不同浓度高糖对原代乳鼠心肌细胞及H9C2细胞系线粒体形态影响的观察
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摘要
目的探讨不同浓度高糖对原代乳鼠心肌细胞及H9C2细胞系线粒体形态及膜电位的影响。方法分离培养原代乳鼠心肌细胞及传代培养H9C2细胞,两种细胞贴壁24 h后分为正常对照组(NC)、甘露醇组(NC+M)、25 mmol/L高糖组(HG25)与35 mmol/L高糖组(HG35),检测各组细胞线粒体形态与线粒体膜电位的变化并测定乳酸脱氢酶(LDH)含量和细胞凋亡率。结果乳鼠心肌细胞及传代培养H9C2细胞中HG25组与HG35组均导致细胞线粒体形态的改变,表现为线粒体面积/周长比及线粒体反圆度降低,两组均使线粒体膜电位水平降低(P<0. 05)。与NC组细胞凋亡率、LDH水平比较,HG25组、NC+M组差异无统计学意义(P>0. 05),HG35组升高,差异有统计学意义(P<0. 05)。结论 25 mmol/L及35 mmol/L两种高糖浓度均能降低心肌细胞线粒体连接度与拉长度,使其由网状向点块状改变,并降低其膜电位水平,影响心肌细胞能量代谢,但25 mmol/L的高糖浓度并未影响心肌细胞凋亡率。
Objective To evaluate the influence of different high glucose concentrations on mitochondrial morphology and membrane potential in primary neonatal rat myocardial cells and H9C2 cell lines.MethodsIsolated primary neonatal rat cardiomyocytes and passaged H9C2 cells were adherent cultured for24 h and then divided into control group(NC),mannitol group(NC + M),25 mmol/L glucose group(HG25)and 35 mmol/L glucose group(HG35). LDH content,cell apoptosis rate,myocardial mitochondrial morphology and mitochondrial membrane potential changes were observed.ResultsAfter treated with25 mmol/L and 35 mmol/L high glucose,neonatal rat myocardial cells and H9C2 cells both exhibited decreased area/perimeter ratio and inverse circularity. Mitochondrial membrane potential decreased significantly in the two high glucose groups(P<0. 05). Cell apoptosis rate and LDH was not statistical different between HG25 group and NC group(P>0. 05). Cell apoptosis rate and LDH were higher in HG35 group(P<0. 05).Cell apoptosis rate and mitochondrial morphology were not influenced in NC+M group.ConclusionHigh glucose with 25 mmol/L and 35 mmol/L concentration reduced myocardial mitochondrial connectivity and length pull,and decreased membrane potential level. Thus affect the myocardial cell energy metabolism due to the mitochondrial morphology changes. But high glucose with 25 mmol/L concentration did not affect myocardial apoptosis rate.
Objective To evaluate the influence of different high glucose concentrations on mitochondrial morphology and membrane potential in primary neonatal rat myocardial cells and H9C2 cell lines.MethodsIsolated primary neonatal rat cardiomyocytes and passaged H9C2 cells were adherent cultured for24 h and then divided into control group(NC),mannitol group(NC + M),25 mmol/L glucose group(HG25)and 35 mmol/L glucose group(HG35). LDH content,cell apoptosis rate,myocardial mitochondrial morphology and mitochondrial membrane potential changes were observed.ResultsAfter treated with25 mmol/L and 35 mmol/L high glucose,neonatal rat myocardial cells and H9C2 cells both exhibited decreased area/perimeter ratio and inverse circularity. Mitochondrial membrane potential decreased significantly in the two high glucose groups(P<0. 05). Cell apoptosis rate and LDH was not statistical different between HG25 group and NC group(P>0. 05). Cell apoptosis rate and LDH were higher in HG35 group(P<0. 05).Cell apoptosis rate and mitochondrial morphology were not influenced in NC+M group.ConclusionHigh glucose with 25 mmol/L and 35 mmol/L concentration reduced myocardial mitochondrial connectivity and length pull,and decreased membrane potential level. Thus affect the myocardial cell energy metabolism due to the mitochondrial morphology changes. But high glucose with 25 mmol/L concentration did not affect myocardial apoptosis rate.
引文
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[12]陈梦楠,卢海龙,李雷,等.体外高糖环境对H9c2心肌细胞中STIM1、Orai1及TRPC1的影响.实用老年医学,2017,31:24-28.
[13]左军,吴霞,汤之铭.高糖诱导H9C2大鼠心肌细胞线粒体功能障碍及其机制.山西医科大学学报,2017,48:405-409.
[14]Neary MT,Ng KE,Ludtmann MH,et al.Hypoxia signaling controls postnatal changes in cardiac mitochondrial morphology and function.J Mol Cell Cardiol,2014,74:340-352.
[15]Galloway CA,Lee H,Nejjar S,et al.Transgenic control of mitochondrial fission induces mitochondrial uncoupling and relieves diabetic oxidative stress.Diabetes,2012,61:2093-2104.
