The Promotion And Mechanism Of Neuropeptide FF System On Neurite Outgrowth Of Neuro 2A Cells

Neurite outgrowth is an important process in neural regeneration and plasticity,especially after neural injury,and recent evidences indicated that several G?i/o protein-coupled receptors played an important role in neurite outgrowth.NPFF system contains two G?i/o protein-coupled receptors-NPFF1 and NPFF2 receptors,which mainly distributes in the central nervous system.One of the aim of the present study was to determine whether NPFF system is involved in neurite outgrowth in Neuro 2A cells.Our results indicated that Neuro 2A cells endogenously expressed NPFF2 receptor,and the NPFF2 receptor agonist dNPA inhibited the cAMP production stimulated by forskolin(5 ?m)in Neuro 2A cells.Furthermore,the present studies demonstrated dNPA dose-dependently induced neurite outgrowth in Neuro 2A cells,which were completely abolished by the NPFF receptor antagonist RF9.Interestingly,pretreatment with the MAPK inhibitor U0126(10 ?m),SP600125(10 ?m)and SB203580(10 ?m)could reduce dNPA-induced neurite outgrowth.In addition,dNPA(1 ?m)increased the phosphorylation levels of ERK,JNK and p38 in Neuro 2A cells,which was completely antagonized by the pretreatment of U0126(10 ?m),SP600125(10 ?m)and SB203580(10 ?m).Taken together,these data suggest that activation of NPFF2 receptor stimulates neurite outgrowth in Neuro 2A cells through an activating MAPK signaling pathway.There are reports indicated that Neuro 2A cells endogenously express the cannabinoid receptor CB1,the CB1 receptor is belong to G?i/o protein-coupled receptor,and activation of CB1 receptor stimulates neurite outgrowth through an activating ERK1/2 and JNK signaling pathway.Moreover,existing research indicated that cannabinoid system and NPFF system have interactions in regulation of pain.So one of the other aim of the present study is to study whether NPFF system ans cannabinoid system have interactions in neurite outgrowth in Neuro 2A cells.The results indicated that the cannabinoid agonist WIN55,212-2 could induce neurite outgrowth in Neuro 2A cells,when Neuro 2A cells was subjected to WIN55,212-21(1 ?m)+ dNPA(1 ?m),the induced neurite outgrowth of each of them was obviously inhibited.Moreover,both of WIN55,212-2(1 ?m)and dNPA(1 ?m)could increase the phosphorylation levels of ERK and JNK in Neuro 2A cells.But when Neuro 2A cells was subjected to WIN55,212-2(1 ?m)+ dNPA(1 ?m),the increased-the phosphorylation levels of ERK and JNK in Neuro 2A cells didn't significantly change compared to dNPA or WIN55,212-2-induced the increasement of the phosphorylation levels of ERK and JNK.So these data suggest that WIN55,212-2 and dNPA had reciprocal inhibition in inducing neurite outgrowth in Neuro 2A cells through an activating non-ERK1/2 and non-JNK signaling pathway.In conclusion,Neuro 2A cells endogenously expressed NPFF2 receptor,and dNPA induce neurite outgrowth through an activating MAPK signaling pathway;WIN55,212-2 and dNPA have reciprocal inhibition in inducing neurite outgrowth through non-ERK1/2 and non-JNK signaling pathways.So,the present study broaden the physiological functions of NPFF system,and we further confirm the reciprocal interactions of NPFF system and cannabinoid system.