Chinese General Practice ›› 2023, Vol. 26 ›› Issue (36): 4565-4574.DOI: 10.12114/j.issn.1007-9572.2023.0145

• Article • Previous Articles     Next Articles

Effect and Mechanism of Pivot Meridian Massage on TLR8/ERK Signaling Pathway and LncRNA-GAS5 in Rats with Neuropathic Pain

  

  1. 1. Guangxi University of Chinese Medicine, Nanning 530041, China
    2. The First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine, Nanning 530023, China
    3. Fangchenggang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine, Fangchenggang 538000, China
  • Received:2023-05-14 Revised:2023-07-10 Published:2023-12-20 Online:2023-07-25
  • Contact: WANG Xiongjiang

枢经推拿对神经病理性疼痛大鼠TLR8/ERK信号通路及LncRNA-GAS5的影响及作用机制研究

  

  1. 1.530041 广西壮族自治区南宁市,广西中医药大学
    2.530023 广西壮族自治区南宁市,广西中医药大学第一附属医院
    3.538000 广西壮族自治区防城港市,广西中医药大学附属防城港医院
  • 通讯作者: 王雄将
  • 作者简介:
    作者贡献:韦宗波负责文章的构思、论文撰写及论文的修订;王雄将负责设计研究方案,进行动物造模;龙炳材、夏天、卢栋明进行整理数据、研究结果的分析;梁英业负责文章的质量控制与审校;唐宏亮提出研究思路,对文章整体负责、监督管理。
  • 基金资助:
    国家自然科学基金资助项目(82205305); 广西自然科学基金资助项目(2019GXNSFBA245074); 广西中青年教师科研基础能力提升项目(2020KY07014); 广西中医药大学校级科研项目(2019QN013)

Abstract:

Background

Pivotal meridian massage for neuropathic pain obtained favorable results in recent years, however, its specific mechanism of action has not been fully elucidated.

Objective

To observe the analgesic effect of pivotal meridian massage on rats according to the research indexes taking rat model of neuropathic pain induced by L5 spinal nerve ligation as the object of observation, to further investigate whether the analgesic effect is achieved by affecting LncRNA-GAS5 and then regulating the apoptosis of neurons in the dorsal horn of the spinal cord.

Methods

The experiment was conducted from January to June 2021 at the Experimental Center for Animal Medicine of Guangxi University of Chinese Medicine and Guangxi University. A total of 120 healthy female SD rats were randomly divided into the normal group, model group, sham-operated group, sham-manipulation group, and meridian manipulation group, with 24 rats in each group. Rat model of neuropathic pain was prepared by ligating the L5 spinal nerve in the model, sham-operated, sham-manipulation and meridian manipulation groups. After modeling for 24 hours, the L5 spinal nerve was exposed for a few minutes without ligation, and the wound was closed layer by layer in the sham-operated group; hind limbs of the rats in the sham-manipulation group were gently stroked for 18 minutes; a self-made massager was used to sequentially stimulate the three acupoints on the bilateral Foot Shaoyang Gallbladder Meridian of Huan Tiao, Yang Ling Quan, and Xuan Zhong, with a stimulation force of 5 N, frequency of 2 Hz, intervention of 1 minute for each acupoint and technique, totaling 18 minutes in the meridian manipulation group. The normal group and model group were fed and observed normally without any intervention. Behavioral tests (mechanical withdrawal threshold and thermal withdrawal latency) were performed before modeling and on days 1, 3, 7, and 14 after modeling. On days 7 and 14 of the intervention, 12 rats were randomly selected for tissue sampling to detect the expression of TLR8/ERK signaling pathway-related proteins (Bcl-2, Caspase-3, ERK, TLR8 protein levels) and the expression levels of LncRNA-GAS5 and miR-21 genes in the spinal cord tissue.

Results

(1) In terms of behavioral observations, the mechanical withdrawal threshold of the model group, sham-manipulation group, and meridian manipulation group was lower than the normal group on days 1, 3, 7, and 14 after modeling (P<0.05) . The mechanical withdrawal threshold of the sham-operated group, sham-manipulation group and meridian manipulation group was higher than the model group on day 14 after modeling (P<0.05) . The mechanical withdrawal threshold of the sham-operated group, meridian manipulation group was higher than the sham-manipulation group on days 7 and 14 after modeling (P<0.05) . The thermal withdrawal latency in the sham-manipulation group and meridian manipulation group was shorter than the normal group on days 1, 3, and 7 after modeling (P<0.05) . The thermal withdrawal latency in the sham-operated group, sham-manipulation group and meridian manipulation group was longer than the model group on days 7 and 14 after modeling (P<0.05) . The sham-operated group, meridian manipulation group had longer thermal withdrawal latency than the sham-manipulation group on day 14 after modeling (P<0.05) . (2) In terms of protein and gene expression levels related to the signaling pathway, on day 7 after modeling, the Bcl-2 protein expression level in the normal group was lower than the other groups (P<0.05) . The Bcl-2 protein expression level in the meridian manipulation group was higher than the model group, while the Caspase-3, ERK, and TLR8 protein expression levels were lower than the model group (P<0.05) . The Bcl-2, Caspase-3, ERK, and TLR8 protein expression levels in the meridian manipulation group were lower than the sham-operated group, sham-manipulation group (P<0.05) . On day 14 after modeling, the Bcl-2 protein expression level in the meridian manipulation group remained higher than the model group, while the Caspase-3 and TLR8 protein expression levels remained lower than the model group, and the ERK protein expression level was higher than the model group (P<0.05) . After 7 days of modeling, the expression level of LncRNA-GAS5 gene in the sham-manipulation group and meridian manipulation group was higher than the model group, while the expression level of miR-21 gene was lower than the model group (P<0.05) . The expression level of LncRNA-GAS5 gene in the meridian manipulation group was higher than the sham-manipulation group, while the expression level of miR-21 gene was lower than the sham-manipulation group (P<0.05) . After 14 days of modeling, the expression level of LncRNA-GAS5 gene in the model group was lower than the normal group, while the expression level of LncRNA-GAS5 gene in the meridian manipulation group and sham-manipulation group was higher than the model group (P<0.05) . The expression level of miR-21 gene in the meridian manipulation group and sham-manipulation group was higher than the model group (P<0.05) .