[16]Croston TL,Thapa D,Holden AA,et al.Functional deficienciesof subsarcolemmal mitochondria in the type 2 diabetichuman heart.Am J Physiol Heart Circ Physiol,2014,307:H54-H65.
[17]Gawlowski T,Suarez J,Scott B,et al.Modulation of dynamin-related protein 1(DRP1)function by increased O-linked-betaNacetylglucosamine modification(O-GlcNAc)in cardiac myocytes.J Biol Chem,2012,287:30024-30034.
[18]Shenouda SM,Widlansky ME,Chen K,et al.Altered mitochondrial dynamics contributes to endothelial dysfunction in diabetes mellitus.Circulation,2011,124:444-453.
[19]张国勇,高蓓蕾,余文军,等.动力相关蛋白1通过诱导线粒体自噬对高糖导致心肌细胞损伤的影响.心脏杂志,2017,29:506-511.
[20]张东亚,陈建昌.葡萄糖浓度对乳鼠心肌细胞自噬的影响.苏州大学学报(医学版),2012,32:193-196.
[2]Yu T,Sheu SS,Robotham JL,et al.Mitochondrial fission mediates high glucose-induced cell death through elevated production of reactive oxygen species.Cardiovasc Res,2008,79:341-351.
[3]Holmstr?m KM,Baird L,Zhang Y,et al.Nrf2 impacts cellular bioenergetics by controlling substrate availability for mitochondrial respiration.Biol Open,2013,2:761-770.
[4]Weng J,Bi Y.Diabetes in China:The challenge now.J Diabetes Investig,2010,1:170-171.
[5]范丽芬,杨啸,姚艳玲.白藜芦醇对高糖诱导乳鼠心肌细胞损伤和凋亡的保护作用.中国心血管病研究,2013,11:533-539.
[6]赵鑫,陈璐,郭政.P物质对高糖孵育心肌细胞缺氧/复氧损伤的影响.麻醉与监护论坛,2014,21:54-57.
[7]廖荣华,李黎,张玲敏,等.高糖微环境对体外心肌细胞增殖活性和氧化应激的影响.中国医院药学杂志,2018,38:1030-1034.
[8]Makino A,Suarez J,Gawlowski T,et al.Regulation of mitochondrial morphology and function by O-GlcNAcylation in neonatal cardiac myocytes.Am J Physiol Regul Integr Comp Physiol,2011,300:R1296-R1302.
[9]周艳芳,郑晓伟,齐国先.高葡萄糖在正常氧及缺氧条件下对乳鼠心肌细胞存活与凋亡的不同影响.中国动脉硬化杂志,2008,6:89-92.
[10]林春喜,林建聪,郭润民,等.硫化氢通过调控JNK通路对抗高糖诱导的心肌细胞氧化应激损伤.中国动脉硬化杂志,2013,21:1-5.
[11]崔桂丽,王瑞玲,马琴,等.自噬在高糖高脂引起心肌细胞系H9C2凋亡中的影响.基础医学与临床,2017,37:1699-1705.
[12]陈梦楠,卢海龙,李雷,等.体外高糖环境对H9c2心肌细胞中STIM1、Orai1及TRPC1的影响.实用老年医学,2017,31:24-28.
[13]左军,吴霞,汤之铭.高糖诱导H9C2大鼠心肌细胞线粒体功能障碍及其机制.山西医科大学学报,2017,48:405-409.
[14]Neary MT,Ng KE,Ludtmann MH,et al.Hypoxia signaling controls postnatal changes in cardiac mitochondrial morphology and function.J Mol Cell Cardiol,2014,74:340-352.
[15]Galloway CA,Lee H,Nejjar S,et al.Transgenic control of mitochondrial fission induces mitochondrial uncoupling and relieves diabetic oxidative stress.Diabetes,2012,61:2093-2104.
[16]Croston TL,Thapa D,Holden AA,et al.Functional deficienciesof subsarcolemmal mitochondria in the type 2 diabetichuman heart.Am J Physiol Heart Circ Physiol,2014,307:H54-H65.
[17]Gawlowski T,Suarez J,Scott B,et al.Modulation of dynamin-related protein 1(DRP1)function by increased O-linked-betaNacetylglucosamine modification(O-GlcNAc)in cardiac myocytes.J Biol Chem,2012,287:30024-30034.
[18]Shenouda SM,Widlansky ME,Chen K,et al.Altered mitochondrial dynamics contributes to endothelial dysfunction in diabetes mellitus.Circulation,2011,124:444-453.
[19]张国勇,高蓓蕾,余文军,等.动力相关蛋白1通过诱导线粒体自噬对高糖导致心肌细胞损伤的影响.心脏杂志,2017,29:506-511.
[20]张东亚,陈建昌.葡萄糖浓度对乳鼠心肌细胞自噬的影响.苏州大学学报(医学版),2012,32:193-196.
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