摘要
目的通过观察不同推拿刺激参数对坐骨神经慢性压迫损伤小鼠脊髓背角胶质纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)及炎症因子白细胞介素-6(Interleukin-6,IL-6)、白细胞介素-1β(Interleukin-1β,IL-1β)、肿瘤坏死因子-α(Tumor Necrosis Factor-alpha,TNF-α)表达的影响,明确推拿发挥镇痛作用的最佳刺激参数及推拿对坐骨神经痛的中枢镇痛机制。方法将56只C57BL/6雄性小鼠按随机数字表法分组为空白组、假手术组、模型组、推拿0.2N组、推拿0.4N组、推拿0.6N组、推拿非经穴组,每组各8只,模型组和各推拿干预组通过手术结扎坐骨神经干制备小鼠坐骨神经慢性压迫损伤(Chronic constriction injury,CCI)模型,假手术组仅暴露坐骨神经干不结扎。造模后第8天开始推拿干预,连续干预7 d。分别于造模前,干预后第3天、第7天、第10天、第14天观察各组小鼠足底表观形态、机械刺激缩足阈值(Mechanical paw withdrawal threshold,PWT)和热缩足潜伏期(Paw withdrawal latencies,PWL)的变化,取材后ELISA法检测脊髓背角组织IL-6、IL-1β、TNF-α蛋白表达水平,免疫荧光染色法检测脊髓背角组织GFAP表达水平。结果造模后,与空白组和假手术组比较,模型组右侧后足足趾始终呈并拢卷曲形态,着地时动作不协调,行走时出现跛行和足外翻,并伴有抬腿、舔足等行为,PWT和PWL值均明显下降(P<0.001),并在第7天到达最低值并持续至实验观察结束,提示CCI模型小鼠造模成功。干预后第3天,与模型组比较,推拿0.4N组右侧后足足趾并拢卷曲形态稍展,但仍有跛行和足外翻,PWT和PWL显著升高(P<0.001),推拿0.2N组、推拿0.6N组、推拿非经穴组与模型组同期比较具有相似的行为和足底形态表现,PWT和PWL均无明显变化(P>0.05)。干预后第7天,与模型组比较,推拿0.4N组右侧后足足趾并拢卷曲形态展开明显,着地时动作协调度提高,行走时跛行和足外翻减轻,PWT和PWL持续显著升高(P<0.001);推拿0.2N组右足足趾并拢卷曲形态稍展,但展开程度不及推拿0.4N组,且仍有跛行和足外翻行为,PWT和PWL稍有上升(P<0.05,P<0.01),而推拿0.6N组、推拿非经穴组PWT和PWL均无明显变化(P>0.05);与推拿0.2N组比较,推拿0.4N组PWT和PWL升高更明显,组间差异有统计学意义(P<0.001)。免疫荧光染色和ELISA结果显示,与空白组和假手术组比较,模型组、推拿0.2N组、推拿0.4N组、推拿0.6N组、推拿非经穴组GFAP、IL-6、IL-1β、TNF-α表达均明显升高(P<0.05);与模型组比较,推拿0.2N组、推拿0.4N组GFAP、IL-6、IL-1β和TNF-α表达均降低(P<0.05);与推拿0.2N组比较,推拿0.4N组GFAP、IL-6、IL-1β和TNF-α表达降低更明显(P<0.05)。结论推拿环跳、阳陵泉穴的刺激参数为0.4N时,可有效下调脊髓背角炎症因子的表达,抑制星形胶质细胞活化,中枢痛觉敏化程度降低最为明显。
Objective To identify the optimal parameters of tuina for relieving pain and the mechanism of tuina in attenuating central sensitization of pain in sciatica by observing the effects of tuina with different parameters on glial fibril⁃lary acidic protein(GFAP)and inflammatory cytokines[interleukin-6(IL-6),interleukin-1 beta(IL-1β),and tumor necrosis factor-alpha(TNF-α)]in the mouse model of chronic constriction injury(CCI)of the sciatic nerve.Meth⁃ods Fifty-six male C57BL/6 mice were assigned by the random number table method into the following groups(n=8):blank,sham,model,tuina at 0.2N(0.2N),tuina at 0.4 N(0.4N),tuina at 0.6 N(0.6N),and tuina at non-meridian acupoints.The mice in the model group and each tuina intervention group were subjected to surgical ligation of sciatic nerve trunk for the modeling of CCI of the sciatic nerve,while those in the sham group only exposed sciatic nerve trunk without ligation.Tuina intervention began on day 8 after modeling and lasted for 7 days.The apparent morphology,mechanical paw withdrawal threshold(PWT),and paw withdrawal latency(PWL)of mice in each group were observed before modeling and on days 3,7,10,and 14 of intervention.Enzyme-linked immunosorbent assay(ELISA)was employed to measure the ex⁃pression levels of IL-6,IL-1β,and TNF-αin the spinal dorsal horn,and immunofluorescence staining was employed to determine the expression of GFAP in the spinal