| 引用本文: | 潘桂萱,魏雪萍,孙维,吴友苹,张升,由振强.二甲双胍通过改善自噬功能障碍保护多柔比星诱导的心肌细胞损伤[J].中国现代应用药学,2022,39(19):2475-2482. |
| PAN Guixuan,WEI Xueping,SUN Wei,WU Youping,ZHANG Sheng,YOU Zhenqiang.Metformin Protects Cardiomyocyte from Injury Induced by Doxorubicin Via Repairing Autophagy Dysfunction[J].Chin J Mod Appl Pharm(中国现代应用药学),2022,39(19):2475-2482. |
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| 摘要: |
| 目的 探讨二甲双胍(metformin，Met)保护DOX诱导的心肌损伤的作用机制。方法 ①H9C2细胞经0.1~10 mmol·L-1Met预处理后加入1 μmol·L-1 DOX处理24 h，MTT法检测细胞存活率，Annexin V-PI染色后流式细胞技术检测细胞凋亡率，使用ROS试剂盒检测细胞内ROS蓄积情况；②使用Western blotting检测0.1，0.3，3 mmol·L-1 Met对DOX激活自噬水平的调节作用，并使用25 μmol·L-1 AG-126考察ERK信号通路调节DOX诱导心肌细胞损伤中的作用；③10 nmol·L-1 BafA1用于探索Met恢复DOX阻断心肌细胞自噬流的研究，流式细胞技术检测吖啶橙细胞内溶酶体pH的变化。结果 DOX(1 μmol·L-1)的接触会使H9C2细胞的存活率显著下降(P<0.05)，同时心肌细胞内ROS累积增加(P<0.05)，凋亡细胞比例升高(P<0.05)，不同浓度的Met可以有效缓解DOX升高的H9C2细胞内ROS和凋亡细胞比率(P<0.05)，从而增加心肌细胞存活率(P<0.05)。DOX(1 μmol·L-1)处理H9C2细胞6 h能够有效激活细胞自噬，损伤心肌细胞。通过AG-126干预可知，DOX诱导的心肌细胞自噬与ERK分子的激活相关，3 mmol·L-1 Met可以下调ERK的磷酸化水平(P<0.05)，从而降低DOX诱导的心肌细胞自噬。吖啶橙染色和Western blotting结果显示，与自噬流阻断剂BafA1处理的H9C2细胞比较，DOX同样增加H9C2细胞内溶酶体pH(P<0.05)，使心肌细胞自噬功能异常，增加细胞凋亡程度。结论 DOX损伤心肌细胞是通过上调自噬水平并诱发自噬功能障碍引起心肌细胞凋亡，Met下调ERK磷酸化水平，缓解DOX破坏的心肌细胞自噬障碍，保护心肌细胞免受DOX诱导的伤害。 |
| 关键词: 多柔比星 二甲双胍 自噬 溶酶体 |
| DOI：10.13748/j.cnki.issn1007-7693.2022.19.007 |
| 分类号:R285.5 |
| 基金项目:浙江省自然科学基金项目(LYY21H310004);浙江省卫生健康科技计划项目(2021KY634) |
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| Metformin Protects Cardiomyocyte from Injury Induced by Doxorubicin Via Repairing Autophagy Dysfunction |
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PAN Guixuan1, WEI Xueping1, SUN Wei1, WU Youping2, ZHANG Sheng1, YOU Zhenqiang1
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1.School of Food Science and Engineering, Hangzhou Medical College, Hangzhou 310013, China;2.The Children's Hospital, Zhejiang University School of Medicine, Hangzhou 310000, China
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| Abstract: |
| OBJECTIVE To investigate the mechanism of action of metformin (Met) to protectively against DOX-induced myocardial injury.METHODS ①H9C2 cells were pretreated with 0.1–10 mmol·L-1 concentration of Met,then treated with 1 μmol·L-1 Dox for 24 h.The cell survival was detected by MTT,the apoptosis rate was detected by flow cytometry after Annexin V-PI staining,and the intracellular ROS accumulation was examined using ROS kit;②The expression of autophagy-related proteins was detected by Western blotting to optimize the autophagy dose and concentration induced by DOX.Western blotting was also used to investigate the effects of 0.1,0.3 and 3 mmol·L-1 Met on the regulation of DOX-activated autophagy,and 25 μmol·L-1 AG-126 was used to investigate the role of ERK signaling pathway in regulating DOX-induced cardiomyocyte injury;③10 nmol·L-1 Bafilomycin A1(BafA1) was used to explore the study of Met restoration of DOX blocked-autophagic flux in cardiomyocyte,and flow cytometric detection of acridine orange intracellular lysosomal pH changes.RESULTS Exposure to 1 μmol·L-1 DOX resulted in a significant decrease in H9C2 cell survival (P<0.05),along with an increase in intracellular ROS accumulation (P<0.05) and an increase in the proportion of apoptotic cells (P<0.05),and different concentrations of Met effectively alleviated the DOX-elevated intracellular ROS and apoptotic cell ratios in H9C2 cells (P<0.05),thereby increasing cardiomyocyte survival.The treatment of H9C2 cells with 1 μmol·L-1 DOX for 6 h was effective in activating autophagy and damaging cardiomyocyte.As shown by AG-126 intervention,DOX-induced cardiomyocyte autophagy was associated with activation of ERK molecules,and 3 mmol·L-1 Met could down-regulate the phosphorylation level of ERK (P<0.05),then decreasing DOX-induced cardiomyocyte autophagy.Acridine orange staining and Western blotting results showed that DOX similarly increased intracellular lysosomal pH in H9C2 cells compared with autophagic flux blocker BafA1 used (P<0.05),caused an abnormal cardiomyocyte autophagy and increased the degree of apoptosis.CONCLUSION DOX damages cardiomyocyte by upregulating autophagy levels and inducing autophagy dysfunction causing apoptosis,Met downregulates ERK phosphorylation levels,alleviates DOX-damaged cardiomyocyte autophagy impairment,and protects cardiomyocyte from DOX-induced injury. |
| Key words: doxorubicin metformin autophagy lysosomal |
