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. 2017 Jul 20;37(7):895-901.
doi: 10.3969/j.issn.1673-4254.2017.07.07.

[Angiotensin-(1-7) protects cardiac myocytes against high glucose-induced injury by inhibiting ClC-3 chloride channels]

[Article in Chinese]
Shao-Ai Ccai  1 , Jing-Fu Chen, Mei-Ji Chen, Jian-Cong Lin, Jian-Qiang Feng, Kai Lin, Xi-Mei Zhi, Wei-Jie Zhang, Wen Wu
Affiliations

[Angiotensin-(1-7) protects cardiac myocytes against high glucose-induced injury by inhibiting ClC-3 chloride channels]

[Article in Chinese]
Shao-Ai Ccai et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To explore whether angiotensin-(1-7) [Ang-(1-7)] protects cardiac myocytes against high glucose (HG)-induced injury by inhibiting ClC-3 chloride channels.

Method: H9c2 cardiac cells were exposed to 35 mmol/L glucose for 24 h to establish a cell injury model. The cells were treated with Ang-(1-7) or the inhibitor of chloride channel (NPPB) in the presence of HG for 24 h to observe the changes in HG-induced cell injury. Cell counter kit 8 (CCK-8) assay was used to test the cell viability, and the morphological changes of the apoptotic cells were detected using Hoechst 33258 staining and fluorescent microscopy. The intracellular level of reactive oxygen species (ROS) was examined by DCFH-DA staining, SOD activity in the culture medium was measured using commercial kits, and the mitochondrial membrane potential (MMP) of the cells was tested with rodamine 123 staining. The expression level of cardiac ClC-3 chloride channels was detected with Western blotting.

Results: Exposure of H9c2 cardiac cells to 35 mmol/L glucose for 24 h markedly enhanced the expressions of cardiac ClC-3 channel protein (P<0.01). Co-treatment of the cells with 1 µmol/L Ang-(1-7) and HG for 24 h significantly attenuated HG- induced upregulation of ClC-3 channel protein expression (P<0.01). Co-treatment of the cells exposed to HG with 1 µmol/L Ang-(1-7) or 100 µmol/L NPPB for 24 h obviously ameliorated HG-induced injuries as shown by increased cell viability, enhanced SOD activity, decreased number of apoptotic cells, and reduced intracellular ROS generation and loss of MMP (P<0.01).

Conclusion: ClC-3 channels are involved in HG-induced injury in cardiac cells. Ang-(1-7) protects cardiac cells against HG-induced injury by inhibiting ClC-3 channels.

目的: 探讨血管紧张素-（1-7）[Ang-（1-7）]能否通过调控ClC-3通道保护心肌细胞对抗高糖引起的损伤。

方法: 应用35 mmol/L葡萄糖处理H9c2心肌细胞24 h建立损伤模型。Ang-（1-7）或氯通道抑制剂与心肌细胞共处理24 h观察对高糖诱发的心肌细胞损伤的影响。应用细胞计数试剂盒8检测细胞存活率；Hoechst33258染色荧光显微镜照相术检测凋亡细胞的形态学改变；双氯荧光素染色荧光显微镜照相术测定细胞内活性氧水平；超氧化物歧化酶试剂盒测定活性；罗丹明123染色荧光显微镜照相术检测线粒体膜电位；Western blot法测定心肌细胞ClC-3通道蛋白的表达水平。

结果: 35 mmol/L葡萄糖处理H9c2心肌细胞24 h明显地增加ClC-3通道蛋白的表达水平（P < 0.01）；1 μmol/LAng（-1-7）与葡萄糖共处理心肌细胞24 h显著地抑制葡萄糖对ClC-3通道蛋白表达的上调作用（P < 0.01）；1 μmol/LAng（-1-7）或100 μmol/L氯通道抑制剂氯通道抑制剂与葡萄糖共处理心肌细胞24 h减轻葡萄糖引起的损伤作用，表现为增加细胞存活率和超氧化物歧化酶活性，减小细胞凋亡数量，胞内活性氧水平及线粒体膜电位丢失（P < 0.01）。

结论: ClC-3通道参与葡萄糖引起的心肌细胞损伤；Ang-（1-7）通过抑制ClC-3通道保护心肌细胞对抗葡萄糖引起的损伤。

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Figures

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Ang-(1-7) 抑制高糖对H9c2心肌细胞ClC-3通道蛋白表达的上调作用 Angiotensin-(1-7) [Ang-(1-7)] attenuates high glucose(HG)-induced up-regulation of ClC-3 chloride channel protein in H9c2 cardiac cells. A: Cells were treated with 35 mmol/L glucose for 1-24 h; B: Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 24 h. **P < 0.01 vs control group; ‡P < 0.01 vs HG group (n=3).
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Ang-(1-7) 和氯通道抑制剂(NPPB)抑制高糖引起的心肌细胞毒性 Co-treatment of H9c2 cardiac cells for 24 h with 1 μmol/L Ang-(1-7) or 100 μmol/L NPPB, an inhibitor of chloride channel, reduces HG-induced cytotoxicity. **P < 0.01 vs control group; ‡P < 0.01 vs HG group (n=6).
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Ang-(1-7) 和氯通道抑制剂保护心肌细胞对抗高糖引起的凋亡 Ang-(1-7) and NPPB protect cardiac cells against HG-induced apoptosis. A-F: Morphological changes of apoptotic cells observed by fluorescent microscopy with Hoechst 33258 nuclear staining. G: Percentages of apoptotic cells in different groups analyzed using a cell counter and ImageJ 1.41 software. **P < 0.01 vs control group; ‡P < 0.01 vs HG group.
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Ang-(1-7) 和氯通道抑制剂保护心肌细胞对抗高糖对MMP的损伤作用 Ang-(1-7) and NPPB protect H9c2 cardiac cells against HG-induced dissipation of MMP. A-F: MMP assessed by using the fluorescent dye Rh123. G: Quantitative analysis of fluorescence intensity in different groups. **P < 0.01 vs control group; ‡ P < 0.01 vs HG group.
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Ang-(1-7) 和氯通道抑制剂保护心肌细胞对抗高糖引起的心肌细胞内ROS生成增多 Ang-(1-7) and NPPB protect H9c2 cardiac cells against HG-induced increase in reactive oxygen species (ROS) production. A-F: Intracellular ROS production level detected using the redox-sensitive fluorescent dye DCFH-DA. G: Quantitative analysis of ROS production in different groups. **P < 0.01 vs control group; ‡P < 0.01 vs HG group.
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Ang-(1-7) 和氯通道抑制剂阻断高糖对心肌细胞SOD活性的抑制作用 Ang-(1-7) and NPPB block the inhibitory effect of HG on SOD activity in H9c2 cardiac cells. **P < 0.01 vs control group; ‡P < 0.01 vs HG group.
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