萸连丸对溃疡性结肠炎小鼠脾脏CD4^(+)T细胞BCL-6/Blimp-1,PD-1/PD-L1表达的调节作用

Yulian Pills(萸连丸)Regulate the Expression of BCL-6/Blimp-1 and PD-1/PD-L1 in Spleen CD4^(+)T Cells in the Mouse Model of Ulcerative Colitis

目的:探讨萸连丸对葡聚糖硫酸钠(DSS)诱导的溃疡性结肠炎(Ulcerative colitis,UC)小鼠脾脏CD4^(+)T细胞转录因子B细胞淋巴瘤因子6/B细胞诱导成熟蛋白1(BCL-6/Blimp-1),程序性死亡受体-1/程序性死亡受体配体-1(PD-1/PD-L1)表达的调节作用。方法:将40只BALB/c雄性小鼠随机分为正常对照组、模型对照组、萸连丸500 mg/kg组以及美沙拉嗪300 mg/kg组。除正常对照组小鼠外,其余小鼠采用DSS复制慢性溃疡性结肠炎模型,造模成功后,各组灌胃给予药物或生理盐水,观察小鼠体质量、结肠长度、结肠质量、肠质量指数、结肠黏膜病理形态及病理组织学损伤评分;ELISA法检测结肠组织中白细胞介素-7(IL-7)、IL-9、IL-23含量;流式细胞术检测小鼠脾脏中CD4^(+)BCL-6^(+)、CD4^(+)Blimp-1^(+)、CD4^(+)PD-1^(+)及CD4^(+)PD-L1^(+)细胞水平;Western blot法检测结肠组织中BCL-6、Blimp-1蛋白表达。结果:与正常对照组相比,模型对照组结肠组织中IL-7、IL-9、IL-23含量明显升高(P<0.05),小鼠脾脏中CD4^(+)BCL-6^(+)、CD4^(+)PD-1^(+)、CD4^(+)PD-L1^(+)细胞水平显著升高(P<0.01),CD4^(+)Blimp-1^(+)细胞水平显著降低(P<0.01),BCL-6蛋白表达明显上调(P<0.05),Blimp-1蛋白表达显著下调(P<0.01);与模型对照组相比,萸连丸500 mg/kg组小鼠体质量和结肠长度明显增加、肠质量指数明显降低(P<0.05),结肠组织中IL-7、IL-9、IL-23含量显著降低(P<0.01),小鼠脾脏中CD4^(+)BCL-6^(+)、CD4^(+)PD-1^(+)、CD4^(+)PD-L1^(+)细胞水平明显降低(P<0.05),CD4^(+)Blimp-1^(+)细胞水平明显升高(P<0.05),BCL-6蛋白表达明显下调(P<0.05),Blimp-1蛋白表达明显上调(P<0.05)。结论:萸连丸可能通过调节小鼠CD4^(+)T细胞表面BCL-6/Blimp-1,PD-1/PD-L1表达水平改善溃疡性结肠炎。

Objective:To investigate the modulating effects of Yulian Pills(萸连丸)on the expression of B-cell lymphoma 6(BCL-6)/B-lymphocyte-induced maturation protein 1(Blimp-1)and programmed death receptor-1(PD-1)/programmed death ligand-1(PD-L1)in spleen CD4^(+)T cells in the mouse model of dextran sodium sulfate(DSS)-induced ulcerative colitis(UC).Methods:Forty SPF-grade BALB/c male mice were randomized into normal control,model control,Yulian Pills(500 mg/kg),and mesalazine(300 mg/kg)groups.The mice in other groups except the normal control group were induced by DSS to establish the UC model.After successful modeling,each group was administered with corresponding drugs or saline by gavage.The body mass,colon length,colon weight,intestinal weight index,pathological morphology of colon mucosa,and histopathological damage score were determined to evaluate the therapeutic effects.The expression levels of interleukin(IL)-7,IL-9,and IL-23 in the colonic tissue were measured by enzyme-linked immunosorbent assay.The levels of CD4^(+)BCL-6^(+),CD4^(+)Blimp-1^(+),CD4^(+)PD-1^(+),and CD4^(+)PD-L1^(+)cells in the mouse spleen were measured by flow cytometry.Western blotting was employed to determine the expression levels of BCL-6 and Blimp-1 in the colon tissue.Results:Compared with the normal control group,the model control group showed elevated levels of IL-7,IL-9 and IL-23 in the colon tissue(P<0.05),increased CD4^(+)BCL-6^(+),CD4^(+)PD-1^(+),and CD4^(+)PD-L1^(+)cells(P<0.01),reduced CD4^(+)Blimp-1^(+)cells(P<0.01),up-regulated BCL-6 protein level(P<0.05),and down-regulated Blimp-1 protein level(P<0.01).Compared with the model control group,Yulian Pills increased the body mass and colon length(P<0.05)and decreased the intestinal weight index(P<0.05).Furthermore,Yulian Pills lowered the levels of IL-7,IL-9,and IL-23 in the colon tissue(P<0.01),reduced CD4^(+)BCL-6^(+),CD4^(+)PD-1^(+),and CD4^(+)PD-L1^(+)cells(P<0.05),increased CD4^(+)Blimp-1^(+)cells(P<0.05),down-regulated the protein level of BCL-6(P<0.05),and up-regulated the protein level of Blimp-1(P<0.05).Conclusion:Yulian Pills may mitigate UC by regulating the expression levels of BCL-6/Blimp-1 and PD-1/PD-L1 on the surface of CD4^(+)T cells in mice.