血管紧张素Ⅱ对高糖培养大鼠血管平滑肌细胞核转录因子κB活化的影响
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摘要
目的
糖尿病血管并发症是糖尿病患者最主要的致死因素。血管平滑肌细胞(Vascular smooth muscle cells，VSMCs)的增殖和迁移在以动脉粥样硬化为特征的糖尿病大血管并发症发生过程中具有重要意义，高糖和血管紧张素Ⅱ是两个VSMCs增殖的独立刺激因素。核因子κB(Nuclear Factor κB，NFκB)是一类多向性氧化应激敏感型转录因子，介导多种刺激促进VSMCs增殖的作用。已有研究表明高糖和血管紧张素Ⅱ均可单独诱导NFκB的活化，为进一步探讨血管紧张素Ⅱ在糖尿病血管并发症中的作用机制，本研究观察了血管紧张素Ⅱ分别对高糖和低糖培养的大鼠VSMCs的NFκB核转移的影响。在NFκB活化过程中，IκBα降解是启动NFκB核转移的重要步骤，因此，本实验也观察了血管紧张素Ⅱ分别对高糖和低糖培养的大鼠VSMCs的IκBα降解影响。
实验方法
8周龄体重范围200～250g雄性Wistar大鼠以10％水合氯醛4ml／kg麻醉后取胸主动脉，按经典组织贴块法培养大鼠胸主动脉血管平滑肌细胞，选取生长良好的第三代细胞进行实验。长满玻片或培养瓶底的VSMCs分别以高糖型和低糖型无血清条件培养液同步24小时后换用含10~(-7)mol／L血管紧张素Ⅱ的高糖和低糖条件培养液处理细胞0，1，2，6，12小时，免疫细胞化学方法观察处理前后不同时间点NFκB核转移情况，Western Blot检测各时间点胞浆IκBα蛋白量变化，Metamorph图像分析系统扫描进行图像分析。
实验结果
倒置相差显微镜下观察原代培养细胞呈长梭形，峰-谷状生长。免疫细胞化学染色显示：低糖培养VSMCs暴露于血管紧张素Ⅱ前胞浆NFκB
gbs染色呈中等程度阳性，血管紧张素＊刺激 1／J’时胞浆 NFKB p65染色转
为弱阳性，2FJ2 ／J’时胞浆 NFKB P65染色呈阴性；高糖培养细胞未暴露于
血管紧张素n前胞浆NF。B P65染色呈弱阳性，血管紧张素＊刺激L2户、
12小时胞浆 NFa p65染色呈阴性，所有处理组未见核染色。
Western Blot检测显示：VSMCs暴露于血管紧张素I前，高糖培养细胞
I妨a蛋白量即显著低于低糖培养细胞，血管紧张素＊处理后，高糖培养细
胞各时间点Ida蛋白量均低于低糖培养细胞。低糖培养细胞和高糖培养
细胞中IKB。蛋白量减少均呈时间依赖趋势，但低糖培养细胞中这种时间
依赖性趋势更为明显。
结 论
1）Aug 11和高糖促使血管平滑肌细胞胞浆中 NFo减低。
2）高糖诱导血管平滑肌细胞I妨a蛋白量降低。
3）血管紧张素＊诱导血管平滑肌细胞Ida蛋白量降低呈时间依赖性
关系，这一关系在高糖和低糖环境中均有表现，且以低糖环境下更为明显。
Diabetic vascular complication is a major death cause for patients suffering from diabetes, proliferation and migration of vascular smooth muscle cells play a very important role during the development of diabetic large vessels complication such as Atherosclerosis and high - glucose and Angiotensin II ( Ang II ) are two independent stimulus of VSMCs proliferation. NFkB is a pleiotropic oxidant -sensitive transcriptional factor and the effect of many stimulus on VSMCs proliferation was mediated by NFkB. Some studies indicated that both high - glucose and Ang II can induce NFkB activation. In order to definitude the role of Ang II in diabetic vascular complication, we studied the effect of Ang II on NFkB translocation in VSMCs cultured in media containing higher or lower glucose. During the course of NFkB activation, the degradation of IkBa: is an initiate step of NFkB nuclear translocation, so we also examined the effect of Ang II on the degradation of IkBa in VSMCs cultured in media containing higher or lowe
r glucose. .
