| There is accumulating evidence that microRNAs are emerging as pivotal regulators in the development and progression of neu-ropathic pain.MicroRNA-15a/16(miR-15a/16)have been reported to play an important role in various diseases and inflammation response processes.However,whether miR-15a/16 participates in the regulation of neuroinflammation and neuropathic pain development remains unknown.In this study,we established a mouse model of neuropathic pain by chronic constriction injury(CCI)of the sciatic nerves.Our results showed that both miR-15 a and miR-16 expression was significantly upregulated in the spinal cord of CCI rats.Downregulation of the expression of miR-15 a and miR-16 by intrathecal injection of a specific inhibitor significantly attenuated the mechanical allodynia and thermal hyperalgesia of CCI rats.Furthermore,inhibition of miR-15 a and miR-16 downregulated the expression of interleukin-1β and tumor-necrosis factor-αin the spinal cord of CCI rats.Bioinformatic analysis predicted that G protein-coupled receptor kinase 2(GRK2),an important regulator in neuropathic pain and inflamma-tion,was a potential target gene of miR-15 a and miR-16.Inhibition of miR-15 a and miR-16 markedly increased the expression of GRK2 while downregulating the activation of p38 mitogen-activated protein kinase and NF-κB in CCI rats.Notably,the silencing of GRK2 significantly reversed the inhibitory effects of miR-15a/16 inhibition in neuropathic pain.In conclusion,our results suggest that inhibition of miR-15a/16 expression alleviates neuropathic pain development by targeting GRK2.These findings provide novel insights into the molecular pathogenesis of neuropathic pain and suggest potential therapeutic targets for preventing neuropathic pain development.Methods1)Chronic constriction injury of sciatic nerve(CCI)was used to construct a neuropathic pain model in mice.Pain threshold measurement of CCI mouse model,namely,ethometrics: ethometrics of thermal pain stimulus and ethometrics of mechanical pain stimulus.2)Intrathecal injection of mir-15 a /16 antagonists(anti-miR-15 a,anti-miR-16,or a mixture of miR-15 a and miR-16).3)Total RNA was extracted with TRIzol reagent.Total RNA was reversely transcribed by m-mlv reverse transcriptase.MiRNA was detected using the TaqMan miRNA reverse transcription kit.4)Enzyme linked immunosorbent assay(ELISA)was used to detect the concentration of il-1 and TNF-proteins in the lumbar spinal cord.5)Real-time fluorescence quantitative PCR was used to further identify the miRNA.6)GRK2 3 ’-utrs fragment was inserted into the pmirGLO double luciferase plasmid and co-transfected with mir-15 a /16 into 293 T cells.7)Luciferase reporter activity was determined by a dual luciferase reporter system.8)MRNA and protein expression levels of target genes were detected by western.Results(1)miR-15a/16 expression is upregulated in the spinal cord of rats after CCI(2)Inhibition of miR-15a/16 alleviates thermal hyperalgesia and mechanical allodynia in CCI rats(3)Inhibition of miR-15a/16 decreases the expression of IL-1β and TNF-α in CCI rats(4)miR-15a/16 binds to the 3’-UTR of GRK2(5)Inhibition of miR-15a/16 upregulates the expression of GRK2 in CCI rats(6)Inhibition of miR-15a/16 suppresses the activation of p38 MAPK/NF-κB(7)Knockdown of GRK2 eliminates the protective effects of miR-15a/16 inhibition in neuropathic pain rats(8)Knockdown of GRK2 reverses the inhibitory effect of miR-15a/16 inhibition on p38 MAPK activation Conclusion:In the course of the occurrence and development of neuropathic pain,mir-15 a /16 is involved in the occurrence and development of the disease through the targeted regulation of GRK2.Inhibition of mir-15 a /16 can promote the expression of GRK2 and inhibit the activation of p38 MAPK to alleviate neuropathic pain and neuroinflammation.These findings provide new insights into the molecular pathogenesis of neuropathic pain and open the possibility of further developing effective therapeutic strategies for the treatment and prevention of neuropathic pain. |
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