、CD4^(+)IL-10+Treg细胞及其转录调控因子(Eomes,Foxp3)、细胞因子IL-10表达水平显著上升(P<0.05,P<0.01);此外,BPYCP抑制结肠炎小鼠Notch信号活化,结肠组织中DLL1、DLL4、Jagged1、Jagged2、Notch1、Notch2和Hes1基因蛋白表达水平显著下降(P<0.05,P<0.01);相关性分析进一步发现Notch1基因水平与Th17细胞数量呈正相关,而与Treg呈负相关,且BPYCP有效逆转了Th17和Treg细胞的Notch1的表达。结论:BPYCP重塑Th17/Treg细胞平衡有效治疗DSS诱导的小鼠UC,其潜在的作用机制与抑制Notch信号密切相关。

Objective:To investigate the mechanism of the intervention of Notch signaling pathway by Bupi Yichang Pills(BPYCP)in the treatment of ulcerative colitis(UC)based on the balance of Th17/Treg cells.Methods:UC was induced in mice by dextran sodium sulfate,and BPYCP and 5-ASA were administered simultaneously;mice were observed for clinical signs and weighed during the experimental period,and colon samples were collected,measured and weighed at the end of the experimental period,and colon tissue injury was evaluated by pathological histological techniques;the percentages of Th17/Treg cells in mesenteric lymph nodes were detected by flow cytometry,and the expression levels of their related nuclear transcription factors and secreted cytokines IL-17A/IL-10 were detected by qPCR and ELISA;Western Blot and qPCR methods were used to detect the expression levels of Notch signalling molecules DLL1,DLL3,DLL4,Jagged1,Jagged2,Notch1,Notch2,Notch3,Hes1,Hes2 and Hes5 in colonic tissues;the correlation analysis was applied to evalute the correlation between Notch1 gene and Th17/Treg cells,and flow cytometry was further used to detect the expression levels of Notch1 in Th17/Treg cells.Results:In this study,BPYCP effectively alleviated DSS-induced experimental colitis in mice in a typical dose-related manner,inhibited weight loss,down-regulated disease activity index,improved survival,and significantly lengthened colon,while colon weight,colon weight/colon length,and colon weight index were significantly reduced,and colonic mucosal ulceration and inflammatory cells infiltration were significantly improved.Compared with the DSS group,the expression levels of CD4^(+)CCR6^(+),CD4^(+)IL-17A+Th17 cells and their key transcription factors(AHR,BATF,RORɑand RORγt)and cytokine IL-17A in mesenteric lymph nodes were significantly decreased in colitis mice treated with medium and high doses of BPYCP administration(P<0.05,P<0.01).Medium and high doses of BPYCP administration significantly increased the expression levels of CD4^(+)CD25^(+),CD4^(+)CD25^(+)Foxp3^(+),CD4^(+)IL-10+Treg cells and their key transcription factors(Eomes,Foxp3),and cytokine IL-10 were significantly increased(P<0.05,P<0.01).In addition,BPYCP inhibited the activation of Notch signaling pathway in colitis mice,and the protein and gene expression levels of DLL1,DLL4,Jagged1,Jagged2,Notch1,Notch2,and Hes1 genes were significantly decreased in colonic tissues(P<0.05,P<0.01).The correlation analysis further revealed that Notch1 gene expression was positively correlated with Th17 cells and negatively correlated with Treg cells,and BPYCP effectively reversed Notch1 expression in Th17 and Treg cells.Conclusion:BPYCP reshapes the balance of Th17/Treg cells to effectively treat DSS-induced UC in mice,and the underlying mechanism of action is closely related to the inhibition of Notch signalling.