基于血液代谢组学技术的三妙丸抗类风湿关节炎作用机制研究

Mechanism study of Sanmiao Pill in the treatment of rheumatoid arthritis based on serum metabolomics technology

目的采用液质联用的血液代谢组学技术研究三妙丸对类风湿关节炎(rheumatoid arthritis,RA)模型大鼠的抗炎作用及机制。方法将大鼠根据体重随机分成空白组、模型组和给药组,每组8只。模型组和给药组大鼠采用臀部多点注射完全弗氏佐剂0.1 ml制备RA大鼠模型。实验第7天开始给药,给药组大鼠灌胃三妙丸溶液0.054 g/ml,按1 ml/100 g大鼠体重灌胃,连续给药7 d。分别于实验第1、14天测定各组大鼠足肿胀程度。末次给药后代谢48 h,采集血液样本进行代谢指纹图谱分析。对非靶向的轮廓质谱数据(Continuum模式)采用无监督的主成分分析处理,通过降维处理后明确RA大鼠与健康大鼠的最大代谢差异,在通过有监督的模式识别分析筛选和锁定差异离子,最终通过权威的代谢组学数据库HMDB等及自动化的分析软件进行结构鉴定和相关代谢通路分析。结果与模型组比较,给药组大鼠足肿胀度[(2.01±0.19)ml比(2.27±0.30)ml]减轻(P<0.01)。给药组大鼠的代谢轮廓主要分布在空白组和模型组间,表明三妙丸对RA模型大鼠有一定的干预作用,整体代谢水平向健康状态发展。通过聚焦分析得到20个与RA相关的血液生物标志物,主要涉及氨酰tRNA生物合成代谢,甘氨酸、丝氨酸和苏氨酸代谢,丙氨酸、天门冬氨酸和谷氨酸代谢,亚油酸代谢,花生四烯酸代谢等代谢通路。结论三妙丸对RA模型大鼠具有一定的干预作用,其作用机制可能与调节丙氨酸、天门冬氨酸和谷氨酸代谢等关键代谢酶及代谢通路有关。

Objective To study the anti-rheumatoid arthritis mechanism of Sanmiao Pill by using liquid chromatography-mass spectrometry.Methods The rats were randomly divided into control group,model group and medicated group based on weight,8 rats in each group.The model group and medicated group were treated by intradermal injection of 0.1 ml complete Freund’s adjuvant on buttock(s).Rats of medicated group were given Sanmiao Pill solution 0.054 g/ml by gavage,1 ml/100 g based on weight from the 7th day of experiment for 7 days.And then to score the degree of feet swelling of rats.After 48 hours of the last medication,to collecte the blood samples for metabolic fingerprint analysis.Then analized and processed the non-targeted contour mass spectrometry data(Continuum model)with unsupervised principal component method;identified the maximum metabolic differences between rheumatoid arthritis rats and healthy rats by dimension reduction technic.The differential ions were screened and locked by supervised pattern recognition analysis.Finally,with the help of the automated analysis software database like HMDB,identifiedthe structures and analized the correlated metabolic pathway.Results Compared with the model group,the swelling degree of the feet(2.01±0.19 ml vs.2.27±0.30 ml)in medicated group significantly decreased(P<0.01).The HMDB data shows that the metabolic profiles of rats were mainly distributed in the control group and the model group,which proved that the rheumatoid arthritis rats treated with Sanmiao Pill had a pullback trend,and the overall metabolic level has a healthy state.With this analysis,20 blood biomarkers related to rheumatoid arthritis were obtained,which involved the metabolic pathways of aminoacyl-tRNA biosynthesis,Glycine,Serine and Threonine metabolism,Alanine,Aspartic acid and Glutamic acid metabolism,Linoleic acid metabolism,Arachidonic acid metabolism.Conclusions Sanmiao Pill has interventional effect on rheumatoid arthritis rats.The mechanism might be related with metabolic enzymes and metabolic pathways such as Alanine,Aspartic acid and Glutamic acid.