目的观察番茄红素(1yc)对缺氧复氧(H/R)引起的小鼠心肌细胞内质网应激及细胞凋亡的影响。方法C57BL/6乳鼠心肌细胞培养72h后随机数字表法分为4组,即正常对照组(对照组)、lyc组(lyc5ixmoL/L预处理)、H/R组(缺氧4h,复氧6h)和lyc+H/R组(1yc5μmol/L预处理4h后H/R处理)。采用CCK-8法检测心肌细胞存活率,AnnexinV—PI检测心肌细胞凋亡率,实时荧光定量聚合酶链反应检测转录激活因子6(ATF6)、真核细胞起始因子2α(eIF2α)和剪切X盒结合蛋白1(sXbp-1)mRNA的表达水平。蛋白印迹法检测葡萄糖调节蛋白78(GRP78)、C/EBP同源蛋白(CHOP)、B细胞淋巴瘤基因-2(Bcl-2)和Bel-2相关X蛋白(Bax)蛋白的表达水平。氧自由基(ROS)试剂盒检测细胞内ROS含量。结果H/R组乳鼠心肌细胞存活率明显低于对照组[(64.28±6.12)%比(100.00±4.98)%,P〈0.01],心肌细胞凋亡率明显高于对照组[(24.42±1.76)%比(5.16±1.31)%,P〈0.01],Bax/Bc]-2蛋白表达水平比值明显高于对照组(2.33±0.20比1.00±0.09,P〈0.01),CHOP蛋白表达水平明显高于对照组(1.98±0.15比1.00±0.12,P〈0.01),GRP78蛋白表达水平明显高于对照组(2.09±0.11比1.00±0.09,P〈0.05),ATF6、eIF2α和sXbp-1mRNA表达水平均明显高于对照组(P均〈0.01),心肌细胞内ROS含量明显高于对照组[(262.13±22.03)%比(100.00±12.35)%,P〈0.01]。而lyc+H/R组乳鼠心肌细胞存活率明显高于H/R组[(81.75±6.85)%比(64.28±6.12)%,P〈0.01],细胞凋亡率明显低于H/R组[(17.24±2.02)%比(24.42±1.76)%,P〈0.01],Bax/Bel-2蛋白表达水平比值明显低于H/R组(1.64±0.13比2.33±0.20,P〈0.01),CHOP蛋白表达水平明显低于H/R组(1.54±0.12比1.98±0.15,P〈0.
Objective To investigate the effects of lycopene on primary cultured neonatal mouse caMiomyocytes with hypoxia/reoxgenation (H/R) injury and explore related mechanism. Methods Primary cultured neonatal mouse cardiomyocytes were randomly divided to control group (control) ; lycopene group (5 μmol/L, lyc) ; H/R group (4 hours hypoxia followed by 6 hours reoxgenation) ; lycopene + H/R group (lyc + H/R, the cardiomyocytes were incubated with 5 μmol/L lycopene for 4 hours before H/R treatment). The cell viability of cardiomyocytes was assessed by CCK-g assay. The apoptotic rate ofcardiomyocytes was evaluated by flow cytometry using AnnexinV-PI double staining. Western blot was used to determine the GRP78, CHOP, Bax and Bcl-2 protein expression in cardiomyocytes. The mRNA expressions of ATF6, eIF2α and sXbp-1 were detected by real-time PCR. The fluorescence intensity for reactive oxygen species (ROS) in cardiomyocytes was measured with Olympus fluorescence microscope. Results Compared to control group, the cell viability of cardiomyocytes was significantly reduced ((64.28±6. 12)% vs. (100.00 ±4.98)%, P〈0.01), the apoptotic rate ((24.42 ± 1.76)% vs. (5.16 ± 1.31)%, P〈0.01) and ratio of Bax/Bcl-2 (2.33 ±0.20 vs. 1.00 ±0.09, P〈0.01) significantly increased, the ATF6, elF2α and sXbp-1 mRNA expression, the CHOP and GRP78 protein expression ( 1.98 ±0. 15 vs. 1.00 ±0. 12, 2. 09 ±0. 11 vs. 1.00 ±0. 09) as well as fluorescence intensity for ROS ( (262. 13 ±22. 03)% vs. ( 100. 00 ± 12. 35)% ) were markedly increased inH/R group (all P 〈 0. 01 ). Compared to the I-I/R group, pretreatment with lyeopene markedly improved the cell viability of cardiomyoeytes ( ( 81.75 ± 6. 85 ) % , P 〈 0.01 ) , significantly decreased the apoptotic rate ( ( 17. 24 ± 2. 02) % , P 〈 0. 01 ) and ratio of Bax/Bcl-2 ( 1.64 ± 0. 13, P 〈 0. 01 ), significantly down-regulated the mRNA expression levels of ATF6, elF2α and sXbp-1,