黄芩苷对兔晶状体上皮细胞的抑制及对NF-κB表达的影响
摘要
目的:研究黄芩苷对兔晶状体上皮细胞增殖及对NF-κB表达的影响,初步探讨黄芩苷对减轻后囊混浊的作用,为后囊膜混浊的药物治疗开辟一条新的途径。
方法:
1.兔晶状体上皮细胞的原代培养及传代:将健康无眼疾成年家兔静脉空气栓塞处死后,取眼球,截取前囊膜剪成1mm×1mm大小的碎片接种于培养瓶内,Ⅰ型胶原酶消化,每2- 3天换液一次,约2周后细胞可长满大部分瓶底。用0.25%胰酶消化后1:2传代,选用2- 3代细胞用于实验
2.设立不同浓度、不同作用时间的药物组及空白对照组
3.MTT法检测各时间各浓度药物对兔晶状体上皮细胞的抑制率
4.流式细胞技术测定作用48h各浓度组细胞早期凋亡率
5.半定量逆转录聚合酶链反应法(RT- PCR)检测不同浓度黄芩苷作用兔晶状体上皮细胞中NF -κB p65亚基的表达。
结果:
1.MTT结果显示黄芩苷能抑制兔晶状体上皮细胞的增殖,抑制率与时间、浓度呈正相关。48小时半数抑制浓度为46.32μg/m1。
2.流式细胞学检测证实黄芩苷能有效诱导兔晶状体上皮细胞的凋亡,细胞早期凋亡率呈现浓度依赖性递增。
3.RT-PCR结果显示,与空白对照组,黄芩苷作用组NF-κB p65 mRNA表达明显降低,差异有统计学意义(P<0.01)。
结论:
1.黄芩苷能有效抑制兔晶状体上皮细胞的增殖,诱导其凋亡。
2.黄芩苷对兔晶状体上皮细胞的抑制可能与NF-κB表达下调有关,NF-κB表达异常可能参与后囊膜混浊的发生发展。
Aim To investigate the effects of Baicalin on proliferation inhibition and apoptosis induction in rabbit lens epithelial cell and to explore its underlying mechanisms.
Methods rabbit lens epithelial cell were exposed to baicalin at diferent dosages and its proliferation inhibition was detected by MTT assay.The ability of baicalin to induce lens epithelial cell apoptosis was examined by Annexin V-FITC/PI double staining analysis.The mRNA expressions of NF-κB P65 were detected by RT-PCR.
Results Baicalin remarkably inhibited the rabbit lens epithelial cell proliferation, with an IC50 value of 46.32μg/m1 .Apoptosis was remarkably induced by baicalin in a dose-dependent manner,its earlier stages were detected by annexin V-FITC/PI double staining analysis.RT-PCR showed that the mRNA expressions of NF-κB p65 in rabbit lens epithelial cell decreased in a dose-dependent manner.
Conclusion Baicalin efficiently induces proliferation inhibition and apoptosis in rabbit lens epithelial cell,which may be related with the down-regulation of NF-κB.
方法:
1.兔晶状体上皮细胞的原代培养及传代:将健康无眼疾成年家兔静脉空气栓塞处死后,取眼球,截取前囊膜剪成1mm×1mm大小的碎片接种于培养瓶内,Ⅰ型胶原酶消化,每2- 3天换液一次,约2周后细胞可长满大部分瓶底。用0.25%胰酶消化后1:2传代,选用2- 3代细胞用于实验
2.设立不同浓度、不同作用时间的药物组及空白对照组
3.MTT法检测各时间各浓度药物对兔晶状体上皮细胞的抑制率
4.流式细胞技术测定作用48h各浓度组细胞早期凋亡率
5.半定量逆转录聚合酶链反应法(RT- PCR)检测不同浓度黄芩苷作用兔晶状体上皮细胞中NF -κB p65亚基的表达。
结果:
1.MTT结果显示黄芩苷能抑制兔晶状体上皮细胞的增殖,抑制率与时间、浓度呈正相关。48小时半数抑制浓度为46.32μg/m1。
2.流式细胞学检测证实黄芩苷能有效诱导兔晶状体上皮细胞的凋亡,细胞早期凋亡率呈现浓度依赖性递增。
3.RT-PCR结果显示,与空白对照组,黄芩苷作用组NF-κB p65 mRNA表达明显降低,差异有统计学意义(P<0.01)。
结论:
1.黄芩苷能有效抑制兔晶状体上皮细胞的增殖,诱导其凋亡。
2.黄芩苷对兔晶状体上皮细胞的抑制可能与NF-κB表达下调有关,NF-κB表达异常可能参与后囊膜混浊的发生发展。
Aim To investigate the effects of Baicalin on proliferation inhibition and apoptosis induction in rabbit lens epithelial cell and to explore its underlying mechanisms.