Methods
8w male wistar rats (weighting 200 -250g) were anesthetized with 10% aquachloral, then thoracic aortae were prepared for VSMCs . Classic tissue expanding method was used to acquire VSMCs, and the third passage cells were used for experiment. After fully grew on slide or culture bottle, VSMCs were made quiescent with serum depletion medium containing higher or lower glucose for 24h then the cells were exposed to 10-7mol/L Ang II for 0, 1, 2, 6, 12h in media containing higher or lower glucose. Immunocytochemistry was employed to observe NFkB nuclear translocation and Western Blot was used to exanimate
IkBa protein quantity change in different time point before and after the treatment, Metamorph image analyses system was employed to take on image analy-
ses.
Results
To be observed with inverted phase contrast microscope, the primary cultured cells show a long fusiform shape, peak - gorge growth pattern. Immunocy-tochernistry stain show that in VSMCs cultured in low glucose type DMEM, before the cells were exposed to Ang II , the NFkB p65 show a median positive stain in cytoplasm, after the cells were exposed to Ang II for 1 h, the NFkB p65 in cytoplasm turn to a weak positive stain; in VSMCs cultured in high glucose type DMEM, the NFkB p65 show a weak positive stain in cytoplasm, before the cells were exposed to Ang II , the NFkB p65 in cytoplasm turn to a negative stain, after the cells were exposed to Ang II for 1 h; both the cells cultured in high glucose type and low glucose type media show a negative stain of NFkB p65 in cytoplasm after be exposed to Ang II for 2, 6, 12h. all the sample show negative stain of NFkB p65 in nuclear. Western Blot indicate that before VSMCs were exposed to Ang II, the IkBo: protein quantity in VSMCs cultured in high glucose type DMEM significantly is lower than that in VSMCs cultured in high glucose type DMEM. After be exposed to Ang II, the IkBa protein quantity in VSMCs cultured in high glucose type DMEM all is lower than that VSMCs cultured in low glucose type DMEM to every time point. The IkBa protein quantity change both in VSMCs cultured in high glucose type DMEM and low glucose type DMEM show time dependent tendency, but this tendency is more significant in VSMCs cultured in low glucose type DMEM.
Conclusions
1) Ang II and higher glucose induce the cytoplasmic NFkB of VSMC decreased.
2) Higher glucose induce IkBa: protein quantity decreased in VSMCs.
3 ) Ang II induce IkBo: protein quantity decreased in VSMCs and the IkBa protein quantity change show a time dependent tendency. This tendency is more significant in VSMCs cultured in low glucose type DMEM.
