基于多元统计分析对秦艽中环烯醚萜苷抗胶原诱导型关节炎作用机制的研究

Research on secoiridoid glucoside extracted from Gentiana Macrophylla Pall. against collagen induced arthritis based on multivariate statistical analysis

目的:测定秦艽中环烯醚萜苷对胶原诱导型关节炎(CIA)大鼠关节滑膜中TGF-β1、IFN-γ、IL-4、VEGF、RANTES、CD3、CD34及CD68的表达水平并进行多元统计分析,以探讨秦艽中环烯醚萜苷对类风湿性关节炎(RA)的治疗作用及相关机制。方法:构建大鼠胶原诱导型关节炎模型,通过免疫组化法检测大鼠踝关节TGF-β1、IFN-γ、IL-4、VEGF、RANTES、CD3、CD34及CD68的表达,HE染色观察踝关节组织的病理学改变,并采用主成分分析法(PCA)和正交偏最小二乘法判别分析法(OPLS-DA)对测定数据进行分析。结果:与正常对照组比较模型组的TGF-β1、IFN-γ、RANTES、VEGF、CD3、CD34和CD68的表达水平明显升高,IL-4表达明显降低。经药物治疗后,与模型组比较,秦艽环烯醚萜苷0.35g/kg组的关节肿胀度、评分、TGF-β1、IFN-γ、CD3、CD34和CD68表达明显降低,VEGF表达降低。HE染色结果显示,秦艽环烯醚萜苷0.35g/kg组的关节软骨破坏有所修复,血管新生减少。在PCA的基础上进行OPLS-DA分析,筛选出VEGF、RANTES和CD34是区分对照组与CIA模型组的关键差异变量。结论:多元统计分析方法可对RA复杂的致病及治疗作用机理中的数据起到更好的解析作用。秦艽环烯醚萜苷可能主要通过下调CD34、VEGF的表达,抑制病理性微血管新生,改善关节滑膜慢性炎症这一机制干预RA的发展。

Objective:To observe the effects of secoiridoid glycosides from Gentiana Macrophylla Pall.on the expressions of TGF-β1,IFN-γ,IL-4,VEGF,RANTES,CD3,CD34 and CD68 in collagen induced rat arthritis and analyze the data by multivariate statistical analysis,to explore the therapeutic effects and related mechanism on RA.Methods:The arthritis model of rats was induced by collagen II.The expressions of TGF-β1,IFN-γ,IL-4,VEGF,RANTES,CD3,CD34 and CD68 in the ankle joint of rats were detected by immunohistochemistry,the pathological changes of ankle joint were observed by HE staining.All data were analyzed by principal component analysis(PCA)and orthogonal partial least square discrimination analysis(OPLS-DA).Results:Compared with the control group,the expressions of TGF-β1,IFN-γ,RANTES,VEGF,CD3,CD34 and CD68 in CIA model group were significantly increased and IL-4 was decreased.After the treatment,compared with the model group,the expressions of TGF-β1,IFN-γ,CD3,CD34 and CD68 in secoiridoid glycosides of Gentiana group(0.35 g/kg)were significantly decreased and VEGF was decreased.HE staining results showed that the degree of articular cartilage destruction was reduced,and the number of angiogenesis decreased in 0.35 g/kg drug group.OPLS-DA was carried out on the basis of PCA,VEGF,RANTES and CD34 were selected as the key difference variables to distinguish the control group from CIA model group.Conclusion:The multivariate statistical analysis method shows advantage to analyze the data from the complex pathogenetic and therapeutic mechanism of RA.This study suggested that secoiridoid glycosides of Gentiana might interfere with the development of RA by down-regulating the expressions of CD34 and VEGF,inhibiting the pathological microangiogenesis and reducing the chronic synovial inflammation.