利用非靶向代谢组学方法探讨甲氨蝶呤治疗类风湿关节炎的作用机制

The mechanism of methotrexate in the treatment of rheumatoid arthritis using untargeted metabolomics

目的通过构建胶原诱导性关节炎(CIA)大鼠模型,利用非靶向代谢组学的研究方法,探讨甲氨蝶呤治疗类风湿关节炎的作用机制。方法采用ELISA测定血清中TNF-α、IL-1β、IL-6、IL-4、IL-10的含量,苏木精-伊红(HE)染色及Masson染色观察各组大鼠关节组织学改变,非靶向气相色谱-质谱代谢组学技术筛查血清差异代谢物表达谱并进行聚类分析和KEGG富集分析,筛查出差异性代谢途径,并采用实时定量聚合酶链反应对差异性代谢途径中的关键酶进行检测。将所有符合正态分布的实验数据,采用单因素方差分析比较组间差异,以P<0.05表示差异具有统计学意义。结果甲氨蝶呤(MTX)可明显改善CIA大鼠的关节炎症反应和关节炎评分(P<0.05),增加CIA大鼠的体质量(P<0.05)。HE和Masson染色结果显示MTX可改善CIA大鼠滑膜组织对关节软骨的侵蚀。ELISA结果显示MTX可显著降低CIA大鼠血清中TNF-α[(191.2±17.4)pg/ml,F=40.31,P<0.001]、IL-1β[(28.4±1.2)pg/ml,F=10.11,P=0.012]和IL-6[(118.7±1.4)pg/ml,F=829.40,P<0.001]的含量,提高血清中IL-4[(49.3±3.3)pg/ml,F=33.44,P<0.001]和IL-10[(30.2±0.7)pg/ml,F=33.44,P<0.001]的含量。非靶向代谢组学结果显示MTX对CIA大鼠血清中的磷酸胆碱、棕榈酸、油酸、胆碱等代谢产物有影响。KEGG通路富集分析结果显示MTX对CIA大鼠的甘油磷脂代谢(P<0.01)和鞘脂代谢(P<0.05)有影响。实时定量聚合酶链反应(qRT-PCR)结果显示MTX可下调CIA大鼠关节滑膜甘油磷脂代谢途径中关键酶磷脂酶B1(Plb1)[(1.00±0.49),F=8.23,P=0.019]、甘油磷胆碱磷酸二酯酶(Gpcpd1)[(1.10±0.09),F=8.19,P=0.019]、胆碱激酶α(Chka)[(1.33±0.19),F=33.00,P<0.001],胆碱激酶β(Chkb)[(2.07±1.21),F=8.20,P=0.019]和磷酸乙醇胺/磷酸胆碱磷酸酶1(Phospho1)[(1.07±0.14),F=13.58,P=0.006]的表达水平,也可下调鞘脂代谢途径中关键酶3-酮二氢鞘氨醇还原酶(Kdsr)[(1.24±0.32),F=13.85,P=0.006]、磷脂磷酸酶(Plpp1)[(1.61±0.32),F=11.95,P=0.003]和鞘脂去饱和酶1(Degs1)[(1.21±0.15),F=46.55,P<0.001]的表达水平。结论MTX治疗RA的生物学机制可能与下调机体的甘油磷脂代谢和鞘脂代谢通路的代谢水平有关。

Objective To investigate the mechanism of methotrexate in the treatment of rheumatoid arthritis(RA)by constructing a rat model of collagen-induced arthritis(CIA)and using non-targeted metabolomics.Methods Enzyme-linked immunosorbent assay(ELISA)was used to determine the contents of TNF-α,IL-1β,IL-6,IL-4 and IL-10 in serum.HE staining and Masson staining were used to observe the histological changes of joints in each group.Non-targeted gas chromatography-mass spectrometry metabolomics technique was used to screen the expression profiles of differential metabolites in serum and cluster analysis and KEGG enrichment analysis were performed to screen the differential metabolic pathways,and real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the key enzymes in the differential metabolic pathways.All experimental data conforming to the normal distribution were compared between groups using one-way ANOVA,and P<0.05 was considered statistically significant.Results MTX significantly improved the joint inflammatory response and arthritis score and increased the body weight of CIA rats.The results of HE and Masson staining showed that MTX could ameliorate the erosion of articular cartilage by synovial tissue in CIA rats.ELISA results showed that MTX significantly decreased the contents of TNF-α[(191.2±17.4)pg/ml,F=40.31,P<0.001],IL-1β[(28.4±1.2)pg/ml,F=10.11,P=0.012]and IL-6[(118.7±1.4)pg/ml,F=829.40,P<0.001]in the serum and increased the contents of IL-4[(49.3±3.3)pg/ml,F=33.44,P<0.001]and IL-10[(30.2±0.7)pg/ml,F=33.44,P<0.001]in the serum of CIA rats.Non-targeted metabolomics technique showed MTX had an effect on metabolites such as phosphocholine,palmitic acid,oleic acid,and choline in the serum of CIA rats.KEGG pathway enrichment analysis showed that MTX had an effect on glycerophospholipid metabolism(P<0.01)and sphingolipid metabolism(P<0.05)in CIA rats.qRT-PCR results showed that MTX could down-regulate the expression of the key enzymes such as Plb1[(1.00±0.49),F=8.23,P=0.019],Gpcpd1[(1.10±0.09),F=8.19,P=0.019],Chka[(1.33±0.19),F=33.00,P<0.001],Chkb[(2.07±1.21),F=8.20,P=0.019]and Phospho1[(1.07±0.14),F=13.58,P=0.006]in the glycerophospholipid metabolic pathway in the synovial membrane of CIA rats,and can also down-regulate the expression of the key enzymes Kdsr[(1.24±0.32),F=13.85,P=0.006],Plpp1[(1.61±0.32),F=11.95,P=0.003]and Degs1[(1.21±0.15,F=46.55,P<0.001]in the sphingolipid metabolic pathway.Conclusion The biological mechanism of MTX in the treatment of rheumatoid arthritis may be related to the down-regulation of glycerophospholipid metabolism and sphingolipid metabolism pathway metabolic levels in the body.