MiR-187-3p mimic alleviates ischemia-reperfusion-induced pain hypersensitivity through inhibiting spinal P2X7R and subsequent mature IL-1β release in mice
MiR-187-3p mimic alleviates ischemia-reperfusion-induced pain hypersensitivity through inhibiting spinal P2X7R and subsequent mature IL-1β release in mice
复制标题
MiR-187-3p 模拟物通过抑制小鼠脊髓 P2X7R 和随后成熟的 IL-1β 释放来减轻缺血再灌注引起的疼痛超敏反应。
DOI：
10.1016/j.bbi.2019.05.021
复制
发表时间：
2019-07-01
影响因子：
15.1
通讯作者：
Ma, Hong
中科院分区：
文献类型：
--
作者：
Li, Xiao-Qian;Yu, Qian;Ma, Hong
背景资料：缺血-再灌注（IR）诱导的疼痛超敏反应具有神经炎症和神经病理性疼痛的特征，伴随着白细胞介素（IL）-1 β的过度产生。在IR期间，多种microRNA（miR）失调;在这些miR中，最近报道miR-187- 3 p通过激活嘌呤能受体家族的成员来驱动视网膜疾病中的IL-1 β释放。然而，miR-187- 3 p在脊髓中的作用尚不清楚。因此，我们研究了miR-187- 3 p是否通过调节P2 X7 R信号和随后的IL-1 β释放参与IR诱导的疼痛超敏反应的发病机制。方法：通过阻断主动脉弓5分钟建立小鼠模型。通过缩爪阈值（PWT）和缩爪潜伏期（PWL）评估疼痛超敏反应。通过RT-PCR和蛋白质印迹法测量miR-187- 3 p、P2 X7 R、裂解的半胱天冬酶-1和成熟IL-1 β的表达水平。通过在单独的实验中用合成miR、选择性激动剂和拮抗剂鞘内处理来探索miR-187- 3 p、P2 X7 R和IL-1 β的体内作用。采用双重免疫荧光染色描绘P2 X7 R和IL-1 β的细胞分布。结果：IR诱导的PWT和PWL值进行性降低与miR-187- 3 p的降低和P2 X7 R表达水平随时间的增加密切相关。通过生物信息学数据库初步预测了功能性miR-187- 3 p/P2 X7 R对，并通过定量分析在体内证实，因为模拟物-187极大地增加了miR-187- 3 p但降低了P2 X7 R表达水平，而抑制剂-187逆转了这些变化。相反，通过模拟物-187或A-438079治疗下调P2 X7 R可增加IR损伤小鼠的PWT和PWL值，而通过抑制剂-187或BzATP治疗上调P2 X7 R可降低假手术小鼠的PWT和PWL值。此外，P2 X7 R和IL-1 β免疫反应性在各组中的变化模式相同。这一发现进一步得到了以下结果的支持，这些结果显示，通过A-438079和IL-1 β中和抗体下调IL-1 β类似地降低了P2 X7 R、切割的半胱天冬酶-1和成熟IL-1 β表达水平，而BzATP处理增加了这些水平。结论：脊髓miR-187- 3 p/P2 X7 R对在小鼠IR模型中起作用。增加miR-187- 3 p可部分通过抑制P2 X7 R活化来保护疼痛超敏反应和成熟IL-1 β过度产生。
Background: Ischemia-reperfusion (IR)-induced pain hypersensitivity shares features of neuroinflammation and neuropathic pain, accompanied by overproduction of interleukin (IL)-1 beta. Multiple microRNAs (miRs) are dys-regulated during IR; among these miRs, miR-187-3p was recently reported to drive IL-1 beta release in retinal disease by activating members of the purinergic receptor family. However, the roles of miR-187-3p in the spinal cord are unclear. Thus, we investigated whether miR-187-3p is involved in the pathogenesis of IR-induced pain hypersensitivity by regulating the P2X7R signal and subsequent IL-1 beta release.Methods: A mouse model was established by 5-min occlusion of the aortic arch. Pain hypersensitivity was assessed by the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). MiR-187-3p, P2X7R, cleaved caspase-1 and mature IL-1 beta expression levels were measured by RT-PCR and Western blotting. The in vivo roles of miR-187-3p, P2X7R and IL-1 beta were explored by intrathecal treatment with synthetic miRs, selective agonists and antagonists in separate experiments. Double immunofluorescence staining was performed to delineate the cellular distribution of P2X7R and IL-1 beta.Results: IR-induced progressively decreased PWT and PWL values were closely related to decreases in miR-187-3p and increases in P2X7R expression levels over time. The functional miR-187-3p/P2X7R pair was preliminarily predicted by a bioinformatic database and confirmed in vivo by quantitative analysis, as mimic-187 greatly increased miR-187-3p but decreased P2X7R expression levels, whereas inhibitor-187 reversed these changes. In contrast, downregulating P2X7R by mimic-187 or A-438079 treatment comparably increased PWT and PWL values in IR-injured mice, while upregulating P2X7R by inhibitor-187 or BzATP treatment decreased PWT and PWL values in sham-operated mice. Moreover, P2X7R and IL-1 beta immunoreactivities in each group were changed in the same patterns. This finding was further supported by results showing that downregulating IL-1 beta by A-438079 and IL-1 beta-neutralizing antibody similarly decreased P2X7R, cleaved caspase-1 and mature IL-1 beta expression levels, whereas BzATP treatment increased these levels.Expectedly, mimic-187 treatment preserved PWT and PWL values, with decreased cleaved caspase-1 and mature IL-1 beta expression levels, whereas inhibitor-187 reversed these effects.Conclusions: The spinal miR-187-3p/P2X7R pair functioned in a mouse IR model. Increasing miR-187-3p protected against pain hypersensitivity and mature IL-1 beta overproduction, partially through inhibiting P2X7R activation.