Involvement Of Src Tyrosine Protein Kinase Within Hypothalamic Arcuate Nucleus In CFA-induced Inflammatory Pain

Objective: We aimed to explore whether in the hypothalamic arcuate nucleus(ARC) non-receptor protein tyrosine kinase Src plays a key role in the regulation of N-methyl-D-aspartate(NMDA) receptor NR2 B subunit in rats with peripheral inflammatory pain.Methods: The inflammation was induced by a subcutaneous injection of Complete Freund’s Adjuvant(CFA) into the plantar surface of left hind paws of male Sprague-Dawley rats. We assessed mechanical allodynia with Von Frey filaments, thermal hyperalgesia with radiant heat and the motor function with Rota-rod test. All agents were administrated into ARC through a guide cannula implanted in ARC. Lentivirus was injected into ARC by stereotaxis technique and retaining the springe for. We analyzed expressions of NMDA NR2 B, pNR2 B, PKA,PKCγ,pCaMKIIα, Src and pSrc in ARC by western blotting. Succesful infections of the virus containing Src sh RNA and cDNA encoding green fluorescence protein in ARC were determined through observations under a fluorescence microscope All data were presented as mean ± S.E.M. Behavioral data were analyzed by using one-way repeated-measures analysis of variance(ANOVA)or student’s t-test. P<0.05 was considered statistically significant.Results:(1) CFA induced mechanical allodynia and thermal hyperalgesia lasted at least for 14 days(P<0.001).(2) Microinjection of MK-801, chelerythrine, ifenprodil, PP2 into ARC reversed the reduction of pain threshold under inflammatory condition(P<0.05, 0.01 or 0.001).(3) Compared with control animals, NR2 B, pNR2 B, Src, pSrc, and PKCγexpressions increased gradually in ARC till day 14 after CFA intraplantar injected(P<0.05,). The expression of pCa MKIIα decreased at day 1, then increased gradually till day 14 after CFA injection(P<0.05). PKA didn’t show any significant changes after CFA injection(P>0.05)(4) Knocking down Src by infection of Src shRNA in ARC not only reduced CFA-induced increases in the expressions of pNR2 B, NR2 B and PKCγ in ARC but also significantly decreased mechanical allodynia and thermal hyperalgesia(P<0.05). In contrast, the Src knock down did not produce any effects on expressions of pCa MKIIα and PKA in ARC following CFA injection.Conclusions:(1) NMDA receptors in ARC played an important role in the regulation of the central sensitization induced by peripheral inflamation.(2) Src kinase was critically involved in this regulation through regulating the tyrosine phosphorylation of NMDA receptor NR2 B subunit in ARC.(3) Thus, modulating the Src activity in ARC was an effective approach controlling inflammation-indcued pain.