Xiao-Qian Li1, Qian Yu2, Zai-Li Zhang1, Xi-Jia Sun1, Hong Ma3. 1. Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning, China. 2. Department of Thoracic Surgery, Fourth Affiliated Hospital, China Medical University, Shenyang 110032, Liaoning, China. 3. Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning, China. Electronic address: mahong5466@126.com.
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.
BACKGROUND:Ischemia-reperfusion (IR)-induced painhypersensitivity 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 painhypersensitivity by regulating the P2X7R signal and subsequent IL-1β release. METHODS: A mouse model was established by 5-min occlusion of the aortic arch. Painhypersensitivity 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 painhypersensitivity and mature IL-1β overproduction, partially through inhibiting P2X7R activation.