电针颈夹脊穴对神经根型颈椎病模型大鼠小胶质细胞、P38丝裂原活化蛋白激酶和炎性因子表达的影响

Effects of Electro-Needling Cervical Jiaji Points on Expressions of Microglia,P38-MAPK and Inflammatory Cytokines in Rats with CSR

目的:观察电针颈夹脊穴对神经根型颈椎病(CSR)模型大鼠疼痛行为学、脊髓背角中小胶质细胞激活标志物补体受体3(CR3)、P38丝裂原活化蛋白激酶(P38-MAPK)和炎性细胞因子表达的影响,探讨电针治疗CSR所致神经病理性疼痛的镇痛机制。方法:将72只雄性SD大鼠随机分为空白组、模型组、假手术组、电针组、胶质细胞抑制剂L-α-氨基己二酸(LAA)组和电针+LAA组,每组12只。空白组不做任何处置;假手术组仅暴露右侧C7脊神经,不结扎,并进行鞘内置管;模型组、电针组、LAA组及电针+LAA组结扎右侧C7脊神经建立神经根型颈椎病模型并进行鞘内置管。空白组不予任何干预;假手术组与模型组仅在电针组干预同时间点进行捆绑操作;电针组于术后第7天开始电针双侧C6、C7颈夹脊穴,20 min/次,1次/d,连续干预14 d;LAA组于术后第7天开始鞘内注射LAA,5 nmol/次,1次/d,连续干预14 d;电针+LAA组于术后第7天开始同电针组与LAA组进行电针双侧C6、C7颈夹脊穴及鞘内注射LAA干预。术后观察并记录大鼠患侧足底热缩足反射潜伏期(PWTL),采用免疫荧光法观察CR3、P38-MAPK表达水平,采用免疫组化法观察炎性细胞因子白介素1β(IL-1β)、白介素6(IL-6)、白介素18(IL-18)与肿瘤坏死因子α(TNF-α)表达水平。结果:造模后第7天,模型组、电针组、LAA组与电针+LAA组PWTL明显下降,差异具有统计学意义(P<0.05);经干预后,电针组、LAA组和电针+LAA组治疗后患侧前足底PWTL均持续升高,术后14 d、21 d患侧前足底PWTL明显高于同时间节点模型组,差异具有统计学意义(P<0.05)。与空白组比较,假手术组大鼠脊髓背角CR3、P38-MAPK、IL-1β、IL-6、IL-18与TNF-α表达差异无统计学意义(P>0.05);与假手术组比较,模型组大鼠脊髓背角CR3、P38-MAPK、IL-1β、IL-6、IL-18与TNF-α表达明显升高,差异具有统计学意义(P<0.05);与模型组比较,电针组、LAA组、电针+LAA组大鼠脊髓背角CR3、P38-MAPK、IL-1β、IL-6、IL-18与TNF-α表达均明显降低,差异具有统计学意义(P<0.05)。结论:电针颈夹脊穴可抑制小胶质细胞激活,下调P38-MAPK表达,减少炎性细胞因子IL-1β、IL-6、IL-18与TNF-α的生成,提示电针可能通过抑制小胶质细胞激活、降低P38-MAPK表达和抑制炎性细胞因子分泌以发挥镇痛作用。

Objective:To observe the effects of electro-needling cervical Jiaji acupoints on pain behavior,expressions of microglial specific markers Complementreceptor 3(CR3),p38 mitogen-activated protein kinase(p38-MAPK)and inflammatory cytokines in spinal dorsal horn of rats with cervical spondylotic radiculopathy(CSR),and to explore the analgesic mechanism of electro-acupuncture(EA)in treatment of neuropathic pain caused by CSR.Methods:72 male SD rats were randomly divided into the blank group,the model group,the sham operation group,the EA group,the glial cell inhibitor L-α-aminoadipate(LAA)group and the EA plus LAA group,with 12 rats in each group.The model of CSR was established by ligating the right C7spinal nerve,whereas no intervention was made in the blank group,and the right C7spinal nerve was not ligated in the sham operation group.The sham operation group and the model group only performed the binding at the same time point of the EA group,and the EA was treated with electro-needling bilateral C6and C7cervical Jiaji points on the 7th day after modeling,20 min per time,once a day for continuous 14 days.On the 7th day after modeling,LAA(5 nmol)was injected in the LAA group once a day for continuous 14 days.The EA plus LAA group was treated with electro-needling bilateral C6and C7cervical Jiaji points and LAA injection on the 7th day after modeling.The paw withdraw thermal latency(PWTL)was observed and recorded after modeling.The expressions of CR3 and P38-MAPK were observed by immunofluorescence,and the expressions of IL-1β,IL-6,IL-18 and TNF-αwere observed by immunohistochemistry.Results:On the 7th day after modeling,PWTL decreased significantly in the model group,the EA group,the LAA group and the EA plus LAA group(P<0.05),which was continuously increased after the intervention in the three groups;PWTL of affected forefoot was significantly higher on 14 days and 21 days after modeling in the three groups than that in the model group at the same time point(P<0.05).There were no statistical differences in the expressions of CR3,P38-MAPK,IL-1β,IL-6,IL-18 and TNF-αin the spinal dorsal horn between the sham operation group and the blank group(P>0.05);compared with those in the sham operation group,the expressions of CR3,P38-MAPK,IL-1β,IL-6,IL-18 and TNF-αin the spinal dorsal horn was significantly increased in the model group(P<0.05);which were significantly decreased after the intervention in the EA group,the LAA group and the EA plus LAA group compared with those in the model group(P<0.05).Conclusion:Electro-needling cervical Jiaji points can inhibit the activation of microglia,down-regulate the expression of P38-MAPK and reduce the productions of inflammatory cytokines of IL-1β,IL-6,IL-18 and TNF-α,suggesting that EA may exert its analgesic effect by inhibiting the activation of microglia,reducing the expression of P38-MAPK and inhibiting the secretion of inflammatory cytokines.