表没食子儿茶素没食子酸酯通过抑制心肌细胞凋亡缓解心肌缺血再灌注损伤的机制研究
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篇名: 表没食子儿茶素没食子酸酯通过抑制心肌细胞凋亡缓解心肌缺血再灌注损伤的机制研究
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摘要: 目的:探讨表没食子儿茶素没食子酸酯(EGCG)对心肌缺血再灌注损伤的保护作用及其可能机制。方法:以叔丁基过氧化氢(TBHP)处理H9C2心肌细胞构建缺血再灌注细胞模型,采用MTS法考察经不同剂量(3.125、6.25、12.5、25、50、100、200 μmol/L)EGCG预处理后细胞的存活情况,并计算细胞存活率;采用Western blotting法检测经不同剂量(100、200 μmol/L)EGCG预处理后细胞中凋亡蛋白(Bcl-2、Bax)的表达情况。将雄性C57BL/6小鼠随机分为假手术组、模型组和EGCG组(5 mg/g),每组15只。假手术组和模型组小鼠均灌胃等体积生理盐水,EGCG组小鼠灌胃相应药物,每日1次,连续7 d。末次给药12 h后,采用前降支结扎法复制心肌缺血再灌注损伤小鼠模型。采用伊文思蓝和TTC双染色法观察各组小鼠的心肌梗死面积,并计算梗死面积占横截面积百分比,采用WST-1法检测其血清超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,采用Western blotting法检测其心肌组织中凋亡蛋白的表达(Bcl-2、Bax)以及通路相关蛋白[磷脂酰肌醇3激酶(PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)]的磷酸化水平。结果:细胞试验结果显示,与对照组比较,模型组细胞存活率、Bcl-2的相对表达量均显著降低,Bax的相对表达量显著升高(P<0.05);与模型组比较,25、50、100、200 μmol/L EGCG组细胞存活率以及100、200 μmol/L EGCG组细胞Bcl-2的相对表达量均显著升高,100、200 μmol/L EGCG组细胞Bax的相对表达量均显著降低(P<0.05)。动物实验结果显示,假手术组小鼠未见心肌组织缺血、心腔扩大等现象;模型组小鼠可见明显的心肌梗死现象,其梗死面积占横截面积百分比、血清MDA含量、心肌组织Bax的相对表达量和p-PI3K/PI3K、p-Akt/Akt值均较假手术组显著升高,SOD活性和Bcl-2的相对表达量均较假手术组显著降低(P<0.05);与模型组比较,EGCG组小鼠心肌梗死面积有所缩小,其梗死面积占横截面积百分比、血清MDA含量、心肌组织Bax的相对表达量和p-PI3K/PI3K、p-Akt/Akt值均显著降低,SOD活性和Bcl-2的相对表达量均显著升高(P<0.05)。结论:EGCG对心肌缺血再灌注损伤具有一定的保护作用,这种作用可能与抑制心肌细胞凋亡、改善机体氧化应激状态、调控凋亡蛋白表达、降低PI3K/Akt通路相关蛋白的磷酸化水平有关。
ABSTRACT: OBJECTIVE: To investigate the protective effect and potential mechanism of epigallocatechin gallate (EGCG) on myocardial ischemia-reperfusion injury. METHODS: H9C2 cardiomyocytes were treated with tert-butyl hydroperoxide (TBHP) to establish ischemia-reperfusion cell model. The cell viability was measured by MTS after pretreated with different doses of EGCG (3.125, 6.25, 12.5, 25, 50, 100, 200 μmol/L), and the survival rate was calculated. The expression of apoptotic proteins (Bcl-2, Bax) in cardiomyocytes pretreated with different doses of EGCG (100, 200 μmol/L) were detected by Western blotting. Male C57BL/6 mice were randomly divided into sham operation group, model group and EGCG group (5 mg/g), with 15 mice in each group. Sham operation group and model group were given constant volume of normal saline intragastrically, while EGCG group was given relevant medicine intragastrically, once a day, for consecutive 7 d. Twelve hours after last medication, myocardial ischemia-reperfusion injury model was established by anterior descending coronary artery ligation. The area of myocardial infarction was observed by double staining of Evan’s blue and TTC; the percentage of infarction area to cross-sectional area was calculated;SOD activity and MDA content in serum were determined by WST-1 assay; the expression of apoptotic proteins (Bcl-2, Bax) in myocardial tissue were detected by Western blotting, while the phosphorylation levels of signaling pathway related proteins (PI3K, p-PI3K, Akt, p-Akt) were also detected. RESULTS: Cell test results showed that, compared with control group, survival rate and relative expression of Bcl-2 were decreased significantly in model group, while relative expression of Bax was increased significantly (P<0.05). Compared with model group, survival rate of cardiomyocyte in 25, 50, 100, 200 μmol/L EGCG groups as well as relative expression of Bcl-2 in 100, 200  μmol/L EGCG groups were increased significantly, while relative expression of Bax in 100, 200 μmol/L EGCG groups were decreased significantly (P<0.05). Animal experiments showed that no ischemia of myocardial tissue and enlargement of cardiac cavity were observed in sham operation group. Myocardial infarction was observed in model group. Compared with sham operation group, percentage of infarction area to cross-sectional area, the serum content of MDA, the relative expression of Bax in myocardial tissue and p-PI3K/PI3K, p-Akt/Akt were increased significantly in model group, while SOD activity and relative expression of Bcl-2 were decreased significantly (P<0.05). Compared with model group, myocardial infarction area of mice in EGCG group was reduced, the percentage of infarction area to cross-sectional area, the serum content of MDA, the relative expression of Bax in myocardial tissue and p-PI3K/PI3K, p-Akt/Akt were significantly decreased, the activity of SOD activity and the relative expression of Bcl-2 were increased significantly (P<0.05). CONCLUSIONS: EGCG can protect against myocardial ischemia-reperfusion injury, the mechanism of which may be associated with inhibiting the apoptosis of myocardial cells, improving oxidation stress, regulating the expression of apoptotic protein, reducing the phosphorylation level of PI3K/Akt signaling pathway-related proteins.
期刊: 2019年第30卷第16期
作者: 符武岛,曾敏,陈娟,冯光球,管频,钟春荣
AUTHORS: FU Wudao,ZENG Min,CHEN Juan,FENG Guangqiu,GUAN Pin,ZHONG Chunrong
关键字: 表没食子儿茶素没食子酸酯;心肌缺血再灌注损伤;凋亡;磷脂酰肌醇3激酶/蛋白激酶B通路;H9C2心肌细胞;C57BL/6小鼠
KEYWORDS: Epigallocatechin gallate; Myocardial ischemia-reperfusion injury; Apoptosis;PI3K/AKT signaling pathway; H9C2 cell; C57BL/6 mice
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