Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning, China.
Department of Thoracic Surgery, Fourth Affiliated Hospital, China Medical University, Shenyang 110032, Liaoning, China.
Brain Behav Immun. 2019 Jul;79:91-101. doi: 10.1016/j.bbi.2019.05.021. Epub 2019 May 14.
Ischemia-reperfusion (IR)-induced pain hypersensitivity shares features of neuroinflammation and neuropathic pain, accompanied by overproduction of interleukin (IL)-1β. Multiple microRNAs (miRs) are dysregulated during IR; among these miRs, miR-187-3p was recently reported to drive IL-1β 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-1β release.
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-1β expression levels were measured by RT-PCR and Western blotting. The in vivo roles of miR-187-3p, P2X7R and IL-1β 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-1β.
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-1β immunoreactivities in each group were changed in the same patterns. This finding was further supported by results showing that downregulating IL-1β by A-438079 and IL-1β-neutralizing antibody similarly decreased P2X7R, cleaved caspase-1 and mature IL-1β 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-1β expression levels, whereas inhibitor-187 reversed these effects.
The spinal miR-187-3p/P2X7R pair functioned in a mouse IR model. Increasing miR-187-3p protected against pain hypersensitivity and mature IL-1β overproduction, partially through inhibiting P2X7R activation.
缺血再灌注（IR）诱导的痛觉过敏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）-1β的过度产生。IR 期间多种 microRNAs（miRs）失调；在这些 miRs 中，miR-187-3p 最近被报道通过激活嘌呤能受体家族成员来驱动视网膜疾病中 IL-1β的释放。然而，miR-187-3p 在脊髓中的作用尚不清楚。因此，我们通过调节 P2X7R 信号和随后的 IL-1β释放，研究了 miR-187-3p 是否参与了 IR 诱导的痛觉过敏的发病机制。
通过主动脉弓 5 分钟闭塞建立小鼠模型。通过足底撤回阈值（PWT）和足底撤回潜伏期（PWL）评估痛觉过敏。通过 RT-PCR 和 Western blot 测量 miR-187-3p、P2X7R、裂解的半胱天冬酶-1 和成熟的 IL-1β 的表达水平。通过鞘内给予合成的 miRs、选择性激动剂和拮抗剂在单独的实验中探索了 miR-187-3p、P2X7R 和 IL-1β 的体内作用。通过双重免疫荧光染色描绘 P2X7R 和 IL-1β 的细胞分布。
IR 诱导的逐渐降低的 PWT 和 PWL 值与 miR-187-3p 的降低和 P2X7R 表达水平随时间的增加密切相关。功能 miR-187-3p/P2X7R 对是通过生物信息学数据库初步预测的，并通过体内定量分析得到证实，因为模拟物-187 大大增加了 miR-187-3p，但降低了 P2X7R 的表达水平，而抑制剂-187 则逆转了这些变化。相比之下，通过模拟物-187 或 A-438079 处理下调 P2X7R 可使 IR 损伤小鼠的 PWT 和 PWL 值同等增加，而通过抑制剂-187 或 BzATP 处理增加 P2X7R 可使 sham 手术小鼠的 PWT 和 PWL 值降低。此外，各组中的 P2X7R 和 IL-1β 免疫反应性也呈现出相同的变化模式。这一发现得到了以下结果的进一步支持：通过 A-438079 和 IL-1β 中和抗体下调 IL-1β 可使 P2X7R、裂解的半胱天冬酶-1 和成熟的 IL-1β 的表达水平降低，而 BzATP 处理则增加了这些水平。预期的是，模拟物-187 处理可维持 PWT 和 PWL 值，降低裂解的半胱天冬酶-1 和成熟的 IL-1β 的表达水平，而抑制剂-187 则逆转了这些作用。
脊髓中的 miR-187-3p/P2X7R 对在小鼠 IR 模型中起作用。增加 miR-187-3p 可预防痛觉过敏和成熟的 IL-1β 过度产生，部分通过抑制 P2X7R 激活。
