六味地黄汤通过树突状细胞Notch信号通路调节T细胞分化干预自身免疫性脑脊髓炎

目的 通过观察树突状细胞（dendritic cells，DCs）Notch信号通路对CD4+ T细胞分化的影响，探究六味地黄汤干预实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis，EAE）的机制。方法 采用主动免疫法诱导2D2小鼠EAE模型，采用磁珠分选收集脾脏和淋巴结中CD4+ T细胞；体外分离培养及诱导C57BL/6（C57）小鼠骨髓来源树突状细胞（bone marrow derived dendritic cells, BMDCs）成熟，分组干预后，与CD4+ T细胞共培养后，被动注射至C57小鼠体内，观察小鼠发病情况。将BMDCs与CD4+ T细胞的共培养细胞分为对照组、六味地黄汤组、γ分泌酶抑制剂（gamma-secretase inhibitor，GSI）组。采用Western blot法检测各组BMDCs的Notch通路蛋白表达水平，采用流式细胞仪分析各组CD4+ T细胞分化情况。结果 与正常组比较，模型组Notch通路相关蛋白Notch1、Jagged1、MAML蛋白表达水平均显著升高（P<0.05）。与模型组比较，六味地黄汤组DCs的Notch1、Jagged1、MAML蛋白水平显著下调（P<0.05）。通过BMDCs诱导的CD4+ T细胞向Treg分化增多，向Th17分化减少，并改善被动免疫诱导的EAE小鼠症状评分。结论 六味地黄汤可通过抑制DCs的Notch信号通路活化，减少CD4+ T细胞分化，进而缓解EAE残疾症状。

Therapeutic Effect of Liuwei Dihuang Decoction on Autoimmune Encephalomyelitis by Regulating T Cell Differentiation via the Notch Signaling Pathway in Dendritic Cells

Objective To investigate the effect of the Notch signaling pathway in dendritic cells (DCs) on T cell differentiation and the mechanism of Liuwei Dihuang Decoction in intervening against experimental autoimmune encephalomyelitis (EAE). Methods Active immunization was used to induce EAE in 2D2 mice, and magnetic-activated cell sorting was used to collect the CD4+ T cells from the spleen and lymph nodes. Bone marrow-derived dendritic cells (BMDCs) from C57BL/6 (C57) mice were isolated, cultured, and induced to maturity in vitro, and after grouping, intervention, and co-culture with CD4+ T cells, BMDCs were passively injected into C57 mice to observe the onset of the disease. The co-cultured cells of BMDCs and CD4+ T cells were divided into control group, Liuwei Dihuang Decoction group, and gamma-secretase inhibitor (GSI) group. Western blot was used to measure the protein expression levels of the Notch signaling pathway, and flow cytometry was used to analyze the differentiation of CD4+ T cells. Results Compared with the normal group, the model group had significant increases in the protein expression levels of Notch1, Jagged1, and MAML associated with the Notch pathway (P<0.05), and compared with the model group, the Liuwei Dihuang Decoction group had significant reductions in the protein expression levels of Notch1, Jagged1, and MAML (P<0.05). After induction with BMDCs, more CD4+ T cells were differentiated into regulatory T cells, and less CD4+ T cells were differentiated into T helper 17 cells, with an improvement in the symptom score of mice with EAE induced by passive immunization. Conclusion Liuwei Dihuang Decoction can alleviate the disability symptom of EAE by inhibiting the activation of the Notch signaling pathway in DCs and reducing the differentiation of CD4+ T cells.