摘要
目的探讨壬二酸对阿霉素诱导的心肌细胞损伤的保护作用及其机制。方法取生长融合度达80%以上的H9c2心肌细胞随机分为正常组、模型组、壬二酸组,正常组以高糖DMEM培养基培养,模型组以5μmol/L阿霉素处理24 h,壬二酸组以10μmol/L壬二酸和5μmol/L阿霉素共处理24 h,采用CCK-8法和微量酶标法检测心肌细胞活力及乳酸脱氢酶(LDH)漏出量,采用化学荧光法检测细胞内活性氧(ROS)水平及线粒体膜电位(ΔΨm)变化,采用荧光素酶法检测H9c2心肌细胞内三磷酸腺苷(ATP)水平,采用透射电子显微镜观察H9c2心肌细胞中自噬体的数量,采用免疫荧光染色分析细胞中Bcl-2/腺病毒E1B相互作用蛋白3(BNIP3)与线粒体荧光探针(Mitotracker)共定位的情况,采用Western blot实验检测H9c2心肌细胞中自噬相关蛋白微管相关蛋白轻链3-Ⅰ(LC3-Ⅰ)、LC3-Ⅱ、泛素结合蛋白(P62)、凋亡相关蛋白B淋巴细胞瘤-2基因相关启动子(Bad)及BNIP3的表达水平。24只C57BL/6J小鼠随机分为正常组、模型组(仅第1天腹腔注射阿霉素10 mg/kg)、壬二酸组(在模型组处理基础上壬二酸20 mg/kg连续灌胃7 d),每组8只小鼠,采用超声心电图检测小鼠心功能的变化,采用Masson染色法检测小鼠心脏组织中心肌纤维化的情况。结果两组细胞LDH漏出量、ROS水平、ATP含量、ΔΨm、BNIP3与Mitotracker的Pearson系数、自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ比值及Bad、BNIP3、P62蛋白表达水平与模型组比较,差异均具有显著性(t=2.54~17.37,P<0.05)。正常组、壬二酸组小鼠与模型组小鼠比较,EF%、FS%差异均具有显著性(t=3.41~6.49,P<0.05)。结论壬二酸对阿霉素诱导的心肌细胞损伤具有保护作用,其作用可能是通过抑制细胞中BNIP3介导的线粒体自噬实现的。
Objective To investigate the protective effect of azelaic acid against doxorubicin-induced cardiomyocyte injury and its mechanism.\Methods\H9c2 cardiomyocytes with a growth confluence of>80%were randomly divided into normal group,model group,and azelaic acid group.The cells in the normal group were cultured with a high-glucose DMEM medium,those in the model group were treated with 5μmol/L doxorubicin for 24 hours,and those in the azelaic acid group were treated with 10μmol/L azelaic acid and 5μmol/L doxorubicin for 24 hours.CCK-8 assay and the microenzyme labeling method were used to measure cardiomyocyte viability and lactate dehydrogenase(LDH)leakage;the chemifluorescence method was used to measure the level of reactive oxygen species(ROS)and the change in mitochondrial membrane potential(ΔΨm)in cells;the luciferase method was used to measure the level of adenosine triphosphate(ATP)in H9c2 cardiomyocytes;a transmission electron microscope was used to observe autophagosomes in H9c2 cardiomyocytes;immunofluorescent staining was used to observe the coloca-lization of Bcl-2/adenovirus E1B interacting protein 3(BNIP3)and mitochondrial fluorescent probe(Mitotracker)in cells;Western blot was used to measure the expression levels of autophagy-related proteins[microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅰ)and microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ)],ubiquitin-binding protein P62,apoptosis-related protein B-cell lymphoma-2 gene-related promoter(Bad),and BNIP3 in H9c2 cardiomyocytes.A total of 24 C57BL/6J mice were randomly divided into normal group,model group(intraperitoneal injection of doxorubicin 10 mg/kg only on day 1),and azelaic acid group(azelaic acid 20 mg/kg by gavage for 7 consecutive days in addition to the treatment in the model group),with 8 mice in each group.Echocardiography was used to measure the change in cardiac function,and Masson staining was used to observe myocardial fibrosis in cardiac tissue.Results There were significant differences between the two groups o
作者
高远真
原阳
叶婷
李梦娇
张钰坤
邢东明
GAO Yuanzhen;YUAN Yang;YE Ting;LI Mengjiao;ZHANG Yukun;XING Dongming(School of Basic Medicine,Qingdao University,Qingdao 266071,China)
出处
《精准医学杂志》
2022年第4期288-293,299,共7页
Journal of Precision Medicine
基金
国家自然科学基金资助项目(32000830)。
