基于PKCθ-Ikk-NF-κB信号通路研究白头翁汤介导NKT细胞抑制分泌IL-13因子治疗UC机制  

Study on the Mechanism of Pulsatilla Decoction Mediating NKT Cell Inhibition of IL-13 in the Treatment of UC in PKCθ-Ikk-NF-κB Pathway

作　　者：王学伟[1] 徐丽君[1] 曹勤[1] 汪涛 许健[1] WANG Xuewei;XU Lijun;CAO Qin;WANG Tao;XU Jian(Division of Gastroenterology,Putuo Hospital,Shanghai University ofTraditional Chinese Medicine,Shanghai 200062,China)

机构地区：[1]上海中医药大学附属普陀医院消化内科,上海200062

出　　处：《甘肃科学学报》2022年第2期43-48,共6页Journal of Gansu Sciences

基　　金：2018年上海市普陀区卫生系统自主创新科研资助项目(ptkwws201806)。

摘　　要：探究白头翁汤通过抑制自然杀伤T细胞(NKT细胞)分泌白细胞介素-13(IL-13)细胞因子治疗溃疡性结肠炎(UC)的作用机制。首先用噁唑酮建立小鼠结肠炎模型,分离培养结肠黏膜NKT细胞。为进一步明确与NKT细胞表达IL-13相关的信号通路和转录因子,采用药理学特异性激酶抑制剂抑制的方法,先分别抑制PLCγ1-Calcineurin-NFAT、MEK-ERK、PI3K-Akt-mTORC1及PKCθ-Ikk-NF-κB信号通路,再用抗CD3和抗CD28单克隆抗体刺激,最后用实时荧光定量PCR(RT-PCR)和酶联免疫吸附实验(ELISA)观察IL-13 mRNA和蛋白表达水平以及用蛋白质免疫印迹实验(Western blot)测定心肌转录因子3(GATA3)、早幼粒细胞白血病锌指蛋白(PLZF)、T盒子转录因子(T-bet)、维甲酸相关孤儿受体γτ(RORγτ)等转录因子的表达变化。结果显示,治疗组和模型组相比,小鼠的生长状况明显好转,小鼠黏血便和黏液减少最后消失,而且小鼠进食增加,体重明显升高,白头翁汤组结肠黏膜组织损伤和疾病活动指数评分(DAI)降低(P<0.05)。RT-PCR和ELISA结果显示,和对照组相比,模型组IL-13的RNA和蛋白质表达水平明显升高(P<0.01),而用白头翁汤治疗后,IL-13在分子和蛋白水平的表达发生了下降(P<0.05)。用1μM AEB071抑制PKCθ蛋白激酶后,模型组NKT细胞表达IL-13分子水平显著降低,而且白头翁组NKT细胞NF-κB的表达水平明显下降,与模型组相比,差异显著(P<0.01)。研究表明白头翁汤可能通过PKCθ-Ikk-NF-κB信号通路降低IL-13因子的表达促使小鼠炎症减轻,肠壁损伤修复,恢复结肠黏膜的分泌等功能,最终使小鼠恢复到正常生理状态。The purpose of this paper is to explore the mechanism of Pulsatilla decoction in treating ulcerative colitis(UC)by inhibiting the secretion of IL-13 cytokines of NKT cells.Firstly,Oxazolidone was used to establish a colitis model in mice,and then NKT cells of colonic mucosa were isolated and cultured.To further clarify the signaling pathways and transcription factors related to the expression of IL-13 in NKT cells,pharmacologically specific kinase inhibitors were used in the following experiments.First,it used inhibitors to supress the PLCγ1-Calcineurin-NFAT,MEK-ERK,PI3K-Akt-mTORC1 and PKCθ-Ikk-NF-κB signaling pathways respectively,and then stimulated with anti-CD3 and anti-CD28 monoclonal antibodies.Finally,the expression levels of IL-13 mRNA and protein were detected singly by real-time quantitative PCR(RT-PCR)and ELISA,and the expression of GATA3,PLZF,T-BET,ROR was determined by Western blot.The results show that compared with the model group,the growth status of rats in Pulsatilla decoction group was significantly improved,the mucous stool and mucous fluid of the rats were reduced and finally disappeared,and the appetite of the rats was improved,the weight of the mice was significantly increased,and the colonic mucosal tissue injury and histopathological score of the mice in the Pulsatilla decoction group were decreased(P<0.05).1μM AEB071 was used to inhibit PKCθkinase,then the expression level of IL-13 in NKT cells in the model group was significantly decreased,and the expression level of NF-κB of the NKT cells in the pulsatillae group was significantly decreased,compared with that in the model group(P<0.01).Therefore,Pulsatilla decoction may reduce the expression of IL-13 in mice through PKCθ-Ikk-NF-κB signaling pathways,which can significantly reduce the inflammation,repair intestinal wall damage,and restore the secretion function of colonic mucosa.

关 键 词：白头翁汤 UC NKT细胞 IL-13 PKCθ-Ikk-NF-κB信号通路 

分 类 号：R737.14[医药卫生—肿瘤]