NF-κB信号通路和脑出血后的继发性脑损伤关系的研究
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摘要
背景：
脑出血(Intracerebral Hemmorhage ICH)是一种致残率及病死率都很高的急性脑血管病。过去认为出血性脑损伤主要由于出血的机械性损伤和血肿直接压迫所致,目前研究结果显示,ICH血肿周围组织的继发性脑损伤可能更为重要。出血性脑损伤不仅包括血肿的占位效应及血肿对周围组织的直接破坏,而且包括血肿周围组织水肿、缺血、炎症、细胞毒性等原因。NF-κB(nuclear factor-Kappa B)是一种多向性转录调节蛋白,当其激活后,能进入细胞核内并与DNA结合,通过对炎性因子如TNF-α、IL-2、3、6和IL-8、转移生长因子、细胞间黏附分子-1(ICAM-1)、iNOS、环氧化酶-2等的转录调控,而在脑血管病的炎症反应中起着中心环节的作用。国内外研究已经证明,慢性炎症、脑缺血和细胞凋亡均与NF-κB的激活密切相关。
本次实验通过建立大鼠自体血注入脑出血模型,用蛋白印迹及免疫组化方法动态观察脑出血后脑组织内NF-κB及抑制因子Iκ-Bs的水平变化,以探讨NF-κB信号通路在脑出血后继发性脑损伤的可能作用及其机制。
材料与方法：
本研究着手调查NF-κB及抑制因子IκBs在脑出血(ICH)模型中的表达并阐明NF-κB信号通路与ICH后的继发性脑损伤的相关性。总共100只雄性SD大鼠被随机分为7组：对照组；2小时组；6小时组；12小时组；1天组；3天组；7天组。对照组大鼠予同侧基底节(IBG)生理盐水注射,其他老鼠予同侧基底节(IBG)自体血注射并于脑出血后2小时(h),6小时(h),12小时(h),1天(d),3天(d)和7天(d)处理。检测同侧基底节(IBG)、对侧基底节(CBG)和小脑(C)的脑水含量。用免疫组化(immunohistochemistry)、Western blot (WB)检测NF-κB及IκBs的表达情况。
结果：
IBG脑水含量在ICH后2h为78.702±0.065%,6h为79.305±0.139%,12h为80.425±0.409%,1d为81.355±0.425%,3d为82.598±0.311%,7d为80.625±0.512%,相对地,在3d时达峰值。在生理盐水注射对照组中NF-κB p65及抑制因子IκBα的免疫活性非常低,而ICH后NF-κB p65及IκBαt蛋白表达水平显著升高。蛋白印迹检测提示：较之生理盐水注射对照组,脑出血后2h血肿周围的NF-κB p65及IκBα蛋白表达水平升高,在出血后1d达到高峰,此后NF-κB表达水平逐渐下降,但直至出血后7天仍可检测出蛋白活性。免疫组化检测提示：脑出血后2h即可在血肿周围组织内发现NF-κB阳性细胞显著增多。相关性分析显示在大鼠ICH模型中,NF-Kb信号通路和继发性脑损伤的发展强相关。
结论：
脑出血后NF-κB p65及抑制因子IκBα表达明显高于正常对照组,可能参与了脑出血后的继发性损伤。
Objective and Background
Intracerebral hemmorhage (ICH) is an acute cerebrovascular disease with high morbidity and mortality.Hemorrhagic brain injury in the past was mainly due to bleeding mechanical injury and compression of hematoma, recent results show, the secondary brain injury induced by perihematomal zone may be more important. Hemorrhagic brain injury includes not only the hematoma mass effect and directly damage to the surrounding tissue, but also the surrounding tissue edema, ischemia, inflammation, cell toxicity and other reasons.NF-icB (nuclear factor-icappaB) is a multi-directional transcriptional regulatory protein, when it activated, can enter the nucleus and bind with DNA,. This results in the transcriptional induction of genes for manyproinflammatory substances, such as TNF-α, IL-2,3,6 and IL-8,transforming growth factor, ICAM-1, the iNOS, epoxide enzyme-2 and so on. Its activation has been played a key role in the inflammatory response in the cerebral diseases. Domestic and international studies have demonstrated that chronic inflammation, cerebral ischemia and apoptosis are closely related to the activation of NF-κB.
