摘要
目的 研究雷公藤红素(CSR)对溃疡性结肠炎(UC)小鼠树突状细胞(DC)与滤泡辅助性T细胞(Tfh)亚群的影响.方法 将48只雄性BALB/c小鼠随机分为健康对照组、模型组和CSR干预组,每组16只.健康对照组给予正常纯净水喂养,模型组和CSR干预组小鼠饮用3％DSS溶液诱导为UC模型.自模型诱导开始,CSR干预组小鼠每日给予1 mg/kg的CSR灌胃,模型组以相同方式给予等量生理盐水.记录各组小鼠体质量、大便性状,评估小鼠的疾病活动指数(DAI).8 d干预结束后,测量小鼠结肠长度,观察组织病理学变化,并计算组织病理学评分.采用流式细胞术检测结肠组织固有层、肠系膜淋巴结和脾脏中DC、经典DC(CD8α+cDC1、CD103+cDC1、cDC2)、浆细胞样DC(pDC)与Tfh亚群百分比.使用多因子检测试剂盒测定结肠组织中DC与Tfh相关细胞因子[白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、白细胞介素21(IL-21)]的表达水平.体外实验检测CSR对初始CD4+T细胞增殖和Tfh分化的影响.结果 建模成功率100％,小鼠均存活.与模型组比较,CSR干预组小鼠体质量更高,DAI更低,结肠长度短缩明显减轻,差异均具有统计学意义(均P＜0.05).模型组小鼠肠黏膜组织结构紊乱,并有大量炎性细胞浸润;CSR干预组小鼠肠黏膜屏障趋近正常结构,炎性细胞较模型组减少,组织病理学评分明显降低(P＜0.05).在结肠固有层中,与模型组相比,CSR干预组小鼠DC、CD8α+cDC1、Tfh百分比均降低,CD103+cDC1百分比升高,差异均有统计学意义(均P＜0.05).在肠系膜淋巴结中,与模型组相比,CSR干预组CD8α+cDC1百分比降低,DC、CD103+cDC1、cDC2、Tfh百分比均升高,差异均有统计学意义(均P＜0.05).在脾脏中,与模型组相比,CSR干预组pDC百分比显著降低(P＜0.05).CSR干预组小鼠结肠组织中IL-6、TNF-α、IL-21表达水平均低于模型组,而IL-10表达水平显著高于模型组,差异均有统计学意义(均P＜0.05).在体外实验中,CSR可抑制初始CD4+T细胞增殖,并抑制Tfh分化,差异均有统计学意义(均P＜0.05).结论 CSR可以缓解UC小鼠肠道损伤,可能与其调节DC与Tfh亚群进而调控局部免疫微环境有关.
Abstract
Objective To investigate the effects of celastrol(CSR)on dendritic cell(DC)and T follicular helper cell(Tfh)subsets in the mouse of ulcerative colitis(UC).Methods Forty-eight male BALB/c mice were randomly divided into healthy control group,model group,and CSR intervention group,with 16 mice in each group.The healthy control group was fed with normal purified water,while the mice in model group and CSR intervention group were fed with 3％DSS solution to induce UC model.Since the induction,the mice in CSR intervention group were gavaged with 1mg/kg of CSR,and the mice in UC group were gavaged with equal volume of saline once a day.The weight and stool characteristics of the mice were recorded,and disease activity index(DAI)were evaluated.After the 8-day intervention,the length of the mouse colon was measured,the histopathological changes were observed,and the histopathological score was evaluated.Flow cytometry was used to detect the percentage of DC,conventional DC(CD8α+cDC1,CD103+cDC1,cDC2),plasmacytoid DC(pDC),and Tfh subsets in colon lamina propria,mesenteric lymph nodes and spleen.Cytometric bead array kit was used to detect the expression levels of DC and Tfh related cytokines[interleukin 6(IL-6),tumor necrosis factor α(TNF-α),interleukin 21(IL-21)]in colon tissue.The influence of CSR on naive CD4+T cell proliferation and Tfh differentiation were validated in vitro experiments.Results The modelling success rate was 100％and all mice survived.Compared with model group,mice in CSR intervention group had heavier weight,lower DAI,and ameliorated colonic length shortening,with all differences being statistically significant(all P＜0.05).The intestinal mucosal structure of mice in model group was disordered,with a large number of inflammatory cell infiltration;the intestinal mucosal barrier of mice in CSR intervention group approached normal structure,with fewer inflammatory cells,and the histopathological scores were significantly decreased(P＜0.05).In the colon lamina propria,compared with model group,the percentages of DC,CD8α+cDC1 and Tfh decreased,while the percentage of CD103+cDC1 increased in CSR intervention group,and these differences were all statistically significant(all P＜0.05).In mesenteric lymph nodes,the percentage of CD8α+cDC1 decreased,while the percentages of DC,CD 103+cDC1,cDC2 and Tfh increased in CSR intervention group compared with model group,and these differences were all statistically significance(all P＜0.05).In the spleen,compared with model group,the percentage of pDC was significantly reduced in CSR intervention group(P＜0.05).The expression levels of IL-6,TNF-α and IL-21 in colon tissues of CSR intervention group were lower,while IL-10 was higher than those of model group,and these differences were statistically significant(all P＜0.05).In vitro experiments,CSR could inhibit naive CD4+T cell proliferation and Tfh differentiation,with statistically significant differences(all P＜0.05).Conclusion CSR can alleviate intestinal damage in UC mice,potentially by modulating the local immune microenvironment through regulating DC and Tfh subsets.
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