番茄红素可改善血管紧张素Ⅱ诱导的H9c2细胞氧化应激

Lycopene attenuates angiotensin Ⅱ induced oxidative stress in H9c2 cells

目的 观察番茄红素（lycopene,Lyc）对血管紧张素Ⅱ（angiotensionⅡ,AngⅡ）诱导的H9c2细胞氧化应激的影响,并探讨其机制.方法 本研究为细胞实验,采用的是大鼠H9c2细胞系.实验将H9c2细胞分为6组,分别为空白对照组（对照组）、单加AngⅡ组（1.μmol/L）（AngⅡ组）、AngⅡ（1μmol/L） ＋Lyc （3.125 nmol/L）组（AngⅡ＋低剂量Lyc组）、AngⅡ（1μmol/L）＋Lyc（6.25nmol/L）组（AngⅡ＋中剂量Lyc组）、AngⅡ（1μmol/L）＋Lyc（12.5 nmol/L）组（AngⅡ＋高剂量Lyc组）和单加Lyc（12.5 nnmol/L）组（Lyc组）.采用不同因素干预H9c2细胞12h后,细胞毒性检测试剂盒（CCK-8）检测不同浓度Lyc和（或）AngⅡ对H9c2活性的影响,酶标仪及荧光显微镜检测Lyc对AngⅡ诱导细胞产生活性氧（ROS）的影响,实时荧光定量聚合酶链反应检测Lyc对AngⅡ诱导的细胞内还原型辅酶Ⅱ（NADPH）氧化酶2（NOX2）、NADPH氧化酶p47^phox亚基（p47^phox）、超氧化物歧化酶1（SODl）及超氧化物歧化酶2（SOD2）基因表达的影响,细胞丙二醛（MDA）检测试剂盒检测MDA的变化.结果 （1）各组H9c2细胞活力的检测结果：AngⅡ组细胞生存率明显低于对照组（P＜0.01）.而AngⅡ＋低剂量Lyc组、AngⅡ＋中剂量Lyc组和AngⅡ＋高剂量Lyc组H9c2细胞生存率均明显高于AngⅡ组（P均＜0.01）,且呈浓度依赖性.（2）各组H9c2细胞中ROS表达水平的检测结果：AngⅡ组H9c2细胞中ROS表达水平明显高于对照组（P＜0.01）.而AngⅡ＋低剂量Lyc组、AngⅡ＋中剂量Lyc组和AngⅡ＋高剂量Lyc组H9c2细胞中ROS表达水平均明显低于AngⅡ组（P均＜0.01）,且呈浓度依赖性.（3）各组H9c2细胞中SOD1、SOD2 mRNA表达水平及MDA含量的检测结果：AngⅡ组H9c2细胞中SOD1、SOD2 mRNA水平均明显低于对照组,而MDA含量明显高于对照组（P均＜0.01）.而AngⅡ＋低剂量Lyc组、AngⅡ＋中剂量Lyc组和AngⅡ＋高剂量Lyc组H9c2细胞中SOD1、SOD2 mRNA水平均明显高于AngⅡ组,MDA含量则均低于AngⅡ组（P均＜0.01）,且均呈浓度依赖性.（4）各组H9c2细胞中NOX2蛋白及其亚单位p47phox^mRNA表达水平的检测结果：AngⅡ组H9c2细胞中NOX2蛋白表达水平及其亚单位p47^phox的mRNA水平均明显高于对照组（P均＜0.01）,而AngⅡ＋低剂量Lyc组、AngⅡ＋中剂量Lyc组和AngⅡ＋高剂量Lyc组二者的表达水平均明显低于AngⅡ组（P均＜0.01）,且呈浓度依赖性.结论 Lyc可改善AngⅡ诱导的H9c2细胞氧化应激,其机制与Lyc可抑制AngⅡ诱导的H9c2细胞中ROS的生成,同时可促进细胞ROS的清除能力有关.

Objective To investigate the effect of Lycopene （Lyc） on Ang Ⅱ induced oxidative stress in H9c2 cell line derived from rat cardiac tissue,and to explore related mechanisms.Methods H9c2 cells were divided into 6 groups：control group,Ang Ⅱ group （1 μmol/L）,Ang Ⅱ （1 μmol/L） ＋ low dose Lyc （3.125 nmol/L） group,AngⅡ （1 μmol/L） ＋ moderate dose Lyc（6.25 nmol/L）group and Ang Ⅱ （1 μmol/L） ＋ high dose Lyc（12.5 nmol/L） group and Lyc group （12.5 nnmol/L）.Cell growth was determined by CCK8 assay,ROS generation was detected using a Microplate reader and Fluorescence microscopy,the expression of NOX2 was determined by Western blot,mRNA expression of p47phox,SOD1 and SOD2 were determined by Real Time-PCR,MDA was detected by ELISA kit.Results Compared to control group,cell survival was significantly reduced and ROS generation was significantly increased post Ang Ⅱ stimulation,cotreatment with Lyc significantly improved cell survival and reduced ROS generation in a dosedependent manner （all P 〈 0.01）.mRNA expression of SOD1 and SOD2 was significantly downregulated while MDA concentration was significantly increased in Ang Ⅱ treated cells,which could be significantly reversed by cotreatment with Lyc in a dose dependent manner （all P 〈 0.01）.Protein expression of NOX2 and mRNA expression of p47^phox were significantly upregulated post Ang Ⅱ and which could be significantly downregulated by cotreatment with Lyc in a dose-dependent manner （all P 〈 0.01）.Conclusion Lyc could attenuate Ang Ⅱ induced oxidative stress and this effect is linked with its capacity of reducing ROS generation and enhancing cellular ROS scavenging ability in H9c2 cells.