We investigated the levels of NF-κB and inhibition of factor IκBs after ICH to discuss the possible mechanism of NF-κB signaling pathway in secondary brain injury after ICH.
Materials and methods
This study was understaken to investigate the expression of NF-κB and inhibition of factor IκBs in ICH model and to clarify the relationship of NF-κB signaling pathway in secondary brain injury. A total of 100 rats were randomly divided into 7 groups:control group; 2 hours,6 hours,12 hours,1 day,3 days and 7 days groups. The animals in control groups were subjected to physiological saline injection into ipsilateral basal ganglia (IBG), and the other animals were subjected to autologous blood injection into IBG and were killed 2 hours,6 hours,12 hours,1 day,3 days and 7 days after ICH. Brain water content of IBG, contralateral basal ganglia (CBG) and cerebellum (C) was measured and the NF-κB P65 and IκBα. expression was assessed by Immunohistochemistry and Western blot (WB)
Results
The brain water content of IBG was found to be 78.702±0.065% at 2h, 79.305±0.139% at 6h,80.425±0.409% at 12h,81.355±0.425% on Id,82.598±0.311% on 3d, and 80.625±0.512% on 7d, respectively, which reached to the peak point on 3d. NF-κB and IκBs Immunoreactivities were very low in the cerebral hemispheres of the control rat.However,NF-KB and IκBs protein levels were increased markedly in the first 2 hours after ICH, peaked at day 1, and were still detcetable at day 7 after ICH.NF-κB and IκBs Positive cells were found in the Perihematomal zone. Correlation analysis revealed that NF-κB signaling pathway is strongly positvely correlated with the development of econdary brain injury in a rat ICH model.
Conclusions
The high expression of NF-κB and IκBs may participate in secondary brain injury after intracerebral hemorrhage
脑出血(Intracerebral Hemmorhage ICH)是一种致残率及病死率都很高的急性脑血管病。过去认为出血性脑损伤主要由于出血的机械性损伤和血肿直接压迫所致,目前研究结果显示,ICH血肿周围组织的继发性脑损伤可能更为重要。出血性脑损伤不仅包括血肿的占位效应及血肿对周围组织的直接破坏,而且包括血肿周围组织水肿、缺血、炎症、细胞毒性等原因。NF-κB(nuclear factor-Kappa B)是一种多向性转录调节蛋白,当其激活后,能进入细胞核内并与DNA结合,通过对炎性因子如TNF-α、IL-2、3、6和IL-8、转移生长因子、细胞间黏附分子-1(ICAM-1)、iNOS、环氧化酶-2等的转录调控,而在脑血管病的炎症反应中起着中心环节的作用。国内外研究已经证明,慢性炎症、脑缺血和细胞凋亡均与NF-κB的激活密切相关。
本次实验通过建立大鼠自体血注入脑出血模型,用蛋白印迹及免疫组化方法动态观察脑出血后脑组织内NF-κB及抑制因子Iκ-Bs的水平变化,以探讨NF-κB信号通路在脑出血后继发性脑损伤的可能作用及其机制。
材料与方法：
本研究着手调查NF-κB及抑制因子IκBs在脑出血(ICH)模型中的表达并阐明NF-κB信号通路与ICH后的继发性脑损伤的相关性。总共100只雄性SD大鼠被随机分为7组：对照组；2小时组；6小时组；12小时组；1天组；3天组；7天组。对照组大鼠予同侧基底节(IBG)生理盐水注射,其他老鼠予同侧基底节(IBG)自体血注射并于脑出血后2小时(h),6小时(h),12小时(h),1天(d),3天(d)和7天(d)处理。检测同侧基底节(IBG)、对侧基底节(CBG)和小脑(C)的脑水含量。用免疫组化(immunohistochemistry)、Western blot (WB)检测NF-κB及IκBs的表达情况。
结果：
IBG脑水含量在ICH后2h为78.702±0.065%,6h为79.305±0.139%,12h为80.425±0.409%,1d为81.355±0.425%,3d为82.598±0.311%,7d为80.625±0.512%,相对地,在3d时达峰值。在生理盐水注射对照组中NF-κB p65及抑制因子IκBα的免疫活性非常低,而ICH后NF-κB p65及IκBαt蛋白表达水平显著升高。蛋白印迹检测提示：较之生理盐水注射对照组,脑出血后2h血肿周围的NF-κB p65及IκBα蛋白表达水平升高,在出血后1d达到高峰,此后NF-κB表达水平逐渐下降,但直至出血后7天仍可检测出蛋白活性。免疫组化检测提示：脑出血后2h即可在血肿周围组织内发现NF-κB阳性细胞显著增多。相关性分析显示在大鼠ICH模型中,NF-Kb信号通路和继发性脑损伤的发展强相关。
结论：
脑出血后NF-κB p65及抑制因子IκBα表达明显高于正常对照组,可能参与了脑出血后的继发性损伤。
Objective and Background
