| Background and Aims:G protein-coupled kinases 6(GRK6)is a crucial protein that accommodates cell signals by phosphorylating specific amino acid residues in the cytoplasmic domain of G protein coupled receptor to desensitize GPCRs(G protein-coupled receptors).Studies have demonstrated that GRK6 is involved in inflammatory pain and neuropathic pain.However,the function and mechanism of GRK6 in chronic visceral pain were not fully understood.The arcuate nucleus(ARC)is a vital brain region for pain modulation.The present study was designed to explore the role and underlying mechanism of GRK6 and Nuclear factor-?B(NF-?B)signaling pathways in ARC of chronic visceral pain.Methods:1.Neonatal maternal deprivation(NMD)was used to induce chronic visceral pain sensitivity in adult rats.2.Behavioral methods:Colorectal distention(CRD)and abdominal withdrawal scores(AWR scores)were used to detect chronic visceral hyperalgesia in adult rats.Paw withdrawal threshold(PWT)in response to von Frey filament(VFF,0.4-15 g)stimulation and thermal paw withdrawal latency(PWL)in response to radiant heat applied to the hindpaw plantar surface were performed.3.Western blotting,Immunofluorescence,Real-time quantitative polymerase chain reaction and nissl staining were employed to determine the expression of GRK6 and NF-?B.4.Pharmacological methods:Pyrrolidine dithiocarbamate(PDTC),a selective antagonist of NF-?B,was injected into ARC by catheterization and microinjection.The pain behavior of rats and the expression of GRK6 in ARC were observed.5.Genetic methods:LV-GRK6(Lentiviral vectors GRK6)or LV-NC(Lentiviral negative control)were injected into ARC by catheterization and microinjection.The pain behavior of rats and the expression of GRK6 with NF-?B in ARC were observed.Results:1.Chronic visceral hypersensitivity was induced by NMD rats manifested by the reduced CRD threshold(***p<0.001)and the increased AWR scores(*p<0.05,**p<0.01).No significant changes of PWL and PWT were observed in NMD group when compared with CON group(p>0.05).The results showed that NMD did not induce somatic mechanical pain sensitivity and heat pain sensitivity in adult rats.2.NMD suppressed GRK6 expression in ARC(*p<0.05).There was no significant difference in GRK6 expression in the basolateral amygdala(BLA)and insular cortex(IC)regions of NMD rats when compared with CON rats(p>0.05).GRK6 was co-expressed predominately in neurons but not in astrocytes,and a little with microglial cells.NMD reduced the percentage of GRK6 positive ARC neurons(*p<0.05).3.NF-?B expression in ARC was upregulated(**p<0.01)and was co-expressed in GRK6 positive neurons of CON rats.There was no significant difference in NF-?B expression in BLA and IC regions of NMD rats when compared with CON rats(p>0.05).4.Single injection of pyrrolidine dithiocarbamate(PDTC)into the ARC area of NMD rats significantly attenuated visceral pain sensitivity of NMD rats(*p<0.05,***p<0.001).Moreover,the effective time of the PDTC group lasted from 5 min to 30 min after injection.Consecutive 7-day injection of PDTC into ARC remarkably attenuated the chronic visceral pain of NMD rats(***p<0.001).This effect lasted from 5 min to 120 min after the drug injection.5.Overexpression of GRK6 in ARC region reversed chronic visceral pain in NMD rats(**p<0.01,***p<0.001).Compared with LV-NC rats,the mechanical and thermal pain thresholds of hindpaw of LV-GRK6 rats had no significant change(p>0.05).Overexpression of GRK6 suppressed NF-? B expression(*p<0.05).There was no significant alteration of GRK6 expression in PDTC-treated group when compared with DMSO group(p>0.05).Conclusions:Present data suggest that NMD suppressed GRK6 expression and enhanced NF-?B expression,thus contributing to visceral hypersensitivity of adult rats.GRK6 might be a key molecule for ARC participated in visceral hyperalgesia in NMD rats,which provided a theoretical basis for GRK6 as a target for the treatment of chronic visceral pain. |
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