龙胆泻肝汤对葡萄膜炎大鼠Notch信号通路的干预作用

目的　探讨龙胆泻肝汤对实验性自身免疫性葡萄膜炎（experimental autoimmune uveitis，EAU）大鼠Notch信号通路相关基因表达的影响。方法　Lewis大鼠用随机数字表法分为正常对照组、EAU模型组和龙胆泻肝汤干预组，后两组诱导EAU模型，龙胆泻肝汤干预组造模后用龙胆泻肝汤灌胃。免疫后12 d分别分离三组大鼠的脾脏和淋巴结收集CD4⁺T细胞，流式细胞仪检测Th17、Treg细胞的表达水平；实时荧光定量PCR（qRT-PCR）检测Notch1、Notch2、Notch3、Notch4基因的表达情况；ELISA检测Notch信号通路相关蛋白的表达水平。结果　流式细胞仪检测发现，与正常对照组相比，EAU模型组大鼠脾脏和淋巴结中Th17细胞水平明显升高，Treg细胞水平降低；与EAU模型组相比，龙胆泻肝汤干预组大鼠的Th17细胞水平明显下降，Treg细胞水平升高，两者比例逐渐恢复均衡；qRT-PCR检测发现龙胆泻肝汤干预组大鼠脾脏和淋巴结中Notch1、Notch2和Notch4基因的表达在免疫后12 d高于正常对照组（均为P<0.01），但显著低于EAU模型组（均为P<0.01），Notch3基因在大鼠脾脏和淋巴结未检测到表达；ELISA检测结果发现，龙胆泻肝汤干预组大鼠脾脏和淋巴结中Notch1、Notch2和Notch4蛋白表达水平在免疫后12 d虽然高于正常对照组（均为P<0.01），但显著低于EAU模型组大鼠（均为P<0.01）。结论　龙胆泻肝汤可有效缓解EAU大鼠中Th17/Treg比例失衡状态，其作用机制可能涉及到Notch信号通路调节的nave CD4⁺T细胞向Th17 和Treg细胞的分化。

Inhibitory effects of Longdan Xiegan Decoction on Notch signaling in rats with experimental autoimmune uveitis

Objective　To investigate the effects of Longdan Xiegan Decoction on the expression of Notch signaling-related genes in rats with experimental autoimmune uveitis (EAU).Methods　Totally 24 Lewis rats were randomly divided into normal control group，EAU model group and Longdan Xiegan Decoction treatment group.A rat model of EAU was established in the EAU model group and Longdan Xiegan Decoction treatment group，followed by intragastrical administration of Longdan Xiegan Decoction in the latter group after successful modeling.On day 12 after different treatments，the spleen and lymph nodes were isolated from rats of all the three groups for the collection of CD4⁺ T cells，and the levels of both Th17 and Treg cells were analyzed by flow cytometry to calculate the ratio of Th17 to Treg cells.Meanwhile the levels of Notch1，Notch2，Notch3，Notch4 mRNAs expression were monitored by qRT-PCR.Moreover，the expressions of Notch signaling-related proteins were further detected using ELISA technique.Results　Flow cytometry showed increased Th17 cell level but decreased Treg cell level in the EAU model group when compared with the normal control group.However，after treatment with Longdan Xiegan Decoction，the Th17 level was decreased，whereas the Treg level was increased，and the ratio of Th17 to Treg gradually restored to equilibrium when compared to EAU model group.qRT-PCR results showed that the expression levels of Notch1，Notch2 and Notch4 mRNAs in Longdan Xiegan Decoction treatment group were significantly higher than those in the normal control group (all P<0.01)，but lower than those in EAU model group on day 12 after treatment (all P<0.01)，without Notch 3 expression in the spleen and lymph nodes.In addition，Notch 1，Notch 2 and Notch 4 protein levels of the spleen and lymph nodes in Longdan Xiegan Decoction treatment group showed the similar tendency as compared to those of Notch 1，Notch 2 and Notch 4 mRNAs (all P<0.01)，and the expression of Notch 3 protein was still not observed.Conclusion　Longdan Xiegan Decoction can effectively relieve the imbalance of Th17/Treg cells in EAU rats，and its mechanism may involve the differentiation of naive CD4⁺T cells into Th17 and Treg cells，which is regulated by Notch signaling.