Intracerebral hemmorhage (ICH) is an acute cerebrovascular disease with high morbidity and mortality.Hemorrhagic brain injury in the past was mainly due to bleeding mechanical injury and compression of hematoma, recent results show, the secondary brain injury induced by perihematomal zone may be more important. Hemorrhagic brain injury includes not only the hematoma mass effect and directly damage to the surrounding tissue, but also the surrounding tissue edema, ischemia, inflammation, cell toxicity and other reasons.NF-icB (nuclear factor-icappaB) is a multi-directional transcriptional regulatory protein, when it activated, can enter the nucleus and bind with DNA,. This results in the transcriptional induction of genes for manyproinflammatory substances, such as TNF-α, IL-2,3,6 and IL-8,transforming growth factor, ICAM-1, the iNOS, epoxide enzyme-2 and so on. Its activation has been played a key role in the inflammatory response in the cerebral diseases. Domestic and international studies have demonstrated that chronic inflammation, cerebral ischemia and apoptosis are closely related to the activation of NF-κB.
We investigated the levels of NF-κB and inhibition of factor IκBs after ICH to discuss the possible mechanism of NF-κB signaling pathway in secondary brain injury after ICH.
Materials and methods
This study was understaken to investigate the expression of NF-κB and inhibition of factor IκBs in ICH model and to clarify the relationship of NF-κB signaling pathway in secondary brain injury. A total of 100 rats were randomly divided into 7 groups:control group; 2 hours,6 hours,12 hours,1 day,3 days and 7 days groups. The animals in control groups were subjected to physiological saline injection into ipsilateral basal ganglia (IBG), and the other animals were subjected to autologous blood injection into IBG and were killed 2 hours,6 hours,12 hours,1 day,3 days and 7 days after ICH. Brain water content of IBG, contralateral basal ganglia (CBG) and cerebellum (C) was measured and the NF-κB P65 and IκBα. expression was assessed by Immunohistochemistry and Western blot (WB)
Results
The brain water content of IBG was found to be 78.702±0.065% at 2h, 79.305±0.139% at 6h,80.425±0.409% at 12h,81.355±0.425% on Id,82.598±0.311% on 3d, and 80.625±0.512% on 7d, respectively, which reached to the peak point on 3d. NF-κB and IκBs Immunoreactivities were very low in the cerebral hemispheres of the control rat.However,NF-KB and IκBs protein levels were increased markedly in the first 2 hours after ICH, peaked at day 1, and were still detcetable at day 7 after ICH.NF-κB and IκBs Positive cells were found in the Perihematomal zone. Correlation analysis revealed that NF-κB signaling pathway is strongly positvely correlated with the development of econdary brain injury in a rat ICH model.
Conclusions
The high expression of NF-κB and IκBs may participate in secondary brain injury after intracerebral hemorrhage
引文
1 Carhuapoma J, Wang P, Beauchamp N, et al. Diffusion weighted MRI and proton MR spectroscopic imaging in the study of secondary neuronal injury after intracerebral hemorrhage. Stroke,2000,31(3):726-732.
2 Barber M, Rodit i G, Stott D, et al. Poor outcome in primary intracerebral hemorrhage:results of a matched comparison. Postgrad Med J,2004,80(940): 89-92.
