高糖对人脐静脉内皮细胞凋亡、NF-κB-iNOS-NO通路的影响及硫辛酸的干预作用
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摘要
目的:糖尿病(diabetes mellitus, DM),是一组以慢性血糖水平增高为特征的代谢疾病,主要病因是胰岛素抵抗和胰岛β细胞功能衰竭。长期慢性的血糖控制不良,以及随之而来的高血脂、高血压,可以导致心,眼,肾,足等多个脏器的并发症,严重影响人们的生活质量。氧化应激信号通路激活会导致胰岛素抵抗和胰岛素分泌受损,氧化损伤主要由活性氧物质(reactive oxygen species, ROS)和活性氮物质(reactive nitrogen species, RNS)所致,它们是细胞代谢过程中产生的含氧或含氮的活性产物,可通过脂质过氧化等途径诱导组织细胞损伤,具有重要的生理和病理意义。氧化应激不仅也参与了2型糖尿病发病过程,而且构成糖尿病晚期并发症的发病机制。动脉粥样硬化是糖尿病大血管病变的病理基础,氧化应激亦在其中发挥重要的作用。英国的前瞻性研究表明,仅仅控制好血糖并不能有效的降低大血管并发症的发病率,这提示我们,虽然血糖是造成糖尿病大血管病变的始动因素,但是不是唯一因素,在大血管病变发展的过程中,还有别的因素参与进来。血糖升高会导致内皮细胞功能不良,凋亡,内皮完整性及保护作用遭到破坏,屏障作用消失,黏附分子表达增加,趋化因子释放,白细胞黏附,巨噬细胞活化,平滑肌细胞迁移和增殖,最终形成动脉斑块。NF-κB作为核转录因子,参与了氧化应激、多种炎症反应和免疫反应。NF-κB可以激活多种免疫和炎症反应有关的基因包括iNOS、白介素-1,6、干扰素、肿瘤坏死因子-a、血管细胞粘附分子-1和胞间粘附分子[1]。NF-κB也可通过调整自身克隆基因和IκB的表达水平以达到自身调节的目的[2]。有学者认为NF-κB是动脉粥样硬化发生的始动机理之一[3]。Linder等使用原位杂交和免疫组织技术研究发现,在小鼠内皮细胞受损伤后45分钟,在其受损的内皮细胞核中NF-κB p65和p50蛋白亚体含量明显较正常为高,3小时后经Northern杂交技术发现VCAM-1mRNA及蛋白的表达增高[4]。大量的实验研究涉及到NF-κB及其所诱导表达的细胞因子在方方面面的应用,本实验就是体外观察高糖对人脐静脉内皮细胞(HUVEC)凋亡及产?生核因子-kappaB(NF-κB)、诱导型一氧化氮合酶(iNOS)及一氧化氮(NO)的影响,探讨NF-κB-iNOS-NO在糖尿病大血管病变发病机制中的作用,并应用硫辛酸干预,探讨该药的可能作用及机制。
方法:
体外培养HUVEC细胞,分为5组:(1)对照组(葡萄糖浓度:5.5mmol/L)(2)高糖组:(葡萄糖浓度:25mmol/L)(3)高糖+硫辛酸(50μmol/L)组(4)高糖+硫辛酸(100μmol/L)组(5)高糖+硫辛酸(200μmol/L)组,分别于0h,6h,12h,24h,48h留取样本进行检测。应用流式细胞术测细胞凋亡,Western Blot测NF-κB的表达,比色法测iNOS,硝酸还原酶法测定NO。
结果:
1高糖组细胞凋亡率及上清液中iNOS、NO较对照组在6h、12h、24h、48h均显著升高(P<0.01)。加入LA干预,48h后,A1组的凋亡率及iNOS、NO表达量未见明显下降,A2、A3组凋亡率及iNOS、NO均较高糖组下降(P<0.01),差异具有统计学意义,呈现药物剂量依赖性。
2高糖组细胞核NF-κB蛋白的表达量随着高糖作用时间的延长而明显增加,而在给予硫辛酸干预后NF-κB蛋白的表达逐渐下降。
结论:
1高糖环境可导致人脐静脉内皮细胞凋亡,NF-κB、iNOS、NO水平显著增加,提示高糖可通过NF-κB-iNOS-NO通路对人脐静脉内皮细胞发挥作用,NF-κB-iNOS-NO通路可能与糖尿病大血管病变的发生有关。
2 iNOS、NO在高糖作用早期(6h)就已表现出升高,提示其可能早期就参与了糖尿病大血管病变。
3硫辛酸注射液能减少细胞凋亡,减轻NF-κB表达,遏制iNOS、NO的表达,从而改善内皮细胞功能,减轻细胞损伤。提示硫辛酸可能通过NF-κB-iNOS-NO途径减轻细胞损伤,从而延缓高血糖对内皮细胞的毒性作用,提示其对糖尿病及其并发症有防治作用,早期硫辛酸应用可使糖尿病患者获益。
Objective: Diabetes (diabetes mellitus, DM), is a group characterized by elevated blood glucose levels of chronic metabolic diseases, which major cause is insulin resistance and pancreaticβ-cell failure. Chronic poorly controlled blood glucose, as well as the ensuing high blood lipids, hypertension, may lead to heart, eyes, kidneys, feet and other organs of the complications of serious impact on people's quality of life. Oxidative stress Signaling pathway causes insulin resistance and impaired insulin secretion, mainly by oxidative damage of ROS (reactive oxygen species, ROS) and reactive nitrogen substances (reactive nitrogen species, RNS), which are the of