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中华危重症医学杂志(电子版)

中华危重症医学杂志(电子版) ›› 2020, Vol. 13 ›› Issue (03) : 170 -175. doi: 10.3877/cma.j.issn.1674-6880.2020.03.003

所属专题： 文献；

论著

麝香通心滴丸对心肌细胞缺氧/复氧损伤的保护作用
刘新文1, 沈男男1, 王佳良1, 傅永平2,†()   
  1. 1. 312000　浙江绍兴，绍兴文理学院附属医院（绍兴市立医院）药剂科
    2. 312000　浙江绍兴，绍兴文理学院附属医院（绍兴市立医院）心血管内科
  • 收稿日期:2020-04-21 出版日期:2020-06-01
  • 通信作者: 傅永平
  • 基金资助:
    浙江省自然科学基金项目(LY18H020008); 浙江省医药卫生科技计划项目(2018KY842、2019RC297); 绍兴市科技计划项目(2018C30031)

Protective effect of shexiang tongxin dropping pills on anoxia-reoxygenation injury in H9c2 cardiomyocytes

Xinwen Liu1, Nannan Shen1, Jialiang Wang1, Yongping Fu2,†()   

  1. 1. Department of Pharmacy, the Affiliated Hospital of Shaoxing University (Shaoxing Municipal Hospital), Shaoxing 312000, China
    2. Department of Cardiovascular Medicine, the Affiliated Hospital of Shaoxing University (Shaoxing Municipal Hospital), Shaoxing 312000, China
  • Received:2020-04-21 Published:2020-06-01
  • Corresponding author: Yongping Fu
  • About author:
    Corresponding author: Fu Yongping, Email:
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引用本文：

刘新文, 沈男男, 王佳良, 傅永平. 麝香通心滴丸对心肌细胞缺氧/复氧损伤的保护作用[J]. 中华危重症医学杂志(电子版), 2020, 13(03): 170-175.

Xinwen Liu, Nannan Shen, Jialiang Wang, Yongping Fu. Protective effect of shexiang tongxin dropping pills on anoxia-reoxygenation injury in H9c2 cardiomyocytes[J]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2020, 13(03): 170-175.

目的

探讨麝香通心滴丸（STDP）对心肌细胞缺氧/复氧的保护作用。

方法

建立心肌缺氧/复氧模型。将H9c2细胞分为5组：对照组（细胞正常培养，不做任何处理）、模型组、STDP组（在复氧前40 min给予STDP 50 mg / L）、3-甲基腺嘌呤（3-MA）组（在复氧前40 min给予3-MA 5 mmol / L）及STDP + 3-MA组（在复氧前40 min分别给予STDP 50 mg / L及3-MA 5 mmol / L）。采用细胞计数试剂盒8（CCK-8）法检测各组细胞存活率，并测定各组细胞乳酸脱氢酶（LDH）、丙二醛及肌酸激酶水平。采用流式细胞仪检测各组细胞的细胞凋亡率，实时荧光定量PCR检测信使RNA（mRNA）自噬相关基因Beclin-1、Atg5的表达水平，并通过Western-blotting检测各组细胞自噬相关蛋白LC3Ⅱ / LC3Ⅰ、低氧诱导因子1α（HIF-1α）、Bcl-2 /腺病毒E1B 19 kDa相互作用蛋白3（BNIP3）、Beclin-1、Atg5的表达水平。

结果

各组细胞存活率比较，差异有统计学意义（F = 85.893，P = 0.028），且STDP组细胞存活率较模型组、3-MA组及STDP + 3-MA组细胞存活率均显著升高[（90 ± 7）%、（63 ± 5）%、（80 ± 5）%、（81 ± 6）%，P均< 0.05]。各组细胞LDH、丙二醛、肌酸激酶水平，细胞凋亡率，Beclin-1 mRNA、Atg5 mRNA以及LC3Ⅱ / LC3Ⅰ、HIF-1α、BNIP3、Beclin-1、Atg5蛋白表达水平的比较，差异均有统计学意义（F = 78.381、23.519、48.376、22.726、6.315、5.294、75.219、116.546、21.125、39.724、65.247，P均< 0.05）。进一步两两比较发现，在STDP组细胞中，LDH[（92 ± 6）、（194 ± 13）、（195 ± 11）、（192 ± 10）μmol / L，P均< 0.05]、丙二醛[（12.5 ± 0.7）、（17.2 ± 1.2）、（18.5 ± 1.6）、（17.9 ± 1.0）μmol / L，P均< 0.05]、肌酸激酶[（19.9 ± 1.0）、（34.3 ± 2.2）、（35.3 ± 2.2）、（34.8 ± 2.5）μmol / L，P均< 0.05]水平，细胞凋亡率[（5.8 ± 0.8）%、（8.8 ± 0.9）%、（7.6 ± 0.7）%、（7.3 ± 0.5）%，P均< 0.05]较模型组、3-MA组及STDP + 3-MA组均明显降低；Beclin-1 mRNA、Atg5 mRNA、LC3Ⅱ / LC3Ⅰ、BNIP3、Beclin-1、Atg5蛋白表达水平较模型组均明显降低，而较3-MA组及STDP + 3-MA组均显著升高（P均< 0.05）；HIF-1α蛋白表达水平较模型组、3-MA组及STDP + 3-MA组均明显升高（P均< 0.05）。

结论

STDP可通过调控HIF-1α / BNIP3信号通路发挥对心肌细胞缺氧/复氧损伤的保护作用。

关键词: 肌细胞，心脏, 缺氧, 麝香通心滴丸
Objective

To study the protective effect of shexiang tongxin dropping pills (STDP) on anoxia-reoxygenation injury in H9c2 cardiomyocytes.

