本文選題：血紅素加氧酶-1　切入點：H9c2心肌細胞　出處：《華中科技大學》2012年博士論文　論文類型：學位論文

【摘要】：近年來,先天性心臟病的救治正向著復雜、危重和幼小化的方向發(fā)展,心肌缺血再灌注損傷已成為困擾臨床療效的重要問題。氧化應激誘導的心肌細胞凋亡是心肌缺血再灌注損傷發(fā)生發(fā)展的重要病理基礎。氧化應激是指由于活性氧過量生成和／或細胞內(nèi)抗氧化防御系統(tǒng)受損,導致超氧陰離子(O2-)、過氧化氫(hydrogen peroxide, H2O2)等氧自由基及其相關代謝產(chǎn)物過量聚集,從而對細胞產(chǎn)生多種毒性作用的病理狀態(tài)。大量研究表明,抑制氧化應激誘導的心肌細胞凋亡是減輕心肌缺血再灌注損傷的有效途徑。 血紅素加氧酶-1(heme oxygenase-1, HO-1)是血紅素降解的起始酶和限速酶,能夠降解血紅素生成一氧化碳(carbon monoxide, CO)、Fe2+和膽紅素(bilirubin)。研究表明,在多種心血管疾病如心肌梗死、心肌缺血再灌注損傷和心力衰竭的研究中,采用藥理學方法或基因方法使HO-1蛋白表達增加可抑制心肌細胞損傷。姜黃素是從姜科植物姜黃根莖中提取的一種酚性色素,具有抑制腫瘤、抗炎以及抗氧化等多種生物學效應。已有研究證實,在體外培養(yǎng)的內(nèi)皮細胞和成纖維細胞中,姜黃素能夠成濃度依賴性的誘導HO-1蛋白表達及活性增加,進而發(fā)揮細胞保護作用。因此,我們推測姜黃素可能對H9c2心肌細胞氧化應激損傷具有保護效應,且該效應與HO-1密切相關。 Akt(protein kinase B, PKB,又稱Akt)是磷脂酰肌醇3激酶(phosphatidylinositol-3kinase, PI3K)的下游信號調(diào)節(jié)激酶之一,在多種物質(zhì)介導的抗凋亡效應中發(fā)揮重要作用。在多種細胞損傷模型中,PI3K/Akt信號通路介導的HO-1蛋白上調(diào)能夠使細胞對抗多種應激刺激。核因子NF-κB是一個多效能的核轉(zhuǎn)錄因子,在不同的細胞模型中可表現(xiàn)為凋亡抑制或凋亡促進作用。 本實驗旨在通過建立H202誘導的H9c2心肌細胞氧化應激損傷模型,在細胞水平上探討姜黃素的心血管保護作用以及HO-1介導姜黃素對抗H9c2心肌細胞氧化應激損傷的機制。 第一部分 HO-1對H9c2心肌細胞氧化應激損傷的保護作用研究 目的探討HO-1對H9c2心肌細胞氧化應激損傷的保護作用及其機制。 方法建立H202誘導的H9c2心肌細胞氧化應激損傷模型,給予HO-1誘導劑hemin預處理,或給予HO-1抑制劑Znpp-IX共孵育。雙波長法檢測HO-1活性；四甲基偶氮唑藍(MTT)法檢測細胞存活率；Hoechst33342染色和caspase-3活性檢測評估細胞凋亡；硫代巴比妥酸顯色法檢測MDA含量,氮藍四唑顯色法檢測總SOD活性；westernblot法檢測NF-κB蛋白表達水平。 結(jié)果與對照組相比,hemin成濃度依賴性的誘導HO-1活性增加。與H202組相比,HO-1活性增加能夠顯著下調(diào)細胞凋亡率和caspase-3活性,降低細胞MDA含量,增加總SOD活性,且上述作用能被Znpp-IX逆轉(zhuǎn)。此外,HO-1活性增加能夠顯著抑制H202誘導的NF-κB激活,且該作用能被Znpp-IX逆轉(zhuǎn)。 結(jié)論Hemin誘導的HO-1活性增加可能通過抑制NF-κB激活,維持細胞氧化還原平衡狀態(tài),抑制H202誘導的H9c2心肌細胞氧化應激損傷。 