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。

1	PANENI F, BECKMAN J A, CREAGER M A, et al. Diabetes and vascular disease: Pathophysiology, clinical consequences, and medical therapy: Part i[J]. European heart journal, 2013, 34: 2436-2443.
2	BROWNLEE M. The pathobiology of diabetic complications: A unifying mechanism[J]. Diabetes, 2005, 54: 1615-1625.
3	RAMASAMY R, GOLDBERG I J. Aldose reductase and cardiovascular diseases, creating human-like diabetic complications in an experimental model[J]. Circulation research, 2010, 106: 1449-1458.
4	ZHANG W, LIU J, TIAN L, et al. Trib3 mediates glucose-induced insulin resistance via a mechanism that requires the hexosamine biosynthetic pathway[J]. Diabetes, 2013, 62: 4192-4200.
5	SUGIYAMA S, MIYATA T, HORIE K, et al. Advanced glycation end-products in diabetic nephropathy[J]. Nephrology dialysis transplantation, 1996, 11 Suppl 5: 91-94.
6	GIACCO F, BROWNLEE M. Oxidative stress and diabetic complications[J]. Circulation research, 2010, 107: 1058-1070.
7	WADA J, NAKATSUKA A. Mitochondrial dynamics and mitochondrial dysfunction in diabetes[J]. Acta medica okayama, 2016, 70: 151-158.
8	REN L, HAN F, XUAN L, et al. Clusterin ameliorates endothelial dysfunction in diabetes by suppressing mitochondrial fragmentation[J]. Free radical biology and medicine, 2019, 145: 357-373.
9	YAMA K, SATO K, ABE N, et al. Epalrestat increases glutathione, thioredoxin, and heme oxygenase-1 by stimulating nrf2 pathway in endothelial cells[J]. Redox biology, 2015, 4: 87-96.
10	DRYGALSKI K, FERENIEC E, ZALEWSKA A, et al. Phloroglucinol prevents albumin glycation as well as diminishes ros production, glycooxidative damage, nitrosative stress and inflammation in hepatocytes treated with high glucose[J]. Biomedicine and pharmacotherapy=Biomédecine and Pharmacothérapie, 2021, 142: 111958.
11	BHATTACHARYYA S, FEFERMAN L, TOBACMAN J K. Distinct effects of carrageenan and high-fat consumption on the mechanisms of insulin resistance in nonobese and obese models of type 2 diabetes[J]. Journal diabetes research, 2019: 9582714.
12	FORBES J M, COOPER M E. Mechanisms of diabetic complications[J]. Physiological reviews, 2013, 93: 137-188.
13	CADE W T. Diabetes-related microvascular and macrovascular diseases in the physical therapy setting[J]. Physical therapy, 2008, 88: 1322-1335.
14	DEVASAGAYAM T P, TILAK J C, BOLOOR K K, et al. Free radicals and antioxidants in human health: Current status and future prospects[J]. The journal of the association of physicians of India, 2004, 52: 794-804.
15	WANG Q, ZHANG M, TORRES G, et al. Metformin suppresses diabetes-accelerated atherosclerosis via the inhibition of drp1-mediated mitochondrial fission[J]. Diabetes, 2017, 66: 193-205.
16	SORRENTINO V, MENZIES K J, AUWERX J. Repairing mitochondrial dysfunction in disease[J]. Annual review of pharmacology and toxicology, 2018, 58: 353-389.
17	ZINOVKIN R A, ROMASCHENKO V P, GALKIN I I, et al. Role of mitochondrial reactive oxygen species in age-related inflammatory activation of endothelium[J]. Aging, 2014, 6: 661-674.
18	CHAN D C. Fusion and fission: Interlinked processes critical for mitochondrial health[J]. Annual review of genetics, 2012, 46: 265-287.
19	YOULE R J, VAN DER BLIEK A M. Mitochondrial fission, fusion, and stress[J]. Science, 2012, 337: 1062-1065.
20	ZANDALINAS S I, MITTLER R. Ros-induced ros release in plant and animal cells[J]. Free radical biology and medicine, 2018, 122: 21-27.
21	ZOROV D B, JUHASZOVA M, SOLLOTT S J. Mitochondrial reactive oxygen species (ros) and ros-induced ros release[J]. Physiological reviews, 2014, 94: 909-950.
22	VÁSQUEZ-TRINCADO C, GARCÍA-CARVAJAL I, PENNANEN C, et al. Mitochondrial dynamics, mitophagy and cardiovascular disease[J]. The journal of physiology, 2016, 594: 509-525.
23	KRZYWANSKI D M, MOELLERING D R, WESTBROOK D G, et al. Endothelial cell bioenergetics and mitochondrial DNA damage differ in humans having african or west eurasian maternal ancestry[J]. Circulation cardiovascular genetics, 2016, 9:26-36.
24	ROSCA M G, HOPPEL C L. Mitochondrial dysfunction in heart failure[J]. Heart failure reviews, 2013, 18: 607-622.
25	ZONG W X, RABINOWITZ J D, WHITE E. Mitochondria and cancer[J]. Molecular cell, 2016, 61: 667-676.
26	CADONIC C, SABBIR M G, ALBENSI B C. Mechanisms of mitochondrial dysfunction in alzheimer's disease[J]. Molecular neurobiology, 2016, 53: 6078-6090.
27	BOSE A, BEAL M F. Mitochondrial dysfunction in Parkinson's disease[J]. Journal of neurochemistry, 2016, 139 Suppl 1: 216-231.
28	PATIL N K, PARAJULI N, LAMACMILLAN-CROW, et al. Inactivation of renal mitochondrial respiratory complexes and manganese superoxide dismutase during sepsis: Mitochondria-targeted antioxidant mitigates injury[J]. American journal of physiology renal physiology, 2014, 306: F734-743.
29	SONG Y, COOK N R, ALBERT C M, et al. Effects of vitamins c and e and beta-carotene on the risk of type 2 diabetes in women at high risk of cardiovascular disease: A randomized controlled trial[J]. The American journal of clinical nutrition, 2009, 90: 429-437.
30	SESSO H D, BURING J E, CHRISTEN W G, et al. Vitamins e and c in the prevention of cardiovascular disease in men: The physicians' health study ii randomized controlled trial[J]. The journal of the American medical association, 2008, 300: 2123-2133.

