TRPV4/NF-κB信号通路探讨点按承山穴调控脊髓胶质细胞活性干预CCD大鼠的镇痛机制研究

Exploring the analgesic mechanism of acupoint pressure at Chengshan（BL57）on CCD rats via modulating spinal glial cell activity based on the TRPV4/NF-κB signaling pathway

关键词:  神经病理性疼痛  点按法  承山  瞬时受体电位香草素受体4型通道蛋白/核因子-κB通路  胶质细胞  背根神经节持续压迫

Key words:  neuropathic pain  acupoint pressure technique  Chengshan（BL57） acupoint  TRPV4/NF-κB pathway  glial cells  chronic compression of dorsal root ganglion

摘要:

[目的] 探讨点按承山穴能否通过瞬时受体电位香草素受体4型通道蛋白（TRPV4）/核因子-κB（NF-κB）信号通路抑制脊髓背角胶质细胞活化从而参与背根神经节持续压迫（CCD）大鼠镇痛的作用机制。[方法] 将36只大鼠随机分为空白组、CCD模型组和推拿组。CCD模型组和推拿组建立CCD模型。推拿组在造模后第4 d起点按术侧“承山”穴，每次15 min，连续干预14 d。应用仪器检测大鼠热缩足潜伏期（PWL）与机械刺激缩足阈值（PWT）；蛋白免疫印迹法（Western blot）检测细胞中TRPV4、NF-κB蛋白表达水平；免疫荧光检测细胞中小胶质细胞标记物（IBA1）、星形胶质细胞标记物（GFAP）表达水平；酶联免疫吸附试验（ELISA）检测细胞中白介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）水平。[结果] 造模后，与空白组相比，CCD模型大鼠的 PWL、PWT值均显著下降（P<0.01），TRPV4、NF-κB、IBA1、GFAP和IL-1β、TNF-α表达均升高（P<0.05）；与CCD模型组相比，7 d组的PWL值无明显变化（P>0.05），PWT值显著升高（P<0.01），TRPV4、NF-κB 表达无差异（P>0.05），IBA1、GFAP表达显著降低（P<0.01），TNF-α表达降低（P<0.05），IL-1β表达显著降低（P<0.01）；11 d组的PWL值升高（P<0.05），PWT值显著升高（P<0.01），NF-κB表达降低（P<0.05），TRPV4表达显著降低（P<0.01），IBA1、GFAP和IL-1β、TNF-α表达均显著降低（P<0.01）；18 d组的PWL、PWT值均显著升高（P<0.01），NF-κB表达降低（P<0.05），TRPV4表达显著降低（P<0.01），IBA1、GFAP及IL-1β、TNF-α表达均显著降低（P<0.01）。[结论] 点按承山穴能有效缓解CCD大鼠神经性疼痛，其镇痛机制可能与抑制TRPV4/NF-κB信号通路，从而抑制小胶质细胞、星形胶质细胞活化及相关的神经炎症有关，且在14 d的干预内，镇痛效果随干预时间的延长而增强。

Abstract:

[Objective] To investigate whether acupoint pressure at Chengshan（BL57） alleviates neuropathic pain in chronic compression of dorsal root ganglion（CCD） model rats by modulating the transient receptor potential vanilloid 4（TRPV4）/nuclear factor kappa B（NF-κB） signaling pathway and inhibiting the activation of spinal dorsal horn glial cells. [Methods] Thirty-six Sprague-Dawley rats were randomly divided into a blank control group，a CCD model group，and an acupoint pressure group（n=12 per group）. The CCD model was established at the L4/L5 level in the model and acupoint pressure groups. Starting from the 4th day post-surgery，rats in the acupoint pressure group received 15 minutes of acupoint pressure at the ipsilateral Chengshan（BL57） acupoint daily for 14 consecutive days. The paw withdrawal latencies and mechanical paw withdrawal threshold were measured before modeling and on days 7，11，and 18 after intervention. Additionally，the protein expression levels of TRPV4 and NF-κB were evaluated through Western blot analysis in the cells. Immunofluorescence staining was used to observe the expression and morphological changes of the microglial marker Iba1 and the astrocytic marker GFAP. The levels of interleukin-1β and tumor necrosis factor-α in spinal cord tissue were measured by enzyme-linked immunosorbent assay. [Results] Compared with the blank control group，the CCD model group showed significantly decreased thermal and mechanical pain thresholds（P<0.01），along with significantly increased protein expression of TRPV4，NF-κB p65，Iba1，and GFAP，as well as elevated levels of inflammatory cytokines in the spinal dorsal horn（P<0.01）. Compared with the model group，after 7 days of intervention，the acupoint pressure group exhibited a significantly increased mechanical pain threshold（P<0.01） and decreased expression of Iba1，GFAP，and inflammatory factors（P<0.05 or P<0.01），while no significant changes were observed in the thermal pain threshold or the expression of TRPV4 and NF-κB p65（P>0.05）. After 11 and 18 days of intervention，both thermal and mechanical pain thresholds were significantly recovered（P<0.05 or P<0.01），and the protein expression of TRPV4，NF-κB p65，Iba1，GFAP，and the levels of inflammatory cytokines in the spinal cord showed a time-dependent significant decrease（P<0.05 or P<0.01）. [Conclusion] Acupoint pressure at Chengshan（BL57） can effectively alleviate neuropathic pain in CCD rats. The analgesic mechanism may be related to the inhibition of the TRPV4/NF-κB signaling pathway in the spinal dorsal horn，thereby suppressing the overactivation of microglia and astrocytes and the associated neuroinflammation. Within the 14-day intervention period，the analgesic effect was enhanced with prolonged intervention time.