Methods rabbit lens epithelial cell were exposed to baicalin at diferent dosages and its proliferation inhibition was detected by MTT assay.The ability of baicalin to induce lens epithelial cell apoptosis was examined by Annexin V-FITC/PI double staining analysis.The mRNA expressions of NF-κB P65 were detected by RT-PCR.
Results Baicalin remarkably inhibited the rabbit lens epithelial cell proliferation, with an IC50 value of 46.32μg/m1 .Apoptosis was remarkably induced by baicalin in a dose-dependent manner,its earlier stages were detected by annexin V-FITC/PI double staining analysis.RT-PCR showed that the mRNA expressions of NF-κB p65 in rabbit lens epithelial cell decreased in a dose-dependent manner.
Conclusion Baicalin efficiently induces proliferation inhibition and apoptosis in rabbit lens epithelial cell,which may be related with the down-regulation of NF-κB.
引文
[1]葛坚.眼科学(八年制)[M].北京:人民卫生出版社,2005:75～76
[2]Ishibashi T. collagen types in human posterior capsule opacification[J]. Cataract Refract Surg. 1994,20(8): 643～648
[3]柳玉霞,李瑞庄.后发性白内障的发生机制及防治研究现状[J].医学综述.2008,l1(14):3266～3269
[4]Nishi O, Nishi K, Yamada Y, et a1. Effect of indomethaein-coated posterior chamber intraocular lenses on postoperative inflanmmtion and posteriorcapsule opacification[J]. J Cataract Reflact Surg. 1995,2(153): 574～578
[5]Chen L, Greene W.C. Shaping the nuclear action of NF-kappaB[J]. Nat Rev Mol Cell Biol.2004,5:392～401
[6]Niranjan Awasthi, Suqin Guo. Posterior Capsular Opacification A Problem Reduced but Not Yet Eradicated[J]. Arch Ophthalmol. 2009,127(4):555～562
[7]宁宏,张劲松等.后发性白内障形成中晶体上皮细胞PCNA的表达[J].中国实用眼科杂志.2002,20(7):508～510
[8]胡世林,冯学锋.黄芩研究的某些新进展[J].中国药学杂志.2001,36(11):728～730
[9]刘菊福,卢长安,廖福龙.不同产地黄芩提取物主要药效作用的比较[J].中国中医药信息杂志.2001,8(3):28～32
[10]董玲婉,吕圭源.浅谈中药黄芩的药理作用[J].浙江中医药大学学报. 2007,11(31):787～788
[11]张黎,胡燕华. Baicalin抑制人血管内皮细胞ECV-304的增殖和迁移[J].江西医学院学报. 2004, 44(2): 11～14
[12]程国强,冯年平,唐琦文等.黄芩甙对眼科常见病原菌的体外抗菌作用[J].中国医院药学杂志. 2001, 21(6):384～385
[13]张黎,胡燕华.Baicalin对兔角膜上皮细胞的毒性分析眼科研究[J]. 2007, 25(7): 537～539
[14]俞燕,杨于.黄芩甙对大鼠感染性脑水肿NF-κB活性的影响[J].中国当代儿科杂志,2000, 12(26): 386～389
[15]张永钦,周井炎.黄芩甙对白内障晶状体作用的研究[J].眼科研究. 1998, 16(9): 173～175
[1] Sen R, Baltimore D. Inducibility of Kappa Immunoglobulin Enhancer binding Protein Nf-kappa B by a Posttranslational Mechanism[J]. Cell. l986,47(6):92l～928
[2] Hoffmann A, Natoli G, and Ghosh, G. Transcriptional regulation via the NF-kappaB signaling module[J]. Oncogene.2006,25(4): 6706～6716
[3]Balwin AS. The NF-κB and IκB proteins:new discoveries and insights [J]. Annu Rev Immunol.1996,14:649～683
[4] Chen L, Greene W.C. Shaping the nuclear action of NF-kappaB[J]. Nat Rev Mol Cell Biol.2004,5:392～401
[5] Chen L, Mu Y and Greene W.C. Acetylation of RelA at discretesites regulates distinct nuclear functions of NF-kappaB[J]. EMBO J.2002,21: 6539～6548
