褐色钟花树中一种新环烯醚萜抑制多柔比星诱导的心肌细胞凋亡

Inhibition of doxorubicin-induced cardiomyocyte apoptosis by a new iridoid from the Tabebuia avellanedae

本研究从褐色钟花树中分离得到一种新环烯醚萜,确定其抗多柔比星诱导的心肌损伤活性,并从抑制炎症、调节氧化应激和抑制凋亡三方面对其机制进行探讨。褐色钟花树内皮用沸水提取后进行液液萃取,通过硅胶/ODS/Sephadex LH-20柱色谱结合高效液相,纯化得到avelladoid I (Avd I),利用核磁共振波谱、高分辨质谱等技术确定其化学结构。建立多柔比星诱导的H9c2心肌细胞损伤模型,用1~40μmol·L^(-1)Avd I处理细胞后,检测细胞活力、细胞上清乳酸脱氢酶(lactate dehydrogenase, LDH)、炎症水平、氧化应激水平、细胞凋亡水平和线粒体凋亡通路关键蛋白。结果显示, Avd I改善心肌细胞损伤的有效浓度为1~20μmol·L^(-1),且1μmol·L^(-1)Avd I可降低白细胞介素-6(interleukin-6, IL-6)和白细胞介素-β (interleukin-1β, IL^(-1)β)水平,显著降低心肌细胞活性氧(reactive oxygen species,ROS)水平,增加超氧化物歧化酶(superoxide dismutase, SOD)水平,极显著降低心肌细胞的凋亡水平,并显著降低心肌细胞B淋巴细胞瘤-2相关X蛋白(B-cell lymphoma-2 associated X protein, Bax)/B淋巴细胞瘤-2 (B-cell lymphoma-2, Bcl-2),活化的半胱氨酸蛋白水解酶3 (cleaved cysteinyl aspartate specific proteinase 3, cleaved caspase 3)/半胱氨酸蛋白水解酶3 (cysteinyl aspartate specific proteinase 3, caspase 3)比率。Avd I能够改善心肌细胞活力,降低LDH水平,抑制炎症水平和氧化应激水平,其机制可能与调控线粒体凋亡途径进而抑制心肌凋亡水平有关。

A new iridoid was isolated from the Tabebuia avellanedae,its anti-myocardial injury activity was determined,and its mechanisms underlying inhibition of inflammation,regulation of oxidative stress and inhibition of apoptosis were explored.The dried inner bark of the Tabebuia avellanedae was extracted with boiling water,separated by liquid-liquid extraction,and purified by silica gel/ODS/Sephadex LH-20 column chromatography coupled with high-performance liquid chromatography(HPLC)to obtain avelladoid I(Avd I).The structure of Avd I was identified by nuclear magnetic resonance spectroscopy(NMR)and high-resolution mass spectrometry(HRMS).Cardiomyocyte injury model was established by 1μg·mL^(-1)doxorubicin.After treatment with 1−40μmol·L^(-1)of Avd I,the cell viability was evaluated by methyl thiazole tetrazolium(MTT)assay,and the lactate dehydrogenase(LDH)was measured.The inflammatory factor levels of interleukin-6(IL-6),interleukin-1β(IL^(-1)β)and tumor necrosis factor-α(TNF-α)as well as the oxidative stress levels of lactate dehydrogenase(LDH),superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione peroxidase(GSH-Px)were detected.DCFH-DA.staining was employed to observe the level of reactive oxygen species(ROS),AnnexinV-FITC/PI double stainings were performed to detect the apoptosis level,JC-1 single staining was used to measure the mitochondrial membrane potential level,and the Incell-Western assay was conducted to determine the apoptosis-related protein expression levels.The results showed that the cardiomyocyte injury was improved by 1−20μmol·L^(-1)of Avd I,and the IL-6 and IL^(-1)βlevels were decreased to near normal cell levels by 1μmol·L^(-1)Avd I.The ROS level was strongly reduced and the SOD level was highly increased by 1μmol·L^(-1)Avd I.In addition,1μmol·L^(-1)Avd I significantly decreased the apoptosis level,the B-cell lymphoma-2 associated X protein(Bax)/B-cell lymphoma-2(Bcl-2)ratio and the cleaved cysteinyl aspartate specific proteinase 3(cleaved caspase 3)/cysteinyl aspartate specific proteinase 3(caspase 3)ratio.Therefore,Avd I could stimulate the cardiomyocyte proliferation,reduce the LDH level and inhibit inflammation levels through regulating the mitochondrial apoptotic pathway.