“夹脊”电针早期干预调控腰髓Toll样受体4/核因子-κB信号通路缓解肌萎缩侧索硬化症小鼠疾病进展

王仕林; 孙远征; 于天洋; 赵广然; 孙妍

doi:10.13702/j.1000-0607.20211379

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“夹脊”电针早期干预调控腰髓Toll样受体4/核因子-κB信号通路缓解肌萎缩侧索硬化症小鼠疾病进展

机制探讨 | 更新时间：2023-08-11

- “夹脊”电针早期干预调控腰髓Toll样受体4/核因子-κB信号通路缓解肌萎缩侧索硬化症小鼠疾病进展
- Early electroacupuncture intervention delays progression of disease in mice with amyotrophic lateral sclerosis by down-regulating TLR4/NF-κB signaling
- 针刺研究 2023年48卷第3期页码：287-293
- 作者机构：
  
  1. 黑龙江中医药大学附属第二医院
  2. 黑龙江中医药大学第二临床医学院
- 作者简介：
- 基金信息：
  
  国家中医药管理局全国名老中医药专家传承工作室建设项目(No.国中医药人教发[2014]20号)
- DOI：10.13702/j.1000-0607.20211379
  中图分类号：
- 纸质出版日期：2023
- 稿件说明：
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王仕林, 孙远征, 于天洋, 等. “夹脊”电针早期干预调控腰髓Toll样受体4/核因子-κB信号通路缓解肌萎缩侧索硬化症小鼠疾病进展[J]. 针刺研究, 2023,48(3):287-293.

WANG Shi-lin, SUN Yuan-zheng, YU Tian-yang, et al. Early electroacupuncture intervention delays progression of disease in mice with amyotrophic lateral sclerosis by down-regulating TLR4/NF-κB signaling[J]. Acupuncture research, 2023, 48(3): 287-293.
王仕林, 孙远征, 于天洋, 等. “夹脊”电针早期干预调控腰髓Toll样受体4/核因子-κB信号通路缓解肌萎缩侧索硬化症小鼠疾病进展[J]. 针刺研究, 2023,48(3):287-293. DOI： 10.13702/j.1000-0607.20211379.

WANG Shi-lin, SUN Yuan-zheng, YU Tian-yang, et al. Early electroacupuncture intervention delays progression of disease in mice with amyotrophic lateral sclerosis by down-regulating TLR4/NF-κB signaling[J]. Acupuncture research, 2023, 48(3): 287-293. DOI： 10.13702/j.1000-0607.20211379.

摘要

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-SOD1(G93A)基因突变的ALS-SOD1

(G93A)

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-SOD1(G93A)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-SOD1

(G93A)

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

0.05)

腰髓Iba-1、TLR4、NF-κB、TNF-α蛋白表达水平均降低(P

0.01

0.05)。且60 d电针组效果优于90 d电针组(P

0.01

0.05)。结论:“夹脊”电针早期干预延缓ALS-SOD1(G93A)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

0.05)

腰髓Iba-1、TLR4、NF-κB、TNF-α蛋白表达水平均降低(P

0.01

0.05)。且60 d电针组效果优于90 d电针组(P

0.01

0.05)。结论:“夹脊”电针早期干预延缓ALS-SOD1

(G93A)

小鼠疾病进展效果优于发病后干预，且早期干预可推迟ALS-SOD1(G93A)小鼠疾病进展效果优于发病后干预，且早期干预可推迟ALS-SOD1

(G93A)

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。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-SOD1(G93A)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。

Abstract

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-SOD1

(G93A)

) mice with SOD1(G93A)) mice with SOD1

(G93A)

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-SOD1(G93A) 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-SOD1

(G93A)

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

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

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-SOD1(G93A) 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

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

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

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-SOD1

(G93A)

mice

which may be related to its functions in inhibiting the excessive activation of microglia

and down-regulating TLR4/NF-κB signaling.

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