The Roles And Underlying Mechanisms Of Spinal MicroRNA-146a-5p/CCL8 In Regulating Visceral Hyperalgesia In Mice With Colitis

ObjectiveVisceral hyperalgesia is the main pathophysiological mechanism of abdominal pain in patients with inflammatory bowel disease(IBD)and the exact molecular mechanisms have not been fully elucidated.Using a mouse model of experimental colitis and in vitro cultured mouse neuroblastoma cells(Neuro-2a),the present study aimed to(1)define the effects and possible mechanism of spinal chemokine CCL8 and its main receptor CCR5 signaling in visceral hyperalgesia.(2)investigate the functional role of spinal miR-146a-5p in visceral pain process and its regulatory effect on CCL8 and its downstream signals.Methods(1)Colitis was induced by intracolonic administration of trinitrobenzene sulphoni cacid(TNBS)in mice.(2)Disease activity index(DAI),colonic myeloperoxidase(MPO)activity and hematoxylin-eosin(HE)staining were performed to identify colonic inflammation.(3)Mice underwent colorectal distension(CRD)and were tested for visceral pain threshold to assess visceral hypersensitivity(visceral hyperalgesia).(4)MiR-146a-5p expression and mRNA levels of CCL8,CCR5,TNF-?,IL-1? and IL-6 were determined using quantitative real-time PCR(qPCR).(5)CCL8 protein level was quantified by enzyme-linked immuno sorbent assay(ELISA).(6)Western blot were conducted to evaluate protein expressions of CCR5 and phosphorylated extracellular signal-regulated kinase(pERK).(7)Immunofluorescence double staining was used to confirm the cellular location localization of CCL8,CCR5 and pERK in spinal dorsal horn.(8)The cellular location of miR-146a-5p in spinal dorsal horn were determined by combined analysis of fluorescence in-situ hybridization(FISH)and immunofluorescence staining.(9)Target genes of miR-146a-5p were predicted by bioinformatics analysis.Dual-luciferase reporter assay was performed to identify the relationship between mir-146a-5p and CCL8.(10)To test the effects of miR-146a-5p on CCL8 and its downstream signal,Neuro-2a cells were transfected with miR-146a-5p mimic or inhibitor.(11)To define the role of spinal CCL8,CCR5,ERK or miRNA-146a-5p in colitis-induced visceral hypersensitivity,visceral pain threshold was assessed after intrathecal administration of CCL8 neutralizing antibody,CCL8 siRNA,CCR5 antagonist DAPTA,ERK kinase(MEK)inhibitor PD98059,recombinant mouse chemokine CCL8,miR-146a-5p agomir or miR-146a-5p antagomir.Results(1)TNBS enema induced colon inflammation,characterized by increase of DAI and colonic MPO activity on day 3,7,10.HE staining of colon tissue in TNBS-treated mice showed impaired mucosa accompanied with numerous inflmmatory cell infiltration.The mRNA expression of proinflammatory cytokines,including TNF-?,IL-1?and IL-6 in colon tissue were significantly increased on day 7 after TNBS.(2)TNBS induced significant visceral hyperalgesia,showing that the visceral pain threshold decreased on day 3,reached the lowest on day 7,remained lower on day 10 and recovered on day 14.(3)CCL8 mRNA and protein level in the lumbosacral spinal cord increased on day 3,peaked on day 7,declined on day 10 and recovered to basal level on day 14 after TNBS enema.The time course of CCL8 expression was parallel to that of pain behavior.The upregulated CCL8 was primarily localized to spinal dorsal horn neurons,but not astrocytes or microglia.A single intrathecal injection of CCL8 neutralizing antibody on day 7 after TNBS instillation increased the visceral pain threshold,which was dose-dependent and time-dependent.Similarly,intrathecal injection of CCL8 siRNA(2 injections,12 h apart)on day 7 after TNBS injection not only inhibited the mRNA and protein expression of spinal cord CCL8,but also significantly attenuated the painful behavior.