oxygen or nitrogen-containing active products in the process of cell metabolism. They can induce tissue and cell injury through lipid peroxidation, have important physiological and pathological significance. Oxidative stress not only participates in the pathogenesis of type 2 diabetes, but also constitutes the pathogenesis of diabetes late complications. The pathological basis of diabetic vascular disease is Atherosclerosis, oxidative stress also play an important role. United Kingdom prospective studies have shown that glucose compliance lonely can not reduce the morbidity of diabetic macrovascular complications, which suggest that the initiating agent of diabetes is glucose, but not the only factor. There are other factors in the process of macroangiopathy development. Now we think that atherosclerosis is possibly caused by the arterial inflammatory diseases. Elevated blood glucose can cause endothelial cell dysfunction, apoptosis, the destruction of endothelial integrity and protective effect of the barrier disappearance, increased expression of adhesion molecules, chemokine release, leukocyte adhesion, macrophage activation, smooth muscle cell migration and proliferation,plaque finally. As a nuclear transcription factor NF-κB involved in a variety of oxidative stress, inflammatory and immune response. NF-κB can activate inducible nitric oxide synthase(iNOS), interleukin-1,6, interferon, tumor filament factor-a, vascular cell adhesion molecule-1 and intercellular adhesion molecule. NF-κB can also be cloned by adjusting its own gene and the expression level of IκB in order to achieve the purpose of self-regulation. Some scholars believe that NF-κB is one of the starting mechanism of atherosclerosis. By in situ hybridization and immunohistochemical study Linder and others found that after endothelial cells in mice injured for 45 minutes, in its damaged endothelial cells NF-κB p65 and p50 protein were significantly higher than normal, After 3 hours It was found that the VCAM-1 mRNA and protein expression increased by Northern hybridization. There are a lot of experimental studies related to NF-κB and cytokines, this study is the in vitro effect of high glucose on human umbilical vein endothelial cells(HUVEC)apoptosis and nuclear factor- kappaB(NF-κB), inducible nitric oxide synthase(iNOS), nitric oxide(NO)to explore the NF-κB-iNOS-NO in the pathogenesis of diabetic vascular disease. And we observe intervention of lipoic acid to explore the possible role and mechanism of the drug.