[1]	曹雯佳, 刘学兵, 罗安果, 钟释敏, 邓岚, 王玉琳, 李赵欢. 超声矢量血流成像对2型糖尿病患者颈动脉壁剪切应力的研究[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(07): 709-717.
[2]	钟雅雯, 王煜, 王海臻, 黄莉萍. 肌苷通过抑制线粒体通透性转换孔开放缓解缺氧/复氧诱导的人绒毛膜滋养层细胞凋亡[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(05): 525-533.
[3]	王杰, 袁泉, 王玥琦, 乔佳君, 谭春丽, 夏仲元, 刘守尧. 溃疡油在糖尿病足溃疡治疗中的应用效果及安全性观察[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(06): 480-484.
[4]	徐志刚, 曹涛, 何亭, 李博奥, 魏婧韬, 张栋梁, 官浩, 杨薛康. 采用抗生素骨水泥治疗糖尿病患者心脏术后胸骨骨髓炎的临床效果观察[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(06): 498-502.
[5]	别瑶, 曹志斌, 辛静, 王健楠, 惠宗光. 应用基质血管成分细胞治疗糖尿病足溃疡的研究进展[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(05): 453-456.
[6]	姜珊, 李湘燕, 田硕涵, 温冰, 何睿, 齐心. 采用优化抗感染治疗模式改善糖尿病足感染预后的临床观察[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(05): 398-403.
[7]	孟令凯, 李大勇, 王宁, 王桂明, 张炳南, 李若彤, 潘立峰. 袖状胃切除术对肥胖伴2型糖尿病大鼠的作用及机制研究[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 638-642.
[8]	李猛, 姜腊, 董磊, 吴情, 贾犇黎. 腹腔镜胃袖状切除术治疗肥胖合并2型糖尿病及脂肪胰的临床研究[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(05): 554-557.
[9]	严虹霞, 王晓娟, 张毅勋. 2 型糖尿病对结直肠癌患者肿瘤标记物、临床病理及预后的影响[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(06): 483-487.
[10]	周学锋, 董哲毅, 冯哲, 蔡广研, 陈香美. 糖尿病肾脏疾病中西医结合诊疗指南计划书[J/OL]. 中华肾病研究电子杂志, 2024, 13(06): 301-305.
[11]	杜军霞, 赵小淋, 王浩然, 高志远, 王曼茜, 万楠熙, 张冬, 丁潇楠, 任琴琴, 段颖洁, 汤力, 朱晗玉. 2 型糖尿病的血液透析患者肠道微生物组学高通量测序分析[J/OL]. 中华肾病研究电子杂志, 2024, 13(06): 313-320.
[12]	邱岭, 朱旭丽, 浦坚, 邢苗苗, 吴佳玲. 糖尿病肾病患者肠道菌群生态特点与胃肠道功能障碍的关联性研究[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(05): 453-458.
[13]	王璇, 娜扎开提·尼加提, 雒洋洋, 蒋升. 皮肤晚期糖基化终末产物浓度与2型糖尿病微血管并发症的相关性[J/OL]. 中华临床医师杂志(电子版), 2024, 18(05): 447-454.
[14]	王星, 陈园, 热孜万古丽·乌斯曼, 郭艳英. T2DM、Obesity、NASH、PCOS共同致病因素相关的分子机制[J/OL]. 中华临床医师杂志(电子版), 2024, 18(05): 481-490.
[15]	曹慧, 刘华, 赵婷奕, 唐茂庆, 韩骐, 于永华. 非酮症高血糖性偏身舞蹈症一例报道及文献回顾[J/OL]. 中华脑血管病杂志(电子版), 2024, 18(05): 501-505.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

The effect and implication of inhibiting glycotoxicity pathway on mitochondrial dysfunction

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

The effect and implication of inhibiting glycotoxicity pathway on mitochondrial dysfunction