基于LC-Q/TOF-MS技术分析电针对类风湿关节炎寒证模型兔尿液代谢产物的影响

Effects of Electroacupuncture on Urine Metabonomics of Rabbits with Rheumatoid Arthritis Cold Syndrome Based on LC-Q/TOF-MS Technology

目的采用液相色谱-四极杆飞行时间质谱(LC-Q/TOF-MS)技术分析电针对类风湿关节炎(RA)寒证模型兔尿液代谢产物的影响,从代谢组学角度探讨其调控RA寒证的作用机制。方法24只新西兰兔随机分为正常组、模型组和电针组,每组8只。采用卵蛋白诱导联合低温冷冻法制备RA寒证模型。电针组在双侧“足三里”予电针干预,1次/d,连续7 d;正常组和模型组不进行干预。干预结束后测定兔膝关节周径和痛阈值;彩超探测膝关节腔,观察滑膜病理改变;收集新鲜尿液,采用LC-Q/TOF-MS检测尿液代谢产物,并对尿液进行代谢组学分析。结果与正常组比较,模型组兔膝关节周径明显增加(P<0.05),痛阈值明显降低(P<0.05);膝关节彩超见滑膜明显增厚,滑膜内有大量线状及点状血流信号,滑膜细胞异常增生,血管翳形成,炎性细胞浸润、关节腔积液明显,关节肿胀;尿液中柠檬酸、琥珀酸、苹果酸、焦谷氨酸、4-羟基苯基丙酮酸水平相对下降,2-羟基丁酸、3-羟基丁酸水平相对升高。与模型组比较,电针组兔膝关节周径明显减少(P<0.05),痛阈值明显升高(P<0.05);滑膜厚度降低,滑膜细胞增生、炎性细胞浸润减轻,关节腔积液、异常血流信号有所改善;尿液中2-羟基丁酸、3-羟基丁酸水平降低,柠檬酸、琥珀酸、苹果酸、焦谷氨酸、4-羟基苯基丙酮酸水平升高。结论电针可改善RA寒证模型兔一般状态,修复关节滑膜,其干预RA机制与调节三羧酸循环、谷胱甘肽循环、酮体代谢、酪氨酸代谢相关。

Objective To investigate the effects of electroacupuncture on urine metabonomics of rheumatoid arthritis(RA)rabbits with cold syndrome based on LC-Q/TOF-MS;To discuss its mechanism of regulating RA cold syndrome from the perspective of metabolomics.Methods A total of 24 New Zealand rabbits were randomly divided into normal group,model group and electroacupuncture group,with 8 rabbits in each group.The model of RA cold syndrome was prepared by ovalbumin induction combined with low-temperature freezing method.The electroacupuncture group was treated with electroacupuncture in“Zusanli”(ST36),1 time a day,a total of 7 d.There was no intervention in normal group and model group.After intervention,knee circumference and pain threshold were measured.Color Doppler ultrasound was used to probe the knee joint cavity and the pathological changes of the synovium.Fresh urine was collected,metabolites in urine were detected by LC-Q/TOF-MS,and metabonomics was analyzed by multivariate statistical analysis.Results Compared with the normal group,the circumference of the knee joint significantly increased in the model group (P<0.05), and the pain threshold was significantly reduced (P<0.05);Color Doppler ultrasound of the knee joint showed obvious thickening of the synovium, a large number of linear and punctate blood flow signals showed in the synovium, abnormal proliferation of synovial cells appeared, pannus formed, inflammatory cell infiltration appeared, joint cavity effusion was obvious, and joint swelled;The levels of citric acid, succinic acid, malic acid, pyroglutamic acid, and 4-hydroxyphenylpyruvate in urine relatively decreased, while the levels of 2-hydroxybutyric acid and 3-hydroxybutyric acid relatively increased. Compared with the model group, the circumference of the knee joint in electroacupuncture group significantly reduced (P<0.05), the pain threshold significantly increased (P<0.05);synovial membrane thickness decreased, synovial cell proliferation, and inflammatory cell infiltration were reduced, and joint cavity effusion and abnormal blood flow signals were improved;the levels of 2-hydroxybutyric acid and 3-hydroxybutyric acid in urine were decreased, the levels of citric acid, succinic acid, malic acid, pyroglutamic acid, and 4-hydroxyphenylpyruvate were increased. Conclusion Electroacupuncture can improve the general morphology of rabbits with RA cold syndrome and repair the synovium of joints. The mechanism of its intervention in RA is related to the regulation of tricarboxylic acid cycle, glutathione cycle, ketone body metabolism, and tyrosine metabolism.