Methods:
The HUVEC cells were cultured and divided into five groups: (1) normal control group (glucose’s concentration: 5.5mmol/l) (2) high glucose groups: (glucose’s concentration: 25mmol/l) (3) high glucose + lipoic acid (50umol/l) group (4) high glucose + lipoic acid (100umol/l) group (5) high glucose + lipoic acid (200umol/l) group, respectively, 0h, 6h, 12h, 24h, 48h specimens from a sample for testing. The cell apoptosis rate is detected by flow cytometry, NF-κB is measured by Western Blot test, the iNOS is measured by colorimetry and the NO is measured by nitrate reduction test.
Results:
1 On high glucose the cell apoptosis rate and the expression of iNOS, NO are significantly increased than the control group at 6h, 12h, 24h, 48h (P <0.01). With LA intervention, 48h later, on Group A1 the apoptosis rate, the expression of iNOS and NO have no significant change with the high glucose group for 48h (P> 0.05); on Group A2, A3 the apoptosis rate, the expression of iNOS and NO are decreased than high glucose group for 48h (P<0.01), showed dose-dependent.
2 On high glucose the expression of NF-κB are significantly increased, With LA intervention, NF-κB are decreased.
Conclusion:
1 High glucose can cause HUVEC cells apoptosis, NF-κB, iNOS, NO increased significantly, It is prompted that high glucose plays a role in human umbilical vein endothelial cells through the NF-κB-iNOS-NO pathway, and the NF-κB-iNOS-NO pathway may be associated diabetic macroangiopathy.
2 High glucose can cause iNOS, NO increased significantly in early times(6h),It is prompted it possibly involve in early diabetic vascular disease.
3 Lipoic Acid injection can reduce cell apoptosis, reduce the NF-κB expression, control the expression of iNOS and NO, and improve endothelial function, reduce cell damage. It is prompted lipoic acid may be reduce cell damage through NF-κB-iNOS-NO pathway, and delay high glucose toxicity on endothelial cell. It is prompted lipoic acid has the effect to prevention and treatment of diabetes and its complications, early application of lipoic acid could benefit diabetes.
方法:
体外培养HUVEC细胞,分为5组:(1)对照组(葡萄糖浓度:5.5mmol/L)(2)高糖组:(葡萄糖浓度:25mmol/L)(3)高糖+硫辛酸(50μmol/L)组(4)高糖+硫辛酸(100μmol/L)组(5)高糖+硫辛酸(200μmol/L)组,分别于0h,6h,12h,24h,48h留取样本进行检测。应用流式细胞术测细胞凋亡,Western Blot测NF-κB的表达,比色法测iNOS,硝酸还原酶法测定NO。
结果:
1高糖组细胞凋亡率及上清液中iNOS、NO较对照组在6h、12h、24h、48h均显著升高(P<0.01)。加入LA干预,48h后,A1组的凋亡率及iNOS、NO表达量未见明显下降,A2、A3组凋亡率及iNOS、NO均较高糖组下降(P<0.01),差异具有统计学意义,呈现药物剂量依赖性。
2高糖组细胞核NF-κB蛋白的表达量随着高糖作用时间的延长而明显增加,而在给予硫辛酸干预后NF-κB蛋白的表达逐渐下降。
结论:
1高糖环境可导致人脐静脉内皮细胞凋亡,NF-κB、iNOS、NO水平显著增加,提示高糖可通过NF-κB-iNOS-NO通路对人脐静脉内皮细胞发挥作用,NF-κB-iNOS-NO通路可能与糖尿病大血管病变的发生有关。
2 iNOS、NO在高糖作用早期(6h)就已表现出升高,提示其可能早期就参与了糖尿病大血管病变。
3硫辛酸注射液能减少细胞凋亡,减轻NF-κB表达,遏制iNOS、NO的表达,从而改善内皮细胞功能,减轻细胞损伤。提示硫辛酸可能通过NF-κB-iNOS-NO途径减轻细胞损伤,从而延缓高血糖对内皮细胞的毒性作用,提示其对糖尿病及其并发症有防治作用,早期硫辛酸应用可使糖尿病患者获益。
Objective: Diabetes (diabetes mellitus, DM), is a group characterized by elevated blood glucose levels of chronic metabolic diseases, which major cause is insulin resistance and pancreaticβ-cell failure. Chronic poorly controlled blood glucose, as well as the ensuing high blood lipids, hypertension, may lead to heart, eyes, kidneys, feet and other organs of the complications of serious impact on people's quality of life. Oxidative stress Signaling pathway causes insulin resistance and impaired insulin secretion, mainly by oxidative damage of ROS (reactive oxygen species, ROS) and reactive nitrogen substances (reactive nitrogen species, RNS), which are the of oxygen or nitrogen-containing active products in