基于差异蛋白质组学研究刮痧干预腰椎间盘突出症的效应机制

Lumbar intervertebral disc herniation (LIDH) is a common and frequently-occurring disease, for which, scraping therapy has obvious effects. However, its mechanism has still been unclear yet. The previous studies confirmed that scraping therapy has the effects of anti-inflammation, immunity improvement, and nerve function regulation. However, these relevant researches need to be furthered. The pathological changes of LIDH is a complex process involving the participation of specific proteins. While proteomics can integrally and systematically explore the mechanism of scraping treating LIDH. Based on the previous studies, this study will make use of the rats with LIDH as models, compare the local tissue's pathological changes and relevant biological indicators among sham operation group, model group, and scraping group and in the different treatment courses in scraping group, in order to deeply explore and discuss the nerve function regulation, anti-inflammation and immunity improvement of scraping. And then observe the dynamic changes of all proteins in the serum and vertebra disc of rats with LIDH after scraping with the methods of proteomics of two-dimensional gel electrophoresis combined with tandem mass spectrometry and liquid chromatography-mass spectrometry using iTRAQ labeling technique, thus in order to explore the network channels and possible target which is closely related with scraping therapy treating LIDH, to identify the material foundation of scraping effects, and further provide the objective experimental evidence for the mechanism of scraping therapy as well as clinical guidance for LIDH treated by scraping therapy.

腰椎间盘突出症（LIDH）手术治疗风险较大，西药治疗有毒副作用，寻求一种安全、有效、简便的中医治疗方法尤为迫切。本课题组前期临床研究证实：刮痧治疗LIDH临床疗效显著，但机制不明。基础研究证实：刮痧具有抗炎、提高免疫力、神经调节功能等效应，但研究有待深入。而LIDH病变过程涉及特异性蛋白质参与的复杂过程。由此可见，蛋白组学可从整体、系统的角度探讨刮痧治疗LIDH的效应机制。本项目旨在既往研究基础上，采用LIDH大鼠模型，用特定的刮痧方法进行干预，深入探讨刮痧治疗LIDH神经调节功能、抗炎、提高免疫力等作用效应；利用蛋白质组学双向凝胶电泳结合串联质谱技术以及iTRAQ标记的液质联用技术，观察刮痧干预LIDH差异蛋白质的动态变化，探讨刮痧治疗LIDH密切相关的网络途径和可能作用靶点，寻找与LIDH相关的标志性内容物，为揭示刮痧治疗效应机制提供客观依据，为临床合理应用刮痧法治疗LIDH提供依据。

刮痧是中医特色的外治技术，腰椎间盘突出症（LDH）是刮痧治疗的优势病种，但机制不明。本课题组前期研究发现，刮痧后大鼠的血清IL-1、IgG含量明显降低，但研究有待深入。而单纯研究一个或几个生物学指标，并不能完全反映刮痧是通过多环节、多途径、多靶点综合作用来产生治疗效应的特点，因此，从整体、系统角度研究刮痧治疗LDH的效应机制方能体现中医治病整体功能调整规律。本研究首先运用传统生物学技术，进一步完善刮痧治疗LDH效应；并借助2种蛋白质组学技术，筛选出刮痧治疗LDH大鼠的血清、DRG差异蛋白，探讨刮痧对差异蛋白及信号通路与网络的调节作用。.结果：（1）运用传统生物学技术检测刮痧后LDH大鼠血清炎性因子、DRG病理形态及NGF、SP表达水平，发现刮痧可显著降低血清促炎因子、促进Th1 / Th2（IFN-γ / IL-4）恢复动态平衡；刮痧能够有效减轻大鼠DRG的病理损伤程度，抑制背根神经节中炎性因子SP的表达，促进背根神经节中NGF的增长。（2）运用iTRAQ 技术检测刮痧后大鼠血清蛋白表达变化，发现Rac1、Orm1、Hpx、Gapdh、Hsp70、Htt等蛋白在刮痧治疗过程中发挥了重要作用，刮痧可能通过调节ERK1/2、NFκB等信号通路，从而参与了细胞形态与器官形态、机体损伤与异常、神经系统发育与功能等信号网络。（3）运用iTRAQ 技术检测刮痧后大鼠DRG蛋白表达变化，发现蛋白参与神经元细胞增殖、凋亡、免疫、代谢等多条通路和亚网络调节，Sod1、Htt、Efna3等上游调节因子可能为刮痧的潜在治疗靶点。（4）借助双向凝胶电泳结合串联质谱技术检测刮痧后大鼠血清蛋白表达变化，发现刮痧治疗能够在三个疗程间持续性地调控LDH大鼠血清中转录激活蛋白Pur-α、IL-17A、C9等14个蛋白的表达，通过相关信号网络，起到修复损伤、抑制炎症、保护细胞等多方面的治疗作用。（5）借助双向凝胶电泳结合串联质谱技术检测刮痧后大鼠DRG蛋白表达变化，发现刮痧治疗能够在三个疗程间持续性地调控LDH大鼠背根神经节中TNFα、TGFβ1、NFκB1等27个蛋白的表达，通过相关信号网络，起到抑制退变、减轻炎症、调控代谢等多方面的治疗作用。