第二部分HO-1介導姜黃素對抗H9c2心肌細胞氧化應激損傷 目的探討姜黃素對H9c2心肌細胞氧化應激損傷的保護效應以及HO-1在該效應中的作用。 方法建立H202誘導的H9c2心肌細胞氧化應激損傷模型,給予姜黃素預處理,或給予HO-1抑制劑Znpp-Ⅸ共孵育。雙波長法檢測HO-1活性；MTT法檢測細胞存活率；流式細胞術Annexin-Ⅴ FITC/PI染色和caspase-3活性檢測評估細胞凋亡；硫代巴比妥酸顯色法檢測MDA含量,氮藍四唑顯色法檢測總SOD活性。western blot檢測HO-1,Bc1-2,Bax, cleaved caspase-3蛋白表達水平。 結(jié)果與對照組相比,姜黃素呈濃度依賴性的上調(diào)HO-1mRNA表達,同時上調(diào)HO-1蛋白表達和活性。與H202組相比,姜黃素能夠顯著上調(diào)細胞存活率,下調(diào)細胞凋亡率和caspase-3活性。同時,姜黃素能夠顯著抑制H202誘導的bcl-2/bax比例下降以及cleaved caspase-3蛋白表達增加。此外,姜黃素能夠顯著抑制H202誘導的細胞MDA含量增加以及總SOD活性下降。姜黃素的上述作用均能被Znpp-Ⅸ部分逆轉(zhuǎn)。 結(jié)論姜黃素可能通過增加HO-1蛋白表達,上調(diào)HO-1活性,維持細胞氧化還原平衡狀態(tài),對抗H202誘導的H9c2心肌細胞氧化應激損傷。 第三邵分 HO-1介導姜黃素對抗H9c2心肌細胞氧化應激損傷的機制研究 目的探討HO-1介導姜黃素對抗H9c2心肌細胞氧化應激損傷的機制。 方法5-15μM姜黃素處理H9c2心肌細胞12h或15μM姜黃素處理H9c2心肌細胞0.5-12h, western blot檢測p-Akt, t-Akt蛋白表達水平。建立H2O2誘導的H9c2心肌細胞氧化應激損傷模型：(1)給予姜黃素預處理,或給予PI3K抑制劑LY294002共孵育,western blot法檢測cleaved caspase-3和HO-1蛋白表達水平。(2)給予姜黃素預處理,或給予HO-1抑制劑.Znpp-Ⅸ共孵育,western blot法檢測核因子NF-κB蛋白表達水平。 結(jié)果5-15μM姜黃素可成濃度依賴性的誘導p-Akt/t-Akt比例增加,15μM姜黃素處理H9c2心肌細胞0.5-12h后,p-Akt/t-Akt比例在30min內(nèi)顯著上升,隨后逐漸下降。在H202誘導的H9c2新2肌細胞氧化應激損傷模型中,姜黃素對cleaved caspase-3蛋白表達的抑制效應能夠被P13K抑制劑LY294002逆轉(zhuǎn)。同時,LY294002能夠顯著抑制姜黃素誘導的HO-1蛋白表達水平上調(diào)。此外,姜黃素預處理能夠顯著抑制H202誘導的NF-κB激活,且該作用能被Znpp-IX部分逆轉(zhuǎn)。 結(jié)論姜黃素可能通過PI3K/Akt信號途徑上調(diào)HO-1蛋白表達,抑制H202誘導的NF-κB激活,發(fā)揮細胞保護作用。
[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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R541.1

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本文編號：1620781