the process of cell metabolism. They can induce tissue and cell injury through lipid peroxidation, have important physiological and pathological significance. Oxidative stress not only participates in the pathogenesis of type 2 diabetes, but also constitutes the pathogenesis of diabetes late complications. The pathological basis of diabetic vascular disease is Atherosclerosis, oxidative stress also play an important role. United Kingdom prospective studies have shown that glucose compliance lonely can not reduce the morbidity of diabetic macrovascular complications, which suggest that the initiating agent of diabetes is glucose, but not the only factor. There are other factors in the process of macroangiopathy development. Now we think that atherosclerosis is possibly caused by the arterial inflammatory diseases. Elevated blood glucose can cause endothelial cell dysfunction, apoptosis, the destruction of endothelial integrity and protective effect of the barrier disappearance, increased expression of adhesion molecules, chemokine release, leukocyte adhesion, macrophage activation, smooth muscle cell migration and proliferation,plaque finally. As a nuclear transcription factor NF-κB involved in a variety of oxidative stress, inflammatory and immune response. NF-κB can activate inducible nitric oxide synthase(iNOS), interleukin-1,6, interferon, tumor filament factor-a, vascular cell adhesion molecule-1 and intercellular adhesion molecule. NF-κB can also be cloned by adjusting its own gene and the expression level of IκB in order to achieve the purpose of self-regulation. Some scholars believe that NF-κB is one of the starting mechanism of atherosclerosis. By in situ hybridization and immunohistochemical study Linder and others found that after endothelial cells in mice injured for 45 minutes, in its damaged endothelial cells NF-κB p65 and p50 protein were significantly higher than normal, After 3 hours It was found that the VCAM-1 mRNA and protein expression increased by Northern hybridization. There are a lot of experimental studies related to NF-κB and cytokines, this study is the in vitro effect of high glucose on human umbilical vein endothelial cells(HUVEC)apoptosis and nuclear factor- kappaB(NF-κB), inducible nitric oxide synthase(iNOS), nitric oxide(NO)to explore the NF-κB-iNOS-NO in the pathogenesis of diabetic vascular disease. And we observe intervention of lipoic acid to explore the possible role and mechanism of the drug.
Methods:
The HUVEC cells were cultured and divided into five groups: (1) normal control group (glucose’s concentration: 5.5mmol/l) (2) high glucose groups: (glucose’s concentration: 25mmol/l) (3) high glucose + lipoic acid (50umol/l) group (4) high glucose + lipoic acid (100umol/l) group (5) high glucose + lipoic acid (200umol/l) group, respectively, 0h, 6h, 12h, 24h, 48h specimens from a sample for testing. The cell apoptosis rate is detected by flow cytometry, NF-κB is measured by Western Blot test, the iNOS is measured by colorimetry and the NO is measured by nitrate reduction test.
Results:
1 On high glucose the cell apoptosis rate and the expression of iNOS, NO are significantly increased than the control group at 6h, 12h, 24h, 48h (P <0.01). With LA intervention, 48h later, on Group A1 the apoptosis rate, the expression of iNOS and NO have no significant change with the high glucose group for 48h (P> 0.05); on Group A2, A3 the apoptosis rate, the expression of iNOS and NO are decreased than high glucose group for 48h (P<0.01), showed dose-dependent.
