基于PKCθ/STAT3信号通路探讨虎杖苷抑制溃疡性结肠炎小鼠Th17细胞的效应

Polydatin Ameliorated Colonic Inflammation via Precluding Phosphorylation of PKCθ/STAT3 Pathway to Inhibit Th17 Cells in Mice with Ulcerative Colitis

目的:探讨虎杖苷对小鼠溃疡性结肠炎(UC)的治疗作用,并通过其调控蛋白激酶Cθ(PKCθ)/信号转导与转录激活因子3(STAT3)通路信号对辅助性T细胞17(Th17)细胞效应的抑制作用探讨其治疗UC的作用机制。方法:32只雄性C57BL/6小鼠随机分为正常组、模型组、虎杖苷组(0.045 g·kg^(-1))及柳氮磺吡啶组(0.5 g·kg^(-1))。采用3%葡聚糖硫酸钠(DSS)溶液给与小鼠自由饮水构建UC模型,虎杖苷和柳氮磺吡啶组于造模前0.5 h灌胃给药,连续7 d,正常组及模型组给予等体积生理盐水灌胃。末次给药后取结肠组织,苏木素-伊红(HE)染色观察结肠组织病理变化,流式细胞术检测结肠黏膜固有层中Th17比例,酶联免疫吸附测定法(ELISA)检测血清中白细胞介素-17A(IL^(-1)7A)的表达。磁珠分选提取C57BL/6小鼠脾脏CD4+T细胞进行体外培养,加入细胞刺激合剂刺激并随机分为正常组、模型组、虎杖苷低、高剂量组。流式细胞术检测虎杖苷对Th17效应的影响及蛋白免疫印迹法(Western blot)检测磷酸化PKCθ和STAT3的蛋白表达。结果:与正常组比较,模型组小鼠体质量明显下降、疾病活动指数(DAI)评分显著升高(P<0.01),结肠黏膜上皮破损并可见黏膜及黏膜下层炎细胞浸润明显,结肠黏膜固有层中Th17的比例显著升高(P<0.01),血清IL^(-1)7A的含量显著增加(P<0.01);与模型组比较,虎杖苷和柳氮磺吡啶组体质量及DAI评分均有显著的改善(P<0.01),结肠组织损伤程度明显改善,结肠黏膜固有层中Th17的比例显著下降(P<0.01),血清中IL^(-1)7A的含量显著下降(P<0.01)。体外实验显示,与正常组比较,模型组Th17细胞比例显著增加(P<0.01),PKCθ和STAT3的磷酸化水平显著升高(P<0.01);与模型组比较,虎杖苷组Th17细胞比例显著下降(P<0.01),PKCθ和STAT3的磷酸化水平显著降低(P<0.01)。结论:虎杖苷能够抑制PKCθ以及STAT3的磷酸化表达,进而抑制Th17细胞分泌IL^(-1)7A,改善肠黏膜炎性病理,对UC起治疗作用。

Objective: To investigate the therapeutic effect of polydatin on ulcerative colitis(UC)in mice and its regulation of protein kinase Cθ(PKCθ)/signal transducer and activator of transcription 3(STAT3)signaling on T helper cell 17(Th17)and its mechanism in the treatment of UC. Method:The 32 male C57 BL/6 mice were randomly divided into normal group,model group,polydatin group(0.045 g·kg^(-1))and sulfasalazine group(0.5 g·kg^(-1)). The UC model was established by giving 3% dextran sodium sulfate(DSS)solution to free drinking water in mice. Polydatin and sulfasalazine groups were given by gavage 0.5 h before modeling for 7 days. The normal group and model group were given the same amount of normal saline. After the last administration,the colonic tissue was taken and hematoxylin-eosin(HE)was used to observe the pathological changes of colonic tissue. Flow cytometry was used to detect the proportion of Th17 in the lamina propria of colonic mucosa. The expression of interleukin-17A(IL^(-1)7A) in serum was detected by enzyme-linked immunosorbent assay(ELISA). Polydatin was added to CD4+T cells purified from spleen of C57 BL/6 mice by magnetic-activated cell sorting(MACS) under the stimulation of cell stimulation cocktail in vitro in order to detect its impact on PKCθ and STAT3 phosphorylation. Result: Compared with normal group,the body weight was significantly decreased,and disease activity index(DAI) scores of the model group was significantly increased(P<0.01),the colonic mucosal epithelium was damaged and inflammatory cells infiltration in the mucosa and submucosa was obvious,the proportion of Th17 in the lamina propria of colonic mucosa was significantly increased(P<0.01),and the content of serum IL^(-1)7A was significantly increased(P<0.01).Compared with the model group, the weight and DAI score of polydatin and sulfasalazine groups were significantly improved(P<0.01),the degree of colon tissue damage was significantly improved,the proportion of Th17 in colon mucosa lamina propria was significantly decreased(P<0.01),and the content of IL^(-1)7A in serum was significantly decreased(P<0.01). In vitro experiments showed that polydatin could significantly inhibit the phosphorylation of PKCθ and STAT3 in Th17(P<0.01) as well as IL^(-1)7A secretion. Conclusion:Polydatin can improve the ulcerative colitis in mice via inhibiting the phosphorylation of PKCθ and STAT3 to preclude IL^(-1)7A secreting in Th17.