【摘要】：心力衰竭是各類心血管疾病的終極歸宿,集高發(fā)病率、高死亡率、可造成沉重經(jīng)濟(jì)負(fù)擔(dān)的特點(diǎn),對(duì)國(guó)民健康形成重要威脅。我們通過斑馬魚心力衰竭模型篩選,發(fā)現(xiàn)并證實(shí)新型小分子AF-HF001可改善心臟擴(kuò)大率及血流動(dòng)力學(xué),體現(xiàn)出治療心力衰竭的潛力。心室重塑是心力衰竭病程的基本機(jī)制,伴隨著心肌細(xì)胞的肥大和缺失。心肌缺血/缺氧引起氧化應(yīng)激誘導(dǎo)的心肌細(xì)胞凋亡是心力衰竭的重要誘因。本研究利用雙氧水和氯化鈷誘導(dǎo)H9c2心肌細(xì)胞凋亡模型,采用Western blot、ELISA和活性氧敏感的DCFH-DA熒光試劑檢測(cè)等方法,研究AF-HF001對(duì)氧化應(yīng)激誘導(dǎo)心肌細(xì)胞凋亡的保護(hù)機(jī)制及對(duì)氧化應(yīng)激下心肌細(xì)胞中活性氧產(chǎn)生的影響;通過基因芯片篩選結(jié)合熒光定量PCR初步鑒定AF-HF001保護(hù)氧化應(yīng)激誘導(dǎo)心肌細(xì)胞凋亡的候選分子。實(shí)驗(yàn)結(jié)果表明:AF-HF001能顯著降低氧化應(yīng)激誘導(dǎo)的細(xì)胞中caspase3的裂解以及DNA的斷裂,且在終濃度為50 n M開始出現(xiàn)抑凋亡效果,濃度為500 n M時(shí)可以完全逆轉(zhuǎn)細(xì)胞凋亡;在50 500 n M濃度范圍內(nèi),隨著AF-HF001添加濃度的增加,抗細(xì)胞凋亡效果越明顯;同時(shí)DCFH-DA熒光檢測(cè)結(jié)果顯示AF-HF001能顯著減少氧化應(yīng)激下H9c2心肌細(xì)胞中活性氧的產(chǎn)生。基因芯片篩選結(jié)果顯示,AF-HF001逆轉(zhuǎn)雙氧水刺激H9c2心肌細(xì)胞引起的相關(guān)基因表達(dá)變化:如Ndufs2,Ndufs3,TPM1,Ry R1和TIMP4,涉及活性氧的生成、心肌收縮力和細(xì)胞外基質(zhì)重塑等;如Araf,MAP2K5,Bax,Hsp27,Apaf1,casp3和casp7涉及MAPK和內(nèi)源性線粒體信號(hào)通路。進(jìn)一步研究表明AF-HF001促進(jìn)H9c2心肌細(xì)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),調(diào)節(jié)抗細(xì)胞凋亡作用。基因芯片篩選研究與鑒定發(fā)現(xiàn)AF-HF001能有效減少雙氧水誘導(dǎo)H9c2細(xì)胞中MDSP的表達(dá),為了進(jìn)一步確認(rèn)在AF-HF001介導(dǎo)的抗細(xì)胞凋亡中MDSP的作用,通過熒光定量PCR實(shí)驗(yàn)發(fā)現(xiàn),雙氧水刺激H9c2心肌細(xì)胞會(huì)以時(shí)間依賴的方式促進(jìn)MDSP的表達(dá),且在刺激24 h達(dá)到表達(dá)高峰;JNK抑制劑(SP600125)明顯增加雙氧水誘導(dǎo)H9c2心肌細(xì)胞中MDSP的表達(dá)。p EGFP-MDSP質(zhì)粒介導(dǎo)外源性過表達(dá)GFP-MDSP融合蛋白,可顯著增加DNA的斷裂;基因沉默H9c2大鼠心肌細(xì)胞中內(nèi)源性MDSP表達(dá)后,可顯著減少雙氧水誘導(dǎo)的caspase3裂解的增加。因此,預(yù)處理AF-HF001激活JNK,顯著逆轉(zhuǎn)雙氧水刺激MDSP表達(dá)量的增加,減少雙氧水誘導(dǎo)的凋亡。綜上所述:我們首次發(fā)現(xiàn)并證實(shí)新型小分子AF-HF001以濃度依賴的方式抑制H2O2誘導(dǎo)的H9c2心肌細(xì)胞的凋亡,并減少胞內(nèi)活性氧的產(chǎn)生;同時(shí)發(fā)現(xiàn)AF-HF001通過減少M(fèi)DSP的表達(dá),保護(hù)H2O2誘導(dǎo)的H9c2心肌細(xì)胞凋亡。因而,深入研究AF-HF001對(duì)氧化應(yīng)激誘導(dǎo)心肌細(xì)胞凋亡的保護(hù)機(jī)制,為其發(fā)展成為臨床藥物奠定理論基礎(chǔ);同時(shí),為心力衰竭的預(yù)防或治療提供全新的靶點(diǎn)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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R541.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Outline of the Report on Cardiovascular Disease in China,2010[J];Biomedical and Environmental Sciences;2012年03期

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本文編號(hào)：2443553