Conclusion

The meridian manipulation technique has a certain analgesic effect on rats with neuropathic pain. It is initially hypothesized that the analgesic mechanism may be achieved by upregulating the expression level of LncRNA-GAS5 to inhibit neuronal apoptosis by adsorbing miR-21 to mediate TLR8/ERK pathway-related proteins. Although the specific mechanism has not been conclusively confirmed, LncRNA-GAS5 is expected to be a new target for future treatment of neuropathic pain in the future.

Key words: Pain, Neuropathic pain, Tuina therapy, Pivot meridian massage, LncRNA-GAS5, Analgesia, Rats

摘要:

背景

近年来,枢经推拿在治疗神经病理性疼痛方面展现出较好的疗效,但其具体作用机制尚未充分阐明。

目的

以L5脊神经结扎而成的神经病理性疼痛大鼠模型为观察对象,根据各项研究指标观察枢经推拿对大鼠的镇痛作用,探讨其是否通过影响长链非编码RNA-生长阻滞特异性转录因子5(LncRNA-GAS5)进而调控脊髓背角神经元凋亡达到镇痛效果。

方法

实验于2021年1—6月在广西中医药大学、广西大学动物医学实验中心完成。选取健康雌性SD大鼠120只,按随机数字表法分为正常组、模型组、假手术组、假推拿组和枢经推拿组,每组24只;模型组、假手术组、假推拿组和枢经推拿组通过结扎L5脊神经来制备神经病理性疼痛大鼠模型。造模24 h后,假手术组暴露L5脊神经几分钟,不予结扎,逐层缝合关闭伤口;假推拿组轻抚大鼠双后肢18 min;枢经推拿组用自备按摩仪循序刺激两侧足少阳胆经上的环跳、阳陵泉、悬钟三穴,刺激力量为5 N,频率2 Hz,每穴、每法干预1 min,双侧6个穴位,3种手法,共计18 min;正常组、模型组正常喂养及观察,不施加任何干预。于造模前及造模后1、3、7、14 d分别进行行为学检测(机械缩足反射阈值、热缩足反射潜伏期);分别于干预7、14 d,随机抽取12只进行取材检测脊髓组织中介导TLR8/ERK信号通路相关蛋白表达情况〔脊髓组织B淋巴细胞瘤2(Bcl-2)、Caspase-3、细胞外调节蛋白激酶(ERK)、Toll样受体8(TLR8)蛋白表达水平,LncRNA-GAS5、miR-21基因表达水平)〕。

结果

(1)行为学方面,模型组、假推拿组、枢经推拿组大鼠造模后1、3、7、14 d机械缩足反射阈值均低于正常组(P<0.05);假手术组、假推拿组、枢经推拿组的机械缩足反射阈值在造模后14 d均高于模型组(P<0.05);假手术组、枢经推拿组造模后7、14 d机械缩足反射阈值高于假推拿组(P<0.05)。假手术组、枢经推拿组在造模后1、3、7 d热缩足反射潜伏期均低于正常组(P<0.05);假手术组、假推拿组、枢经推拿组在造模后7、14 d热缩足反射潜伏期均长于模型组(P<0.05);假手术组、枢经推拿组在造模后14 d热缩足反射潜伏期长于假推拿组(P<0.05)。(2)信号通路相关蛋白以及基因表达水平方面,造模后7 d,正常组Bcl-2蛋白表达水平低于其余各组(P<0.05);枢经推拿组的Bcl-2蛋白表达水平高于模型组,Caspase-3、ERK、TLR8蛋白表达水平均低于模型组(P<0.05);枢经推拿组的Bcl-2、Caspase-3、ERK、TLR8蛋白表达水平低于假手术组、假推拿组(P<0.05);造模后14 d,枢经推拿组的Bcl-2蛋白表达水平仍高于模型组,Caspase-3、TLR8蛋白表达水平仍低于模型组,而ERK蛋白表达水平高于模型组(P<0.05)。造模后7d,假推拿组和枢经推拿组LncRNA-GAS5基因表达水平均高于模型组,miR-21基因表达水平均低于模型组(P<0.05);枢经推拿组LncRNA-GAS5基因表达水平高于假推拿组,miR-21基因表达水平低于假推拿组(P<0.05);造模后14 d,模型组LncRNA-GAS5基因表达水平低于正常组,枢经推拿组与假推拿组lncRNA-GAS5基因表达水平高于模型组(P<0.05);枢经推拿组与假推拿组miR-21基因表达水平高于模型组(P<0.05)。

结论

枢经推拿手法对神经病理性疼痛大鼠起到一定的镇痛效果,初步推测镇痛作用机制可能是通过升高LncRNA-GAS5的表达来吸附miR-21介导TLR8/ERK通路相关蛋白抑制神经元凋亡,虽然其具体机制未明确证实,但LncRNA-GAS5有望成为以后治疗神经病理性疼痛的新靶点。

关键词: 疼痛, 神经病理性疼痛, 推拿疗法, 枢经推拿, 长链非编码RNA-生长阻滞特异性转录因子5, 镇痛, 大鼠