山萘酚和芒柄花黄素对缺氧条件下H9c2心肌细胞内活性氧水平的影响及其机制研究

发布时间：2018-01-01 01:18

  本文关键词：山萘酚和芒柄花黄素对缺氧条件下H9c2心肌细胞内活性氧水平的影响及其机制研究　出处：《北京中医药大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 活性氧 芒柄花黄素 NAPDH氧化酶 山萘酚

【摘要】：心力衰竭是多种心血管疾病的终末阶段,发病率高,死亡率高,预后差,成为世界医学难题。氧化应激是心衰的主要病理环节。前期研究发现,芪参颗粒能够有效的抗氧化应激,调控NADPH氧化酶活性,从而改善心功能。山萘酚和芒柄花黄素是其调控NADPH氧化酶、抗氧化应激的潜在效应成分。研究目的从氧化应激入手,采用体外培养的H9c2心肌细胞系,揭示山萘酚和芒柄花黄素改善心功能、降低活性氧水平的主要作用环节。为研发新的、可用于临床心力衰竭防治的、低毒的、高效的NADPH氧化酶抑制剂提供可用的实验依据。研究方法与内容1MTT法检测细胞活性:体外培养H9c2心肌细胞,以细胞密度1×105个/ml接种于细胞培养板中,适应性培养至孔底80%-90%时,通过加入不同浓度梯度(10-4-10-8M)的山萘酚和芒柄花黄素,比较对细胞生长活性的影响;同样的方法比较同时给药和预给药的不同。细胞缺氧造模,即正常组不变,其余组均转入Earle's平衡盐溶液中,并放入缺氧小室中培养,确定最佳造模时间,确定山萘酚和芒柄花黄素的有效作用浓度。2 DCFH-DA法观察活性氧水平:选取缺氧造模的H9c2细胞,用DCFH-DA进行染色。倒置荧光显微镜随机选取5个不重复区域进行拍片记录。比较各实验组之间的差别。3流式细胞仪检测细胞内活性氧水平:选取缺氧造模的H9c2细胞,用0.05%胰蛋白酶消化细胞,10μM DCFH-DA重悬细胞进行染色。清洗后,用200μl PBS缓冲液重悬细胞,200目-400目尼龙网过滤,上机检测。4 Real time PCR 检测 NADPH(nicotinamide adenine dinucleotide phosphate)氧化酶相关亚基Nox4、p67phox、p47phox、p22phox的mRNA变化:收取造模完成的H9c2细胞,Trizol裂解,提取RNA进行反转录,得到cDNA,实时荧光定量PCR仪上检测基因含量。5Western blot检测NADPH氧化酶相关亚基Nox4、p67phox、p47phox、p22phox的蛋白表达量的变化:RIPA裂解液裂解并用细胞刮刀收取造模完成的H9c2细胞,超声裂解提取蛋白,进行电泳、电转、显色等操作,对蛋白条带进行拍照分析。研究结果1MTT实验结果显示:与正常组相比,给予山萘酚和芒柄花黄素10-5M及以上的浓度对细胞的生长有抑制作用(P0.01);预给药24h的细胞生长状态优于同时给药组,二组细胞的生长活性之间有统计学差异(P0.05);缺氧造模随着时间的增长,细胞生长状态受到抑制,正常组与缺氧造模8h、12h、24h及其各组之间的都有显著的统计学差异(P0.01);相同的缺氧造模时间,不同给药浓度作用于细胞,确定山萘酚和芒柄花黄素浓度在10-6M对细胞的生长状态最有益。2DCFH-DA法观察活性氧水平结果显示:H9c2细胞经缺氧造模8h后,倒置荧光显微镜下观察到模型组细胞内有高强度绿色荧光,胞质内部黑颗粒增多,胞体四周卷曲变亮,细胞状态较正常组有明显改变,细胞数量明显减少;经山萘酚和芒柄花黄素预给药处理的细胞与模型组相比有明显好转,由此可见山萘酚和芒柄花黄素对心肌细胞有一定的保护作用,能够改善H9c2心肌细胞的生长状态,增加细胞存活率和贴壁程度。3流式细胞仪结果显示:与正常组相比,模型组荧光强度显著增强,有显著的统计学差异(P0.01)。与模型组相比,山萘酚组、芒柄花黄素组、阳性药夹竹桃组荧光强度显著降低(P0.01),说明山萘酚、芒柄花黄素能降低细胞内活性氧水平。4 Real time PCR结果显示:H9c2细胞经缺氧造模4h,与正常组相比,模型组的NOX4、p67phox、p47phoxmRNA 显著增加(P0.01),经山萘酚干预后,NOX4、p67phox的mRNA的量较模型组分别下降了 33%(P0.05)、70%(P0.01),经芒柄花黄素干预后,NOX4、p67phox的mRNA的量较模型组分别下降了 13%(P0.05)、69%(P0.01),说明山萘酚和芒柄花黄素能够下调NOX4、p67phox的mRNA的表达量。另外一个时间点,经缺氧造模8h,与正常组相比,模型组的NOX4、p67phox、p47phoxmRNA 显著增加(P0.01),经山萘酚干预后,NOX4、p67phox、p47phox 的mRNA 的量较模型组分别下降了 80%(P0.01)、53%(P0.01)、45%(P0.05),经芒柄花黄素干预后,NOX4、p67phox、p47phox的mRNA的量较模型组分别下降了 80%(P0.01)、18%(P0.05)、55%(P0.05),说明山萘酚和芒柄花黄素能够下调 NOX4、p67phox、p47phox 的 mRNA 的表达量。5 Western blot结果显示:经缺氧造模4h,与正常组相比,模型组的p67phox、p47phox蛋白表达量显著增加(P0.01),经山萘酚干预后,NOX4、p67phox、p47phox的蛋白量较模型组分别下降了 21%(P0.05)、19%(P0.05)、26%(P0.05),经芒柄花黄素干预后,NOX4、p67phox的蛋白量较模型组分别下降了 9%(P0.05)、55%(P0.01),说明山萘酚和芒柄花黄素能够下调NOX4、p67phox的蛋白表达量。另一个时间点,经缺氧造模8h,与正常组相比,模型组的NOX4、p67phox、p47phox蛋白表达量显著增加(P0.01),经山萘酚干预后,NOX4、p67phox、p47phox的蛋白量较模型组分别下降了 32%(P0.05)、22%(P0.05)、30%(P0.05),经芒柄花黄素干预后,NOX4、p67phox、p47phox的蛋白量较模型组分别下降了 35%(P0.01)、65%(P0.01)、24%(P0.05),说明山萘酚和芒柄花黄素能够下调NOX4、p67phox、p47phox的蛋白表达量。结论经缺氧诱导的H9c2心肌细胞细胞活力下降,细胞内活性氧水平显著升高,缺氧8h时表现最明显。山萘酚、芒柄花黄素浓度为10-6M,预给药24h处理,可明显改善细胞的生长和增殖状态,可以明显降低细胞内活性氧水平;山萘酚可以下调Nox4、p67phox、p47phox的mRNA和蛋白水平,芒柄花黄素能够下调p67phox、p47phox的mRNA,明显下调Nox4、p67phox蛋白的水平。结果提示山萘酚和芒柄花黄素可降低细胞内活性氧水平,抗氧化应激,保护心肌细胞,其作用机制与下调Nox4、p67phox、p47phox的表达水平相关。本研究为临床调控氧化应激治疗心力衰竭提供了实验依据和新的思路。
