血红素加氧酶-1介导姜黄素对抗H9c2心肌细胞氧化应激损伤的实验研究

发布时间：2018-03-16 16:46

本文选题：血红素加氧酶-1　切入点：H9c2心肌细胞　出处：《华中科技大学》2012年博士论文　论文类型：学位论文

【摘要】：近年来,先天性心脏病的救治正向着复杂、危重和幼小化的方向发展,心肌缺血再灌注损伤已成为困扰临床疗效的重要问题。氧化应激诱导的心肌细胞凋亡是心肌缺血再灌注损伤发生发展的重要病理基础。氧化应激是指由于活性氧过量生成和／或细胞内抗氧化防御系统受损,导致超氧阴离子(O2-)、过氧化氢(hydrogen peroxide, H2O2)等氧自由基及其相关代谢产物过量聚集,从而对细胞产生多种毒性作用的病理状态。大量研究表明,抑制氧化应激诱导的心肌细胞凋亡是减轻心肌缺血再灌注损伤的有效途径。血红素加氧酶-1(heme oxygenase-1, HO-1)是血红素降解的起始酶和限速酶,能够降解血红素生成一氧化碳(carbon monoxide, CO)、Fe2+和胆红素(bilirubin)。研究表明,在多种心血管疾病如心肌梗死、心肌缺血再灌注损伤和心力衰竭的研究中,采用药理学方法或基因方法使HO-1蛋白表达增加可抑制心肌细胞损伤。姜黄素是从姜科植物姜黄根茎中提取的一种酚性色素,具有抑制肿瘤、抗炎以及抗氧化等多种生物学效应。已有研究证实,在体外培养的内皮细胞和成纤维细胞中,姜黄素能够成浓度依赖性的诱导HO-1蛋白表达及活性增加,进而发挥细胞保护作用。因此,我们推测姜黄素可能对H9c2心肌细胞氧化应激损伤具有保护效应,且该效应与HO-1密切相关。 Akt(protein kinase B, PKB,又称Akt)是磷脂酰肌醇3激酶(phosphatidylinositol-3kinase, PI3K)的下游信号调节激酶之一,在多种物质介导的抗凋亡效应中发挥重要作用。在多种细胞损伤模型中,PI3K/Akt信号通路介导的HO-1蛋白上调能够使细胞对抗多种应激刺激。核因子NF-κB是一个多效能的核转录因子,在不同的细胞模型中可表现为凋亡抑制或凋亡促进作用。本实验旨在通过建立H202诱导的H9c2心肌细胞氧化应激损伤模型,在细胞水平上探讨姜黄素的心血管保护作用以及HO-1介导姜黄素对抗H9c2心肌细胞氧化应激损伤的机制。第一部分 HO-1对H9c2心肌细胞氧化应激损伤的保护作用研究目的探讨HO-1对H9c2心肌细胞氧化应激损伤的保护作用及其机制。方法建立H202诱导的H9c2心肌细胞氧化应激损伤模型,给予HO-1诱导剂hemin预处理,或给予HO-1抑制剂Znpp-IX共孵育。双波长法检测HO-1活性；四甲基偶氮唑蓝(MTT)法检测细胞存活率；Hoechst33342染色和caspase-3活性检测评估细胞凋亡；硫代巴比妥酸显色法检测MDA含量,氮蓝四唑显色法检测总SOD活性；westernblot法检测NF-κB蛋白表达水平。结果与对照组相比,hemin成浓度依赖性的诱导HO-1活性增加。与H202组相比,HO-1活性增加能够显著下调细胞凋亡率和caspase-3活性,降低细胞MDA含量,增加总SOD活性,且上述作用能被Znpp-IX逆转。此外,HO-1活性增加能够显著抑制H202诱导的NF-κB激活,且该作用能被Znpp-IX逆转。结论Hemin诱导的HO-1活性增加可能通过抑制NF-κB激活,维持细胞氧化还原平衡状态,抑制H202诱导的H9c2心肌细胞氧化应激损伤。第二部分HO-1介导姜黄素对抗H9c2心肌细胞氧化应激损伤目的探讨姜黄素对H9c2心肌细胞氧化应激损伤的保护效应以及HO-1在该效应中的作用。方法建立H202诱导的H9c2心肌细胞氧化应激损伤模型,给予姜黄素预处理,或给予HO-1抑制剂Znpp-Ⅸ共孵育。双波长法检测HO-1活性；MTT法检测细胞存活率；流式细胞术Annexin-Ⅴ FITC/PI染色和caspase-3活性检测评估细胞凋亡；硫代巴比妥酸显色法检测MDA含量,氮蓝四唑显色法检测总SOD活性。western blot检测HO-1,Bc1-2,Bax, cleaved caspase-3蛋白表达水平。结果与对照组相比,姜黄素呈浓度依赖性的上调HO-1mRNA表达,同时上调HO-1蛋白表达和活性。与H202组相比,姜黄素能够显著上调细胞存活率,下调细胞凋亡率和caspase-3活性。同时,姜黄素能够显著抑制H202诱导的bcl-2/bax比例下降以及cleaved caspase-3蛋白表达增加。此外,姜黄素能够显著抑制H202诱导的细胞MDA含量增加以及总SOD活性下降。姜黄素的上述作用均能被Znpp-Ⅸ部分逆转。结论姜黄素可能通过增加HO-1蛋白表达,上调HO-1活性,维持细胞氧化还原平衡状态,对抗H202诱导的H9c2心肌细胞氧化应激损伤。第三邵分 HO-1介导姜黄素对抗H9c2心肌细胞氧化应激损伤的机制研究目的探讨HO-1介导姜黄素对抗H9c2心肌细胞氧化应激损伤的机制。方法5-15μM姜黄素处理H9c2心肌细胞12h或15μM姜黄素处理H9c2心肌细胞0.5-12h, western blot检测p-Akt, t-Akt蛋白表达水平。建立H2O2诱导的H9c2心肌细胞氧化应激损伤模型：(1)给予姜黄素预处理,或给予PI3K抑制剂LY294002共孵育,western blot法检测cleaved caspase-3和HO-1蛋白表达水平。(2)给予姜黄素预处理,或给予HO-1抑制剂.Znpp-Ⅸ共孵育,western blot法检测核因子NF-κB蛋白表达水平。结果5-15μM姜黄素可成浓度依赖性的诱导p-Akt/t-Akt比例增加,15μM姜黄素处理H9c2心肌细胞0.5-12h后,p-Akt/t-Akt比例在30min内显著上升,随后逐渐下降。在H202诱导的H9c2新2肌细胞氧化应激损伤模型中,姜黄素对cleaved caspase-3蛋白表达的抑制效应能够被P13K抑制剂LY294002逆转。同时,LY294002能够显著抑制姜黄素诱导的HO-1蛋白表达水平上调。此外,姜黄素预处理能够显著抑制H202诱导的NF-κB激活,且该作用能被Znpp-IX部分逆转。结论姜黄素可能通过PI3K/Akt信号途径上调HO-1蛋白表达,抑制H202诱导的NF-κB激活,发挥细胞保护作用。
