乌头汤缓解神经病理性疼痛的炎症网络调控机制研究

Investigation on the inflammation network mechanisms of Wutou decoction acting on neuropathic pain

痛痹要方乌头汤临床治疗神经病理性疼痛(neuropathic pain,NP)疗效确切,但作用机制尚不完全清楚,本研究旨在探究乌头汤缓解NP的炎症网络调控机制。前期基于脊神经结扎(spinal nerve ligation,SNL)小鼠模型,通过全基因表达谱分析发现SNL发病相关基因集及乌头汤干预SNL药效相关基因集。本研究中"疾病基因-药物效应基因"互作网络及通路富集分析结果表明,乌头汤的镇痛网络靶标参与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的富集显著性最高(P=4.04E-12),其中AKT激酶、MAP激酶的激酶4(MAP kinase kinase 4,MKK4)、终端激酶(c-Jun N-terminal kinase,JNK)和转录因子AP-1(JUN)与NP发生、发展中具有重要意义的神经炎症密切相关。基于SNL大鼠模型的药效学实验结果表明,乌头汤可显著升高SNL大鼠的机械痛阈值(P<0.01)和冷痛阈值(P<0.05);机制层面的验证结果表明,SNL大鼠术后脊髓组织中AKT、MKK4、JNK和JUN磷酸化蛋白的表达量均显著升高(P<0.001);乌头汤可上调具有神经保护作用的AKT磷酸化蛋白的表达量(P<0.001),并降低促进神经炎症的MKK4、JNK和JUN磷酸化蛋白的表达量(P<0.01)。上述结果表明,乌头汤可有效缓解SNL大鼠的疼痛程度,其作用机制可能是通过调节AKT-MKK4-JNK-JUN信号轴而缓解神经炎症。本研究丰富了经方乌头汤治疗痛痹的科学内涵,也为基于NP发病机制的多靶点镇痛药物的研发提供了方法学示范。本研究中的动物实验获得中国中医科学院实验动物伦理委员会批准。

Wu-tou decoction(WTD)was originally recorded in the synopsis of the golden chamber and it had been widely used for the treatment of neuropathic pain(NP)with exact therapeutic efficacy.However,the underlying molecular mechanisms still remain unclarified.Thus,in this research,we aimed at clarifying the underlying molecular mechanisms of WTD against NP by combining network analysis and experimental validation based on the spinal nerve ligation(SNL)model.Firstly,the network analysis indicated that key targets of WTD were significantly involved in the MAPK signaling pathway(P=4.04 E-12)and four important components of the above pathway,AKT kinase(AKT),MAP kinase kinase 4(MKK4),c-Jun N-terminal kinase(JNK)and transcription factor AP-1(JUN)had been reported to play a vital role in neuroinflammation during the disease process of NP.Then,experimental validation results proved that WTD markedly reduce the severity of mechanical allodynia(P<0.01)and cold hypersensitivity(P<0.05)of SNL rats.In addition,Western blot results provided evidence that the phosphorylated protein expression levels of AKT,MKK4,JNK and JUN in the superficial lamina of spinal cord of SNL rats were markedly increased(P<0.001),and WTD could improve the phosphorylated protein expression level of AKT(P<0.001)which was reported to be nerve protective and attenuate the phosphorylated protein expression levels of MKK4,JNK and JUN(P<0.01)which were closely involved into neuroinflammation.In conclusion,this study indicated that WTD might exert anti-hyperalgesia action through the inhibition of neuroin‐flammation mediated by AKT-MKK4-JNK-JUN which belong to the MAPK signaling pathway.These findings also provided scientific evidences that WTD might be a promising candidate for NP.Animal experiments in this study were approved by the Ethics Committee of Experimental Animals of the China Academy of Chinese Medical Sciences.