Methods

A model of myocardial anoxia-reoxygenation was established and the H9c2 cardiomyocytes were divided into 5 groups: a control group (with normal H9c2 cardiomyocytes), a model group, a STDP group (given 50 mg / L STDP 40 min before reoxygenation), a 3-methyladenine (3-MA) group (given 5 mmol / L 3-MA 40 min before reoxygenation), and a STDP + 3-MA group (given 50 mg / L STDP + 5 mmol / L 3-MA 40 min before reoxygenation). The cell survival rate was detected by cell counting kit-8, and the levels of lactate dehydrogenase (LDH), malonaldehyde, and creatine kinase were compared among the five groups. The apoptosis rate of cardiomyocytes was detected by flow cytometry. The messenger RNA (mRNA) expression levels of Beclin-1 and Atg5 were examined by real-time fluorescent quantitative PCR. The protein expression levels of LC3Ⅱ / LC3Ⅰ, hypoxia-inducible factor-1alpha (HIF-1α), Bcl-2 / adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), Beclin-1, and Atg5 were detected by Western-blotting.

Results

The cell survival rate was significantly different among the five groups (F = 85.893, P = 0.028), and it was much higher in the STDP group than in the model group, 3-MA group and STDP + 3-MA group [(90 ± 7)%, (63 ± 5)%, (80 ± 5)%, (81 ± 6)%; all P < 0.05]. The LDH, malonaldehyde, creatine kinase, apoptosis rate, Beclin-1 mRNA, Atg5 mRNA, LC3Ⅱ / LC3Ⅰ protein, HIF-1α protein, BNIP3 protein, and Beclin-1 protein were significantly different among the five groups (F = 78.381, 23.519, 48.376, 22.726, 6.315, 5.294, 75.219, 116.546, 21.125, 39.724, 65.247; all P < 0.05). In the STDP group, the levels of LDH [(92 ± 6), (194 ± 13), (195 ± 11), (192 ± 10) μmol / L], malonaldehyde [(12.5 ± 0.7), (17.2 ± 1.2), (18.5 ± 1.6), (17.9 ± 1.0) μmol / L], and creatine kinase [(19.9 ± 1.0), (34.3 ± 2.2), (35.3 ± 2.2), (34.8 ± 2.5) μmol / L] and the apoptosis rate of cardiomyocytes [(5.8 ± 0.8)%, (8.8 ± 0.9)%, (7.6 ± 0.7)%, (7.3 ± 0.5)%] were much lower than those in the model group, 3-MA group and STDP + 3-MA group, and the HIF-1α protein level was much higher (all P < 0.05); meanwhile, the levels of Beclin-1 mRNA, Atg5 mRNA, LC3Ⅱ / LC3Ⅰ protein, BNIP3 protein, Beclin-1 protein, and Atg5 protein were much lower than those in the model group, but were much higher than those in the 3-MA group and STDP + 3-MA group (all P < 0.05).

Conclusion

The STDP can protect cardiomyocytes from anoxia-reoxygenation injury by regulating the HIF-1α / BNIP3 signaling pathway.

Key words: Myocytes, cardiac, Anoxia, Shexiang tongxin dropping pill
表1 各组H9c2细胞LDH、丙二醛及肌酸激酶水平的比较（μmol / L，±s）
组别 细胞数（个/ mL） LDH 丙二醛 肌酸激酶
对照组 1 × 106 64 ± 6 5.0 ± 0.6 14.7 ± 1.1
模型组 1 × 106 194 ± 13ab 17.2 ± 1.2ab 34.3 ± 2.2ab
STDP组 1 × 106 92 ± 6 12.5 ± 0.7 19.9 ± 1.0
3-MA组 1 × 106 195 ± 11b 18.5 ± 1.6b 35.3 ± 2.2b
STDP + 3-MA组 1 × 106 192 ± 10b 17.9 ± 1.0b 34.8 ± 2.5b
F值 ? 78.381 23.519 48.376
P值 ? 0.021 0.019 0.036
表2 各组H9c2细胞凋亡率的比较（%，±s）
组别 细胞数（个/ mL） 细胞凋亡率
对照组 1 × 106 3.6 ± 0.4
模型组 1 × 106 8.8 ± 0.9ab
STDP组 1 × 106 5.8 ± 0.8
3-MA组 1 × 106 7.6 ± 0.7b
STDP + 3-MA组 1 × 106 7.3 ± 0.5b
F值 ? 22.726
P值 ? 0.025
表3 各组H9c2细胞间Beclin-1、Atg5 mRNA表达水平的比较（±s）
组别 细胞数（个/ mL） Beclin-1 mRNA Atg5 mRNA
对照组 1 × 106 1.05 ± 0.04 1.031 ± 0.011
模型组 1 × 106 1.90 ± 0.06ab 1.698 ± 0.028ab
STDP组 1 × 106 1.37 ± 0.05 1.325 ± 0.016
3-MA组 1 × 106 1.17 ± 0.04b 1.181 ± 0.034b
STDP + 3-MA组 1 × 106 1.10 ± 0.04b 1.191 ± 0.027b
F值 ? 6.315 5.294
P值 ? 0.023 0.031
图1 各组H9c2细胞LC3Ⅱ / LC3Ⅰ、HIF-1α、BNIP3、Beclin-1、Atg5蛋白表达水平的比较
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