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中华细胞与干细胞杂志(电子版) ›› 2023, Vol. 13 ›› Issue (01) : 10 -18. doi: 10.3877/cma.j.issn.2095-1221.2023.01.002

论著

达格列净改善高糖诱导的人脐静脉内皮细胞功能的机制研究
罗丽芳1, 刘哲夫2, 董兵2, 刘晓玲2, 丘雨旻2, 周喆2, 何江2, 夏文豪2,()   
  1. 1. 510080 广州,广东药科大学附属第一医院皮肤科
    2. 510080 广州,中山大学附属第一医院高血压血管病科;510080 广州,国家卫生健康委员会辅助循环重点实验室;510080 广州,血管疾病诊断与治疗国家-广东省联合工程实验室
  • 收稿日期:2022-10-24 出版日期:2023-02-01
  • 通信作者: 夏文豪
  • 基金资助:
    国家自然科学基金(82270458); 国家重点研发计划项目(2020YFC2008005); 广州市重点研发项目(202206080004); 广东省省企联合基金(2021A1515220019); 广东省中医药管理局(20201196); 广东省医学科研基金(A2020339)

Mechanism of dapagliflozin improving the function of human umbilical endothelial cells induced by high glucose

Lifang Luo1, Zhefu Liu2, Bing Dong2, Xiaoling Liu2, Yumin Qiu2, Zhe Zhou2, Jiang He2, Wenhao Xia2,()   

  1. 1. Department of Dermatology, the First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou 510080, China
    2. Department of Hypertension and Vascular Disease, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China; Key Laboratory on Assisted Circulation Ministry of Health,Guangzhou 510080, China; Key Laboratory on Assisted Circulation Ministry of Health, Guangzhou 510080, China
  • Received:2022-10-24 Published:2023-02-01
  • Corresponding author: Wenhao Xia
引用本文:

罗丽芳, 刘哲夫, 董兵, 刘晓玲, 丘雨旻, 周喆, 何江, 夏文豪. 达格列净改善高糖诱导的人脐静脉内皮细胞功能的机制研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2023, 13(01): 10-18.

Lifang Luo, Zhefu Liu, Bing Dong, Xiaoling Liu, Yumin Qiu, Zhe Zhou, Jiang He, Wenhao Xia. Mechanism of dapagliflozin improving the function of human umbilical endothelial cells induced by high glucose[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2023, 13(01): 10-18.

目的

探讨达格列净对高糖诱导的人脐静脉内皮细胞(HUVECs)增殖、迁移、成管和凋亡的影响及其可能的分子机制。

方法

将HUVECs分别采用5.5 mmol/L (对照)、25 mmol/L葡萄糖(高糖)、达格列净和高糖+达格列净干预细胞,用脂质体转染法将pcDNA、pcDNA-JAK2转入HUVECs,后用达格列净和(或)高糖处理。采用CCK8法检测细胞增殖活性,Transwell实验检测细胞迁移能力,血管形成实验检测细胞成管能力,流式细胞术检测细胞凋亡率,Western blot检测JAK2、p-JAK2、STAT3和p-STAT3蛋白表达情况。采用析因设计方差分析或单因素方差分析进行组间比较。

结果

与对照比较,高糖干预细胞活力(0.37±0.05比0.69±0.07)、迁移数、管型形成数均降低,细胞凋亡率[(20.06±5.87)%比(1.95±0.56)%]、p-JAK2 (2.16±0.18比1.06±0.12)和p-STAT3均升高(P均< 0.05),达格列净干预可逆转高糖引起的上述变化。与pcDNA比较,pcDNA-JAK2的细胞活力(0.31±0.03比0.69± 0.10)、迁移数和管型形成数均降低,凋亡率[(20.59±2.81)%比(1.63±0.16)%]、p-JAK2(2.15±0.10比1.03±0.07)和p-STAT3蛋白表达均升高(P均< 0.05),过表达JAK2后高糖干预细胞活力、迁移数和管型形成数进一步降低,凋亡率、p-JAK2和p-STAT3蛋白表达进一步升高。与pcDNA-JAK2比较,pcDNA-JAK2+达格列净处理的细胞活力(0.65±0.06比0.31±0.03)、迁移数和管型形成数均升高,细胞凋亡率[(9.68±0.83)%比(20.59±2.81)%]、p-JAK2 (1.32±0.04比2.15±0.10)和p-STAT3蛋白的表达均降低(P均< 0.05)。与pcDNA-JAK2+高糖处理比较,pcDNA-JAK2+高糖+达格列净处理的HUVECs活力、体外迁移数量和管型形成数量升高,凋亡率、p-JAK2和p-STAT3蛋白表达水平均降低(P均< 0.05)。

结论

达格列净促进高糖诱导的HUVECs增殖、迁移和成管能力的恢复,抑制细胞凋亡,其机制可能与抑制JAK2/STAT3信号通路有关。

Objective

To investigate the effect of dapagliflozin on the function of human umbilical vein endothelial cells (HUVECs) induced by high glucose and its molecular mechanism.