[6]Sato S, Sanjo H, Takeda K, et al. Essential function for the kinaseTAK1 in innate and adaptive immune responses[J]. Nat Immunol.2005, 6: 1087～1095
[7] Senftleben U, Cao Y, Xiao G, et al. Activation by IKKalpha of a second evoluti- -onary conservedNF-kappa B signaling pathway[J]. 2001. Science 293, 1495～1499
[8]Matthew S, Hayden and Sankar G.Shared Principles in NF-κB Signaling [J].Cell.2008,132(3):344～362
[9] Basak S, Kim H, Kearns J.D.et al.A fourth IkappaB protein within the NF-kappaB signaling module[J].Cell.2007,128:369～381
[10] Covert M W, Leung T H, Gaston J E,et a1. Achieving Stability of Lipopoly- -saccharide induced NF-kappaB Activation[J]. Science. 2005, 309(5742): l854～1857
[11] Wertz I E, O’Rourke K M, Zhou H, et a1. De-ubiquitination and Ubiquitin Ligase Domains of A20 Downregulate NF-kappaB Signalling[J]. Nature, 2004,430(7000):694～699
[12] Werner S L, Barken D, Hoffmann A. Stimulus Specificity of Gene Expression Programs Determined by Temporal Controlof IKK Activity [J]. Science. 2005,309(5742):1857～1861
[13]葛坚.眼科学(八年制)[M].北京:人民卫生出版社,2005:75～76
[14]孙红,惠延年,王琳,等.晶状体上皮细胞凋亡与白内障发生的分子生物学机制[J].中国临床康复. 2003,3(7):398～401
[15] Alexander G, Carlsen H, Blomhoff R. Strong in vivo activation of NF-kappaB in mouse lenses by classic stressors[J]. Invest Ophthalmol Vis Sci. 2003 , 44(6): 2683～2688
[16]徐周兴,胡建章,林鸿,等.年龄相关性白内障晶状体上皮细胞的超微结构研究.国际眼科杂志.2004,4(4):631～632
[17] Dudek EJ, Shang F, Taylor A. H2O2-mediated oxidative stress activates NF -kappa B in lens epithelial cells. Free Radic Biol Med. 2001 ,31(5):651～658
[18] Wu M, Bian Q,Liu Y, Fernandes AF, et al. Sustained oxidative stress inhibits NF-kappaB activation partially via inactivating the proteasome. Free Radic Biol Med. 2009 ,46(1):62～69
[19]赵宏伟,唐罗生,游志鹏.PDTC对过氧化氢诱导鼠晶状体上皮细胞NF-κB活化表达的影响.眼科新进展.2004,24(1): 26～30
[20] Lee SJ, Bae S, Seomun Y, et al. The role of nuclear factor kappa B in lens epithelial cell proliferation using a capsular bag model[J]. Ophthalmic Res. 2008, 40(5):273～278
[21] Yadav UC, Ighani-Hosseinabad Prevention of posterior capsular opacification through aldose reductase inhibition. Invest Ophthalmol Vis Sci. 2009, 50(2): 752～759
[22] Nambu H, Kubo E, Takamura Y,et al. Attenuation of aldose reductase gene suppresses high-glucose-induced apoptosis and oxidative stress in rat lens epith- -elial cells. Diabetes Res Clin Pract. 2008, 82(1):18～24
[23]张晶,赵桂秋,车成业.大鼠顿挫性眼外伤所致白内障动物模型中晶状体上皮细胞IKKα的表达[J].眼科研究.2008,26(7):147～151
[24]赵宏伟,唐罗生.大鼠外伤性白内障晶体上皮细胞NF-κB mRNA表达的实验研究[J].创伤外科杂志.2OO4,6(3):200～202
[25]赵宏伟,唐罗生.鼠外伤性白内障晶体上皮细胞NF-κB活化表达的实验研究.Chinese J.of Today’S Med. 2004,4(2):2～4
[26] Yang Qian ,Zhao Gui xiang. Expression of nuclear factor-κB in traumatic catarac. Chinese Journal of Traumatology. 2006,9(2):86～90
[27] Bakunowicz, Arczyk A, Moniuszko T, et al. Levels of selected cytokines in aqueous humor of patients with cataract extraction[J]. K1in Oczna .1997,99:235～237
