大黄酚对大鼠肾上腺髓质嗜铬细胞瘤分化株细胞缺氧损伤的保护作用

摘要

目的 观察大黄酚对缺氧引起的大鼠肾上腺髓质嗜铬细胞瘤分化株(PC12)细胞损伤的保护作用. 方法 培养PC12细胞,采用自制的密闭三气培养箱建立缺氧所致的PC12细胞损伤模型,根据不同条件,分为对照组、模型组、大黄酚组(包括1μg/ml组和5μg/ml组).于缺氧处理前及处理后1、2、6、12、24h,每个时间点取6个复孔.采用四甲基偶氮唑盐法(MTT)检测PC 12细胞增殖活性,高倍显微镜下观察PC 12细胞核形态,测定各组上清液中超氧化物歧化酶(SOD)含量,免疫组化法测定各组线虫抗凋亡基因的人类同源基因表达蛋白(BCL-2)的表达,实时定量PCR测定p38丝裂原活化蛋白激酶(MAPK)和含半胱氨酸天冬氨酸蛋白酶3(Caspase-3)mRNA的表达. 结果 ①缺氧处理后12h,MTT法测定大黄酚1 μg/ml组的细胞活力高于模型组；缺氧处理24 h,大黄酚1μg/ml组和5 μg/ml组的细胞活力均高于模型组,差异均有统计学意义(P＜0.05).②从缺氧处理后1h开始,大黄酚1 μg/ml组和5μg/ml组的SOD值高于模型组,差异有统计学意义(P＜0.05).③从缺氧后2h开始,大黄酚1μg/ml组和5μg/ml的BCL-2阳性率均高于模型组.④从缺氧处理2h开始,模型组p38MAPK mRNA表达量高于大黄酚1μg/ml组和5μg/ml组,差异有统计学意义(P＜0.05).从缺氧处理6h开始,模型组Caspase-3mRNA表达量高于大黄酚1μg/ml组和5μg/ml组,差异有统计学意义(P＜0.05).⑤缺氧处理后12、24 h,模型组细胞的细胞核皱缩、形态不清,内见颗粒样物质形成增多；大黄酚组的细胞核形态较清楚,少见颗粒样物质形成. 结论 大黄酚可以减轻缺氧所致PC12细胞的损伤,对PC12细胞有保护作用.%Objective To observe the protective effect of chrysophanol on hypoxic induced cell injury of rat adrenal medulla pheochromocytoma cells (PC12). Methods PC12 cells were cultured. A model of hypoxic caused PC12 cell injury was induced by using self-made airtight 3 gas box. The rats were divided into control, model and chrysophanol ( 1 μg/mL or 5 μg/mL) groups according to the different conditions. The proliferation activities of PC12 cells were determined by the tetrazolium salt (MTT) assay at 1, 2, 6 , 12 , and 24 hours before and after hypoxia treatment. The nuclear morphology of PC 12 cells was observed under the oil microscope. Superoxide dismutase (SOD) content in the supernatant in each group was detected. Immunohistochemical methods were used to detect the human homologous protein ( Bcl-2) expression of the C. Elegans anti-apoptotic gene in each group. Real-time quantitative PCR was used to detect the expressions of mitogen-activated protein kinase (p38 MAPK) and cysteine aspartic protease 3 (caspase-3) mRNA. Results ① The cell viability in the chrysophanol 1 μg/mL group detect by MTTrnassay at 12 hours after hypoxia treatment was higher than that in the model group; the cell viability of chry-sophanol 1 μg/mL group and chrysophanol 5 μg/mL group at 24 hours after hypoxia was higher than that in the model group. There were significant differences (P < 0.05). ②Beginning from hypoxia treatment for 1 hour, the SOD value in chrysophanol 1 μg/mL and 5|μg/mL groups was higher than that in the model group. There were significant differences (P<0.05). ③Beginning from 2 hours after hypoxia, the Bcl-2 positive rate in the chrysophanol 1 (μg/mL and 5 μg/mL groups was higher than that in the model group. ④Beginning from 2 hours after hypoxia treatment, the p38MAPK mRNA expression in the model group was higher than that in the chrysophanol 1 μg/mL and 5 μg/mL groups, there were significant differences ( P < 0. 05 ) . Beginning from 6 hour after hypoxia treatment, the caspase-3 mRNA expression in the model group was higher than that in the chrysophanol 1 μg/mL and chrysophanol 5 μg/mL groups. There were significant differences (P < 0. 05).⑤At 12 and 24 hours after hypoxia treatment, the nucleus shrunk, the cell morphology was unclear, and the formation of granular materials increased and became white in the model group; the nuclear morphology was clearer in the chrysophanol 1 μg/mL and 5 μg/mL groups, the formation of granular materials was rare. Conclusion Chrysophanol may reduce the hypoxia-induced PC12 cell injury and have a protective effect on PC12 cells.