龙胆泻肝汤抑制Notch信号通路活化对实验性自身免疫性葡萄膜炎大鼠Th1、Th2细胞分化的影响

目的　探讨龙胆泻肝汤（LXD）对Notch信号通路活化的抑制作用及其对实验性自身免疫性葡萄膜炎（EAU）大鼠Th1、Th2细胞分化的影响。方法　将30只Lewis大鼠随机分为正常对照组、EAU模型组和LXD干预组，其中EAU模型组、LXD干预组大鼠均诱导EAU，LXD干预组大鼠造模后使用LXD每天灌胃处理，EAU模型组和正常对照组大鼠给予等量生理盐水灌胃。干预后12 d分离三组大鼠的脾脏、淋巴结和眼组织，Q-PCR检测Rbpj基因的表达，ELISA检测Rbpj、γ干扰素（IFN-γ）和白细胞介素-4（IL-4）蛋白的表达，流式细胞仪检测各组织中Th1、Th2细胞的表达水平，分析Th1/Th2细胞比例的变化。结果　干预后12 d，Q-PCR检测发现，与正常对照组大鼠相比， Rbpj mRNA在EAU模型组大鼠脾脏、淋巴结和眼组织中均呈显著上调表达(均为P<0.001)；与EAU模型组相比，LXD干预组大鼠脾脏、淋巴结和眼组织中Rbpj mRNA相对表达水平均显著降低(均为P<0.01)。ELISA检测结果发现，EAU模型组大鼠脾脏、淋巴结和眼组织中Rbpj和IFN-γ蛋白表达水平均明显高于正常对照组，IL-4蛋白表达水平均明显低于正常对照组（均为P＜0.05)；相比于EAU模型组，LXD干预组大鼠脾脏、淋巴结以及眼组织中Rbpj IFN-γ蛋白表达水平均显著降低，IL-4蛋白表达水平均显著升高（均为P＜0.05)。流式细胞仪检测发现，与正常对照组相比，EAU模型组大鼠脾脏、淋巴结和眼组织中Th1细胞水平均明显升高，Th2细胞水平均明显降低，Th1/Th2细胞比例失衡（均为P＜0.05）；与EAU模型组相比，LXD干预组大鼠各组织中Th1细胞水平均明显下降，Th2细胞水平均明显升高，两者细胞比例逐渐恢复均衡（均为P<0.01)。结论　LXD可通过下调EAU大鼠Notch信号通路转录因子Rbpj的表达水平抑制Notch信号通路的活化，显著促进EAU大鼠中Th1/Th2细胞比例恢复平衡并改善免疫微环境，从而达到治疗葡萄膜炎的目的。

Effects of Longdan Xiegan Decoction on Th1 and Th2 cell differentiation by inhibiting Notch pathway activation in experimental autoimmune uveitis rats

Objective　To investigate the inhibition effect of Longdan Xiegan Decoction (LXD) on Notch signaling activation and Th1 and Th2 cell differentiation in experimental autoimmune uveitis (EAU) rats.Methods　Totally 30 Lewis rats were randomly divided into normal control (NC) group, EAU model group and LXD intervention group. A model of uveitis was induced in rats of the EAU model group and LXD intervention group. The rats in the LXD intervention group were given LXD intragastrically every day after modeling, while the rats in EAU model group and the NC group were treated with the same volume of normal saline intragastrically. After 12 days of the intervention, the spleen, lymph nodes and eye tissues of the rats in the three groups were isolated. Furthermore, Q-PCR was used to detect the expression of Rbpj mRNA, while enzyme-linked immunosorbent assay (ELISA) was used to measure the protein expression of Rbpj, IFN-γ and IL-4. In addition, the levels of Th1 and Th2 cells in each tissue were detected by flow cytometry, and the proportional changes of Th1/Th2 cell were further analyzed.Results　On day 12 after intervention, compared with the NC group, the result of Q-PCR showed that Rbpj mRNA expression was significantly upregulated in spleen, lymph node and eye tissues in the EAU model group (all P<0.001); compared to the EAU model group, the relative expression levels of Rbpj mRNA in spleen, lymph nodes and eye tissues in LXD intervention group was significantly decreased (all P<0.01). ELISA results revealed that compared to NC group, the expression levels of Rbpj and IFN-γ in spleen, lymph nodes and eye tissues in the EAU model group were significantly elevated, whereas the expression levels of IL-4 were significantly decreased (all P<0.05). Compared with the EAU model group, the levels of Rbpj and IFN-γ protceins in spleen, lymph nodes and eye tissues in the LXD intervention group were significantly decreased, whereas the levels of IL-4 were significantly increased (all P<0.05). The result of flow cytometry showed that compared with the NC group, the expression levels of Th1 in spleen, lymph node and eye tissues in the EAU model group were significantly increased, whereas the expression levels of Th2 were significantly decreased, and the ratio of Th1/Th2 showed at an unbalanced expression state (all P< 0.05); compared with the EAU model group, the Th1 cell levels decreased and the Th2 cell levels increased in the tissues of the LXD intervention group, and the proportion of Th1/Th2 gradually returned to the balance (all P<0.01).Conclusion　LXD can inhibit the activation of Notch signaling pathway by down-regulating the expression level of Rbpj, and significantly restore the balance of Th1/Th2 ratio and improve the immune microenvironment in EAU rats, thereby achieving the purpose of treating uveitis.