Objective: To observe the effect of early electroacupuncture (EA) intervention on Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway in mice with amyotrophic lateral sclerosis (ALS), so as to explore its mechanisms underlying alleviation of ALS.
Methods: Fifty-four ALS (ALS-SOD1G93A) mice with SOD1G93A gene mutation identified by PCR were randomly divided into model group, 60 day(d) EA group and 90 d EA group(n=18 mice in each group), and other 18 ALS-SOD1G93A negative mice were used as the control group. At the age of 60 and 90 days, mice of the two EA groups received EA (2 Hz, 1 mA) stimulation of bilateral "Jiaji" (EX-B2) of L1-L2 and L5-L6 for 20 min, twice a week for 4 weeks,respectively. When being 60 days old, the mice in the model and control groups were subjected to the same binding as that in the two EA groups but without EA intervention. The tail suspension test was used to judge the onset time of disease and the survival period, and rotary rod fatigue test was used to evaluate the hind limb motor function. Nissl staining method was used to observe the content of Nissl bodies in the anterior horn of the lumbar spinal cord. Immunohistochemical staining was used to observe the expression of ionized calcium binding adaptor molecule-1 (Iba-1) in the anterior horn of the lumbar spinal cord, and Western blot was used to detect the relative expression of TLR4, NF-κB and tumor necrosis factor-α (TNF-α) in the lumbar spinal cord.
Results: The disease onset time apparently delayed in the 60 d EA group than in the model group (P<0.01). The survival time was apparently shorter in the model group than in the control group (P<0.01), and obviously prolonged in the 60 d EA and 90 d EA groups than in the model group (P<0.01). The rotatory rod time was obviously shorter in the model group than in the control group (P<0.05), and apparently longer in the 60 d EA group than in the model group and 90 d EA group (P<0.05). Compared with the control group, the model group had a decrease in the number of Nissl bodies in the anterior horn of the lumbar spinal cord (P<0.01), and an increase in the expression levels of Iba-1, TLR4, NF-κB and TNF-α in the lumbar spinal cord (P<0.01). In contrast to the model group, both 60 d EA and 90 d EA groups had an apparent increase in the number of Nissl bodies and a marked decrease in the expression levels of Iba-1, TLR4, NF-κB and TNF-α in the lumbar spinal cord (P<0.05, P<0.01). The therapeutic effects of 60 d EA group were evidently superior to those of 90 d EA group in delaying the onset time of disease, prolonging the survival time and rotatory rod time, increasing the number of Nissl bodies, and in down-regulating the expression of Iba-1, TLR4, NF-κB and TNF-α (P<0.05, P<0.01).
Conclusion: The early intervention of EX-B2 EA is more effective in delaying the progression of ALS than post-onset intervention in ALS-SOD1G93A mice, which may be related to its functions in inhibiting the excessive activation of microglia, and down-regulating TLR4/NF-κB signaling.
目的：探讨“夹脊”电针早期干预通过调控Toll样受体4(TLR4)/核因子-κB(NF-κB)信号通路缓解肌萎缩侧索硬化症(ALS)小鼠疾病进展的作用机制。方法：将经PCR鉴定后超氧化物歧化酶1（SOD1）G93A基因突变的ALS-SOD1G93A小鼠随机分为模型组、60 d电针组和90 d电针组，每组18只；18只ALS-SOD1G93A阴性小鼠作为对照组。60 d电针组和90 d电针组分别在小鼠60日龄和90日龄时予双侧腰(L)1—L2、L5—L6“夹脊”穴电针刺激，20 min/次，2次/周，连续治疗4周。悬尾实验判断小鼠发病期及生存期；转棒疲劳实验评估小鼠后肢运动功能；尼氏染色法观察小鼠腰髓前角尼氏小体数量；免疫组织化学法观察小鼠腰髓前角离子钙结合衔接分子1(Iba-1)蛋白表达水平；Western blot法检测小鼠腰髓组织中TLR4、NF-κB、肿瘤坏死因子-α(TNF-α)蛋白的相对表达量。结果：模型组发病时间为（90.17±0.90) d，与对照组比较，生存期缩短(P<0.01)，转棒时间自发病后明显降低(P<0.05)，腰髓前角尼氏小体数量减少(P<0.01)，腰髓Iba-1、TLR4、NF-κB、TNF-α蛋白表达水平升高(P<0.01)。与模型组比较，60 d电针组发病时间推迟(P<0.01)，转棒时间延长(P<0.05)；60 d和90 d电针组生存期均延长(P<0.01)，腰髓前角尼氏小体数量均增加(P<0.01，P<0.05)，腰髓Iba-1、TLR4、NF-κB、TNF-α蛋白表达水平均降低(P<0.01，P<0.05)。且60 d电针组效果优于90 d电针组(P<0.01，P<0.05)。结论：“夹脊”电针早期干预延缓ALS-SOD1G93A小鼠疾病进展效果优于发病后干预，且早期干预可推迟ALS-SOD1G93A小鼠发病时间，其作用机制可能与抑制小胶质细胞过度活化、下调TLR4/NF-κB信号通路有关。.
Keywords: Amyotrophic lateral sclerosis; Electroacupuncture; Inflammatory factor; Motor function; Survival time; Treatment opportunity.