3 Xi G, Keep RF, JT H. Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol.,2006,5(1):53-63.
4 Sarnico I, Lanzillotta A, Benarese M, et al. NF-kappaB dimers in the regulation of neuronal survival. Int Rev Neurobiol,2009,85:351-362.
5 Yamamoto M,Takeda K. Role of nuclear IkappaB proteins in the regulation of host immune responses. J Infect Chemother.,2008,14(4):265-269.
6 Hickenbottom S, Grotta J, Strong R, et al. Nuclear factor-κB and cell death after experimental intracerebral hemorrhage in rats. Stroke,1999,30(11): 2472-2477.
7 Xi G, Wagner KR, Keep RF, et al. Role of blood clot formation on early edema development after experimental intracerebral hemorrhage. Stroke,2000, 29(12):2580-2586.
8 Hua Y, Keep RF, Hoff JT, et al. Brain injury after intracerebral hemorrhage: the role of thrombin and iron. Department of Neurosurgery,2007,38(2): 759-762.
9 Bergqvist S, Ghosh G, Komives E. The IkappaBalpha/NF-kappaB complex has two hot spots, one at either end of the interface. Protein Sci,2008,17(12): 2051-2058.
10 Jian W,Sylvain D. Heme oxygenase 2 deficiency increases brain swelling and inflammation after intracerebral hemorrhage. Neuroscience,2008,155(4): 1133-1141.
11 Wu H, Cong Y, Wang D, et al. Correlation of macrophage inflammatory protein-2 expression and brain edema in rats after intracerebral hemorrhage. Int J Clin Exp Pathol,2009,2(1):83-90.
1 Carhuapoma J, Wang P, Beauchamp N, et al. Diffusion weighted MRI and proton MR spectroscopic imaging in the study of secondary neuronal injury after intracerebral hemorrhage. Stroke,2000,31(3):726-732.
2 Barber M, Rodit i G, Stott D, et al. Poor outcome in primary intracerebral hemorrhage:results of a matched comparison. Postgrad Med J,2004,80(940): 89-92.
3 Xi G, Keep RF, JT H. Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol.,2006,5(1):53-63.
4 Sarnico I, Lanzillotta A, Benarese M, et al. NF-kappaB dimers in the regulation of neuronal survival. Int Rev Neurobiol,2009,85:351-362.
5 Yamamoto M,Takeda K. Role of nuclear IkappaB proteins in the regulation of host immune responses. J Infect Chemother.,2008,14(4):265-269.
6 Hickenbottom S, Grotta J, Strong R, et al. Nuclear factor-KB and cell death after experimental intracerebral hemorrhage in rats. Stroke,1999,30(11): 2472-2477.
7 Xi G, Wagner KR, Keep RF, et al. Role of blood clot formation on early edema development after experimental intracerebral hemorrhage. Stroke,2000, 29(12):2580-2586.
8 Hua Y, Keep RF, Hoff JT, et al. Brain injury after intracerebral hemorrhage: the role of thrombin and iron. Department of Neurosurgery,2007,38(2): 759-762.
9 Bergqvist S, Ghosh G, Komives E. The IkappaBalpha/NF-kappaB complex has two hot spots, one at either end of the interface. Protein Sci,2008,17(12): 2051-2058.
10 Jian W,Sylvain D. Heme oxygenase 2 deficiency increases brain swelling and inflammation after intracerebral hemorrhage. Neuroscience,2008,155(4): 1133-1141.
11 Wu H, Cong Y, Wang D, et al. Correlation of macrophage inflammatory protein-2 expression and brain edema in rats after intracerebral hemorrhage. Int J Clin Exp Pathol,2009,2(1):83-90.
12 Kyung Jin J, Eun Kyung G, Ji Young K, et al. Suppression of age-related renal changes in NF-κB and its target gene expression by dietary ferulate. J Nutr Biochem,2009,20(5):378-388.
13 Zhang X, Li H, Hu S, et al. Brain edema after intracerebral hemorrhage in rats:the role of inflammation. Neurol India,2006,54(4):402-407.