2 On high glucose the expression of NF-κB are significantly increased, With LA intervention, NF-κB are decreased.
Conclusion:
1 High glucose can cause HUVEC cells apoptosis, NF-κB, iNOS, NO increased significantly, It is prompted that high glucose plays a role in human umbilical vein endothelial cells through the NF-κB-iNOS-NO pathway, and the NF-κB-iNOS-NO pathway may be associated diabetic macroangiopathy.
2 High glucose can cause iNOS, NO increased significantly in early times(6h),It is prompted it possibly involve in early diabetic vascular disease.
3 Lipoic Acid injection can reduce cell apoptosis, reduce the NF-κB expression, control the expression of iNOS and NO, and improve endothelial function, reduce cell damage. It is prompted lipoic acid may be reduce cell damage through NF-κB-iNOS-NO pathway, and delay high glucose toxicity on endothelial cell. It is prompted lipoic acid has the effect to prevention and treatment of diabetes and its complications, early application of lipoic acid could benefit diabetes.
引文
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2 Siebenlist U ,Franzoso G,Brown K. Structure ,regulation and function of NF-κB[J]. Annu Rev Cell Biol,1994,10:405-455
3 Collins T. Endothelial nuclear factor-κB and the initiation of the atherosclerotic lesion[J ] . Lab Invest ,1993 ,68 :499-508
4 Lindner V ,Collins T. Expression of NF-κB and IκB-αby aortic endothelium in a arterial injury model[J].Am J Pathol, 1996, 148:472
5 Hussain A, Claussen B, Ramachandran A,et al. Prevention of type 2 diabetes: a review [J]. Diabetes Res Clin Pract, 2007,76(3):317
6 Zandi E, Rothwaif DM, Delhase M,et al.The IKB Kinase complex(IKK) containst two kinase subunits, IKK2 and IKK, necessary for IKBphosphoralation and NF-KB activation Cell,1997,91:243
7 Guevara NV, et al. Apoptosis in atherosclerosis: pathological and pharmacological implications. Pharmacol Res,2001,44(2):59-71
8 Aoki M , Nata T , Morishita R , et al . Endothelial apoptosis induced by oxidative stress through activation of NF-kappaB : antiapoptotic effect of antioxidant agents on endothelial cells[J] . Hypertension , 2001 ,38 (1) :48-55
9 Sheu ML , Ho FM , Yang RS , et al . High glucose induces human endo- thelial cell apoptosis through a phosphoinositide 3-kinase-regulated cycloo- xygenase-2 pathway [J] . Arterioscler Thromb Vasc Biol , 2005 ,25 (3): 539 -545
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3 Collins T. Endothelial nuclear factor-κB and the initiation of the atherosclerotic lesion[J ] . Lab Invest ,1993 ,68 :499-508
4 Lindner V ,Collins T. Expression of NF-κB and IκB-αby aortic endothelium in a arterial injury model[J].Am J Pathol, 1996, 148:472
5 Hussain A, Claussen B, Ramachandran A,et al. Prevention of type 2 diabetes: a review [J]. Diabetes Res Clin Pract, 2007,76(3):317
6 Zandi E, Rothwaif DM, Delhase M,et al.The IKB Kinase complex(IKK) containst two kinase subunits, IKK2 and IKK, necessary for IKBphosphoralation and NF-KB activation Cell,1997,91:243
7 Guevara NV, et al. Apoptosis in atherosclerosis: pathological and pharmacological implications. Pharmacol Res,2001,44(2):59-71
8 Aoki M , Nata T , Morishita R , et al . Endothelial apoptosis induced by oxidative stress through activation of NF-kappaB : antiapoptotic effect of antioxidant agents on endothelial cells[J] . Hypertension , 2001 ,38 (1) :48-55
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