糖尿病血管并发症是糖尿病患者最主要的致死因素。血管平滑肌细胞(Vascular smooth muscle cells，VSMCs)的增殖和迁移在以动脉粥样硬化为特征的糖尿病大血管并发症发生过程中具有重要意义，高糖和血管紧张素Ⅱ是两个VSMCs增殖的独立刺激因素。核因子κB(Nuclear Factor κB，NFκB)是一类多向性氧化应激敏感型转录因子，介导多种刺激促进VSMCs增殖的作用。已有研究表明高糖和血管紧张素Ⅱ均可单独诱导NFκB的活化，为进一步探讨血管紧张素Ⅱ在糖尿病血管并发症中的作用机制，本研究观察了血管紧张素Ⅱ分别对高糖和低糖培养的大鼠VSMCs的NFκB核转移的影响。在NFκB活化过程中，IκBα降解是启动NFκB核转移的重要步骤，因此，本实验也观察了血管紧张素Ⅱ分别对高糖和低糖培养的大鼠VSMCs的IκBα降解影响。
实验方法
8周龄体重范围200～250g雄性Wistar大鼠以10％水合氯醛4ml／kg麻醉后取胸主动脉，按经典组织贴块法培养大鼠胸主动脉血管平滑肌细胞，选取生长良好的第三代细胞进行实验。长满玻片或培养瓶底的VSMCs分别以高糖型和低糖型无血清条件培养液同步24小时后换用含10~(-7)mol／L血管紧张素Ⅱ的高糖和低糖条件培养液处理细胞0，1，2，6，12小时，免疫细胞化学方法观察处理前后不同时间点NFκB核转移情况，Western Blot检测各时间点胞浆IκBα蛋白量变化，Metamorph图像分析系统扫描进行图像分析。
实验结果
倒置相差显微镜下观察原代培养细胞呈长梭形，峰-谷状生长。免疫细胞化学染色显示：低糖培养VSMCs暴露于血管紧张素Ⅱ前胞浆NFκB
gbs染色呈中等程度阳性，血管紧张素＊刺激 1／J’时胞浆 NFKB p65染色转
为弱阳性，2FJ2 ／J’时胞浆 NFKB P65染色呈阴性；高糖培养细胞未暴露于
血管紧张素n前胞浆NF。B P65染色呈弱阳性，血管紧张素＊刺激L2户、
12小时胞浆 NFa p65染色呈阴性，所有处理组未见核染色。
Western Blot检测显示：VSMCs暴露于血管紧张素I前，高糖培养细胞
I妨a蛋白量即显著低于低糖培养细胞，血管紧张素＊处理后，高糖培养细
胞各时间点Ida蛋白量均低于低糖培养细胞。低糖培养细胞和高糖培养
细胞中IKB。蛋白量减少均呈时间依赖趋势，但低糖培养细胞中这种时间
依赖性趋势更为明显。
结 论
1）Aug 11和高糖促使血管平滑肌细胞胞浆中 NFo减低。
2）高糖诱导血管平滑肌细胞I妨a蛋白量降低。
3）血管紧张素＊诱导血管平滑肌细胞Ida蛋白量降低呈时间依赖性
关系，这一关系在高糖和低糖环境中均有表现，且以低糖环境下更为明显。
Diabetic vascular complication is a major death cause for patients suffering from diabetes, proliferation and migration of vascular smooth muscle cells play a very important role during the development of diabetic large vessels complication such as Atherosclerosis and high - glucose and Angiotensin II ( Ang II ) are two independent stimulus of VSMCs proliferation. NFkB is a pleiotropic oxidant -sensitive transcriptional factor and the effect of many stimulus on VSMCs proliferation was mediated by NFkB. Some studies indicated that both high - glucose and Ang II can induce NFkB activation. In order to definitude the role of Ang II in diabetic vascular complication, we studied the effect of Ang II on NFkB translocation in VSMCs cultured in media containing higher or lower glucose. During the course of NFkB activation, the degradation of IkBa: is an initiate step of NFkB nuclear translocation, so we also examined the effect of Ang II on the degradation of IkBa in VSMCs cultured in media containing higher or lowe
r glucose. .
Methods
8w male wistar rats (weighting 200 -250g) were anesthetized with 10% aquachloral, then thoracic aortae were prepared for VSMCs . Classic tissue expanding method was used to acquire VSMCs, and the third passage cells were used for experiment. After fully grew on slide or culture bottle, VSMCs were made quiescent with serum depletion medium containing higher or lower glucose for 24h then the cells were exposed to 10-7mol/L Ang II for 0, 1, 2, 6, 12h in media containing higher or lower glucose. Immunocytochemistry was employed to observe NFkB nuclear translocation and Western Blot was used to exanimate
IkBa protein quantity change in different time point before and after the treatment, Metamorph image analyses system was employed to take on image analy-
ses.
