Cell-penetrating Peptide Tat-FynP Inhibits NR2B Phosphorylation In Inflammatory Pain Rats

BackgroundInflammatory pain is a main type of chronic pain with high incidence among people,but current treatments are inadequate.According to a great deal of studies,central sensitization is vital in the development and maintenance of chronic pain.N-methyl-D-aspartate receptor is one of the key mediators of central sensitization.Phosphorylation of NR2B in spinal cord horn plays an important role in the maintenance of chronic pain.Therefore,inhibition of NR2B phosphorylation can attenuate pain significantly.However,clinical use of NR2B antagonists is limited by numerous intolerable side effects due to the essential functional contribution of NR2B throughout the central nervous system.Exploring novel ways of inhibiting NR2B phosphorylation without receptor blockade is a hot area of pain research.Researches have shown that under noxious circumstances,the association between Fyn and PSD-95 promotes NR2B tyrosine phosphorylation,resulting in chronic pain state.We previously identified the interaction peptide sequence based on peptide array technique and synthesized the cell-penetrating peptide Tat-FynP and mismatched Tat-FynP.Experiments in vitro have shown that Tat-FynP can inhibit the combination of Fyn and PSD-95,reducing the levels of NR2B phosphorylation in primary neuronal cells of rats.This study assesses the effects of disrupting the protein-protein interaction between Fyn and PSD-95,thus inhibiting the phosphorylation of NR2B and relieving pain sensitivity in rat models of inflammatory pain.MethodsRespective his-tagged drugs were administrated to rats by intrathecal injection.Immunohistochemistry was performed to detect the penetrability of peptides,then we calculated staining scores of the His-tagged peptides expression in spinal dorsal horn of rats.Proteins were extracted from L4-L6 spinal cord of naive rats and rats at the indicated time after the injection of CFA.Immunoblotting was performed to detect the level of p-NR2B in the development of CFA-induced inflammatory pain in rats.Different doses of Tat-FynP and Tat-mFynP were administrated intrathecally 1d after CFA injection.Spinal cord tissues were collected for co-immunoprecipitation and western blotting to detect the interaction between Fyn and PSD-95 and the levels of p-NR2B.Mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured before CFA injection and 1,3,5,7,10,14 days after CFA injection.The reflexes for surface righting,placing and stepping were tested to evaluate the influence of intrathecal drug application on motor functions.ResultsCompared with group FynP,the scores of positive neuronal cells in spinal dorsal horn of rats in the group Tat-FynP and group Tat-mFynP were up-regulated(P<0.01).There is no significant difference between group blank and group FynP(P>0.05).The levels of NR2B phosphorylation at 3,6 and 12 hours and 1,3,7 days were significantly upregulated after CFA treatment when compared with the baseline control levels in saline rats(normalized by NR2B)(P<0.01).Tat-FynP can disrupt Fyn-PSD95 protein-protein interaction.Compared with group CFA,p-NR2B was down-regulated(P<0.01)in group Tat-FynP,MWT was increased significantly and TWL was prolonged after Tat-FynP administration in group Tat-FynP(P<0.01).There is no adverse effect on motor function in experimental rats.ConclusionsCell-penetrating peptide Tat-FynP based on peptide array technique can penetrate into neuronal cells in spinal dorsal horn of rats.Intrathecal injection of Tat-FynP can targetedly reduce phosphorylation level of NR2B in SCDH by perturbing the interaction between Fyn and PSD-95 in inflammatory pain rats,thus can attenuate pain hyperalgesia without affecting motor function,which provides powerful evidence for pain treatment.