基于miR-146a调控NF-κB信号通路探讨芍药甘草汤治疗颈型颈椎病兔颈后肌炎症损伤的作用机制

Effect of Shaoyao Gancao Decoction on Inflammatory Injury of Posterior Cervical Muscle Based on miR-146a Regulation of NF-κB Signaling Pathway

目的:从组织学及分子学层面探讨芍药甘草汤对兔颈后肌炎症损伤的作用及其可能的机制,为临床运用芍药甘草汤治疗颈型颈椎病提供实验依据。方法:将30只6月龄的新西兰白兔随机分为高剂量组、中剂量组、低剂量组、模型组、正常组,每组6只,参照课题组前期成功制备的寒湿型兔颈型颈椎病模型进行造模。造模8周后,高剂量组、中剂量组、低剂量组分别予以16、8、4 g/kg的芍药甘草汤进行灌胃治疗,正常组和模型组给予正常饲养。于治疗4周后用ELISA法检测5组血清中炎症因子IL-1β、IL-6、TNF-α的表达水平。HE染色法观察5组兔颈后肌组织形态的变化。Western blot法检测5组兔颈后肌组织中核转录因子(NF)-κB信号通路关键节点髓样分化因子88(MyD88)、NF-κB P65蛋白表达水平。RT-qPCR法检测5组兔颈后肌TLR4、miR-146a及其下游靶基因TRAF-6的mRNA表达水平。结果:芍药甘草汤可降低兔血清炎症因子IL-1β、IL-6、TNF-α的含量(P<0.01);明显改善受炎症损伤的兔颈后肌细胞形态结构;降低MyD88、NF-κBP65蛋白的表达(P<0.05);同时提高颈后肌组织中miR-146a的表达水平(P<0.05),降低TLR4、TRAF-6的表达水平(P<0.05)。结论:服用芍药甘草汤可以减轻颈型颈椎病模型兔颈后肌的炎症损伤,其作用机制可能是通过改变模型兔颈后肌组织中miR-146a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,从而改善颈型颈椎病模型兔颈部炎症损伤,起到治疗颈型颈椎病的作用。

Objective:To investigate the effect of Shaoyao Gancao decoction on the injury of rabbit cervical posterior myoinflammation and its possible mechanism from histology and molecular level,and provided experimental basis for the clinical application of Shaoyao Gancao decoction in the treatment of cervical spondylopathy.Methods:A total of thirty 6-month-old New Zealand white rabbits were randomly divided into high dose group,medium dose group,low dose group,model group and normal group,with 6 rabbits in each group.The model was constructed according to the cold and wet rabbit cervical spondylopathy model prepared by the research group.Eight weeks after modeling,high-dose,medium-dose,and low-dose groups were given 16,8,4 g/kg Shaoyao Gancao decoction,and the normal and model groups were fed normally.The levels of IL-1β,IL-6 and TNF-αin serum were detected by ELISA after 4 weeks of treatment.The changes of cervical muscle morphology were observed by HE staining.Western blot was used to detect the expression of myeloid differentiation factor 88(MyD88)and nuclear factor(NF)-κB P65 protein in the key nodes of rabbit posterior cervical muscle tissue.The expression levels of miR-146a,TLR4 and TRAF-6 in posterior cervical muscle of rabbits were detected by RT-qPCR.Results:Shaoyao Gancao decoction could reduce the contents of serum inflammatory factors IL-1β,IL-6 and TNF-αin rabbits(P<0.01).It can obviously improve the morphology and structure of the muscle cells of the rabbit posterior neck damaged by inflammation.The expression levels of MyD88,NF-κB P65 were decreased(P<0.05).At the same time,the expression level of miR-146a in cervical posterior muscle tissue was increased(P<0.05),and the expression levels of TLR4 and TRAF-6 were decreased(P<0.05).Conclusion:Shaoyao Gancao decoction can reduce the inflammation and injury of the posterior cervical muscle in rabbits.The mechanism may be that by changing the expression of miR-146a in the posterior cervical muscle tissue of rabbits with cervical spondylosis,it inhibits the release of TNF-α,IL-1β,IL-6 and other inflammatory factors in the NF-κB signaling pathway,so as to improve the inflammatory injury of the neck and treat neck type of cervical spondylosis.