[28] Yao J, Liu Y . UVB radiation induces human lens epithelial cell migration via NADPH oxidase-mediated generation of reactive oxygen species and up regula- -tion of matrix metalloproteinases. Int J Mol Med. 2009,24(2):153～159
[29] Boileau TW, Bray TM, Bomser JA. Ultraviolet radiation modulates nuclear factor kappa B activation in human lens epithelial cells[J]. J Biochem Mol Toxicol. 2003,17(2):108～113
[2]Ishibashi T. collagen types in human posterior capsule opacification[J]. Cataract Refract Surg. 1994,20(8): 643～648
[3]柳玉霞,李瑞庄.后发性白内障的发生机制及防治研究现状[J].医学综述.2008,l1(14):3266～3269
[4]Nishi O, Nishi K, Yamada Y, et a1. Effect of indomethaein-coated posterior chamber intraocular lenses on postoperative inflanmmtion and posteriorcapsule opacification[J]. J Cataract Reflact Surg. 1995,2(153): 574～578
[5]Chen L, Greene W.C. Shaping the nuclear action of NF-kappaB[J]. Nat Rev Mol Cell Biol.2004,5:392～401
[6]Niranjan Awasthi, Suqin Guo. Posterior Capsular Opacification A Problem Reduced but Not Yet Eradicated[J]. Arch Ophthalmol. 2009,127(4):555～562
[7]宁宏,张劲松等.后发性白内障形成中晶体上皮细胞PCNA的表达[J].中国实用眼科杂志.2002,20(7):508～510
[8]胡世林,冯学锋.黄芩研究的某些新进展[J].中国药学杂志.2001,36(11):728～730
[9]刘菊福,卢长安,廖福龙.不同产地黄芩提取物主要药效作用的比较[J].中国中医药信息杂志.2001,8(3):28～32
[10]董玲婉,吕圭源.浅谈中药黄芩的药理作用[J].浙江中医药大学学报. 2007,11(31):787～788
[11]张黎,胡燕华. Baicalin抑制人血管内皮细胞ECV-304的增殖和迁移[J].江西医学院学报. 2004, 44(2): 11～14
[12]程国强,冯年平,唐琦文等.黄芩甙对眼科常见病原菌的体外抗菌作用[J].中国医院药学杂志. 2001, 21(6):384～385
[13]张黎,胡燕华.Baicalin对兔角膜上皮细胞的毒性分析眼科研究[J]. 2007, 25(7): 537～539
[14]俞燕,杨于.黄芩甙对大鼠感染性脑水肿NF-κB活性的影响[J].中国当代儿科杂志,2000, 12(26): 386～389
[15]张永钦,周井炎.黄芩甙对白内障晶状体作用的研究[J].眼科研究. 1998, 16(9): 173～175
[1] Sen R, Baltimore D. Inducibility of Kappa Immunoglobulin Enhancer binding Protein Nf-kappa B by a Posttranslational Mechanism[J]. Cell. l986,47(6):92l～928
[2] Hoffmann A, Natoli G, and Ghosh, G. Transcriptional regulation via the NF-kappaB signaling module[J]. Oncogene.2006,25(4): 6706～6716
[3]Balwin AS. The NF-κB and IκB proteins:new discoveries and insights [J]. Annu Rev Immunol.1996,14:649～683
[4] Chen L, Greene W.C. Shaping the nuclear action of NF-kappaB[J]. Nat Rev Mol Cell Biol.2004,5:392～401
[5] Chen L, Mu Y and Greene W.C. Acetylation of RelA at discretesites regulates distinct nuclear functions of NF-kappaB[J]. EMBO J.2002,21: 6539～6548
[6]Sato S, Sanjo H, Takeda K, et al. Essential function for the kinaseTAK1 in innate and adaptive immune responses[J]. Nat Immunol.2005, 6: 1087～1095
[7] Senftleben U, Cao Y, Xiao G, et al. Activation by IKKalpha of a second evoluti- -onary conservedNF-kappa B signaling pathway[J]. 2001. Science 293, 1495～1499
[8]Matthew S, Hayden and Sankar G.Shared Principles in NF-κB Signaling [J].Cell.2008,132(3):344～362
