基于miR-146a调控NF-kb信号通路探讨芍药甘草汤治疗颈型颈椎病的作用机制

we previously determined the effect of Shaoyao Gancao Decoction treatment to cervical spondylosis, but its mechanism is not entirely clear. The pathological characteristics of cervical spondylosis are local muscle spasm, edema, inflammatory. The present research confirmed that the effects of Shaoyao Gancao Decoction for anti-inflammation and analgesic, however, there are no research to fully demonstrate the concrete is what kind of factors. Shaoyao Gancao Decoction intervention of cervical spondylosis animal model, which changes in muscle morphology observation of the neck, detecting the expression of serum inflammatory factors TNF-a, IL-1 beta, IL-6, inflammatory lesions clearly peony and Licorice Decoction to improve the neck type of cervical spondylosis; detection of NF-kb signal pathway key nodes NF-kb, IKK, IKB, IL-1 beta, IL-6 protein levels and phosphorylation levels, and the detection of NF-kb nuclear translocation, reveal the role of NF-kb signaling pathway in Shaoyao Gancao Decoction in the treatment of neck type of cervical spondylosis; blocking agent and miR-146a mimics blocking the NF-kb signaling pathway by PDTC, observe the Shaoyao Gancao Decoction for anti-inflammatory; to clarify the anti-inflammatory mechanism of the Shaoyao Gancao Decoction mediated miR-146a muscle model NF-kb signaling pathway for the treatment of neck type cervical spondylosis.

前期我们运用芍药甘草汤治疗颈型颈椎病疗效确切，但其具体作用机制尚未完全清楚。颈型颈椎病主要病理改变为局部肌肉痉挛、水肿、炎症浸润。目前研究证实芍药甘草汤抗炎镇痛作用显著，其作用与抑制多种炎症因子有关，仍未有文献充分论证过具体是哪种因子起作用。本课题拟芍药甘草汤干预颈型颈椎病动物模型，通过观察其颈后肌形态学变化情况，检测血清炎症因子TNF-a、IL-1β、IL-6的表达，明确芍药甘草汤改善颈型颈椎病的炎症损伤；检测NF-kb信号通路关键节点NF-kb、IKK、IKB、IL-1β、IL-6的蛋白水平和磷酸化水平，并检测NF-kb的核转位，揭示NF-kb信号通路在芍药甘草汤治疗颈型颈椎病中作用；利用PDTC阻断剂和miR-146a mimics阻断其NF-kb信号通路，观察其芍药甘草汤抗炎效应指标；阐明芍药甘草汤介导miR-146a调控NF-kb信号通路治疗颈型颈椎病模型颈肌的抗炎机制。

本课题的前期研究发现芍药甘草汤治疗颈型颈椎病有明确疗效，在此基础上针对芍药甘草汤对兔颈型颈椎病模型颈椎间盘和颈肌组织形态学的改变情况、炎症相关因子TNF-α、IL-1β、IL-6表达水平的改变情况，以及颈椎间盘和颈肌组织miR-146a/-155-NF-κB信号通路相关因子表达的影响等问题进行探讨。采用ELISA检测炎症因子TNF-α、IL-1β、IL-6表达水平。HE染色观察形态学的变化；Western blotting 法检测MyD88 、NF-κB P65的蛋白水平；RT-qPCR检测TLR4、TRAF-6、miR-146a、miR-155的表达情况。结果：在形态学上，椎间盘和颈后肌组织退变程度均由轻至重依次是正常组、高剂量组、中剂量组、低剂量组、模型组；血清炎症因子TNF-α、IL-1β、IL-6表达水平比较：干预后，高、中、低剂量组的表达水平均明显下降；椎间盘和颈后肌组织中MyD88、NF-κB P65蛋白表达水平比较：干预后，低、中、高剂量组的表达均降低。椎间盘和颈后肌组织中miR-146a、miR-155、TLR4、TRAF-6表达水平的比较：干预后，低、中、高剂量组miR-146a的表达水平均升高，miR-155、TLR4、TRAF-6的表达水平均降低。结论：高、中、低不同剂量的芍药甘草汤均可缓解兔颈型颈椎病的炎症损伤，且其效果呈剂量依赖性：高剂量组﹥中剂量组﹥低剂量组﹥模型组。芍药甘草汤对兔颈型颈椎病的治疗作用机制之一可能是通过改变颈型颈椎病兔颈椎间盘和颈后肌组织中miR-146a、miR-155的表达而抑制NF-κB信号通路释放TNF-α 、IL-1β 、IL-6等炎症因子，从而改善颈部炎症损伤，治疗颈型颈椎病。为临床上运用芍药甘草汤治疗颈型颈椎病提供了更加充足的科学理论依据。