dorsal horn.Results Compared with the blank group and the sham group,the model group showed closed and curled toes of the right posterior paw,uncoordinated movement during landing,lameness and paw eversion during walking,and leg lifting and paw licking behaviors.In addition,the model group showed decreased PWT and PWL(P<0.001),which reached the lowest values on day 7 and maintained until the end of the experiment.The results above indicated that the CCI model was successfully established.On day 3 of intervention,compared with the model group,the 0.4N group showed slightly folded toes of the right posterior paw,presence of lameness and paw eversion,and increased PWT and PWL(P<0.001).The 0.2N,0.6N,and tuina at non-meridian acupoints groups showed similar be⁃haviors and plantar morphology compared with the model group at the same time points and no significant changes in PWT and PWL(P>0.05).On day 7 of intervention,compared with the model group,the 0.4N group showcased unfolded toes of the right posterior paw,coordinated movement during landing,alleviated lameness and paw eversion during walking,and in⁃creased PWT and PWL(P<0.001).The 0.2N group showed slightly unfolded toes of right posterior paw(which was not comparable to that in the 0.4N group),presence of lameness and paw eversion,and increased PWT and PWL(P<0.05,P<0.01).The PWT and PWL of the 0.6N group and the tuina at non-meridian acupoints group were not significantly changed compared with those in the model group(P>0.05).The 0.4N group had higher PWT and PWL than the 0.2N group(P<0.001).Immunofluorescence staining and ELISA results showed that compared with the blank and sham groups,the model,0.2N,0.4N,0.6N,and tuina at non-meridian acupoints groups showed up-regulated expression of GFAP,IL-6,IL-1β,and TNF-α(P<0.05).Compared with the model group,the 0.2N and 0.4N groups showed down-regulated expression of GFAP,IL-6,IL-1β,and TNF-α(P<0.05).The 0.4N group had lower levels of GFAP,IL-6,IL-1β,and TNF-αthan the 0.2N group(P<0.05).Conclusion Tuina at Huantiao(GB30)and Yanglingquan(GB)at 0.4N can down-regulate the expression of inflammatory cytokines in the spinal dorsal horn,inhibit the activation of astrocytes,and attenuate the central sensitization of pain.
作者
童华卿
谷俊刚
肖鸿伟
林荣
朱丹
何坚
TONG Hua-qing;GU Jun-gang;XIAO Hong-wei;LIN Rong;ZHU Dan;HE Jian(College of Rehabilitation Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou Fujian 350122;Zhan-gzhou Health Vocational College,Zhangzhou Fujian 363000)
出处
《世界中西医结合杂志》
2024年第12期2396-2402,2432,共8页
World Journal of Integrated Traditional and Western Medicine
基金
国家重点研发计划(2022YFC2009700)。
关键词
推拿
坐骨神经慢性压迫损伤
小鼠
炎症因子
胶质纤维酸性蛋白
Tuina
Chronic Constriction Injury of Sciatic Nerve
Mice
Inflammatory Cytokines
Glial Fibrillary A⁃cidic Protein
作者简介
通信作者:何坚,Email:591003659@qq.com。
