蕨麻多酚抗缺氧诱导的大鼠心肌细胞凋亡作用研究

Study on the Inhibitory Effect of Potentilla Anserina L.Polyphenols on the Apoptosis Induced by Hypoxia in Primary Cardiomyocytes of Rats

目的:探究蕨麻多酚在缺氧条件下对心肌细胞的抗凋亡作用。方法:取新生1~3 d SD大鼠心肌细胞进行原代培养,随机分成5组:正常对照组、缺氧损伤模型组、蕨麻多酚低(0.11 mg·ml^-1)、中(0.22 mg·ml^-1)、高(0.44 mg·ml^-1)浓度组。细胞在5%CO2-95%N2环境下37℃培养6 h,建立缺氧损伤模型;流式细胞术检测细胞凋亡率;Hoechst-PI荧光染色法观察缺氧损伤后心肌细胞凋亡情况;逆转录-聚合酶链式反应(RT-PCR)检测各组细胞p53、B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bax)mRNA的表达水平;蛋白质印迹法(Western Blotting)检测各组细胞p53、Bcl-2、Bax蛋白的表达水平。结果:与正常对照组相比,缺氧损伤模型组细胞凋亡率显著升高(P<0.01),Hoechst-PI染色可见呈亮蓝色荧光的早期凋亡细胞及大量亮红色荧光的晚期凋亡或死亡细胞;p53、Bax mRNA及蛋白表达显著升高(P<0.01),Bcl-2 mRNA及蛋白表达显著下降(P<0.01);蕨麻多酚干预后细胞较缺氧损伤模型组凋亡率显著降低(P<0.01),Hoechst-PI染色亮红色荧光的晚期凋亡或死亡细胞数量减少。蕨麻多酚各浓度组p53、Bax mRNA及蛋白表达均显著降低(P<0.01),Bcl-2 mRNA及蛋白表达均显著升高(P<0.01)。结论:缺氧时蕨麻多酚能够通过下调心肌细胞p53和Bax水平,上调Bcl-2表达,抑制心肌细胞凋亡,从而减轻缺氧导致的心肌损伤。

Objective:To investigate the anti-apoptotic effect of Potentilla anserina L.polyphenols on cardiomyocytes under hypoxic conditions.Methods:The primary cardiomyocytes of newborn SD rats in 1-3 days were randomly divided into five groups:the normal control group,hypoxia injury model group,and three groups of Potentilla anserina L.polyphenols(0.44,0.22 and 0.11 mg·ml^-1).The hypoxia injury model of cardiomyocytes was established by culturing the cells in an incubator with 5%CO2-95%N2at 37℃for 6 h.Flow cytometry was used to detect the cell apoptosis rate.Hoechst-PI fluorescence staining was used to observe the cell morphological changes in each group after hypoxia.RT-PCR was used to detect B-cell lymphoma-2(Bcl-2),Bcl2-associated X Protein(Bax)and p53 mRNA in each group.The expressions of Bcl-2,Bax and p53 protein in each group were detected by Western blotting.Results:Compared with that of the normal control group,the apoptosis rate of the hypoxia injury model group was significantly increased(P<0.01).Hoechst-PI staining showed early apoptotic cells with bright blue fluorescence,and a large number of late red fluorescence with late fluorescence dead or dead cells;p53,Bax mRNA and protein expressions increased significantly(P<0.01),Bcl-2 mRNA and protein expressions decreased significantly(P<0.01);the apoptosis rate was significantly reduced after the treatment with Potentilla anserina L.polyphenols when comapred with that of hypoxia injury model group(P<0.01),and the number of late apoptotic or dead cardiomyocytes with Hoechst-PI staining and bright red fluorescence decreased.The expressions of p53,Bax mRNA and protein were significantly reduced(P<0.01),and the expressions of Bcl-2 mRNA and protein were significantly increased(P<0.01).Conclusion:Potentilla anserina L.Polyphenols can inhibit the hypoxia-induced cardiomyocytes apoptosis by down-regulating the expressions of p53 and Bax,and up-regulating the expression of Bcl-2 in primary cardiomyocytes of neonatal rats.