针刺足三里对CFA大鼠尾壳核中A1R/cAMP/p-CREB信号通路的影响

Effect of acupuncture on A1R/cAMP/p-CREB signaling pathway in caudate putamen of CFA rats

目的:观察针刺对完全弗氏佐剂(CFA)大鼠尾壳核(CPu)中的腺苷A1受体(A1R)/环磷酸腺苷(cAMP)/cAMP反应元件结合蛋白(CREB)信号通路的影响,探讨针刺治疗炎性痛的潜在机制。方法:将64只6~8周龄雄性Wistar大鼠运用随机数字法随机分为盐水组、模型组(CFA组)、CFA+手针针刺(MA)组、CFA+溶剂二甲基亚砜(DMSO)组、CFA+A1R激动剂2-氯-N6-环戊基腺苷(CCPA)组、CFA+A1R拮抗剂8-环戊基-1,3-二丙基黄嘌呤(DPCPX)组、CFA+MA+DMSO组和CFA+MA+DPCPX组,每组8只。采用右侧足底皮内注射CFA制作关节炎炎性痛模型。针刺组于造模后第2天针刺大鼠双侧足三里穴,每次30 min,每天1次,共7 d。采用足底热辐射痛阈值评判大鼠疼痛反应;ELISA检测CPu脑区cAMP含量变化;Western blot检测CPu脑区PKA和CREB蛋白表达及磷酸化水平的变化;免疫荧光染色检测CPu脑区A1R表达情况。结果:与盐水组比较,CFA造模显著降低大鼠热痛阈值(P<0.01);与CFA组比较,CFA+MA组和CFA+CCPA组大鼠的热痛阈值均显著升高(P<0.05或P<0.01);与CFA+MA+DMSO组比较,CFA+MA+DPCPX组大鼠的热痛阈值显著降低(P<0.05)。与盐水组和CFA组比较,CFA+MA组大鼠CPu脑区中的A1R蛋白相对表达量和阳性细胞数均显著增加(P<0.05或P<0.01)。与盐水组相比,CFA+MA组大鼠CPu脑区中的cAMP含量显著减少,p-CREB蛋白水平显著降低(P<0.05);与CFA+DMSO组相比,CFA+MA+DMSO组和CFA+CCPA组的cAMP含量显著减少,p-CREB蛋白水平显著下降(P<0.01);与CFA+MA+DMSO组相比,CFA+MA+DPCPX组cAMP含量显著增加(P<0.01),p-PKA和p-CREB蛋白水平显著升高(P<0.05或P<0.01)。结论:针刺双侧足三里可以缓解CFA大鼠的炎性疼痛,其机制可能与A1R/cAMP/p-CREB信号通路有关。

AIM:To observe the effect of acupuncture on adenosine A1 receptor(A1R)in the caudate putamen(CPu)of complete Freund's adjuvant(CFA)rats,and to explore the potential mechanism of acupuncture in treatment of inflammatory pain.METHODS:Sixty-four 6^8-week-old male Wistar rats were randomly divided into saline group,model group(CFA group),CFA+manual acupuncture(MA)group,CFA+solvent dimethyl sulfoxide(DMSO)group,CFA+A1R agonist 2-chloro-N6-cyclopentyladenosine(CCPA)group,CFA+A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine(DPCPX)group,CFA+MA+DMSO group and CFA+MA+DPCPX group.In MA groups,on the 2nd day after modeling,the rats were needled at Zusanli points on both sides,30 min at a time,once per day,for 7 d.Pain threshold of plantar thermal radiation was used to observe the pain response of the rats.The content of cyclic adenosine monophosphate(cAMP)in the CPu was detected by ELISA.The protein expression and phosphorylation levels of protein kinase A(PKA)and cAMP response element-binding protein(CREB)were detected by Western blot.The expression of A1R in the CPu was detected by immunofluorescence staining.RESULTS:Compared with saline group,CFA modeling significantly lowered the thermal pain threshold of the rats(P<0.01).Compared with CFA group,the thermal pain threshold of the rats in CFA+MA group and CFA+CCPA group was significantly increased(P<0.05 or P<0.01).Compared with CFA+MA+DMSO group,the thermal pain threshold of the rats in CFA+MA+DPCPX group was decreased(P<0.05).Compared with CFA group,A1R protein relative expression level and positive cells in the CPu of the rats in CFA+MA group were increased(P<0.05 or P<0.01).Compared with saline group,cAMP content and p-CREB protein level in the CPu of the rats in CFA+MA group were decreased(P<0.05).Compared with CFA+DMSO group,cAMP content and p-CREB protein level in CFA+MA+DMSO and CFA+CCPA groups were significantly decreased(P<0.01).Compared with CFA+MA+DMSO group,the levels of cAMP,p-PKA and p-CREB in CFA+MA+DPCPX group were significantly increased(P<0.05or P<0.01).CONCLUSION:Acupuncture on bilateral Zusanli can relieve inflammatory pain in CFA rats,and its mechanism may be related to A1R/cAMP/p-CREB signaling pathway.