养阴清热方通过下调JAK/STAT信号通路调节Treg/Th17平衡的实验研究

Yangyin Qingre Formula Regulates Treg/Th17 Balance by Down-Regulating JAK/STAT Signaling Pathway

    摘要
  • 摘要:
      目的  研究养阴清热方(YHF)对脂多糖(LPS)导致的小鼠Treg/Th17失衡的影响及机制。
      方法  磁珠分选法提取小鼠脾脏CD4+ T细胞, 用LPS刺激CD4+ T细胞, 再以1、2、4 mg · mL-1的YHF水提液分别进行干预。MTT法检测细胞增殖情况; 流式细胞术检测CD4+ T细胞亚群Treg/Th17比例; ELISA法检测细胞上清中细胞因子IL-10、TGF-β、IL-6、IL-12p70的含量; 蛋白免疫印迹法(Western blot)检测细胞中Foxp3、RORγt及信号分子JAK/STAT的表达。
      结果  HPLC检测鉴定出YHF水提液中8种有效成分, 分别为羟基红花黄色素A、虎杖苷、芦丁、2, 3, 5, 4'-四羟基二苯乙烯葡萄糖苷、白藜芦醇、槲皮素、大黄素和姜黄素。YHF水提液可以剂量依赖性地抑制LPS刺激的小鼠CD4+ T细胞的增殖(P < 0.05), 增加Treg的比例(P < 0.05), 降低Th17的比例(P < 0.01), 促进培养上清中Treg相关的抗炎因子IL-10和TGF-β的分泌, 抑制Th17相关的致炎因子IL-6和IL-12p70的分泌。1、2、4 mg · mL-1 YHF均能增加Foxp3蛋白的表达(P < 0.05), 减少RORγt蛋白的表达(P < 0.01)。1、2、4 mg · mL-1 YHF均能降低JAK2、STAT3及其磷酸化蛋白的表达(P < 0.05)。4 mg · mL-1 YHF与JAK2特异性抑制剂AG490联用可以增加培养上清IL-10和TGF-β含量(P < 0.05), 降低IL-6和IL-12p70含量(P < 0.05), 而且可以显著降低p-JAK2、p-STAT3和RORγt蛋白表达(P < 0.01，P < 0.001), 增加Foxp3蛋白表达(P < 0.01)。
      结论  YHF可以有效抑制LPS诱导的小鼠CD4+ T细胞增殖, 通过抑制JAK2和STAT3信号分子表达, 增加Foxp3的表达和Treg数量, 减少RORγt的表达和Th17数量, 调节Treg/Th17平衡及抗炎细胞因子和促炎细胞因子的分泌。

     

    Abstract:
      OBJECTIVE  To investigate the effect and mechanism of Yangyin Qingre formula (YHF) on Treg/Th17 imbalance induced by lipopolysaccharide (LPS) in mice.
      METHODS  CD4+ T cells from mouse spleen were extracted by magnetic bead sorting method and CD4+ T cells differentiation was stimulated by LPS followed by intervention with 1, 2, 4 mg · mL-1 YHF. Cells proliferation was detected by MTT method. The ratio of Treg/Th17 of CD4+ T cells subsets was detected by flow cytometry. The expression of cytokines (IL-10, TGF-β, IL-6, IL-12p70) in cells supernatant was measured by ELISA. The expression of Foxp3, RORγt, and the signalling molecules JAK/STAT in the cells were detected by Western blot.
      RESULTS  HPLC identified eight active ingredients of YHF, namely hydroxy saffron yellow pigment A, polydatin, rutin, 2, 3, 5, 4'-tetrahydroxystilbene glucoside, resveratrol, quercetin, emodin and curcumin. YHF dose-dependently inhibited the proliferation of LPS-stimulated mouse CD4+ T cells (P < 0.05), increased the proportion of Treg (P < 0.05) and decreased the proportion of Th17 (P < 0.01), promoted the secretion of Treg-related anti-inflammatory factors IL-10 and TGF-β in culture supernatant and inhibited the secretion of Th17-related pro-inflammatory factors IL-6 and IL-12p70 secretion. 1, 2, 4 mg · mL-1 YHF increased the expression of Foxp3 protein (P < 0.05) and decreased the expression of RORγt protein (P < 0.01). 1, 2, 4 mg · mL-1 YHF decreased the expression of JAK2, STAT3 and their phosphorylated proteins (P < 0.05). The combination of 4 mg · mL-1 YHF with the JAK2-specific inhibitor AG490 increased culture supernatant IL-10 and TGF-β levels (P < 0.05), decreased IL-6 and IL-12p70 levels (P < 0.05) and more significantly decreased p-JAK2, p-STAT3 and RORγt protein expression (P < 0.01, P < 0.001) and increased Foxp3 protein expression (P < 0.01).
      CONCLUSION  YHF can effectively inhibit the proliferation of inflammatory LPS-induced CD4+ T cells by suppressing the expression of JAK2 and STAT3 signaling molecules, increase the expression of Foxp3 and the number of Treg, decrease the expression of RORγt and the number of Th17, increase the secretion of anti-inflammatory cytokines IL-10 and TGF-β and inhibit the secretion of pro-inflammatory cytokines IL-6 and IL-12p70.

     

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