补脾益肠丸重塑CD4^(+)T细胞亚群稳态缓解溃疡性结肠炎的机制研究

Study on mechanism of Bupi Yichang pill in alleviating experimental ulcerative colitis by restoring the homeostasis of CD4^(+)T cell subpopulations

目的探究补脾益肠丸(BPYCP)对实验性溃疡性结肠炎(UC)小鼠CD4^(+)T细胞亚群的调控作用。方法48只C57BL/6小鼠采用随机数字表法分为对照组(Control组,10只)、模型组(DSS组,13只)、模型+补脾益肠丸组(DSS+BPYCP组,13只)、模型+5-ASA组(DSS+5-ASA组,12只);采用自由饮用2.5%葡聚糖硫酸钠(DSS)溶液诱导小鼠UC模型,DSS+BPYCP组和DSS+5-ASA组分别采用BPYCP或美沙拉嗪(5-ASA)连续灌胃2周。观察小鼠粪便黏稠度及便血情况,测量结肠长度,结肠称质量并计算结肠质量指数及单位结肠质量指数;苏木素-伊红(HE)染色法观察结肠病理变化,并进行病理损伤评分;流式细胞术检测肠系膜淋巴结中的CD4^(+)T细胞亚群水平;酶联免疫吸附试验检测干扰素(INF)-γ、白细胞介素(IL)-4、IL-17A、IL-10及IL-21表达水平;实时荧光定量PCR检测结肠组织T-框蛋白21(T-bet)、GATA结合蛋白3(GATA-3)、维甲酸相关核孤儿受体γt(RORγt)、B细胞淋巴瘤-6(Bcl-6)及大鼠叉头蛋白P3(Foxp3)mRNA表达水平。结果与DSS组比较,DSS+BPYCP组和DSS+5-ASA组UC小鼠腹泻、便血等症状得到改善,小鼠体质量及结肠长度均增加,且结肠质量、结肠质量指数及单位结肠质量指数均下降,黏膜上皮较完整,腺体排列更规整,炎性细胞浸润更少,病理组织损伤评分显著降低,肠系膜淋巴结中的Th2细胞比例降低,Th17细胞比例及IL-17A水平降低,结肠组织T-bet、GATA-3、RORγt、Bcl-6 mRNA水平降低(P<0.05);DSS+BPYCP组的Th1细胞比例降低,CD4^(+)CD25^(+)Treg、CD4^(+)CD25^(+)Foxp3^(+)Treg细胞比例及IL-10水平均升高,CD4^(+)CXCR5^(+)Tfh细胞比例和IL-21水平降低,Foxp3 mRNA水平升高(P<0.05);DSS+5-ASA组的Th1细胞比例及IFN-γ水平降低(P<0.05)。结论BPYCP可能通过重塑肠道组织的CD4^(+)T细胞亚群稳态缓解实验性UC。

Objective To investigate the regulatory effect of Bupi Yichang pill(BPYCP)on CD4^(+)T cell subsets of ulcerative colitis(UC)mice.Methods Forty-eight C57BL/6 mice were randomly divided into 4 groups:the control group(n=10),the model group(DSS group,n=13),the model+BPYCP group(DSS+BPYCP group,n=13)and the model+mesalazine(5-ASA)group(DSS+5-ASA group,n=12).The mouse UC model was induced by 2.5%dextrosan sulfate(DSS)solution.The DSS+BPYCP group and the DSS+5-ASA group were given BPYCP or 5-ASA for 2 weeks,respectively,and fecal viscosity and blood in stool were observed.The colon length was measured.Colonic mass index and unit colonic mass index were calculated.Hematoxylin-eosin(HE)staining was used to observe pathological changes of colon and to score the pathological tissue damage.The level of CD4^(+)T cell subsets in mesenteric lymph nodes was detected by flow cytometry.The expression levels of cytokines interferon-γ(INF-γ),interleukin(IL-4),IL-17A,IL-10 and IL-21 secreted by CD4^(+)T cell subsets in colon tissue were detected by ELISA.Real-time fluorescence quantitative PCR was used to detect colon tissue CD4^(+)T cell subset nuclear transcription factors,mRNA expression levels of T-frame protein 21(T-bet),GatA-binding protein 3(GATA-3),retinoa-associated nuclear orphan receptorγt(RORγt),B cell lymphoma-6(Bcl-6)and Foxp3 in rats.Results Compared with the DSS group,the diarrhea and hematostoecium symptoms of UC mice in the DSS+BPYCP group and the DSS+5-ASA group were significantly improved,body weight and colon length of mice were increased,and colon mass,colon mass index and unit colon mass index were decreased(P<0.05).The mucosal epithelium was more complete than that in the DSS group,and gland arrangement was more regular.The inflammatory cell infiltration was less,and the pathological tissue damage score was significantly decreased(P<0.01).The proportion of Th2 cells in mesenteric lymph nodes was decreased,the proportion of Th17 cells and the level of IL-17A were decreased,and the mRNA levels of T-bet,GATA-3,RORγt and Bcl-6 in colon tissue were decreased(P<0.05).In the DSS+BPYCP group,the proportion of Th1 cells decreased,the proportion of CD4^(+)CD25+Treg cells,CD4^(+)CD25+Foxp3+Treg cells and the level of IL-10 increased,and the proportion of CD4^(+)CXCR5+Tfh cells and the level of IL-21 decreased.The level of Foxp3 mRNA increased(P<0.05).The proportion of Th1 cells and the level of IFN-γwere decreased in the DSS+5-ASA group(P<0.05).Conclusion BPYCP may alleviate UC by remodeling the homeostasis of CD4^(+)T cell subpopulations.