14 Jian W,Sylvain D. Inflammation after intracerebral hemorrhage. J Cereb Blood Flow Metab,2007,27(5):894-908.
15 Aronowski J,Hall C. New horizons for primary intracerebral hemorrhage treatment:experience from preclinical studies. Neurol Res,2005,27(3): 268-279.
16 Wu S, Fang C, Kim J, et al. Enhanced pulmonary inflammation following experimental intracerebral hemorrhage. Exp Neurol,2006,200(1):245-259.
17 Muir E, Adcock K, Morgenstern D, et al. Matrix metalloproteases and their inhibitors are produced by overlapping populations of activated astrocytes. Brain Res Mol Brain Res,2002,100(1-2):103-117.
18 Hsieh H, Wu C, Yang C. Bradykinin induces matrix metalloproteinase-9 expression and cell migration through a PKC--dependent ERK/Elk-1 pathway in astrocytes. Glia,2008,56(6):619-632.
19 Mori T, Wang X, Aoki T, et al. Down regulation of matrix metalloproteinase-9 and attenuation of edema via inhibition of ERK mitogen activated protein kinase in traumatic brain injury. J Neurotrauma,2002,19(11):1411-1419.
20 Kawai N, Kawanishi M, Nagao S. Treatment of cold injury-induced brain edema with a nonspecific matrix metalloproteinase inhibitor MMI270 in rats. Acta Neurochir Suppl,2003,86:291-295.
21 吕磊,蔡定芳.线粒体依赖的凋亡通路与脑出血后神经损伤.国际神经病学神经外科学杂志,2008,35(2)：132-136.
22 KJ Y, DJ K, SH K, et al. Anti-inflammatory effects of sinapic acid through the suppression of inducible nitric oxide synthase, cyclooxygase-2, and proinflammatory cytokines expressions via nuclear factor-kappaB inactivation. J Agric Food Chem,2008,56(21):10265-10272.
23 Campos-Esparza M, Sanchez-Gomez M, Matute C. Molecular mechanisms of neuroprotection by two natural antioxidant polyphenols. Cell Calcium 2009, 45(4):358-368.
2 Barber M, Rodit i G, Stott D, et al. Poor outcome in primary intracerebral hemorrhage:results of a matched comparison. Postgrad Med J,2004,80(940): 89-92.
3 Xi G, Keep RF, JT H. Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol.,2006,5(1):53-63.
4 Sarnico I, Lanzillotta A, Benarese M, et al. NF-kappaB dimers in the regulation of neuronal survival. Int Rev Neurobiol,2009,85:351-362.
5 Yamamoto M,Takeda K. Role of nuclear IkappaB proteins in the regulation of host immune responses. J Infect Chemother.,2008,14(4):265-269.
6 Hickenbottom S, Grotta J, Strong R, et al. Nuclear factor-κB and cell death after experimental intracerebral hemorrhage in rats. Stroke,1999,30(11): 2472-2477.
7 Xi G, Wagner KR, Keep RF, et al. Role of blood clot formation on early edema development after experimental intracerebral hemorrhage. Stroke,2000, 29(12):2580-2586.
8 Hua Y, Keep RF, Hoff JT, et al. Brain injury after intracerebral hemorrhage: the role of thrombin and iron. Department of Neurosurgery,2007,38(2): 759-762.
9 Bergqvist S, Ghosh G, Komives E. The IkappaBalpha/NF-kappaB complex has two hot spots, one at either end of the interface. Protein Sci,2008,17(12): 2051-2058.
10 Jian W,Sylvain D. Heme oxygenase 2 deficiency increases brain swelling and inflammation after intracerebral hemorrhage. Neuroscience,2008,155(4): 1133-1141.
11 Wu H, Cong Y, Wang D, et al. Correlation of macrophage inflammatory protein-2 expression and brain edema in rats after intracerebral hemorrhage. Int J Clin Exp Pathol,2009,2(1):83-90.
1 Carhuapoma J, Wang P, Beauchamp N, et al. Diffusion weighted MRI and proton MR spectroscopic imaging in the study of secondary neuronal injury after intracerebral hemorrhage. Stroke,2000,31(3):726-732.
2 Barber M, Rodit i G, Stott D, et al. Poor outcome in primary intracerebral hemorrhage:results of a matched comparison. Postgrad Med J,2004,80(940): 89-92.
