基于代谢组学的灯盏花素抗阿霉素心脏毒性作用机制研究

Mechanisms of breviscapine against doxorubicin-induced cardiotoxicity based on metabolomics

目的通过体内外实验研究灯盏花素对阿霉素诱导心脏毒性的保护作用及机制。方法体内实验中,将C57BL/6小鼠随机分为对照组、模型组、右雷佐生(12 mg/kg)组和灯盏花素低、中、高剂量(4、8、16 mg/kg)组,给予药物干预3周后,采用苏木素-伊红(HE)染色观察小鼠心肌组织的病理变化;采用ELISA法检测各组小鼠血浆N端B型利钠肽前体(amino-terminal pro-B-type natriuretic peptide,NT-proBNP)水平;采用超高效液相色谱/四极杆飞行时间质谱(UHPLC/Q-TOF MS)研究其代谢途径和主要代谢产物。体外实验中,将大鼠心肌细胞H9c2随机分为对照组、阿霉素组、右雷佐生组和灯盏花素组,给予药物处理后,检测丙二醛(malondialdehyde,MDA)和谷胱甘肽(glutathione,GSH)水平,观察H9c2细胞抗氧化能力;采用TUNEL及Annexin V-FITC/PI双染法检测各组细胞凋亡情况;采用Western blotting法检测H9c2细胞核因子E2相关因子2(nuclear factor E2 related factor 2,Nrf2)、腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)和Ras同源蛋白A(Ras homolog gene family member A,RhoA)蛋白表达情况。结果与对照组比较,模型组小鼠血浆NTproBNP水平显著升高(P<0.05),心肌组织发生肌原纤维紊乱、破裂以及心肌纤维的波状变性;与模型组比较,灯盏花素组和右雷佐生组心肌组织的形态损伤减轻,血浆NT-proBNP水平显著降低(P<0.05);代谢组学分析筛选出16个显著改变的代谢物作为潜在的生物标志物,涉及氨基酸代谢、脂质代谢及炎症因子调节等代谢路径。在H9c2细胞中,阿霉素组GSH水平显著降低(P<0.05),MDA水平显著升高(P<0.05),心肌细胞凋亡率显著升高(P<0.05),RhoA和Nrf2蛋白表达水平均显著降低(P<0.05),AMPK蛋白表达水平显著升高(P<0.05);与阿霉素组比较,灯盏花素组和右雷佐生组细胞存活率升高(P<0.05),MDA水平显著降低(P<0.05),GSH水平显著升高(P<0.05),心肌细胞凋亡率和ROS生成均显著降低(P<0.05),RhoA和Nrf2蛋白表达水平均显著升高(P<0.05),AMPK蛋白表达水平显著降低(P<0.05)。结论灯盏花素通过调节脂质代谢、氨基酸代谢以及炎症因子水平维持细胞氧化还原状态和炎症水平,恢复心肌细胞稳态,抑制了阿霉素诱导的细胞损伤和凋亡,缓解心脏毒性,此外灯盏花素可能通过调节神经活性配体受体相互作用保护受损心脏功能。灯盏花素可能是一种很有前途的心脏保护剂。

Objective To investigate the effects and mechanism of breviscapine against doxorubicin-induced cardiotoxicity in vivo and in vitro.Methods In vivo,C57BL/6 mice were randomly divided into control group,model group,dexrazoxane(12 mg/kg)group and breviscapine low-,medium-and high-dose(4,8,16 mg/kg)groups,drugs were given for intervention for 3 weeks,hematoxylineosin(HE)staining was used to observe the pathological changes of myocardial tissue in mice;ELISA method was used to detect the amino-terminal pro-B-type natriuretic peptide(NT-proBNP)level in plasma;Metabolic pathways and major metabolites were studied by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry(UHPLC/Q-TOF MS).In vitro experiments,rat cardiomyocytes H9c2 were randomly divided into control group,adriamycin group,dexrazoxane group and breviscapine group.After treatment,malondialdehyde(MDA)and glutathione(GSH)levels were detected to observe the antioxidant capacity of H9c2 cells;TUNEL and Annexin V-FITC/PI double staining were used to detect the apoptosis of cells in each group;Western blotting was used to detect nuclear factor E2 related factor 2(Nrf2),adenylate-activated protein kinase(AMPK)and Ras homolog gene family member A(RhoA)protein expressions of H9c2 cells.Results Compared with control group,NT-proBNP level in plasma of mice in model group was significantly increased(P<0.05),myofibrils were disordered,ruptured and wavy degeneration of myocardial fibers occurred in myocardial tissue;Compared with model group,morphological damage of myocardial tissue in breviscapine group and dexrazoxane group was alleviated,and NT-proBNP level in plasma was significantly decreased(P<0.05).Metabolomics analysis screened out 16 significantly altered metabolites as potential biomarkers,involving metabolic pathways such as amino acid metabolism,lipid metabolism and regulation of inflammatory factors.In H9c2 cells,GSH level was significantly decreased(P<0.05),MDA level was significantly increased(P<0.05),apoptosis rate of cardiomyocytes was significantly increased(P<0.05),RhoA and Nrf2 protein expressions in adriamycin group were significantly decreased(P<0.05),AMPK protein expression was significantly increased(P<0.05).Compared with doxorubicin group,survival rate of cells in breviscapine group and dexrazoxane group was increased(P<0.05),MDA level was significantly decreased(P<0.05),GSH level was significantly increased(P<0.05),apoptosis rate and ROS generation of cardiomyocytes were significantly decreased(P<0.05),RhoA and Nrf2 protein expressions were significantly increased(P<0.05),AMPK protein expression was significantly decreased(P<0.05).Conclusion Breviscapine maintains cellular redox state and inflammatory level by regulating lipid metabolism,amino acid metabolism and inflammatory factor levels,restores myocardial cell homeostasis,inhibits doxorubicin-induced cell damage and apoptosis,and relieves cardiac toxicity,and breviscapine may protect impaired cardiac function through neuroactive ligand-receptor interactions.Breviscapine may be a promising cardioprotective agent.