组蛋白去甲基化酶UTX调控辅助型T细胞分化在针刺治疗神经病理痛中的机制研究

Despite of the high prevalence of neuropathic pain, current therapies for treating this type of pain are ineffective. Our previous study has found that acupuncture can relieve chronic constriction injury (CCI) induced pain hypersensitivity by inhibiting spinal interferon-γ (IFN-γ) over-expression. However, the mechanisms underlying the inhibitory effect of acupuncture on IFN-γ expression remains unknown. Type 1 helper T (Th1) cells are the main sources of IFN-γ, while histone demethylase UTX is involved in the the differentiation of Th cells by regulating master transcription factors in Th cells. Based on all above, we proposed that acupuncture may increase the methylation level in transcriptional regulation regions of master transcription factors by inhibiting histone demethylase UTX of T cells, which consequently causes transcription reduction of master transcription factors such as IFN-γ and T-bet，then finally inhibits Th1 cell differentiation to relieve neuropathic pain. In this project, we intend to use behavioral test，flow cytometry and Chip-qPCR to investigate the effect of acupuncture on pain threshold in CCI mouse, as well as the differentiation of Th cell and histone methylation level in T cells. Furthermore, we plan to use interventions like Th cell histone methylation modulators and UTX conditional knockout to further verify the hypothesis. This research will reveal new mechanisms of acupuncture treatment in the perspective of epigenetic modulation in neuro-immune system, and also provide theoretical basis for optimizing the acupuncture treatment of neuropathic pain.

神经病理痛发病率高，缺乏有效治疗手段。我们前期工作发现针刺治疗能抑制脊髓背角γ-干扰素(IFN-γ)过度释放，缓解坐骨神经慢性缩窄损伤(CCI)引起的痛觉过敏，但具体机制不清。I型辅助T细胞(Th1)是IFN-γ的主要来源，而组蛋白去甲基化酶UTX能影响Th细胞分化相关重要因子的转录，调节其分化。据此我们提出假说：针刺治疗抑制T细胞组蛋白去甲基化酶UTX，使Th细胞分化关键因子转录调控区甲基化修饰水平增高，抑制IFN-γ、T-bet等关键因子转录，减少Th1细胞分化以缓解神经病理痛。本项目拟通过行为学测试、流式细胞分析、Chip-qPCR等手段检测针刺对CCI小鼠痛阈、Th1细胞分化及T细胞组蛋白甲基化修饰的影响，并通过调节T细胞组蛋白甲基化修饰水平及UTX条件敲除等干预手段验证假说正确性。本研究从表观遗传免疫调控的角度探讨针刺治疗神经病理痛的机制，为神经病理痛治疗提供新的靶点及思路。

针刺治疗能够通过调节慢性坐骨神经结扎模型小鼠辅助T细胞分化减少CCI术后脊髓IFN-γ释放并缓解病理性疼痛，然而针刺治疗如何调节Th细胞分化并不清楚。本课题研究证明了针刺治疗能够改变T细胞分化重要因子启动子结合区的组蛋白甲基化修饰水平，并抑制Th1细胞分化相关重要因子的转录，最终抑制Th1细胞分化，缓解外周神经压迫引起的慢性病理性疼痛。我们在研究中发现，针刺治疗CCI模型小鼠7天后外周血及脾脏Th1细胞在CD4+ T细胞中的比例显著降低。进一步Chip-qPCR结果表明，电针治疗后CCI模型小鼠脾脏CD4+ T细胞中促进Th1细胞分化的IFN-γ及T-bet转录启动区组蛋白第27位赖氨酸三甲基修饰结合增加，相应的，组蛋白去甲基化酶UTX在IFN-γ及T-bet转录启动区结合减少，IFN-γ及T-bet转录被抑制。更重要的是，CD4+ T细胞UTX特异敲除小鼠在CCI术后，针刺治疗缓解小鼠机械痛觉过敏效力显著下降，且针刺治疗也未能抑制CD4+ T细胞中UTX缺失小鼠在CCI术后Th1细胞及相关因子IFN-γ的增加。本项目研究结果表明，针刺治疗能够通过表观遗传调节免疫细胞分化，发挥治疗慢性病理性疼痛的作用，这为阐释中医针刺治疗的机制提供了全新的视角，也为我们后期从表观遗传调节及免疫调节的角度探索慢性病理性疼痛治疗的新靶点提供思路。