| Objective: In the present study,we investigate which member(s)of Src family kinases in hypothalamic arcuate nucleus(ARC)involved in the hypersensitivity induced by peripheral inflammation.Methods: The peripheral inflammation was induced by an injection of Complete Freund's adjuvat(CFA)into the hind paw of rats.We assessed the mechanical allodynia by Von Frey tests and the thermal hyperalgesia by radiant heat.We analyzed the protein expression of SFK-pY416,Src,Fyn and Lyn in ARC by western blot analysis.We detected the phosphorylation of Src,Fyn and Lyn at the site Y416 immunoprecipitated from the ARC at day 3 after the CFA injection.Src was knockdown by sh-Src transfection.We further examined the effect of sh-Src injected in the ARC on rat pain behaviors.Results:(1)Three days after CFA injection,the mechanical withdraw threshold(MWT)and thermal withdraw latency(TWL)of rats were significantly reduced(MWT: 1.48 ± 0.33,p < 0.001;TWL: 6.89 ± 0.08,p < 0.001;vs.Saline group).(2)Compared with na?ve(control),the expression of activated SFKs in ARC was significantly increased at day 1 after the CFA injection and lasted for 7 days(1.63 ± 0.13,p < 0.01 at CFA day1;1.68 ± 0.11,p < 0.05 at CFA day3;1.62 ± 0.18,p < 0.05 at CFA day7).Also,the expression of Src was also robusted increased at days 3 and 7 after the CFA injection(1.35 ± 0.05,p < 0.01 at CFA day3;1.19±0.03,p < 0.01 at CFA day7).The expression of Fyn and Lyn were not significantly affected in CFA-injected rats.(3)Compared with the saline group,the phosphorylation level of Src was significantly increased(1.30 ± 0.09,p < 0.05),while no significant change was found in Fyn as well as Lyn.(4)Intra-ARC injection of sh-Src decreased the protein expression of Src in ARC(0.38 ± 0.06,p < 0.001),but did not induce any changes in the expression profile of Lyn(1.04 ± 0.07)or Src either in the cortex(0.97 ± 0.08)or spinal dorsal horn(0.98 ± 0.08).(5)Knockdown of Src prevented the CFA-induced increase in Src,and abolished the increased expression of phosphorylated SFKs at Y416 in the ARC(For activated SFKs: 1.04 ± 0.13,p < 0.05 at day 1,1.05 ± 0.04,p < 0.001 at day3,0.90 ± 0.05,p < 0.001 at day7).(6)In the ARC area,knocking down Src prevented CFA injection-induced increases in expressions of GluN2 B and GluN2B-pY1472(For GluN2B: 1.05±0.04,p < 0.05 at day3;0.88±0.02,p < 0.05 at day7;For GluN2B-pY1472: 0.88±0.11,p < 0.05 at day3;0.88±0.10,p < 0.05 at day7).(7)Application of the SFKs inhibitor SU6656 systemically diminished the behavior changes induced by CFA.The effects produced by SU6656 in rats which received the intra-ARC injection of Src shRNA,were significantly reduced when compared with those in rats which did not receive any intra-ARC injections or received the sh-NC injection(MWT relative changes in uninfected group: 160 ± 37%,at 1h;230 ± 31%,at 3h;MWT relative changes in sh-NC group: 380 ± 120%,at 1h;250±100% at 3h;MWT relative changes in sh-Src group: 60±15%,at 1h;21 ± 5%,at 3h;TWL relative changes in uninfected group: 48 ± 5%,at 1h;50 ± 3%,at 3h;TWL relative changes in sh-NC group: 35 ± 3%,at 1h;38 ± 1%,at 3h;TWL relative changes in sh-Src group: 14±2%,at 1h;16±2%,at 3h).Conclusions:(1)The SFKs in ARC were significantly activated by CFA.The protein expression of Src as well as its phosphorylated form(p–Src)were significantly increased.(2)The injection of sh-Src reduced the expressions of SFKs and abolished the effects of the SFKs inhibitor SU6656 after CFA injection,which indicated that Src in the ARC area is a key factor in the facilitation of pain hypersensitivity following peripheral inflammation.(3)The injection of sh-Src into the ARC reduced the expressions of the GluN2 B and the phosphorylated GluN2 B incduced by the CFA injection.The Src-GluN2 B signalling pathway in ARC might be involved in pain hypersensition. |
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