| Objective:The hypothalamic arcuate nucleus(ARC)plays a key role in pain processing.NMDA receptor(NMDAR)and Src are involved in the modulation of nociception and pain in the ARC with inflammatory.The aim of the present study is to explore the possible regulation way of miR-137-3p and miR-195-5p on Src and NMDA receptor in complete Freund's adjuvant(CFA)-induced inflammatory pain rat model by detecting the expression level of miR-137-3p,miR-195-5p,Src and NMDA receptor subunits(GluNl,GluN2A and GluN2B),and analyzing their interrelations,to find new therapy target for chronic inflammatory pain in gene level.Methods:(1)Using bioinformatics tools to identify the potential targeted relationship between miR-137-3p and Src as well as GluN2A gene.The same for miR-195-5p and GluN1 as well as GluN2B gene.(2)Using the Freund's adjuvant(CFA)-induced chronic inflammatory pain model,we assessed mechanical allodynia with Von Frey filaments,thermal hyperalgesia with radiant heat.(3)Then,we investigated the expression profiles of miR-137-3p,miR-195-5p,GluN1 GluN2A,GluN2B and Src in male Sprague-Dawley rat ARC using quantitative real-time PCR and Western Blot at time points of 0 d,Id,3d,5d,7d and 14d,respectively.The relationship of miR-137-3p and Src,GluN2A expression was analyzed by linear regression.The same for miR-195-5p and GluNl as well as GluN2B gene.(4)We further examined the expression of Src,GluN2A combined with that of miR-137-3p in the same ARCs by in situ hybridization and immunohistochemistry.(5)The reporter vectors including the pGL3-control-Src-137-WT,pGL3-control-GluN2A-137-WT with target site sequence and the pGL3-control-Src-137-MT,pGL3-control-GluN2A-137-MT without target site sequence were constructed,then together with the miR-137-3p mimic(100 nM)introduced into the HEK293T.To confirm the targeted relationship between GluN2A,Src gene and miR-137-3p by dual luciferase reporter gene assay.(6)We transfected miR-137-3p mimic or inhibitor into primary neurons of arcuate nucleus cells using lipofectamine 3000.We investigated the expression of miR-137-3p and Src,GluN2A mRNA after transfection by the same way as real-time PCR.Src,GluN2A protein level expression in cell culture after transfection was detected by Western Blot.(7)ARC administration was accomplished using a microinjection syringe connected to the guide cannula implanted in ARC.The complexes containing miR-137-3p agomir or antagomir were administered in 0.5?L volume once daily for 2 days beginning 3days after CFA induced chronic inflammatory pain.After ARC administration we assessed mechanical allodynia and thermal hyperalgesia.The expression of Src,GluN2A protein and mRNA was detected by Western blotting and real-time PCR at the same time.Results:(1)The bioinformatics tools initially identified the potencial targrted relationship between Src and GluN2A gene and miR-137-3p.The same for GluN1 and GluN2B gene as well as miR-195-5p.(2)CFA induced mechanical allodynia and thermal hyperalgesia lasted at least for 14days(P<0.001).(3)Src in CFA-induced inflamed rats significantly increased comparing with control rats in Id,3d,5d,7d and 14d in protein level.GluN1 expression significantly increased comparing with control rats in 3d,5d and 7d.GluN2A expression significantly increased in 3d,5d and GluN2B expression significantly increased in 3d,5d,14d in protein level.Meanwhile the relative amount of Src in CFA-induced inflamed rats significantly increased comparing with control rats in 3d,5d and 7d.GluN1 expression significantly increased comparing with control rats in 5d and 7d.GluN2A expression significantly increased in 3d,5d and GluN2B expression significantly increased in 5d,14d.The results showed that miR-137-3p expression significantly decreased in 3d,5d and 7d after CFA injection and miR-195-5p expression significantly decreased in Id,3d and 7d after CFA injection and then gradually returned to basal level at 14d after CFA injection.Linear regression analysis showed that miR-137-3p expression level was inversely related with Src expression level(R2=0.2562,P<0.01)and GluN2A expression level(R2=0.2562,P<0.01).Otherwise,the expression level of miR-195 was not related with GluN1(R2=0.0641,P>0.05)or GluN2B(R2=0.0623,p>0.05).(4)Expression of miR-137-3p,Src and GluN2A in the same ARC tissues before and after inflammatory pain were observed using the method of in situ hybridization and immunohistochemistry.(5)After transfection with miR-137-3p(400nM,600nM)using lipofectamine2000 for 24h or 48h,compared with NC group,the expression of miR-137-3p was significantly reduced(P<0.001),while the level of Src,GluN2A mRNA changed according to the different dose and time.The expression of Src,GluN2A increased accordingly by western blot,as same as the result of real-time PCR(P<0.01).(6)For luciferase activity assays,when the wild-type 3'UTR of Src and GluN2A was present,miR-137-3p induced significant reduction in the relative luciferase activity,by more than 40%and 35%.This reduction was sequence-specific because the relative luciferase activity of a 3'UTR containing the mutant binding site did not drop as sharply as that of the 3'UTR containing the wild-type binding site.Taken together,these results suggest that miR-137-3p could dramatically downregulate GluN2A and Src expression by directly targeting the 3'UTR of GluN2A and Src.(7)Since day 1 after drugs administration,thermal hyperalgesia and mechanical allodynia induced by CFA were attenuated by ARC administration with miR-137-3p agomir,but not NS control.The results showed miR-137-3p agomir inhibited both Src,GluN2A protein and mRNA expression.However,miR-137-3p antagomir did not affect the thermal hyperalgesia and mechanical allodynia of normal rats.Conclusion:Here,we found that CFA-induced chronic inflammation pain significantly reduced miR-137-3p and miR-195-5p expression in rat ARC.Furthermore,the expression of GluN2A and Src in ARC,an experimentally validated target of miR-137-3p,were increased in CFA rats.Since day 1 after drugs administration,thermal hyperalgesia and mechanical allodynia induced by CFA were attenuated by ARC administration with miR-137-3p agomir.Together,we conclude that miR-137-3p expression regulates chronic inflammatory pain by targeting Src and the subunit GluN2A of NMDAR. |
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