基于毒效评价和代谢组学的铁棒锤抗类风湿关节炎的作用机制研究

Study on mechanism of Aconitum pendulum in treating rheumatoid arthritis based on toxicity efficacy evaluation and metabonomics

研究藏族药铁棒锤及其炮制品对类风湿关节炎(rheumatoid arthritis, RA)的影响,探讨其炮制减毒存效作用及机制。应用牛Ⅱ型胶原诱导类风湿关节炎(typeⅡcollagen-induced arthritis, CIA)大鼠模型;给药后记录大鼠体质量,计算心脏指数、免疫器官指数和关节炎指数;酶联免疫吸附测定(ELISA)法检测大鼠血清中肌酸激酶(creatine kinase, CK)、心肌特异性肌钙蛋白T(cardiac troponin T,cTnT)等多种因子水平;苏木素-伊红染色观察心脏组织及踝关节组织病理学形态变化;免疫组化法检测心肌连接蛋白43(connexin 43,Cx43)在大鼠心脏的表达;超高效液相色谱-串联四极杆飞行时间高分辨质谱(UPLC-Triple-TOF-MS)检测大鼠血清内源性代谢物水平,筛选潜在生物标志物并分析相关代谢通路。结果表明,铁棒锤生品能使大鼠心脏组织局部心肌纤维变性坏死,心脏指数显著增高,心肌组织Cx43表达平均阳性面积明显降低,血清中CK、cTnT等含量明显增高;大鼠免疫器官指数、血清中白细胞介素-6(interleukin-6,IL-6)等炎症因子含量减低、受损滑膜及关节面改善,效毒共存;糌粑制铁棒锤未见明显心脏毒性反应,同时显著降低大鼠关节炎指数、免疫器官指数及血清中IL-6等多种炎症因子含量,改善受损滑膜及关节面,减毒存效作用确切。血清代谢组学研究共筛选出19个铁棒锤生品及糌粑制铁棒锤抗RA的潜在生物标志物,主要涉及甘油磷脂代谢,甘氨酸、丝氨酸和苏氨酸代谢,精氨酸和脯氨酸代谢及类固醇激素合成等。综上,藏族药铁棒锤具有防治RA的作用,生品有一定心脏毒性,糌粑制后减毒存效作用确切,其治疗RA的机制与抗炎,调节甘油磷脂代谢、氨基酸代谢有关。

The study aims to investigate the effects and potential mechanism of raw and processed Aconitum pendulum Busch on rheumatoid arthritis(RA)and analyze their toxicity attenuating and efficacy retaining effects.The bovine typeⅡcollagen-induced arthritis(CIA)rat model was established.The weight,cardiac index,immune organ index,and arthritis index of the rats were recorded and calculated after administration.ELISA was used to measure the expressions of creatine kinase(CK),cardiac troponin T(cTnT),and multiple factors.The pathological morphological changes in heart tissue and ankle joint tissue were observed by hematoxylin-eosin staining.Connexin 43(Cx43)expression in the hearts of CIA rats was detected via immunohistochemical method.The levels of endogenous metabolites in the serum of CIA rats were detected by UPLC-Q-TOF-MS.Potential biomarkers were screened,and related metabolic pathways were analyzed.The results showed that raw A.pendulum could induce local myocardial fiber degeneration and necrosis,increase the cardiac index,decrease the average positive area of Cx43 expression significantly,and increase the expressions of CK and cTnT in cardiac tissue of rats.Meanwhile,raw A.pendulum could decrease the immune organ index,interleukin-6(IL-6),and other inflammatory cytokine contents in the serum and improve the damaged synovium and joint surface of CIA rats,with toxicity and efficacy coexisting.The Zanba stir-fired A.pendulum could reduce the index of arthritis,immune organ index,and content of IL-6 and inflammatory cytokines in serum and improve damaged synovium and joint surface of CIA rats with no obvious cardiac toxicity,showing significant toxicity attenuating and efficacy retaining effects.A total of 19 potential biomarkers of raw A.pendulum and Zanba stir-fired A.pendulum against RA were screened by serum metabolomics,including glycerophospholipid metabolism,glycine,serine,and threonine metabolism,arginine and proline metabolism,and steroid hormone synthesis.In conclusion,Xizang medicine A.pendulum is preventive and curative for RA.Raw A.pendulum has certain cardiotoxicity,and Zanba stir-fired A.pendulum has significant toxicity attenuating and efficacy retaining effects.The anti-RA mechanism may be related to the regulation of glycerophospholipid and amino acid metabolism.