[9] Basak S, Kim H, Kearns J.D.et al.A fourth IkappaB protein within the NF-kappaB signaling module[J].Cell.2007,128:369～381
[10] Covert M W, Leung T H, Gaston J E,et a1. Achieving Stability of Lipopoly- -saccharide induced NF-kappaB Activation[J]. Science. 2005, 309(5742): l854～1857
[11] Wertz I E, O’Rourke K M, Zhou H, et a1. De-ubiquitination and Ubiquitin Ligase Domains of A20 Downregulate NF-kappaB Signalling[J]. Nature, 2004,430(7000):694～699
[12] Werner S L, Barken D, Hoffmann A. Stimulus Specificity of Gene Expression Programs Determined by Temporal Controlof IKK Activity [J]. Science. 2005,309(5742):1857～1861
[13]葛坚.眼科学(八年制)[M].北京:人民卫生出版社,2005:75～76
[14]孙红,惠延年,王琳,等.晶状体上皮细胞凋亡与白内障发生的分子生物学机制[J].中国临床康复. 2003,3(7):398～401
[15] Alexander G, Carlsen H, Blomhoff R. Strong in vivo activation of NF-kappaB in mouse lenses by classic stressors[J]. Invest Ophthalmol Vis Sci. 2003 , 44(6): 2683～2688
[16]徐周兴,胡建章,林鸿,等.年龄相关性白内障晶状体上皮细胞的超微结构研究.国际眼科杂志.2004,4(4):631～632
[17] Dudek EJ, Shang F, Taylor A. H2O2-mediated oxidative stress activates NF -kappa B in lens epithelial cells. Free Radic Biol Med. 2001 ,31(5):651～658
[18] Wu M, Bian Q,Liu Y, Fernandes AF, et al. Sustained oxidative stress inhibits NF-kappaB activation partially via inactivating the proteasome. Free Radic Biol Med. 2009 ,46(1):62～69
[19]赵宏伟,唐罗生,游志鹏.PDTC对过氧化氢诱导鼠晶状体上皮细胞NF-κB活化表达的影响.眼科新进展.2004,24(1): 26～30
[20] Lee SJ, Bae S, Seomun Y, et al. The role of nuclear factor kappa B in lens epithelial cell proliferation using a capsular bag model[J]. Ophthalmic Res. 2008, 40(5):273～278
[21] Yadav UC, Ighani-Hosseinabad Prevention of posterior capsular opacification through aldose reductase inhibition. Invest Ophthalmol Vis Sci. 2009, 50(2): 752～759
[22] Nambu H, Kubo E, Takamura Y,et al. Attenuation of aldose reductase gene suppresses high-glucose-induced apoptosis and oxidative stress in rat lens epith- -elial cells. Diabetes Res Clin Pract. 2008, 82(1):18～24
[23]张晶,赵桂秋,车成业.大鼠顿挫性眼外伤所致白内障动物模型中晶状体上皮细胞IKKα的表达[J].眼科研究.2008,26(7):147～151
[24]赵宏伟,唐罗生.大鼠外伤性白内障晶体上皮细胞NF-κB mRNA表达的实验研究[J].创伤外科杂志.2OO4,6(3):200～202
[25]赵宏伟,唐罗生.鼠外伤性白内障晶体上皮细胞NF-κB活化表达的实验研究.Chinese J.of Today’S Med. 2004,4(2):2～4
[26] Yang Qian ,Zhao Gui xiang. Expression of nuclear factor-κB in traumatic catarac. Chinese Journal of Traumatology. 2006,9(2):86～90
[27] Bakunowicz, Arczyk A, Moniuszko T, et al. Levels of selected cytokines in aqueous humor of patients with cataract extraction[J]. K1in Oczna .1997,99:235～237
[28] Yao J, Liu Y . UVB radiation induces human lens epithelial cell migration via NADPH oxidase-mediated generation of reactive oxygen species and up regula- -tion of matrix metalloproteinases. Int J Mol Med. 2009,24(2):153～159
[29] Boileau TW, Bray TM, Bomser JA. Ultraviolet radiation modulates nuclear factor kappa B activation in human lens epithelial cells[J]. J Biochem Mol Toxicol. 2003,17(2):108～113