龙胆泻肝汤抑制葡萄膜炎大鼠Notch信号通路活化的作用

目的探讨龙胆泻肝汤(Longdan Xiegan Decoction,LXD)对实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis,EAU)大鼠Notch信号通路活化的抑制作用及其对辅助性T细胞17(T helper 17,Th17)和调节性T细胞(regulatory T cell,Treg)表达水平的影响。方法随机将雌性Lewis大鼠分为正常对照(NC)组、EAU模型组、LXD干预组。EAU模型组和LXD干预组大鼠诱导EAU,免疫后LXD干预组大鼠每天给予LXD灌胃处理,EAU模型组大鼠给予等量生理盐水灌胃。免疫后12 d观察大鼠眼部炎症表现,取三组大鼠眼球进行病理切片,观察病理学变化;实时荧光定量PCR(quantitative polymerase chain reaction,QT-PCR)和酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)检测免疫后12 d三组大鼠脾脏、淋巴结及眼组织中Notch1、DLL4、白细胞介素10(interleukin 10,IL-10)和IL-17 mRNA及蛋白的表达;流式细胞仪检测三组大鼠各组织中Th17、Treg细胞的表达。结果病理检查结果表明,LXD对EAU大鼠眼部组织结构有明显的保护作用。QT-PCR和ELISA检测结果发现,与NC组相比,LXD干预组大鼠脾脏、淋巴结和眼组织中Notch1、DLL4、IL-10、IL-17 mRNA和蛋白表达水平均升高,但除IL-10外,其他明显低于EAU模型组(均为P<0.05);流式细胞仪检测结果发现,EAU模型组大鼠的各组织中Th17/Treg比例均高于NC组,经LXD干预后,Th17细胞表达水平下降,Treg表达水平升高,两者比例趋向平衡。结论 LXD可有效降低EAU大鼠脾脏、淋巴结、眼组织中Notch1、DLL4、IL-10和IL-17 mRNA和蛋白的表达水平,改善Th17/Treg细胞比例的平衡,从而有效减轻EAU大鼠的眼部炎症,保护眼部组织结构,调节全身及眼部的免疫状态。

Inhibitory role of longdanxiegan decoction in the activation of notch signaling pathway in treating experimental autoimmune uveitis rats

Objective　To investigate the inhibitory role of Longdan Xiegan Decoction (LXD) in the activation of Notch signaling pathway in experimental autoimmune uveitis (EAU) rats and its effect on the levels of T helper 17 (Th17),regulatory T (Treg) cells.Methods　Female Lewis rats were randomly divided into normal control (NC) group,EAU model group and LXD intervention group.Rats in both EAU model group and LXD intervention group were induced EAU,and then the rats in LXD intervention group received intragastric administration of LXD every day,while those in EAU group were treated with the same volume of normal saline.The ocular fundus inflammation was observed at 12 d after immunization.The pathological sections of the rat eyeballs from three groups were extracted to observe pathological changes.Further,quantitative polymerase chain reaction (QT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expressions of Notch1,DLL4,interleukin-10 (IL-10) and IL-17 at mRNA and protein levels in spleen,lymph nodes and ocular tissues of three groups at 12 d after immunization.While the expressions of Th17 and Treg cells in different tissues in three groups were also detected by flow cytometry.Results　The pathological results showed that LXD exhibited obvious protective effect on the ocular tissue structure of EAU rats.QT-PCR and ELISA analyses showed that the expressions of Notch1,DLL4,IL-10 and IL-17 at mRNA and protein levels in spleen,lymph node and ocular tissues in LXD intervention group were higher than those of normal control group,whereas significantly lower than those of the EAU model group except IL-10 (all P<0.05).Flow cytometry measurement showed that the ratios of Th17/Treg in three tissues in EAU model group were higher than in NC group.LXD intervention apparently decreased the levels of Th17 cells,whereas the levels of Treg cells were increased,the ratio of Th17/Treg tended to balance.Conclusion　LXD can effectively attenuate the expressions of Notch1,DLL4,IL-10 and IL-17 at mRNA and protein levels in spleen,lymph nodes and ocular tissues in EAU rats,improve the balance of Th17/Treg cell ratio,and thus effectively reduce the severity of eye inflammation in EAU rats,protect the eye tissue structure,thereby regulating the immune state of the whole body and the eye environment.