基于UPLC-Q-Orbitrap MS/MS技术研究膝痹宁对KOA模型大鼠的软骨保护效应

Study on Cartilage Protective Effect of Xibining on KOA Model Rats Based on UPLC-Q-Orbitrap MS/MS Technique

    摘要
  • 摘要:
      目的  运用超高效液相色谱-四极杆-静电场轨道阱串联质谱(UPLC-Q-Orbitrap MS/MS)技术分析膝痹宁的药物活性成分, 通过干预膝骨关节炎(KOA)模型大鼠代谢因素探讨膝痹宁软骨保护效应的作用机制。
      方法  制备膝痹宁水提物, UPLC-Q-Orbitrap MS/MS技术分析膝痹宁的活性成分。将大鼠分为空白组、KOA组、膝痹宁组, 提取大鼠软骨组织, HE染色、番红O-固绿染色观察组织结构形态; qPCR和Western blot法检测过氧化物酶体增殖物激活受体(PPARγ)、PPARγ辅助激活因子1α(PGC1α)及基质金属蛋白酶(MMP)3、MMP13的mRNA和蛋白表达水平; 取各组大鼠血清完成代谢组学分析。
      结果  经鉴定, 膝痹宁含活性成分56种; 组织学切片提示膝痹宁具有软骨保护作用, 并能上调KOA大鼠软骨组织PPARγ、PGC1α的mRNA和蛋白表达水平(P < 0.05, P < 0.01), 降低MMP3、MMP13的mRNA和蛋白表达水平(P < 0.05, P < 0.01);此外, 代谢组学研究发现, 膝痹宁对13种KOA差异代谢物存在干预作用, 涉及10条代谢通路。
      结论  膝痹宁具有多种药物活性成分, 其KOA软骨保护效应与药物活性成分对KOA的代谢调控有关。

     

    Abstract:
      OBJECTIVE  To analyze the active components of Xibining and its pathological mechanism of cartilage protective effect by interfering with metabolic factors in knee osteoarthritis (KOA) model rats using ultra-performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UPLC-Q-Orbitrap MS/MS) technique.
      METHODS  The water extract of Xibining was prepared and the active components were analyzed by UPLC-Q-Orbitrap MS/MS. Rats were divided into blank group, KOA group and Xibining group. Cartilage tissue was extracted, and the structure and morphology of the tissue were observed by HE staining and Safranin O staining. The qPCR and Western blot were used to detect the protein and mRNA expression of peroxisome proliferator-activated receptor-γ (PPARγ), PPARγ co-activator-1α (PGC1α) and matrix metalloproteinase (MMP) 3, MMP13, which were representative matrix metalloproteinases of cartilage degradation. Besides, the serum of each group was collected to complete the metabonomics analysis.
      RESULTS  A total of 56 active components were identified in Xibining. Histological sections suggested that Xibining had a protective effect on cartilage, and could up-regulate the gene and protein expression levels of PPARγ and PGC1α, and decrease the gene and protein expression of MMP3 and MMP13 in cartilage tissue of KOA rats. In addition, Xibining had intervention effects on 13 different metabolites of KOA, involving 10 metabolic pathways.
      CONCLUSION  Xibining has a cartilage protective effect of KOA, the mechanism of which may be related to the metabolic regulation of KOA by its active substance.

     

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