Methods

HUVECs were treated with 5.5mmol/L glucose (ctrl), high glucose, high glucose+dapagliflozin. The cells were transfected with pcDNA or pcDNA-JAK2 by liposome-based transfection technology, and then treated with dapagliflozin and/or high glucose. Cell proliferation and apoptosis rate were detected by CCK8 and flow cytometry, respectively. CCK8, Transwell and Tube formation assay determined cell proliferation, migration and tube formation capacity. Proteins expression of JAK2/STAT3 was detected by western blotting. The differences between groups were compared by t-test and ANOVA compared the differences groups.

Results

Compared with the control, the high glucose-treated cells showed obviously lower cell proliferation (0.37±0.05 vs 0.69±0.07) , migration and tube formation. However, the apoptosis rate (20.06±5.87 vs 1.95±0.56) , p-JAK2 (2.16±0.18 vs 1.06±0.12) , and p-STAT3/STAT3 proteins expression were significantly higher, which can be reversed by dapagliflozin treatment. Compared with the pcDNA, the pcDNA-JAK2 significantly decreased-cell proliferation (0.31±0.03 vs 0.69±0.10) , migration and tube formation capacity, while the apoptosis rate (20.59±2.81 vs 1.63±0.16) , p-JAK2 (2.15±0.10 vs 1.03±0.07) and p-STAT3 proteins expression were significantly increased, and the difference was statistically significant (all P < 0.05) . Overexpression of JAK2 further inhibited cell proliferation, migration and tube formation in high glucose-induced cells, but upregulated apoptosis rate, protein levels of p-JAK2 and p-STAT3 (all P < 0.05) . Compared with the pcDNA-JAK2, the cell proliferation (0.65±0.06 vs 0.31±0.03) , migration and tube formation capacity of pcDNA-JAK2+ dapagliflozin-treated cells were significantly increased, while the apoptosis rate (9.68±0.83 vs 20.59±2.81) , p-JAK2 (1.32±0.04 vs 2.15±0.10) and p-STAT3 proteins expression were significantly decreased, and the difference was statistically significant (all P < 0.05) .Compared to JAK2+high glucose, the cell proliferation, migration and tube formation were increased in the cells treated with pcDNA+high glucose+dapagliflozin, wheras the apoptosis rate, p-JAK2 and p-STAT3 were decreased (all P < 0.05) .

Conclusion

Dapagliflozin restores diabetes-associated decline in cell proliferation, migration and tube formation of HUVECs, which may be related to the inhibition of JAK2/STAT3 signal path way.

图1 达格列净对高糖诱导的HUVECs凋亡率的影响注:a图为对照;b图为高糖;c图为达格列净;d图为高糖+达格列净
表1 达格列净对高糖诱导的HUVECs活力和凋亡的影响( ± s
图2 显微镜下观察达格列净对高糖诱导的HUVECs体外迁移能力和管型形成能力的影响(×200)注:a、e图为对照;b、f图为高糖;c、g图为达格列净;d、h图为高糖+达格列净;a~d为结晶紫染色检测各组内皮细胞迁移能力,并统计细胞迁移数量;e ~ h为Matrigel胶检测各组内皮细胞管型形成能力,并统计管型形成数量
表2 达格列净对高糖诱导的HUVECs体外功能的影响( ± s,个)
图3 各组HUVECs中JAK2/STAT3信号通路蛋白质表达
表3 达格列净对高糖诱导的HUVECs中JAK2/STAT3信号通路蛋白表达的影响( ± s
图4 达格列净对JAK2过表达诱导的HUVECs凋亡率的影响注:a图为pcDNA;b图为pcDNA+高糖;c图为pcDNA-JAK2;d图为pcDNA-JAK2+高糖;e图为pcDNA-JAK2+达格列净;f图为pcDNA-JAK2+高糖+达格列净
表4 达格列净对JAK2过表达诱导的HUVECs活力和凋亡的影响( ± s
图5 显微镜下观察达格列净对JAK2过表达诱导的HUVECs体外迁移能力的影响(结晶紫染色,×200)注:a图为pcDNA;b图为pcDNA+高糖;c图为pcDNA-JAK2;d图为pcDNA-JAK2+高糖;e图为pcDNA-JAK2+达格列净;f图为pcDNA-JAK2+高糖+达格列净;a ~ f为结晶紫染色检测内皮细胞迁移能力,并统计细胞迁移数量
图6 达格列净对JAK2过表达诱导的HUVECs体外管型形成能力的影响(×200)注:a图为pcDNA;b图为pcDNA+高糖;c图为pcDNA-JAK2;d图为pcDNA-JAK2+高糖;e图为pcDNA-JAK2+达格列净;f图为pcDNA-JAK2+高糖+达格列净;a ~ f为Matrigel胶检测内皮细胞管型形成能力,并统计管型形成数量
表5 达格列净对JAK2过表达诱导的HUVECs体外功能的影响( ± s,个)
图7 达格列净对JAK2过表达后HUVECs中JAK2/STAT3信号通路的影响注:1为pcDNA;2为pcDNA+高糖;3为pcDNA-JAK2;4为pcDNA-JAK2+高糖;5为pcDNA-JAK2+达格列净;6为pcDNA-JAK2+达格列净+高糖
表6 达格列净对JAK2过表达诱导的HUVECs中JAK2/STAT3信号通路表达的影响( ± s
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