电针对坐骨神经分支选择性损伤大鼠脊髓CaMK Ⅱ-CREB通路的影响  

Effect of Electroacupuncture Intervention on CaMK Ⅱ-CREB Pathway in Spinal Cord in Rats with Spared Nerve lnjury

作　　者：闫丽萍[1] 侯保权 李守栋[1] 王玲玲[1] 马骋[1] 

机构地区：[1]南京中医药大学江苏省针灸学重点实验室,南京210029 [2]南京凯基生物科技发展有限公司,南京210012

出　　处：《针刺研究》2015年第5期358-363,共6页Acupuncture Research

基　　金：国家自然科学基金面上项目(No.81173329)

摘　　要：目的：通过观察坐骨神经分支选择性损伤（SNI）大鼠脊髓磷酸化钙-钙调蛋白依赖性蛋白激酶Ⅱ（CaMKⅡ）与环磷腺苷反应元件结合蛋白（CREB）的变化和电针干预对其的影响,探讨电针干预神经病理性痛的脊髓机制。方法：SD大鼠随机分为5组,每组12只。假手术组仅分离坐骨神经,但不进行结扎和剪断;模型组采用SNI法制备神经病理性痛模型;电针组于造模术后第11~17天电针大鼠损伤侧＂委中＂穴与＂环跳＂穴,刺激时间30min,1次/d;AP-5（N-甲基-D-天冬氨酸受体拮抗剂）组和L-NAME（一氧化氮合酶抑制剂）组同样于术后第11~17天实施药物干预。于造模前、造模后10d和16d时测定机械痛阈。造模后17d时,采用Western blot法测定脊髓磷酸化CaMKⅡ（pCaMKⅡ）与磷酸化CREB（pCREB）蛋白的表达,采用荧光定量-聚合酶链反应法测定脊髓CREB mRNA的表达。结果：与基础值比较,除假手术组外的其余4组SNI后机械痛阈均降低（P〈0.01）;与SNI后10d比较,电针组、AP-5组和L-NAME组SNI后16d时机械痛阈均升高（P〈0.01,P〈0.05）;与假手术组比较,模型组机械痛阈降低（P〈0.01）,其脊髓组织pCaMKⅡ与pCREB的蛋白印迹表达量均升高（P〈0.05）,CREB mRNA的表达亦升高（P〈0.05）;与模型组比较,电针组机械痛阈升高（P〈0.01）,同时脊髓组织pCaMKⅡ与pCREB蛋白印迹及其CREB mRNA表达均被逆转（均P〈0.05）,AP-5组和L-NAME组机械痛阈亦升高（P〈0.05）,但脊髓组织pCaMKⅡ与pCREB蛋白印迹及其CREB mRNA表达均无明显变化（P〉0.05）。结论：电针减轻大鼠神经病理性痛的机制之一,可能与其有效地下调脊髓CaMKⅡ-CREB通路的功能有关。Objective To observe the effect of electroacupuncture（EA）stimulation of＂Weizhong＂（BL 40）-＂Huantiao＂（GB 30）on expression of phosphorylated calcium/calmodulin dependent protein kinaseⅡ（p-CaMKⅡ）and cAMP response element binding protein（p-CREB）in the spinal cord in rats with spared nerve injury（SNI）,so as to explore its mechanism underlying easing neuropathic pain.Methods Sixty SD rats were randomly divided into five groups：control（sham-operation）,model,EA,AP-5（a NMDA receptor antagonist）and L-NAME（a non-selective nitric oxide synthase,NOS inhibitor）（n=12in each group）.The neuropathic pain model was established by sectioning the right tibal nerve and common peroneal nerve.EA intervention（2Hz,1mA,increasing 1mA/10min）was applied to＂Weizhong＂（BL 40）and＂Huantiao＂（GB 30）on the injured side for30 min,once a day for 7days.Rats of the AP-5and L-NAME groups were treated by intragastric administration of AP-5（0.7mg·kg-1·d-1）and L-NAME（60mg·kg-1·d-1）respectively from the 11 th day after operation,once daily for 7days.The mechanical pain thresholds were measured before the SNI procedure（baseline）and at the 10 th and 16 th day after the procedure.The expression of p-CaMKⅡ protein and p-CREB protein and gene of the spinal cord（L4-L6segments）was determined by Western blot and fluorescence quantitative-polymerase chain reaction（PCR）,separately.Results In comparison to the control group,the mechanical pain threshold was significantly decreased in the model group（P〈0.01）.After EA intervention,the mechanical pain thresholds of the EA,AP-5and L-NAME groups were obviously increased（P〈0.01,P〈0.05）on day 16 post SNI procedure.The expression levels of p-CaMKⅡand p-CREB proteins and CREB mRNA in the spinal cord were significantly higher in the model group than in the control group（P〈0.05）.Compared with the model group,the expression levels of spinal p-CaMKⅡand pCREB proteins and CREB mRNA were obviously down-regulated

关 键 词：电针干预 神经病理性痛 脊髓 钙-钙调蛋白依赖性蛋白激酶Ⅱ 环磷腺苷反应元件结合蛋白 

分 类 号：R245.97[医药卫生—针灸推拿学]