Results
To be observed with inverted phase contrast microscope, the primary cultured cells show a long fusiform shape, peak - gorge growth pattern. Immunocy-tochernistry stain show that in VSMCs cultured in low glucose type DMEM, before the cells were exposed to Ang II , the NFkB p65 show a median positive stain in cytoplasm, after the cells were exposed to Ang II for 1 h, the NFkB p65 in cytoplasm turn to a weak positive stain; in VSMCs cultured in high glucose type DMEM, the NFkB p65 show a weak positive stain in cytoplasm, before the cells were exposed to Ang II , the NFkB p65 in cytoplasm turn to a negative stain, after the cells were exposed to Ang II for 1 h; both the cells cultured in high glucose type and low glucose type media show a negative stain of NFkB p65 in cytoplasm after be exposed to Ang II for 2, 6, 12h. all the sample show negative stain of NFkB p65 in nuclear. Western Blot indicate that before VSMCs were exposed to Ang II, the IkBo: protein quantity in VSMCs cultured in high glucose type DMEM significantly is lower than that in VSMCs cultured in high glucose type DMEM. After be exposed to Ang II, the IkBa protein quantity in VSMCs cultured in high glucose type DMEM all is lower than that VSMCs cultured in low glucose type DMEM to every time point. The IkBa protein quantity change both in VSMCs cultured in high glucose type DMEM and low glucose type DMEM show time dependent tendency, but this tendency is more significant in VSMCs cultured in low glucose type DMEM.
Conclusions
1) Ang II and higher glucose induce the cytoplasmic NFkB of VSMC decreased.
2) Higher glucose induce IkBa: protein quantity decreased in VSMCs.
3 ) Ang II induce IkBo: protein quantity decreased in VSMCs and the IkBa protein quantity change show a time dependent tendency. This tendency is more significant in VSMCs cultured in low glucose type DMEM.
引文
1. Hechmidt AM, Yan SD, Wautier JL, et al. Activation of receptor for advanced glycation end products: a mechanism for chronic vascular dysfunction in diabetic vasculopathy and atherosclerosis. Circ Res. 1999 Mar 19 ;84(5): 489-97.
2. Nakagawa K, Chen YX, Ishibashi H, et al. Angiogenesis and its regulation: roles of vascular endothelial cell growth factor. Semin Thromb Hemost. 2000 ;26 (1): 61-6.
3. Hadrava V, Kruppa U, Russo RC, et al. Vascular smooth muscle cell proliferation and its therapeutic modulation in hypertension. Am Heart J. 1991 Oct; 122(4 Pt 2): 1198-203.
4. Luft FC. Angiotensin, inflammation, hypertension, and cardiovascular disease. Curr Hypertens Rep. 2001 Feb; 3 (1): 61-7.
5. Albert S. Baldwin, Jr. The transcription factor NF- kB and human disease. The Journal of Clinical Investigation. 2001. January Vol. 107 (1): 3-6
6. Kiran Kumar V. Yerneni, Wei Bai, et al. Hyperglycemia -Induced Activation of Nuclear Transcription Factor B in Vascular Smooth. Muscle Cells. Diabetes, 1999 ,vol. 48: 855-864.
7. Tummala PE, Chen XL, Sundell CL, Laursen JB, et al. Angiotensin II induces vascular cell adhesion molecule - 1 expression in rat vasculature: A potential link between the renin- angiotensin system and atherosclerosis. Circulation. 1999 Sep 14;100(11): 1223-9
8．司徒镇强，吴军正．细胞培养西安：世界图书出版社，1999．107
9．徐叔云，卞如濂，陈修．药理实验方法学第三版北京：人民卫生出版社，2002．1900
10. Rainer DM, Martina H, Renate HW, et al. The Transcription Factor NF -kB and the Regulation of Vascular Cell Function. Arterioscler Thromb Vase Biol. 2000 ;20: e83-e88.
11. Veenstra TD. Electrospray ionization mass spectrometry: a promising new
technique in the study of protein/DNA noncovalent complexes. Biochem Biophys Res Commun. 1999 Apr 2 ;257 (1): 1-5.
12. van den Berg R, Haenen GR, van den Berg H, et al. Transcription factor NF - kappaB as a potential biomarker for oxidative stress. Br J Nutr. 2001 Aug;86 Suppl 1: S121-7.
