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摘要：
目的 评价番茄红素对缺氧/复氧（hypoxia/reoxygenation，H/R）引起的小鼠心肌细胞内质网应激和凋亡的影响。方法 建立C57BL/6乳鼠心肌细胞缺氧/复氧模型，采用随机数字表法将C57BL/6乳鼠心肌细胞随机分为正常对照组（C组）、番茄红素组（Lyc组，含5μmol/L番茄红素的培养基预处理4h）、缺氧复氧组（H/R组，缺氧4h复氧6h）和番茄红素+缺氧复氧组（Lyc+H/R组，给予5μmol/L番茄红素预处理4h后行H/R处理）。采用水溶性四氮唑-8（Cell Counting Kit-8, CCK-8）法检测心肌细胞存活率，倒置显微镜下观察细胞搏动频率，TUNEL法检测细胞凋亡率，二氯荧光素法 (dichlomnuorescein diacetate，DCFH-DA)检测细胞内活性氧(reactive oxygen species，ROS)含量，实时荧光定量PCR (Real-time PCR) 检测葡萄糖调节蛋白78（glucose-regulated protein 78, GRP78）、C/EBP同源蛋白（C/EBP homologous protein, CHOP）及半胱天冬氨酸蛋白酶-12 (cysteme aspartate specific protease-12, Caspase-12) mRNA的表达，免疫印记法(western blot)分析剪切的半胱天冬氨酸蛋白酶-12(cleaved cysteme aspartate specific protease-12, Cleaved-Caspase-12)和剪切的半胱天冬氨酸蛋白酶-3 (cleaved cysteme aspartate specific protease-3, Cleaved-Caspase-3)的表达。结果 与C组比较，H/R组心肌细胞存活率显著降低[(100.00±5.22)%，(68.80±5.70)%] (P＜0.01)，搏动频率显著降低[(94.4±6.2)min-1，(28.4±4.9)min-1] (P＜0.01)，心肌细胞凋亡率[(4.87±1.54)%，(25.62±2.61)%]和ROS含量[(100.00±10.55)%，(226.03±10.08)%]显著升高(P＜0.01)；CHOP mRNA [(1.00±0.10)，(2.60±0.19)] 和GRP78 mRNA [(1.00±0.18)，(4.12±0.23)]表达水平显著升高(P＜0.05)；Caspase-12 mRNA [(1.00±0.09)，(1.79±0.14)]、Cleaved-Caspase-12 [(1.00±0.08)，(1.85±0.10)]和Cleaved-Caspase-3 [(1.00±0.07)，(1.89±0.14) ] 表达水平显著升高(P＜0.05)。与H/R组比较，Lyc+H/R组心肌细胞存活率明显升高[(68.80±5.70)%，(84.47±6.72)%] (P＜0.05)，搏动频率增加[(28.4±4.9)min-1，(73.2±6.3)min-1](P＜0.05)，凋亡率显著降低[(25.62±2.61)%，(18.18±2.18)%] (P＜0.05)，细胞内ROS含量[(226.03±10.08)%，(139.57±15.65)%]明显降低，CHOP mRNA [(2.60±0.19)，(1.71±0.14)]和GRP78 mRNA [(4.12±0.23)，(1.98±0.19)] 表达水平显著降低(P＜0.05)；Caspase-12 mRNA [(1.79±0.14)，(1.38±0.11)]、Cleaved-Caspase-12 [(1.85±0.10)，(1.26±0.12) ]和Cleaved-Caspase-3 [(1.89±0.14)，(1.36±0.12)]表达水平显著降低(P＜0.05)。结论 番茄红素可抑制缺氧复氧过程中的内质网应激及其凋亡信号途径而减轻心肌细胞损伤。
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Abstract: Objective To evaluate whether the protective mechanism of lycopene is relative to inhibition of endoplasmic reticulum (ER) stress induced apoptosis in primary cultured neonatal mouse cardiomyocytes with hypoxia/reoxgenation(H/R). Methods Primary neonatal C57BL/6 cardiomyocytes were used to establish H/R model and divided by a random number method into control group (group C), lycopene group (Lyc, preconditioned with 5µmol/L lycopene for 4h), H/R group (4h hypoxia followed by 6h reoxgenation), lycopene+H/R group (Lyc+H/R, preconditioned with 5µmol/L lycopene for 4 h before H/R treatment). The cell viability was measured by CCK-8 assay, A Leica inverted microscope was used to observe the cardiomyocytes’ morphology and beating frequency. TUNEL assay was used to assess cardiomyocytes apoptosis. The generation of reactive oxygen species (ROS) was measured by dichlorofluorescein diacetate (DCFH-DA) method．The protein levels of cleaved-caspase-12 and cleaved-caspase-3 were assessed by Western blot. Real-time PCR was used to determine the mRNA expression of the GRP78, CHOP and caspase-12. Results Compared with group C, the cell viability [(100.00±5.22)%, (68.80±5.70)%] and beating frequency [(94.4±6.2)min-1, (28.4±4.9)min-1] were markedly decreased in H/R group(P＜0.01). The apoptotic rate [(4.87±1.54)%, (25.62±2.61)%] and the production of ROS [(100.00±10.55)%, (226.03±10.08)%] in H/R group were significantly increased compared with group C (P＜0.01). The mRNA levels of CHOP [(1.00±0.10), (2.60±0.19)], GRP78 [(1.00±0.18), (4.12±0.23)] and Caspase-12 [(1.00±0.09), (1.79±0.14)], the protein levels of Cleaved-Caspase-12[(1.00±0.08), (1.85±0.10)] and Cleaved-Caspase-3 [(1.00±0.07), (1.89±0.14) ] in H/R group were markedly increased compared with group C (P＜0.05). Compared with H/R group, the cell viability [(68.80±5.70)%, (84.47±6.72)%] and the beating frequency[(28.4±4.9)min-1, (73.2±6.3)min-1] in Lyc+H/R group were significantly increased(P＜0.05). The apoptotic rate of cardiomyocytes [(25.62±2.61)%, (18.18±2.18)%] and the generation of ROS [(226.03±10.08)%, (139.57±15.65)%] in Lyc+H/R group were markedly decreased (P＜0.05) compared with H/R group. The mRNA levels of CHOP [(2.60±0.19), (1.71±0.14)], GRP78 [(4.12±0.23), (1.98±0.19)] and Caspase-12 [(1.79±0.14), (1.38±0.11)] in Lyc+H/R group were significantly decreased compared with H/R group (P＜0.05). Compared with H/R group, the expression of Cleaved-Caspase-12 [(1.85±0.10), (1.26±0.12)] and Cleaved-Caspase-3 [(1.89±0.14), (1.36±0.12)] in Lyc+H/R group significantly decreased (P＜0.05). Conclusion Lycopene may protect the primary neonatal mouse cardiomyocytes against hypoxia/reoxygenation-injury through inhibiting the ER stress and the relative apoptotic pathway.
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