新型小分子AF-HF001抑制氧化应激诱导心肌细胞凋亡研究
发布时间：2019-03-19 13:12
【摘要】：心力衰竭是各类心血管疾病的终极归宿,集高发病率、高死亡率、可造成沉重经济负担的特点,对国民健康形成重要威胁。我们通过斑马鱼心力衰竭模型筛选,发现并证实新型小分子AF-HF001可改善心脏扩大率及血流动力学,体现出治疗心力衰竭的潜力。心室重塑是心力衰竭病程的基本机制,伴随着心肌细胞的肥大和缺失。心肌缺血/缺氧引起氧化应激诱导的心肌细胞凋亡是心力衰竭的重要诱因。本研究利用双氧水和氯化钴诱导H9c2心肌细胞凋亡模型,采用Western blot、ELISA和活性氧敏感的DCFH-DA荧光试剂检测等方法,研究AF-HF001对氧化应激诱导心肌细胞凋亡的保护机制及对氧化应激下心肌细胞中活性氧产生的影响;通过基因芯片筛选结合荧光定量PCR初步鉴定AF-HF001保护氧化应激诱导心肌细胞凋亡的候选分子。实验结果表明:AF-HF001能显著降低氧化应激诱导的细胞中caspase3的裂解以及DNA的断裂,且在终浓度为50 n M开始出现抑凋亡效果,浓度为500 n M时可以完全逆转细胞凋亡;在50 500 n M浓度范围内,随着AF-HF001添加浓度的增加,抗细胞凋亡效果越明显;同时DCFH-DA荧光检测结果显示AF-HF001能显著减少氧化应激下H9c2心肌细胞中活性氧的产生。基因芯片筛选结果显示,AF-HF001逆转双氧水刺激H9c2心肌细胞引起的相关基因表达变化:如Ndufs2,Ndufs3,TPM1,Ry R1和TIMP4,涉及活性氧的生成、心肌收缩力和细胞外基质重塑等;如Araf,MAP2K5,Bax,Hsp27,Apaf1,casp3和casp7涉及MAPK和内源性线粒体信号通路。进一步研究表明AF-HF001促进H9c2心肌细胞凋亡初期JNK的磷酸化(p-T183/Y185),抑制凋亡晚期P38的活化(p-T180),调节抗细胞凋亡作用。基因芯片筛选研究与鉴定发现AF-HF001能有效减少双氧水诱导H9c2细胞中MDSP的表达,为了进一步确认在AF-HF001介导的抗细胞凋亡中MDSP的作用,通过荧光定量PCR实验发现,双氧水刺激H9c2心肌细胞会以时间依赖的方式促进MDSP的表达,且在刺激24 h达到表达高峰;JNK抑制剂(SP600125)明显增加双氧水诱导H9c2心肌细胞中MDSP的表达。p EGFP-MDSP质粒介导外源性过表达GFP-MDSP融合蛋白,可显著增加DNA的断裂;基因沉默H9c2大鼠心肌细胞中内源性MDSP表达后,可显著减少双氧水诱导的caspase3裂解的增加。因此,预处理AF-HF001激活JNK,显著逆转双氧水刺激MDSP表达量的增加,减少双氧水诱导的凋亡。综上所述:我们首次发现并证实新型小分子AF-HF001以浓度依赖的方式抑制H2O2诱导的H9c2心肌细胞的凋亡,并减少胞内活性氧的产生;同时发现AF-HF001通过减少MDSP的表达,保护H2O2诱导的H9c2心肌细胞凋亡。因而,深入研究AF-HF001对氧化应激诱导心肌细胞凋亡的保护机制,为其发展成为临床药物奠定理论基础;同时,为心力衰竭的预防或治疗提供全新的靶点或方向。
[Abstract]:Heart failure is the ultimate destination of all kinds of cardiovascular diseases. It has the characteristics of high morbidity and high mortality, which can cause heavy economic burden and pose an important threat to the national health. By screening the zebra fish heart failure model, we found and confirmed that the novel small molecule AF-HF001 can improve the heart enlargement rate and hemodynamics, and show the potential of treatment for heart failure. Ventricular remodeling is the basic mechanism of heart failure, accompanied by hypertrophy and loss of cardiomyocytes. Cardiomyocyte apoptosis induced by oxidative stress caused by myocardial ischemia / hypoxia is an important inducement of heart failure. In this study, H9c2 cardiomyocyte apoptosis model was induced by hydrogen peroxide and cobalt chloride. Western blot,ELISA and reactive oxygen species sensitive DCFH-DA fluorescence reagent were used to detect the apoptosis of cardiomyocytes. To study the protective mechanism of AF-HF001 on cardiomyocyte apoptosis induced by oxidative stress and its effect on the production of reactive oxygen species (Ros) in cardiomyocytes under oxidative stress. The candidate molecules of AF-HF001 protecting cardiomyocyte apoptosis induced by oxidative stress were preliminarily identified by gene chip screening combined with fluorescence quantitative PCR (FQ-PCR). The results showed that AF-HF001 could significantly reduce the cleavage of caspase3 and the break of DNA in the cells induced by oxidative stress, and inhibited the apoptosis at the final concentration of 50 nm, and could completely reverse the apoptosis at the concentration of 500 nm. In the concentration range of 50 渭 500nm, the anti-apoptosis effect was more obvious with the increase of AF-HF001 concentration, and the results of DCFH-DA fluorescence showed that AF-HF001 could significantly reduce the production of reactive oxygen species (Ros) in H9c2 cardiomyocytes under oxidative stress. The results of gene chip screening showed that AF-HF001 reversed the related gene expression changes induced by hydrogen peroxide in H9c2 cardiomyocytes, such as Ndufs2,Ndufs3,TPM1,Ry R1 