抑制糖毒性通路对细胞线粒体功能障碍的影响和潜在意义

doi:10.3877/cma.j.issn.2095-5820.2023.02.001

目的

探讨不同糖毒性抑制途径对细胞线粒体的影响，为糖尿病血管并发症提供新的治疗策略。

方法

采用高糖刺激人脐静脉内皮细胞（HUVEC），分别在高糖刺激前后加入葡萄糖毒性途径抑制剂依帕司他、氮杂丝氨酸、氨基胍、索他霉素来抑制多元醇途径、己糖胺途径、晚期糖基化终末产物（AGE）和蛋白激酶C（PKC）通路。使用流式细胞仪分析测定HUVEC线粒体活性氧（ROS）水平，激光共聚焦显微镜观察线粒体分裂情况。

结果

采用高糖刺激，能明显升高HUVEC线粒体ROS水平及促进线粒体分裂；在高糖培养后，加入葡萄糖毒性途径抑制剂，细胞线粒体ROS水平和线粒体分裂无明显降低；然而在高葡萄糖刺激前，预先加入葡萄糖毒性途径抑制剂，能有效降低HUVEC中线粒体ROS水平和线粒体分裂情况。

结论

高糖刺激细胞线粒体分裂和ROS增加后，即使抑制糖毒性途径，也无法有效缓解细胞线粒体损伤。但是，当提前抑制糖毒性途径，细胞线粒体损伤可得到有效缓解。明确不同糖毒性抑制方式对细胞线粒体的影响，有望为糖尿病血管并发症的有效治疗提供更佳的治疗策略。

关键词: 内皮细胞, 活性氧, 糖尿病, 高血糖

Objective

To explore the effects of various glycotoxicity pathway inhibitors on cellular mitochondria, and provide better therapeutic strategy for the effective treatment of diabetic vascular complications.

Methods

In this study, we inhibited polyol, hexosamine, AGE and PKC pathway with epalrestat, azaserine, aminoguanidine, sotrastaurin, respectively in human umbilical vein endothelial cell (HUVEC) before and after high glucose treatment. The mitochondria ROS was determined by flow cytometry and mitochondrial division was observed by confocal laser microscopy.

Results

The mitochondrial ROS and fragmentation of HUVEC were significantly increased by high glucose stimulation. After high glucose incubation, the mitochondrial ROS level and fragmentation did not decrease significantly. However, pre-addition of glucose-toxic pathway inhibitors before hyperglucose stimulation can effectively reduce mitochondrial ROS levels and mitochondrial division in HUVEC.

Conclusions

Inhibition of the glucotoxic pathway after high glucose stimulation cannot effectively alleviate mitochondrial damage. When the glycotoxic pathway is inhibited in advance, mitochondrial damage can be effectively alleviated. It is expected to provide better therapeutic strategies for the effective treatment of diabetic vascular complications by clarifying the effects of various glucotoxic pathway inhibitors on cellular mitochondria.

Key words: endothelial cell, reactive oxygen species, diabetes mellitus, hyperglycemia

图1 HG诱导HUVEC线粒体ROS产生增加注：1A. 流式细胞术观察细胞MitoSOX；1B. 3组MitoSOX阳性细胞的百分比和线粒体平均荧光强度统计图，与HG组比较，^aP＜0.05；1C. Mito Tracker荧光探针着染线粒体，激光共聚焦显微镜观察线粒体形态改变；标尺为20 μmol/L。

图2 HG刺激后，加入依帕司他、氮杂丝氨酸、氨基胍和索曲霉素对HUVEC线粒体ROS生成的影响注：2A. 流式细胞术观察细胞MitoSOX；2B. 各组PE MitoSOX阳性细胞的百分比和线粒体平均荧光强度统计图，与NG组比较，^aP＜0.05；2C. Mito Tracker荧光探针着染线粒体，激光共聚焦显微镜观察线粒体形态改变；标尺为20 μmol/L。

图3 HG刺激前，加入依帕司他、氮杂丝氨酸、氨基胍和索他霉素对HUVEC线粒体ROS生成的影响注：3A. 流式细胞术观察细胞MitoSOX；3B. 各组PE MitoSOX阳性细胞的百分比和线粒体平均荧光强度统计图，与NG组比较，^aP＜0.05；3C. Mito Tracker荧光探针着染线粒体，激光共聚焦显微镜观察线粒体形态改变；标尺为20 μmol/L。

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The effect and implication of inhibiting glycotoxicity pathway on mitochondrial dysfunction

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The effect and implication of inhibiting glycotoxicity pathway on mitochondrial dysfunction