针刺对抑郁症小胶质细胞M1/M2型极化紊乱诱导神经炎症的调控机制研究

Recently, the role of neuroinflammation in the study of depression has got widespread attention. However, the mechanisms underlying the effect of acupuncture on regulating the neuroinflammation mediated by microglia M1/M2 polarization disorder are still unclear. Accordingly, this present study proposes a hypothesis that the antidepressant effect of acupuncture might be based on regulating the neuroinflammation mediated by microglia M1/M2 polarization disorder, which exerts antidepressant effect..Research contents: ① The depression model will be established by chronic restraint stress (CRS). The expression of microglia M1-type and M2-type markers of CD86 and Arg-1 will be detected by immunofluorescence method. And the expression of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-β (IL-1β), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) will be observed by western blot (WB) and enzyme-linked immunosorbent assay (ELISA). To investigate the effect of acupuncture on regulating the neuroinflammation mediated by microglia M1/M2 polarization disorder in CRS rats. ② Based on toll-like receptor 4 (TLR4) signaling pathway, TAK-242, the selective inhibitors of TLR4 signaling pathway, will be selected to conduct intracerebroventricular injection. The expression of TLR4, myeloid differentiation primary response gene 88 (MyD88), CD86 and Arg-1 in the hippocampus will be detected by immunofluorescence method. The expression of TLR4, MyD88, NF-κBp65 and TNF-α will be observed by western blot and quantitative polymerase chain reaction (Q-PCR). Conclusively, this study aims to explore the mechanisms underlying the effect of acupuncture on regulating the neuroinflammation mediated by microglia M1/M2 polarization disorder.

目前，神经炎症在抑郁机制症研究中备受关注。而小胶质细胞M1/M2型极化紊乱诱导神经炎症的机制及针刺调控机制尚不清楚。因此，基于前期基础提出假说：针刺通过诱导小胶质细胞表型从M1向M2型转变、调节通过TLR4信号通路介导的神经炎症，产生抗抑郁效应。.研究内容：①采用慢性束缚应激(CRS)抑郁模型，免疫荧光法检测海马M1、M2型标志物CD86和Arg-1表达；WB和ELISA观察TNF-α、IL-6、IL-1β、IL-10和TGF-β表达，探究针刺对CRS大鼠小胶质细胞M1/M2型极化的影响。②基于TLR4信号通路，采用其抑制剂TAK-242侧脑室注射，免疫荧光法检测海马TLR4、MyD88、CD86和Arg-1表达，WB和Q-PCR检测海马TLR4、MyD88、NF-κBp65、TNF-α等表达，从整体耦合视角，探究针刺对抑郁症小胶质细胞M1/M2型极化紊乱诱导神经炎症的调控机制和科学证据。

目前，神经炎症在抑郁机制症研究中备受关注。而小胶质细胞M1/M2型极化紊乱诱导神经炎症的机制及针刺调控机制尚不清楚。因此，基于前期基础提出假说：针刺通过诱导小胶质细胞表型从M1向M2型转变、调节通过TLR4信号通路介导的神经炎症，产生抗抑郁效应。采用慢性束缚应激(CRS)模拟抑郁大鼠模型，主要研究内容：①TLR4信号通路和NLRP3炎性小体介导的免炎性反应在针刺抗抑郁中的作用：采用免疫荧光法观察海马小胶质细胞活化情况；采用ELISA法检测各组大鼠血清炎性因子表达变化；采用免疫荧光法观察海马小胶质细胞M1/M2极化标志物变化情况；采用WB观察不同组别大鼠海马TLR4信号通路关键蛋白和NLRP3炎性小体关键蛋白表达变化；应用q-PCR技术观察关键mRNA表达变化，探究小胶质细胞活化、TLR4信号通路和NLRP3炎性小体介导的免炎性反应在针刺抗抑郁中的作用。②针刺对小胶质细胞调控TLR4信号通路诱导神经炎症的效应机制研究：加以TLR4信号通路的选择性抑制剂TAK-242对比观察大鼠海马TLR4信号通路关键蛋白的表达变化，应用Golgi染色观察海马内神经元树突分支数和树突棘密度的影响，探究针刺对小胶质细胞极化调控TLR4信号通路诱导神经炎症的效应机制。③针刺调节CRS大鼠肠道菌群与免疫炎症的抗抑郁机制研究：基于16S rDNA高通量测序技术对大鼠肠道菌群进行基因丰度表达分析、物种与功能注释分析，探究针刺对CRS模型大鼠肠道菌群的调节效应及作用途径。④针刺调节CRS模型大鼠海马HMGB1介导的神经炎症机制：采用免疫荧光染色法观察海马小胶质细胞活化及HMGB1的表达变化。结果显示：小胶质细胞活化、TLR4信号通路和NLRP3炎性小体介导的免炎性反应在抑郁症病理过程具有重要作用，针刺干预对小胶质细胞M2型极化调节更明显；TLR4信号通路的选择性抑制剂TAK-242、针刺、针刺+TAK242干预在一定程度上逆转了慢性束缚应激诱导海马TLR4信号通路激活介导的神经炎症，提示针刺可能是通过调节TLR4信号通路激活介导的神经炎症而产生抗抑郁效应；针刺对CRS模型大鼠表现出的抗抑郁作用可能与调节肠道菌群，改善肠道通透性，减轻炎症反应有关。