(4)CCR5 mRNA and protein level in the lumbosacral spinal cord of TNBS mice increased significantly on day 3,7,10 and returned to normal on day 14.CCR5 in the spinal dorsal horn was mainly expresssed in neurons,but not astrocytes or microglial cells.A single intrathecal injection of DAPTA,a CCR5 antagonist on day 7 after TNBS enema attenuated colitis-induced visceral hyperalgesia in a dose-dependent and time-dependent manner.(5)pERK expression in the lumbosacral spinal cord increased on day 7 after TNBS enema and was located in neurons.TNBS-induced visceral hypersensitivity was partially reversed by a single intrathecal ERK kinase(MEK)inhibitor PD98059.Moreover,intrathecal CCL8 neutralizing antibody or CCR5 antagonist DAPTA on day 7 after TNBS,significantly reversed the upregulation of spinal pERK in TNBS mice.(6)Intrathecal injection of recombinant mouse CCL8 induced the development of visceral hyperalgesia and the activation of spinal ERK in Naive mice.Pretreatment with intrathecal CCR5 antagonist DAPTA partially prevented recombinant CCL8-induced visceral pain as well as the upregulation of spinal pERK.Intrathecal MEK inhibitor PD98059 partially alleviated the decrease of visceral pain threshold induced by recombinant CCL8.(7)Recombinant mouse chemokine CCL8 induced the activation of ERK in cultured Neuro-2a cells.Preincubation with CCR5 antagonist DAPTA decreased CCL8-induced high expression of pERK.(8)The expression of miR-146a-5p in the lumbosacral spinal cord gradually increased from day 3 to day 14 after TNBS enema and it was mainly expressed in spinal dorsal horn neurons.(9)Bioinformatics analyses predicted CCL8 as a potential target gene of miR-146a-5p.Dual-luciferase reporter assays confirmed that miR-146a-5p suppressed CCL8 expression by directly targeting the 3'-untranslated region(3'-UTR)of CCL8 mRNA.(10)Overexpression of miR-146a-5p by transfection of miR-146a-5p mimic inhibited TNF-?-induced upregution of CCL8 mRNA and protein level in Neuro-2a cells.Conversely,transfection of miR-146a-5p inhibitor induced high expression of CCL8 mRNA and protein and the activation of ERK in Neuro-2a cells.(11)Repetitive intrathecal administration of miR-146a-5p agomir(daily from day 4 to day 6 after TNBS enema)which increased the levels of endogenous miR-146a-5p in the spinal cord,not only inhibited the high expression of CCL8 and the activation of ERK but also attenuated TNBS-induced visceral hyperalgesia.(12)Inhibition of miR-146a-5p by repetitive intrathecal administration of miR-146a-5p antagomir(daily for three consecutive days)increased CCL8 expression in the spinal cord,activate ERK signal and induced the development of visceral pain in na?ve mice,Intrathecal CCL8 neutralizing antibody after repetitive injection of miR-146a-5p antagomir,partially alleviated miR-146a-5p antagomir-induced visceral hypersensitivity and inhibited the activation of ERK.Conclusions(1)Colitis induces visceral hyperalgesia,accompanied by increased expression of CCL8 and its receptor CCR5 in spinal cord neurons.Upregulaiton of CCL8 is involved in colitis-induced visceral pain by activating ERK signaling pathway through CCR5.Inhibition of spinal CCL8/CCR5/ERK axis alleviates visceral hyperalgesia induced by colitis.(2)Colitis induces progressive increase of miR-146a-5p expression in the spinal cord neurons.CCL8 is identified as a potential target gene for miR-146a-5p.Overexpression of miR-146a-5p has a negative regulatory effect on colitis-induced visceral hyperalgesia through targeted inhibition of CCL8 expression and its downstream ERK signaling in the spinal cord.(3)The results of the present study elucidate for the first time the regulatory effects and mechanisms of spinal miRNA-146a-5p and its target gene CCL8 on colitis-induced visceral pain,which may provide a theoretical basis for revealing the central mechanism of abdominal pain in IBD patients.MiRNA-146a-5p and CCL8 may serve as potential therapeutic targets for abdominal pain in IBD condition.