and TIMP4, involved in the production of reactive oxygen species, myocardial contractility and extracellular matrix remodeling, and so on. For example, Araf,MAP2K5,Bax,Hsp27,Apaf1,casp3 and casp7 are involved in MAPK and endogenous mitochondrial signaling pathways. Further studies showed that AF-HF001 promoted the phosphorylation of JNK (p-T183/Y185) at the early stage of apoptosis, inhibited the activation of P38 (p-T180) in the late apoptotic stage and regulated the anti-apoptosis effect of H9c2 cardiomyocytes. Gene chip screening and identification showed that AF-HF001 could effectively reduce the expression of MDSP in H9c2 cells induced by hydrogen peroxide. In order to further confirm the role of MDSP in anti-apoptosis mediated by AF-HF001, fluorescence quantitative PCR assay was performed. Hydrogen peroxide stimulated H9c2 cardiomyocytes increased the expression of MDSP in a time-dependent manner, and reached the peak at 24 h after stimulation. JNK inhibitor (SP600125) significantly increased the expression of MDSP in H9c2 cardiomyocytes induced by hydrogen peroxide. Exogenous over-expression of GFP-MDSP fusion protein mediated by p-EGFP-MDSP plasmid significantly increased the break of DNA. The expression of endogenous MDSP in cardiomyocytes of H9c2 rats induced by gene silencing significantly decreased the increase of caspase3 cleavage induced by hydrogen peroxide. Therefore, pretreatment of AF-HF001 activated JNK, significantly reversed the increase of MDSP expression induced by hydrogen peroxide and decreased the apoptosis induced by hydrogen peroxide. In conclusion, we found and confirmed for the first time that AF-HF001, a novel small molecule, inhibited H2O2-induced apoptosis and decreased the production of reactive oxygen species (Ros) in H9c2 cardiomyocytes in a concentration-dependent manner. At the same time, it was found that AF-HF001 could protect H9c2 cardiomyocytes from apoptosis induced by H2O2 by decreasing the expression of MDSP. Therefore, to study the protective mechanism of AF-HF001 on cardiomyocyte apoptosis induced by oxidative stress, to lay a theoretical foundation for its development into a clinical drug, and to provide a new target or direction for the prevention or treatment of heart failure.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541.6
本文编号：2443553
[Abstract]:Heart failure is the ultimate destination of all kinds of cardiovascular diseases. It has the characteristics of high morbidity and high mortality, which can cause heavy economic burden and pose an important threat to the national health. By screening the zebra fish heart failure model, we found and confirmed that the novel small molecule AF-HF001 can improve the heart enlargement rate and hemodynamics, and show the potential of treatment for heart failure. Ventricular remodeling is the basic mechanism of heart failure, accompanied by hypertrophy and loss of cardiomyocytes. Cardiomyocyte apoptosis induced by oxidative stress caused by myocardial ischemia / hypoxia is an important inducement of heart failure. In this study, H9c2 cardiomyocyte apoptosis model was induced by hydrogen peroxide and cobalt chloride. Western blot,ELISA and reactive oxygen species sensitive DCFH-DA fluorescence reagent were used to detect the apoptosis of cardiomyocytes. To study the protective mechanism of AF-HF001 on cardiomyocyte apoptosis induced by oxidative stress and its effect on the production of reactive oxygen species (Ros) in cardiomyocytes under oxidative stress. The candidate