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柚皮素对缺氧损伤大鼠心肌细胞的保护作用
李磊,胡立群*,邹丽绢,赵鑫扬,彭晟,吴刚
0
(武汉市第四医院(华中科技大学同济医学院附属普爱医院)心内科, 武汉 430033
*通信作者)
摘要:
目的 探究柚皮素对缺氧损伤的大鼠心肌细胞是否有保护作用。方法 用大鼠心肌细胞系H9c2细胞制备心肌细胞缺氧损伤模型,并用乳酸脱氢酶(LDH)细胞毒性检测试剂盒进行模型鉴定。实验分为5组:对照组,模型组,以及柚皮素低、中、高剂量组(20、40、80 μmol/L)。用CCK-8检测细胞增殖抑制率,间接免疫荧光法检测细胞肥大状况,流式细胞术检测细胞凋亡水平,蛋白质印迹法检测凋亡相关蛋白Bcl-2、Bcl-2关联X蛋白(Bax)、caspase 3的表达水平,qRT-PCR检测血管内皮细胞生长因子(VEGF)、NK2型同源异形框基因5(Nkx2.5)、α-平滑肌肌动蛋白(α-SMA)的mRNA表达水平。结果 模型组细胞培养上清中LDH活性高于对照组(P<0.01),表明缺氧损伤心肌细胞建模成功。柚皮素各剂量组细胞增殖抑制率均低于模型组(P<0.05、P<0.01)。与模型组比较,柚皮素各剂量组H9c2心肌细胞肥大状况有所缓解(P<0.05、P<0.01)。柚皮素中、高剂量组凋亡细胞比例均较模型组降低(P均<0.01)。与模型组比较,柚皮素各剂量组凋亡蛋白caspase 3、Bax表达均降低,凋亡抑制蛋白Bcl-2表达均升高(P<0.05、P<0.01)。与模型组比较,柚皮素高剂量组Nkx2.5 mRNA表达水平升高(P<0.01),柚皮素中、高剂量组VEGF mRNA表达水平均升高(P<0.05、P<0.01),柚皮素各剂量组α-SMA mRNA表达水平均降低(P均<0.01)。结论 柚皮素对缺氧损伤的大鼠心肌细胞有保护作用。
关键词:  柚皮素  心肌细胞  细胞增殖  细胞凋亡  凋亡调节蛋白质类
DOI:10.16781/j.0258-879x.2021.06.0657
投稿时间:2020-03-17修订日期:2021-05-17
基金项目:2018年武汉市卫生健康委员会项目(WZ18Q10,WX18Q02).
Protective effect of naringenin on hypoxia-injured myocardial cells in rats
LI Lei,HU Li-qun*,ZOU Li-juan,ZHAO Xin-yang,PENG Sheng,WU Gang
(Department of Cardiology, Wuhan Fourth Hospital (Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology), Wuhan 430033, Hubei, China
*Corresponding author)
Abstract:
Objective To investigate the protective effect of naringenin on hypoxia-injured myocardial cells. Methods A hypoxia-injured myocardial cell model was established with rat myocardial cell line H9c2 cells and identified by lactate dehydrogenase (LDH) cytotoxicity test kit. The experiment was divided into 5 groups: control group, model group, and naringenin low-, medium- and high-dose groups (20, 40 and 80 μmol/L). Cell proliferation inhibition rate was detected by cell counting kit 8; cell hypertrophy was detected by indirect immunofluorescence; cell apoptosis was detected by flow cytometry; the protein expression of apoptosis-related proteins B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax) and caspase 3 was detected by Western blotting; and the mRNA expression of vascular endothelial growth factor (VEGF), NK2 homeobox 5 (Nkx2.5) and alpha-smooth muscle actin (α-SMA) in H9c2 cells was detected by qRT-PCR. Results Compared with the control group, the activity of LDH in cell supernatant of the model group was significantly increased (P<0.01), indicating that the hypoxia-injured myocardial cell model was successfully constructed. Compared with the model group, the inhibition rate of cell proliferation was significantly decreased in each dose group of naringenin (P<0.05, P<0.01); the hypertrophy of H9c2 cells in each dose group of naringenin was significantly alleviated (P<0.05, P<0.01); and the proportion of apoptotic cells was significantly decreased in the medium- and high-dose naringenin groups (both P<0.01). Compared with the model group, the expression levels of apoptotic proteins caspase 3 and Bax in each dose group of naringenin were significantly decreased, while the expression levels of apoptosis inhibition-related protein Bcl-2 were significantly increased (P<0.05, P<0.01). Compared with the model group, the mRNA expression level of Nkx2.5 in high-dose naringenin group was significantly increased (P<0.01), the mRNA expression levels of VEGF were significantly increased in medium- and high-dose naringenin groups (P<0.05, P<0.01), and the mRNA expression levels of α-SMA were significantly decreased in each dose group of naringenin (all P<0.01). Conclusion Naringenin has a protective effect on hypoxia-injured myocardial cells of rats.
Key words:  naringenin  cardiomyocytes  cell proliferation  apoptosis  apoptosis regulatory proteins