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目的:利用液质联用(LC-MS)技术研究热补针法对类风湿关节炎(RA)模型家兔血液代谢物谱的影响,探讨热补针法治疗RA的特异性机制。方法:40只清洁级青紫蓝家兔随机分为正常组、模型组、平补平泻组、捻转补法组和热补针法组,每组8只。除正常组外,余组以卵蛋白诱导法联合低温冷冻法复制RA寒证模型。正常组、模型组不予针刺干预,平补平泻组、捻转补法组和热补针法组于“足三里”分别施以平补平泻、捻转补法、热补针法针刺,留针30 min,每日1次,共7 d。干预结束后测定各组痛阈值以及膝关节表面温度;心脏采血制备血清,对血清进行代谢组学检测;并对数据进行主成分分析(PCA)和偏最小二乘法-判别分析(PLS-DA),通过变量重要性投影值(VIP>1)和P值(P<0.05)筛选出血清中的差异代谢物。结果:与正常组相比,模型组家兔痛阈、膝关节表面温度降低(均P<0.05);与模型组比较,各针刺组干预后痛阈、膝关节表面温度升高(均P<0.05);热补针法组优于平补平泻组、捻转补法组(均P<0.05)。与正常组相比,模型组血液代谢物中肉碱、Lyso PC(14∶0)、Lyso PC(18∶3)、Lyso PE(0∶0/20∶5)、Lyso PE(0∶0/22∶1)、癸酸、硬脂酸以及乳酸的含量均升高,亮氨酸、缬氨酸、谷氨酰胺、焦谷氨酸、α-酮戊二酸、琥珀酸、延胡索酸、苹果酸、半乳糖、甘露糖含量均降低,上述代谢物具体涉及到脂肪酸、氨基酸、三羧酸循环以及糖类代谢。各针刺组干预后上述代谢物均出现不同程度的回调。热补针法组较平补平泻组和捻转补法组乳酸的含量降低,琥珀酸、延胡索酸、苹果酸、半乳糖、甘露糖的含量升高。结论:热补针法治疗RA的特异性体现在对三羧酸循环与糖代谢的调控。
OBJECTIVE: To study the effect of heat-acupuncture on blood metabolites in rabbits with rheumatoid arthritis (RA) by liquid chromatography-mass spectrometry (LC-MS) and to explore the specific mechanism of heat-acupuncture on RA. Methods: Forty cleansing grade blue-purple blue rabbits were randomly divided into normal group, model group, tonifying-tonifying and diarrhea group, twisting-tonifying group and heat-acupuncture group, with 8 in each group. In addition to the normal group, the remaining group to ovalbumin induction combined with cryogenic replication RA cold syndrome model. In the normal group and model group, no acupuncture intervention was performed. The rats in the group of supplementing and relieving diarrhea, twisting and supplementing group and heat supplementing and acupuncture group were treated with “Zusanli” Acupuncture, needle retention 30 min, 1 day, a total of 7 d. After the intervention, the pain threshold and knee surface temperature of each group were measured. The blood samples were collected from the heart to make the metabolomics test. The data were analyzed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) , Differential metabolites in serum were screened by variable importance projection values (VIP> 1) and P values (P <0.05). Results: Compared with the normal group, the pain threshold and the knee joint surface temperature in the model group decreased (all P <0.05). Compared with the model group, the pain threshold and the knee joint surface temperature increased <0.05). The hot-fill-acupuncture group was better than the flat-fill-diarrhea group and the twirling-supplementation group (all P <0.05). Compared with the normal group, carnitine, Lyso PC (14:0), Lyso PC (18: 3), Lyso PE (0: 0/20: 5), Lyso PE 22: 1), the contents of capric acid, stearic acid and lactic acid were all increased, leucine, valine, glutamine, pyroglutamic acid, α-ketoglutaric acid, succinic acid, fumaric acid, malic acid , Galactose, mannose content are reduced, the above metabolites specifically related to fatty acids, amino acids, tricarboxylic acid cycle and carbohydrate metabolism. The acupuncture group after intervention of the above metabolites showed varying degrees of callback. The content of lactic acid in hot-fill acupuncture group was lower than that in flat-fill and flatulence group, and the content of succinic acid, fumaric acid, malic acid, galactose and mannose increased. CONCLUSION: The specificities of RA for RA are reflected in the regulation of TCA and glucose metabolism.