MiR-187-3p mimic alleviates ischemia-reperfusion-induced pain hypersensitivity through inhibiting spinal P2X7R and subsequent mature IL-1β release in mice

Abstract

Background: 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.

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-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.

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-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.

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-1β overproduction, partially through inhibiting P2X7R activation.

Keywords: Interleukin-1β; Ischemia-reperfusion injury; MicroRNAs; P2X7 purinergic receptor; Pain hypersensitivity.