[Abstract]:Heart failure is the end stage of many cardiovascular diseases, high incidence, high mortality and poor prognosis, become the world medical problem. Oxidative stress is the main pathological process of heart failure. The preliminary study found that Shenqi granule can effectively regulate oxidative stress, NADPH oxidase activity, so as to improve the cardiac function of kaempferol and Ononis. The regulation of NADPH oxidase flavin, potential effects of oxidative stress. Objective to study components from oxidative stress by H9c2 of myocardial cells were cultured in vitro, revealing the kaempferol and formononetin improve heart function, the main role low level of reactive oxygen species. For the development of new, can be used for clinical prevention and treatment of heart failure. Low toxicity, and provide experimental evidence available NADPH oxidase inhibitor. Efficient cell activity detection research method and content 1MTT method: H9c2 myocardial cells were cultured in vitro, the cell density of 1 * 105 / Ml cells seeded in culture plate, adaptability cultivation to the bottom of hole 80%-90%, by adding different concentration gradient (10-4-10-8M) of kaempferol and formononetin, compare the effects on cell growth activity; the same method is also administered and pre administration. Different cell hypoxia model, normal group constant, the other groups were transferred into Earle's balanced salt solution, and placed in the hypoxic chamber culture, determine the best modeling time, determine the level of reactive oxygen species observed effective concentrations of.2 DCFH-DA kaempferol and formononetin: H9c2 cells from hypoxia model, using DCFH-DA staining. Fluorescence microscope randomly selected 5 do not repeat regional shooting record. The comparison between the experimental group and the difference of.3 flow cytometry was used to detect intracellular ROS levels: select hypoxia model of H9c2 cells with 0.05% trypsin digestion cells, 10 M DCFH-DA heavy suspension The cells were stained. After cleaning, the cells with 200 L PBS buffer suspension, 200 -400 mesh nylon net filter, the detection of.4 Real time (nicotinamide adenine dinucleotide NADPH PCR detection phosphate) oxidase related subunit Nox4, p67phox, p47phox, mRNA, p22phox changes: charge modeling of H9c2 cells, Trizol cracking, the extraction of RNA by reverse