3 Xi G, Keep RF, JT H. Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol.,2006,5(1):53-63.
4 Sarnico I, Lanzillotta A, Benarese M, et al. NF-kappaB dimers in the regulation of neuronal survival. Int Rev Neurobiol,2009,85:351-362.
5 Yamamoto M,Takeda K. Role of nuclear IkappaB proteins in the regulation of host immune responses. J Infect Chemother.,2008,14(4):265-269.
6 Hickenbottom S, Grotta J, Strong R, et al. Nuclear factor-KB and cell death after experimental intracerebral hemorrhage in rats. Stroke,1999,30(11): 2472-2477.
7 Xi G, Wagner KR, Keep RF, et al. Role of blood clot formation on early edema development after experimental intracerebral hemorrhage. Stroke,2000, 29(12):2580-2586.
8 Hua Y, Keep RF, Hoff JT, et al. Brain injury after intracerebral hemorrhage: the role of thrombin and iron. Department of Neurosurgery,2007,38(2): 759-762.
9 Bergqvist S, Ghosh G, Komives E. The IkappaBalpha/NF-kappaB complex has two hot spots, one at either end of the interface. Protein Sci,2008,17(12): 2051-2058.
10 Jian W,Sylvain D. Heme oxygenase 2 deficiency increases brain swelling and inflammation after intracerebral hemorrhage. Neuroscience,2008,155(4): 1133-1141.
11 Wu H, Cong Y, Wang D, et al. Correlation of macrophage inflammatory protein-2 expression and brain edema in rats after intracerebral hemorrhage. Int J Clin Exp Pathol,2009,2(1):83-90.
12 Kyung Jin J, Eun Kyung G, Ji Young K, et al. Suppression of age-related renal changes in NF-κB and its target gene expression by dietary ferulate. J Nutr Biochem,2009,20(5):378-388.
13 Zhang X, Li H, Hu S, et al. Brain edema after intracerebral hemorrhage in rats:the role of inflammation. Neurol India,2006,54(4):402-407.
14 Jian W,Sylvain D. Inflammation after intracerebral hemorrhage. J Cereb Blood Flow Metab,2007,27(5):894-908.
15 Aronowski J,Hall C. New horizons for primary intracerebral hemorrhage treatment:experience from preclinical studies. Neurol Res,2005,27(3): 268-279.
16 Wu S, Fang C, Kim J, et al. Enhanced pulmonary inflammation following experimental intracerebral hemorrhage. Exp Neurol,2006,200(1):245-259.
17 Muir E, Adcock K, Morgenstern D, et al. Matrix metalloproteases and their inhibitors are produced by overlapping populations of activated astrocytes. Brain Res Mol Brain Res,2002,100(1-2):103-117.
18 Hsieh H, Wu C, Yang C. Bradykinin induces matrix metalloproteinase-9 expression and cell migration through a PKC--dependent ERK/Elk-1 pathway in astrocytes. Glia,2008,56(6):619-632.
19 Mori T, Wang X, Aoki T, et al. Down regulation of matrix metalloproteinase-9 and attenuation of edema via inhibition of ERK mitogen activated protein kinase in traumatic brain injury. J Neurotrauma,2002,19(11):1411-1419.
20 Kawai N, Kawanishi M, Nagao S. Treatment of cold injury-induced brain edema with a nonspecific matrix metalloproteinase inhibitor MMI270 in rats. Acta Neurochir Suppl,2003,86:291-295.
21 吕磊,蔡定芳.线粒体依赖的凋亡通路与脑出血后神经损伤.国际神经病学神经外科学杂志,2008,35(2)：132-136.
22 KJ Y, DJ K, SH K, et al. Anti-inflammatory effects of sinapic acid through the suppression of inducible nitric oxide synthase, cyclooxygase-2, and proinflammatory cytokines expressions via nuclear factor-kappaB inactivation. J Agric Food Chem,2008,56(21):10265-10272.
23 Campos-Esparza M, Sanchez-Gomez M, Matute C. Molecular mechanisms of neuroprotection by two natural antioxidant polyphenols. Cell Calcium 2009, 45(4):358-368.