银杏叶提取物对热应激致鸡心肌细胞氧化损伤的保护作用研究

本试验旨在研究银杏叶提取物（Ginko biloba extract，GBE）对热应激致鸡心肌细胞氧化损伤的保护作用。取12日龄AA肉鸡心脏制备鸡原代心肌细胞，向原代心肌细胞培养板中分别加入0、5、10、50、100、150 mg/mL GBE，CCK-8试剂盒检测心肌细胞存活率；将原代心肌细胞以1×10⁴个/孔接种于96孔板，培养48 h，分别加入0、5、10、50、100、200、500、1 000 μg/mL GBE，Hoechst 33258检测心肌细胞凋亡率，确定最适GBE浓度。42℃热应激处理（0、0.5、1、2和4 h）建立心肌细胞损伤模型，ELISA试剂盒检测乳酸脱氢酶（LDH）、细胞丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH-Px）含量，以及超氧化物歧化酶（SOD）活性；Western blotting检测Hsp72蛋白表达。结果显示，高浓度GBE具有细胞毒性，50、100、150 mg/mL GBE能极显著抑制细胞生长（P<0.01），50 μg/mL GBE能够极显著提高细胞增殖率（P<0.01）。热应激后细胞发生凋亡，细胞内MDA、LDH含量增加，SOD活性、GSH-Px含量减少，产生氧化损伤；GBE可以降低热应激时细胞LDH和MDA的含量，增加SOD活性和GSH-Px的含量，热应激2、4 h，GBE处理效果明显。此外，GBE可增加心肌细胞中Hsp72蛋白的表达。综上所述，GBE能降低热应激状态下心肌细胞的氧化损伤，其机制可能与提高抗氧化酶活性及提高Hsp72的表达有关。

Protective Effects of Ginkgo biloba Extract on Oxidative Damage Induced by Heat Stress in Cardiomyocytes of Chicken

The study was conducted to study the protective effect of Ginko biloba extract (GBE) on oxidative damage in cardiomyocytes of chicken induced by heat stress. The primary cardiomyocytes of 12-day-old AA broiler were prepared,0,5,10,50,100,150 mg/mL GBE were added and the cell vability was tested by CKK-8 method.And then the cardiomyocytes were inoculated and cultured for 48 h in 96-well plates,0,5,10,50,100,200,500 and 1 000 μg/mL GBE were added and the cell apoptosis were studied by Hoechst 33258 method to identify the proper concentration of GBE.The chicken cardiomyocyte heat stress model was established at 42℃ for 0,0.5,1,2 and 4 h,respectively. The LDH,GSH-Px,MDA and SOD were measured by ELISA method.Western blotting was used to detect the expression of Hsp72 protein.The results showed that high concentration of GBE was cytotoxic and 50,100,150 mg/mL GBE could extremely significantly inhibit cell growth (P<0.01),and cell proliferation rate was extremely significantly increased by 50 μg/mL GBE (P<0.01).Heat stress could induce cell apoptosis and oxidative damage through increasing the levels of MDA and LDH and reducing the GSH-Px level and SOD activity. GBE could reduce the levels of LDH and MDA and increase the SOD activity and GSH-Px level after heat stress,and the effect was more obvious when heat stress for 2 and 4 h. Furthermore,Western blotting results showed that GBE enhanced the expression of Hsp72.In conclusion,GBE could protect cardiomyocytes from oxidative damage induced by heat stress by increasing activities of antioxidative enzymes and Hsp72 expression.