molecules of AF-HF001 protecting cardiomyocyte apoptosis induced by oxidative stress were preliminarily identified by gene chip screening combined with fluorescence quantitative PCR (FQ-PCR). The results showed that AF-HF001 could significantly reduce the cleavage of caspase3 and the break of DNA in the cells induced by oxidative stress, and inhibited the apoptosis at the final concentration of 50 nm, and could completely reverse the apoptosis at the concentration of 500 nm. In the concentration range of 50 渭 500nm, the anti-apoptosis effect was more obvious with the increase of AF-HF001 concentration, and the results of DCFH-DA fluorescence showed that AF-HF001 could significantly reduce the production of reactive oxygen species (Ros) in H9c2 cardiomyocytes under oxidative stress. The results of gene chip screening showed that AF-HF001 reversed the related gene expression changes induced by hydrogen peroxide in H9c2 cardiomyocytes, such as Ndufs2,Ndufs3,TPM1,Ry R1 and TIMP4, involved in the production of reactive oxygen species, myocardial contractility and extracellular matrix remodeling, and so on. For example, Araf,MAP2K5,Bax,Hsp27,Apaf1,casp3 and casp7 are involved in MAPK and endogenous mitochondrial signaling pathways. Further studies showed that AF-HF001 promoted the phosphorylation of JNK (p-T183/Y185) at the early stage of apoptosis, inhibited the activation of P38 (p-T180) in the late apoptotic stage and regulated the anti-apoptosis effect of H9c2 cardiomyocytes. Gene chip screening and identification showed that AF-HF001 could effectively reduce the expression of MDSP in H9c2 cells induced by hydrogen peroxide. In order to further confirm the role of MDSP in anti-apoptosis mediated by AF-HF001, fluorescence quantitative PCR assay was performed. Hydrogen peroxide stimulated H9c2 cardiomyocytes increased the expression of MDSP in a time-dependent manner, and reached the peak at 24 h after stimulation. JNK inhibitor (SP600125) significantly increased the expression of MDSP in H9c2 cardiomyocytes induced by hydrogen peroxide. Exogenous over-expression of GFP-MDSP fusion protein mediated by p-EGFP-MDSP plasmid significantly increased the break of DNA. The expression of endogenous MDSP in cardiomyocytes of H9c2 rats induced by gene silencing significantly decreased the increase of caspase3 cleavage induced by hydrogen peroxide. Therefore, pretreatment of AF-HF001 activated JNK, significantly reversed the increase of MDSP expression induced by hydrogen peroxide and decreased the apoptosis induced by hydrogen peroxide. In conclusion, we found and confirmed for the first time that AF-HF001, a novel small molecule, inhibited H2O2-induced apoptosis and decreased the production of reactive oxygen species (Ros) in H9c2 cardiomyocytes in a concentration-dependent manner. At the same time, it was found that AF-HF001 could protect H9c2 cardiomyocytes from apoptosis induced by H2O2 by decreasing the expression of MDSP. Therefore, to study the protective mechanism of AF-HF001 on cardiomyocyte apoptosis induced by oxidative stress, to lay a theoretical foundation for its development into a clinical drug, and to provide a new target or direction for the prevention or treatment of heart failure.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541.6
【参考文献】
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1 ;Outline of the Report on Cardiovascular Disease in China,2010[J];Biomedical and Environmental Sciences;2012年03期
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