大鼠海马miR-181c在神经痛中的作用及其表达调控机制研究

Recent evidences indicate that the hippocampus has a role in modulation of pain perception. A miRNA181c is highly expressed in hippocampus and involved in inflammatory response via down-regulation of TNF-α and TLR4 expression. We found that intra-hippocampal CA1 injection of miRNA181c suppressed allodynia caused by peripheral nerve injury. However, it is not clear the role of hippocampus miRNA181c in the development of neuropathic pain the regulatory mechanism of miRNA181c expression. To further investigate this phenomenon and its possible mechanism, some methods include morphology, ethology and molecular biologytechniques are applied to investigate the behavioral responses and the relationships between the expression of miRNA181c, TNF-α, TLR4, NF-Κb, IKK and CTCF in hippocampus of neuropathic pain rats; to speculate whether down-regulating the TNF-αand TLR4 is involved in miRNA-181c-mediated analgesia; to investigate whether NF-Κb activation may negative regulate the expression of miRNA181c via down-regulating the expression of CTCF, which result allodynia after peripheral nerve injury. The research works will provide some evidence for developing effective analgesics beses on microRNA as the new therapeutic targetegy and provide new ideas and methods for clinical treatment of pathological pain.

近年研究表明，海马参与了外周神经损伤后的痛觉感受。miRNA-181c表达在海马，通过下调TNF-α和TLR4表达参与损伤后的炎症反应。我们在海马CA1区注射miRNA-181c模拟物减轻了神经痛大鼠的痛敏症状，但miRNA-181c调节痛觉感受的机制及miRNA-181c的表达调控方式并不清楚。故本项目拟在已往疼痛研究基础上，采用在体和离体实验，综合应用行为学、形态学和分子生物学方法，观察：①外周神经损伤大鼠行为学变化、海马miRNA-181c、TNF-α、TLR4、NF-κB、IKK及CTCF表达；②海马miRNA181c是否通过下调TNF-α和TLR4信号减轻炎症因子表达发挥镇痛效应；③TNF-α和TLR4下游的转录因子NF-κB 是否通过抑制CTCF表达，下调miRNA-181c表达，促进痛敏。该项目旨在为研发针对miRNA靶点的新型镇痛药提供理论基础，为疼痛治疗提供思路。

海马参与了外周神经损伤后的痛觉感受。miRNA-181c表达在海马，通过下调TNF-α和TLR4表达参与损伤后的炎症反应。我们在海马CA1区注射miRNA-181c模拟物减轻了神经痛大鼠的痛敏症状，但相关机制并不清楚。故本项目在已往疼痛研究基础上，采用在体和离体实验，综合应用行为学、形态学和分子生物学方法，探讨海马miRNA-181c/miR-29c是否通过调节TNF-α/TNFR1信号轴参与神经痛的神经化学机制。结果表明：（1）海马背侧注射小胶质细胞活性抑制剂米诺环素剂可以影响慢性坐骨神经结扎（CCI）大鼠热痛阈、机械痛阈、抑郁样行为及炎症相关基因表达，并对筛选出的相关基因做了PCR验证。（2）海马注射miR-181c激动剂减轻CCI大鼠热痛敏可能是由于miR-181c下调TLR4和TNF-α表达导致。CCI大鼠海马miR-181c表达下调可能与CTCF低表达有关。（3）海马CA1 区给予miR-181c 可以减轻CCI大鼠机械痛敏，抑制海马TRIM2 表达，促进NF-L 与NR1 表达上调，减轻海马神经元损伤。（4）海马CA1 区给予miR-29c 可以减轻CCI大鼠机械痛敏，抑制海马TNFR1表达，抑制ROS生成，减轻海马神经元损伤。（5）miR-29c减轻TNF-α诱导的小鼠海马神经元HT22细胞损伤，其机制可能与抑制TNFR1信号通路激活导致的细胞凋亡有关；(6)此外，我们还观察到，在培养的脊髓背角星形胶质细胞，LPS 通过 TLR4 途径激活转录因子 NF-κBp65/AP-1入核， NF-κBp65 结合到 MCP-1 启动子 254~261bp 序列，NF-κBp50 结合 MCP-1 启动子 389~398bp 序列起促进其转录生成；AP-1 分别结合到 CXCL1 启动子 323~329bp 序列和 Cx43 启动子区域 251~257bp 序列促进 CXCL1 和 Cx43 的转录生成；NF-κBp65 和AP-1 之间的crosstalk参与了MCP-1、 CXCL1和 Cx43的转录过程。本项目的实施为我们理解慢性疼痛的发生提供和新药研发提供理论基础，为神经病理性疼痛的治疗提供新思路。