MiR-325-5p-CCL2 Contributes To Visceral Hypersensitivity In Rat Induced By Neonatal Colonic Inflammation (NCI)

Objective:Chronic visceral hypersensitivity is an important type of chronic pain with unknown etiology and pathophysiology.Recent studies have shown that epigenetic regulation which includes microRNAs play an important role in the development of chronic pain conditions.However,the role of miRNA-325-5p in chronic visceral pain remains unknown.The present study was designed to determine the roles and mechanism of miRNA-325-5p in a rat model of chronic visceral pain.Chronic visceral pain model was induced by neonatal colonic inflammation(NCI).Methods:1.The chronic visceral pain model of neonatal colitis inflammation(NCI)in SD male rats was established by injecting 0.5%dilute acetic acid(0.2 mL)into the colorectum.Pain behavior in NCI rats was assessed by the colorectal distention(CRD)threshold after 6 weeks of adult development.2.The mRNA expression level of miR-325-5p and C-C motif ligand 2(CCL2)at different time points was detected by RT-PCR.The protein expression of CCL2 was detected by western blotting.3.The intrathecal injection of miR-325-5p agomir overexpressed miR-325-5p to detect visceral pain in NCI rats.4.The intrathecal injection of the inhibitor of CCL2 Bindarit to detect visceral pain in NCI rats.5.The localization of MCP1 in neurons of spinal dorsal root ganglion was detected by immunofluorescence.Fluorescence in situ hybridization(FISH)was used to detect the localization of miR-325-5p and co localization with chemokine CCL2.Results:1.NCI led to a significant reduction in expression of miR-325-5p in colon related dorsal root ganglion(DRGs),starting to decrease at the age of 4 weeks and maintaining to the age of 8 weeks2.Intrathecal administration of miR-325-5p-agomir significantly enhanced colorectal distention(CRD)threshold in a time-dependent manner.3.NCI also markedly increased expression of CCL2 in colon related DRGs at mRNA and protein levels when compared with age-matched control rats.The expression of CXCL12,IL33,SFRS7,and LGI1 was not significantly altered in NCI rats.4.CCL2 was co-expressed in NeuN-positive DRGs neurons but not in glutamine synthetase-positive glial cells.Furthermore,CCL2 was mainly expressed in isolectin B4-binding-and calcitonin gene-related peptide-positive DRGs neurons but in few NF200-positive cells.5.More importantly,CCL2 was co-expressed in miR-325-5p-positive DRGs neurons6.Intrathecal injection of miR-325-5p agomir remarkedly reduced the upregulation of CCL2 in NCI rats.7.Administration of Bindarit,an inhibitor of CCL2,markedly enhanced CRD threshold in NCI rats in a dose-and time-dependent manner.Conclusion:These data suggest that NCI suppressed miR-325-5p expression and enhanced CCL2 expression,thus contributing to visceral hypersensitivity of adult rats and provided a theoretical basis for the microRNA-325-5p-CCL2 signaling pathway as a target for the treatment of chronic visceral pain.