基于代谢/蛋白质组学技术及组学间系统生物信息学交联分析探讨艾灸干预AS多靶点效应机制

Ankylosing spondylitis often seriously impaired the patient's physical function and even leads to the occurrence of disability. Moxibustion has a positive effect on AS, but the existing studies only discuss the anti-AS mechanism of moxibustion from a wide of immunity, inflammation or bone metabolism, which does not reflect the multi-target characteristics of moxibustion based on the TCM "holistic concept". Metabolism/proteomics is a newly emerging research method with holistic concept of life sciences. Its holistic and systematic characteristics coincide with the multi-target characteristics of moxibustion based on holistic concept, and become a new method to explore the multi-target mechanism of moxibustion intervention in AS. Thus, on the basis of the early researches, this project aims to use RP/HILIC UPLC-TOF/MS metabolomics and iTRAQ proteomics methods to comprehensively, continuously and quantitatively investigate the profile characteristics of differential metabolites and dynamic changes of differential proteins in serum, urine and ligament tissues of moxibustion-targeted AS mice, hoping to systematically screen out the endogenous metabolites and proteomics of moxibustion-targeted AS mice. At the same time, the system bioinformatics between omics was innovatively used to further clarify the relationship between upstream protein and downstream metabolites of moxibustion, and to construct a multi-target network regulation model of moxibustion intervention for AS. Thus, our research can provide the evidence for clinical application of moxibustion for AS.

强直性脊柱炎(AS)严重危害患者躯体功能甚至导致残疾发生。艾灸治疗AS疗效确切，但现有研究仅从免疫，炎症或者骨代谢的某一环节探讨了艾灸抗AS的机制，未体现艾灸基于“整体观念”的多靶点特征。代谢/蛋白质组学是新涌现的具有生命科学整体观念的研究方法，其整体性、系统性特点与艾灸基于“整体观念”多靶点特征相契合，成为探究艾灸干预AS多靶点机制的新方法。因此，本项目拟在前期研究工作基础上，运用RP/HILIC UPLC-TOF/MS代谢组学和iTRAQ蛋白质组学方法，全面、连续和定量考察艾灸靶向AS小鼠血清，尿液以及韧带组织的差异代谢物轮廓特征以及差异蛋白的动态变化，以期系统筛选出艾灸治疗AS的内源性代谢产物和蛋白组群。同时，创新运用组学间系统生物信息学交联分析，进一步明确艾灸靶向AS作用的上游蛋白和下游代谢物间的相互关系，构建艾灸干预AS多靶点网络调控模型，从而为临床运用艾灸治疗AS提供客观依据。

强直性脊柱炎(AS) 严重危害患者躯体功能甚至导致残疾发生。艾灸治疗AS疗效确切，但现有研究仅从免疫，炎症或者骨代谢的某一环节探讨了艾灸抗AS的机制，未体现艾灸基于“整体观念”的多靶点特征。运用代谢组学和蛋白质组学等现代分子生物学技术，在中医理论指导下，全面探究艾灸靶向AS小鼠“免疫-炎症-骨代谢”致病网络的代谢分子和蛋白质，探查上游蛋白和下游代谢产物彼此间的关联，从系统生物学角度揭示艾灸干预AS的多靶点作用网络。重要结果如下：（1）代谢组学：艾灸可以通过调控肠道菌群SCFA和MCFA代谢物的浓度，修复肠道机械屏障，提高肠道对机体“免疫-炎症”的防御能力；抑制花生四烯酸代谢激活导致炎症性的骨破坏，保护AS关节软骨。同时，艾灸可以通过上调肉碱类代谢分子的浓度，提高AS机体对脂肪酸β-氧化应激损伤的抵御作用。（2）蛋白质组学：艾灸可以通过调控AS蛋白酶体和UPR免疫信号途径相关蛋白（Rnf7和Erp44）的异常表达，改善MHC-I错误折叠增加引起的内质网应激，调节Ifi44和Ighg等肠粘膜免疫球蛋白的表达维持肠-骨轴稳态，抑制Rala和Mrps7核糖体蛋白介导免疫相关的Ras和NF-κB信号通路活化等多个途径，调节“免疫-炎症”引起的蛋白稳态失衡，抑制炎症因子的级联反应。（3）组学间系统生物信息学交联分析：艾灸能够下调糖酵解/糖异生中相关上游蛋白和下游代谢分子的表达，阻断蛋白酶体，抑制UPR以及ER stress，纠正能量代谢异常引起的缺氧微环境，调控AS“免疫-炎症”过程。同时，艾灸通过调节MIF蛋白-肠道氨基酸代谢分子参与的苯丙氨酸代谢途径以及调控载脂蛋白APOE介导的Wnt/β-catenin信号通路，靶向AS肠-骨轴和脂代谢-骨代谢轴，对AS起到一定的骨保护作用。综上，艾灸可以回调代谢分子，逆转AS代谢病理状态，并向正常健康状态进行转归；纠正蛋白的紊乱，维持蛋白的稳态平衡，并针对AS的核心作用靶标发挥多靶点的网络调控效应。