[Abstract]:In recent years, the treatment of congenital heart disease is complex, critical and young in the direction of development, myocardial ischemia reperfusion injury has become an important issue. The clinical curative effect of myocardial cell apoptosis induced by oxidative stress is an important pathological basis of the occurrence and development of myocardial ischemia reperfusion injury. Oxidative stress is due to the excessive production of reactive oxygen species and / or cellular antioxidant defense system is damaged, resulting in superoxide anion (O2-), hydrogen peroxide (hydrogen peroxide, H2O2), oxygen free radicals and related metabolites of excessive aggregation, so as to produce the pathological state of cell toxicity. Many studies showed that inhibition of myocardial cell apoptosis induced by oxidative stress is an effective way to reduce myocardial ischemia reperfusion injury.
Heme oxygenase -1 (heme oxygenase-1 HO-1) and rate limiting enzyme of heme degradation, can degrade into carbon monoxide (carbon, monoxide, CO, Fe2+) and bilirubin (bilirubin). The results show that in many cardiovascular diseases such as myocardial infarction, heart failure and myocardial injury of ischemia reperfusion in the pharmacology the method or method to make the increased expression of HO-1 gene can inhibit myocardial cell injury. Curcumin is a phenolic pigment extracted from Zingiberaceae turmeric rhizome, inhibiting tumor, anti-inflammatory and anti oxidative and other biological effects. Many studies have confirmed that in vitro cultured endothelial cells and fibroblasts, curcumin can become to increase the concentration dependence of the induced expression and activity of HO-1 protein, which play a cytoprotective role. Therefore, we speculate that curcumin on oxidative stress in myocardial cells H9c2 The damage has a protective effect, and the effect is closely related to HO-1.
Akt (protein kinase B PKB, also called Akt) is phosphatidylinositol 3 kinase (phosphatidylinositol-3kinase, PI3K) of the downstream signal regulated kinase, plays an important role in the anti apoptosis effect mediated by a variety of substances. In a variety of cell injury model, PI3K/Akt signaling pathway mediated upregulation of HO-1 protein can cause cells to fight a variety of stress. Nuclear factor kappa B NF- is a multifunctional transcription factor in different cell models can be manifested in apoptosis or apoptosis.
The aim of this study is to establish H202 induced oxidative stress injury model of H9c2 cardiomyocytes, to explore the protective effect of curcumin on the cell level and the mechanism of HO-1 mediated curcumin against oxidative stress injury in H9c2 cardiomyocytes.