transcription, cDNA, real time fluorescent quantitative PCR detection blot detection.5Western gene content related NADPH oxidase subunit Nox4, p67phox, p47phox, the changes of the expression of p22phox protein: RIPA lysate and cell lysis for modeling the scraper H9c2 cells, sonication extraction protein. By electrophoresis, electroporation, color and other operations, with a picture of protein analysis. The results showed that 1MTT test results: compared with normal group, kaempferol and formononetin was given yellow 10-5M and above concentration on Cell growth inhibition (P0.01); pre administration of 24h cell growth state is superior to the drug group at the same time, there were significant differences between the growth activity of cells in two groups (P0.05); hypoxia model with time increasing, the cell growth was inhibited, the normal group and the hypoxia model of 8h, 12h, 24h and each group had statistically significant difference (P0.01); the same time hypoxia model, different concentration on the cell, kaempferol and formononetin in concentrations of 10-6M on cell growth was most beneficial results.2DCFH-DA method to observe the level of reactive oxygen species determined: H9c2 cells were treated with hypoxia model after 8h. Cells were observed under inverted fluorescence microscope in the model group with high intensity of green fluorescence, cytoplasmic internal black particles increased, the cell body curled around the light, cells compared with normal group changed obviously, the number of cells was significantly reduced; the kaempferol and Ononis Emodin pretreatment treated cells compared with the model group has significantly improved, thus kaempferol and formononetin has protective effects on myocardial cells, the growth state of H9c2 can improve the myocardial cells, increase cell viability and attachment of.3 flow cytometry results showed: compared with normal group, model the fluorescence intensity of group increased significantly, there was statistically significant difference (P0.01). Compared with the model group, kaempferol group, formononetin group, positive drug group significantly reduced oleander fluorescence intensity (P0.01), shows that kaempferol, Ononis Huang Suneng reduced the level of ROS were.4 Real time PCR showed that H9c2 cells were treated with hypoxia model of 4h, compared with the normal group, model group, NOX4, p67phox, p47phoxmRNA increased significantly (P0.01), by kaempferol intervention, NOX4, p67phox mRNA were compared with the model group decreased by 33% (P0.05), 70% (P0.01), by Ononis 鑺遍粍绱犲共棰勫悗,NOX4,p67phox鐨刴RNA鐨勯噺杈冩ā鍨嬬粍鍒嗗埆涓嬮檷浜，

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