夹脊电针对ALS-SOD1^(G93A)小鼠腰髓中p38 MAPK/NF-κB信号通路的影响研究

The effect of Jiaji Electro-acupuncture on p38 MAPK/NF-κB signaling pathway in Lumbar Spinal Cord of ALS-SODl^(G93A) Mice

目的探究p38 MAPK/NF-κB信号通路在夹脊电针治疗肌萎缩侧索硬化-超氧化物歧化酶1^(G93A)(ALS—SOD1^(G93A))转基因小鼠的作用机制。方法选取45只ALS-SOD1^(G93A)转基因阳性雌性小鼠为研究对象,随机分为模型组、手针组、电针组,每组各15只,另取15只同窝野生型雌性小鼠为对照组,小鼠60 d龄时开始实验:对照组、模型组每周给予捆绑固定20 min 2次,手针组给与普通针、电针组采用全能脉冲电疗仪给与电针治疗,每周2次刺激双侧L_(1～2)、L_(5～6)夹脊穴,共治疗4周。120 d取小鼠腰膨大L_(1～6)腰髓,采用尼氏染色、透射电镜观察进行形态学检测,采用免疫组织化学法检测p38、p-p38表达,免疫荧光检测p65核移位,蛋白免疫印迹法检测全称肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、白介素-1β(IL-1β)、核因子-κB抑制因子(IκB-α)、p38、p65、p-IκB-α、p-p38、p-p65的表达,实时定量PCR(qPCR)检测TNF-α、IL-6、IL-1βmRNA的表达。结果模型组神经元数目明显减少(P<0.05),手针组与电针组形态学均有较大改善、电针组改善更佳。模型组炎症因子TNF-α、IL-6、IL-1β蛋白及mRNA水平上升,与模型组及手针组比较,电针组TNF-α、IL-6、IL-1β蛋白及mRNA明显降低(P<0.05)。模型组检出大量p38、p-p38阳性细胞分布于脊髓灰质前角,与模型组比较,手针组与电针组p-p38表达减弱,电针组减弱程度更大;与对照组比较,模型组p-IκB-α、p-p65含量升高(P<0.05);与模型组相比,手针组与电针组p-IκB-α、p-p65含量下降(P<0.05)。手针组与电针组均抑制p65入核过程,其中电针组抑制更加明显。结论夹脊电针通过抑制p38 MAPK/NF-κB信号通路,对腰髓前角运动神经元产生保护作用,缓解ALS病情的进展,为夹脊电针临床推广提供了理论依据。

Objective Objective To explore the mechanism of p38 MAPK/NF-κB signaling pathway in the treatment of ALS-SODI^G93 A transgenic mice with electro-acupuncture. Methods 45 ALS-SOD1^G93 A transgenic positive female mice were randomly divided into model group, the hand needle group, electro-acupuncture group, 15 mice in each group. 15 wild type female mice were selected as the negative control group. The mice with 60 days old in the control group and model group were gave bind fixed for 20 min each time, 2 times per week. The mice in the hand needle group were gave ordinary needle. The mice in the electro-acupuncture group were received electro-acupuncture treatment with all-around pulse electrotherapy apparatus, and bilateral L1-2, L5-6 jiaji acupoints were stimulated twice a week for a total of 4 weeks. The L1-6 lumbar medulla of the mice with L1-6 lumbar enlargement were collected at the 120 th day, and their morphological were detected by Nissl staining and transmission electron microscopy. The expressions of p38 and p-p38 were detected by immunohistochemistry. The nuclear translocation of p65 were detected by immunofluorescence. The expressions of p38, p-p38, p65 and TNF-α, IL-6, IL-1β, IκB-α, p38, p65, p-IκB-α, p-p38 and p-p65 were detected by Western blot. The mRNA expressions of TNF-α, IL-6 and IL-1β were detected by qPCR. Results The number of neurons was significantly reduced in the model group(P<0.05). The morphology of the hand acupuncture group and the electro-acupuncture group was significantly improved, and the electro-acupuncture group was better. The levels of protein and mRNA expression of TNF-α, IL-6, IL-1β in the model group were increased, and the one in the electro-acupuncture group were significantly decreased compared with the model group and the hand acupuncture group(P<0.05). A large number of p38 and p-p38 positive cells were detected in the anterior horn of the spinal gray matter in the model group. The contents of p-IκB-α and p-p65 in the model group were higher than those in the control group(P<0.05). Compared with the model group, the contents of p-IκB-α and p-p65 were decreased(P<0.05). The process of p65 nucleation in the hand acupuncture group and the electro-acupuncture group were inhibited, and the inhibition was more obvious in the electro-acupuncture group. Conclusion By inhibiting the p38 MAPK/NF-κB signaling pathway, electro-acupuncture can protect the motoneurons of the anterior horn of lumbar spinal cord and alleviate the progress of ALS, which providing theoretical basis for the clinical promotion of electro-acupuncture treatment.