Part one
Protective effect of HO-1 on oxidative stress injury in H9c2 cardiomyocytes
Objective to investigate the protective effect of HO-1 on oxidative stress injury in H9c2 cardiomyocytes and its mechanism.
Methods H9c2 cells oxidative stress injury model induced by H202, given HO-1 inducer hemin pretreatment or inhibitor of HO-1 were cultured with Znpp-IX. HO-1 activity was detected by double wavelength spectrophotometric method; four methyl thiazolyl tetrazolium (MTT) assay cell viability detection; Hoechst33342 staining and caspase-3 activity assessment of apoptosis; thiobarbituric acid colorimetric method to detect MDA content, nitrogen blue four color of the total SOD activity was measured; to detect the expression of NF- kappa B protein Westernblot.
Results compared with the control group, hemin induced concentration dependent increase of HO-1 activity. Compared with the H202 group, the HO-1 activity increased significantly reduced cell apoptosis rate and caspase-3 activity, decreased the cell MDA content, increase the total SOD activity, and the effect was reversed by Znpp-IX. In addition, the increase of HO-1 activity can significantly inhibit H202 induced NF- K B activation, and the effect was reversed by Znpp-IX.
Conclusion the increase of HO-1 activity induced by Hemin may inhibit the activation of NF- kappa B, maintain the redox balance of cells, and inhibit the oxidative stress induced by H202 in H9c2 cardiomyocytes.
Second part HO-1 mediates curcumin against oxidative stress injury in H9c2 cardiomyocytes
Objective to investigate the protective effect of curcumin on oxidative stress injury in H9c2 cardiomyocytes and the role of HO-1 in this effect.
Methods H9c2 cells oxidative stress injury model induced by H202, curcumin pretreatment or HO-1 inhibitor Znpp- IX co incubation. HO-1 activity was detected by double wavelength spectrophotometric method; cell viability was measured by MTT assay; flow cytometry Annexin- V FITC/PI staining and caspase-3 activity assay. Apoptosis; thiobarbituric acid colorimetric method detection of MDA content, nitrogen blue four color of the total SOD activity was measured in.Western blot Bc1-2, Bax, HO-1 detection, cleaved expression level of caspase-3 protein.
Results compared with the control group, curcumin showed concentration dependent upregulation of HO-1mRNA expression, and the expression and activity of HO-1 protein increased. Compared with H202 group, curcumin can be a significant increase in cell survival rate, apoptosis rate and caspase-3 activity. At the same time, curcumin can significantly inhibit H202 induced bcl-2/bax and cleaved decreased caspase-3 protein expression increased. In addition, curcumin can inhibit the cell content of MDA induced by H202 and increase the total SOD activity decreased. The effect of curcumin can be partially reversed Znpp- IX.
Conclusion curcumin may increase the expression of HO-1 protein, up regulate HO-1 activity, maintain the redox balance of cells, and resist oxidative stress injury induced by H202 in H9c2 cardiomyocytes.
Third Shaozi
Mechanism of HO-1 mediated curcumin against oxidative stress injury in H9c2 cardiomyocytes
Objective to investigate the mechanism of HO-1 mediated curcumin against oxidative stress injury in H9c2 cardiomyocytes.
Methods 5-15 M curcumin treated myocardial cells of H9c2 12h or 15 M curcumin treated myocardial cells of H9c2 0.5-12h, Western blot detection of p-Akt, t-Akt protein expression levels. The establishment of oxidative stress in myocardial cells H9c2 injury model induced by H2O2 (1): Curcumin pretreatment, or inhibitor of PI3K LY294002 co incubated with Western blot method, the level of detection of cleaved caspase-3 and HO-1 protein expression. (2) curcumin pretreatment or HO-1 inhibitor.Znpp- IX co incubation, the expression level of Western blot was used to detect the NF- nuclear factor kappa B protein.
Results 5-15 M curcumin can become a concentration dependent increase induced by p-Akt/t-Akt ratio, 15 M curcumin H9c2 myocardial cells after 0.5-12h, p-Akt/t-Akt increased significantly in 30min, then decreased gradually. In H202 induced H9c2 oxidative stress injury of 2 new muscle cell model, the inhibitory effect of curcumin on expression of cleaved caspase-3 protein can be the P13K inhibitor LY294002 reversed. At the same time, LY294002 can significantly inhibit curcumin induced HO-1 protein expression level increased. In addition, curcumin pretreatment can significantly inhibit H202 induced NF- kappa B activation, which can be used as Znpp-IX partially reversed.
Conclusion curcumin may increase the expression of HO-1 protein through PI3K/Akt signal pathway, inhibit the activation of NF- kappa B induced by H202 and play the role of cell protection.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R541.1

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