13. Deptala A, Bedner E, Gorczyca W, Darzynkiewicz Z. Activation of nuclear factor kappa B (NF- kappaB ) assayed by laser scanning cytometry (LSC). Cytometry. 1998 Nov 1 ;33(3): 376-82
14. Marta RO, Oscar L, Mo'nica R, et al. Angiotensin II Activates Nuclear Transcription Factor kB Through AT1 and AT2 in Vascular Smooth Muscle Cells. Molecular Mechanisms. Circ Res. 2000 ;86: 1266-1272.
15. Frantz S, Fraccarollo D, Wagner H, et al. Sustained activation of nuclear factor kappa B and activator protein 1 in chronic heart failure. Cardiovasc Res. 2003 Mar;57(3): 749-56.
16. Rouet- Benzineb P, Gontero B, Dreyfus P, et al. Angiotensin Ⅱ induces nuclear factor- kappa B activation in cultured neonatal rat cardiomyocytes through protein kinase C signaling pathway. J Mol Cell Cardiol. 2000 Oct; 32(10): 1767-78
2. Nakagawa K, Chen YX, Ishibashi H, et al. Angiogenesis and its regulation: roles of vascular endothelial cell growth factor. Semin Thromb Hemost. 2000 ;26 (1): 61-6.
3. Hadrava V, Kruppa U, Russo RC, et al. Vascular smooth muscle cell proliferation and its therapeutic modulation in hypertension. Am Heart J. 1991 Oct; 122(4 Pt 2): 1198-203.
4. Luft FC. Angiotensin, inflammation, hypertension, and cardiovascular disease. Curr Hypertens Rep. 2001 Feb; 3 (1): 61-7.
5. Albert S. Baldwin, Jr. The transcription factor NF- kB and human disease. The Journal of Clinical Investigation. 2001. January Vol. 107 (1): 3-6
6. Kiran Kumar V. Yerneni, Wei Bai, et al. Hyperglycemia -Induced Activation of Nuclear Transcription Factor B in Vascular Smooth. Muscle Cells. Diabetes, 1999 ,vol. 48: 855-864.
7. Tummala PE, Chen XL, Sundell CL, Laursen JB, et al. Angiotensin II induces vascular cell adhesion molecule - 1 expression in rat vasculature: A potential link between the renin- angiotensin system and atherosclerosis. Circulation. 1999 Sep 14;100(11): 1223-9
8．司徒镇强，吴军正．细胞培养西安：世界图书出版社，1999．107
9．徐叔云，卞如濂，陈修．药理实验方法学第三版北京：人民卫生出版社，2002．1900
10. Rainer DM, Martina H, Renate HW, et al. The Transcription Factor NF -kB and the Regulation of Vascular Cell Function. Arterioscler Thromb Vase Biol. 2000 ;20: e83-e88.
11. Veenstra TD. Electrospray ionization mass spectrometry: a promising new
technique in the study of protein/DNA noncovalent complexes. Biochem Biophys Res Commun. 1999 Apr 2 ;257 (1): 1-5.
12. van den Berg R, Haenen GR, van den Berg H, et al. Transcription factor NF - kappaB as a potential biomarker for oxidative stress. Br J Nutr. 2001 Aug;86 Suppl 1: S121-7.
13. Deptala A, Bedner E, Gorczyca W, Darzynkiewicz Z. Activation of nuclear factor kappa B (NF- kappaB ) assayed by laser scanning cytometry (LSC). Cytometry. 1998 Nov 1 ;33(3): 376-82
14. Marta RO, Oscar L, Mo'nica R, et al. Angiotensin II Activates Nuclear Transcription Factor kB Through AT1 and AT2 in Vascular Smooth Muscle Cells. Molecular Mechanisms. Circ Res. 2000 ;86: 1266-1272.
15. Frantz S, Fraccarollo D, Wagner H, et al. Sustained activation of nuclear factor kappa B and activator protein 1 in chronic heart failure. Cardiovasc Res. 2003 Mar;57(3): 749-56.
16. Rouet- Benzineb P, Gontero B, Dreyfus P, et al. Angiotensin Ⅱ induces nuclear factor- kappa B activation in cultured neonatal rat cardiomyocytes through protein kinase C signaling pathway. J Mol Cell Cardiol. 2000 Oct; 32(10): 1767-78