NADPH氧化酶NOX1、NOX2和DUOX2在溃疡性结肠炎中的表达分析
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摘要
目的探讨NADPH氧化酶家族(nicotinamide adenine dinucleotide phosphate oxidases,NOXs)的主要成员NOX1、NOX2和双氧化物酶(dual oxidase,DUOX)2(人:NOX1、NOX2和DUOX2;小鼠:Nox1、Nox2和Duox2)在人炎症性肠病和小鼠肠炎模型结肠中的表达。方法人结肠标本选自于通过结肠镜活检或手术切除的炎症性肠病组织及距离病变组织边缘5 cm以上正常组织;同时选用8~10周龄的C57BL/6雌性小鼠建立结肠炎模型,采用掷硬币法将其随机分为对照组和慢性肠炎组,慢性肠炎组先自由饮用1.0%(质量分数)葡聚糖硫酸钠7 d,然后自由饮水14 d,循环3次;通过体质量变化和HE染色方法评估肠道炎性反应程度。采用实时定量PCR技术和免疫组织化学方法分别检测结肠组织中NOX1、NOX2及DUOX2 mRNA和蛋白表达情况,并分析在人炎症性肠病中三者表达情况与临床特征之间的关系。结果 NOX1、NOX2和DUOX2 mRNA在人炎症性肠病结肠组织中的表达量均显著高于自身正常对照组织,分别增高2.8、2.0和3.3倍(P均<0.01);与人不同,Nox1和Duox2 mRNA在小鼠慢性肠炎结肠组织中的表达量差异无统计学意义,Nox2 mRNA增加2.4倍(P<0.01)。Nox1蛋白在正常结肠上皮细胞刷状缘和胞质中表达;Nox2蛋白主要表达于浸润的吞噬细胞和中性粒细胞;Duox2蛋白在正常结肠黏膜组织中低表达;三者在人炎症性肠病结肠组织中表达均上调(均P<0.01);在小鼠慢性肠炎模型中,Nox2和Duox2蛋白上调(均P<0.01),而Nox1无变化。除了NOX2 mRNA在男性病人表达高于女性病人外,未发现这些氧化酶与病人临床特征之间有关联。结论NOX1、NOX2和DUOX2不但在维持机体正常生理功能过程中发挥重要作用,而且在炎症性肠病初期阶段的发病中也起一定的作用。
Objective To investigate the expression of NOX1,NOX2 and DUOX2,the members of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase family,in large intestine of patients with inflammatory bowel disease(IBD) and mouse colitis and analyze the relationship between these enzymes and IBD. Methods The samples were collected from large intestine of patients with IBD. Mouse chronic colitis model was established by using eight to ten-week-old C57BL/6 mice treated with three cycles of drinking 1. 0% dextran sulfate sodium(DSS) for 7 days plus drinking water for 14 days. Mouse body weight change and hematoxylin-eosin(HE) staining of colon sections were used to evaluate the severity of inflammatory lesions. The expression of NOX1,NOX2 and DUOX2 gene and protein were detected by real-time quantitative reverse transcription polymerase chain reaction(RT-PCR) and immunohistochemistry,respectively. Results The expression of NOX1,NOX2 and DUOX2 mRNA significantly increased in human inflamed tissues compared to paired normal tissues(all P < 0. 01). NOX2 mRNA higher in male patients than female. Unlike human,only Nox2 mRNA increased by 2. 4 times in chronic colitis mice,Nox1 and Duox2 mRNA remained unchanged. These three proteins in IBD patients increased(all P < 0. 01); in chronic colitis miceNox2 and Duox2 proteins increased(both P < 0. 01),but Nox1 did not. Conclusion NOX1,NOX2 and DUOX2 not only play an important role in maintaining the normal physiological function,but also were associated with early onset IBD.
Objective To investigate the expression of NOX1,NOX2 and DUOX2,the members of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase family,in large intestine of patients with inflammatory bowel disease(IBD) and mouse colitis and analyze the relationship between these enzymes and IBD. Methods The samples were collected from large intestine of patients with IBD. Mouse chronic colitis model was established by using eight to ten-week-old C57BL/6 mice treated with three cycles of drinking 1. 0% dextran sulfate sodium(DSS) for 7 days plus drinking water for 14 days. Mouse body weight change and hematoxylin-eosin(HE) staining of colon sections were used to evaluate the severity of inflammatory lesions. The expression of NOX1,NOX2 and DUOX2 gene and protein were detected by real-time quantitative reverse transcription polymerase chain reaction(RT-PCR) and immunohistochemistry,respectively. Results The expression of NOX1,NOX2 and DUOX2 mRNA significantly increased in human inflamed tissues compared to paired normal tissues(all P < 0. 01). NOX2 mRNA higher in male patients than female. Unlike human,only Nox2 mRNA increased by 2. 4 times in chronic colitis mice,Nox1 and Duox2 mRNA remained unchanged. These three proteins in IBD patients increased(all P < 0. 01); in chronic colitis miceNox2 and Duox2 proteins increased(both P < 0. 01),but Nox1 did not. Conclusion NOX1,NOX2 and DUOX2 not only play an important role in maintaining the normal physiological function,but also were associated with early onset IBD.
引文
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[15]Jones R M,Luo L,Ardita C S,et al.Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species[J].EMBO J,2013,32(23):3017-3028.
[16]杨茉莉,李宏谦,李小珍,等.NADPH氧化酶Nox1和Duox2在小鼠肠炎中的表达和意义[J].世界华人消化杂志,2015,23(10):1560-1567.
[17]Kato M,Marumo M,Nakayama J,et al.The ROS-generating oxidase Nox1 is required for epithelial restitution following colitis[J].Exp Anim,2016,65(3):197-205.
[18]Yokota H,Tsuzuki A,Shimada Y,et al.NOX1/NADPH oxidase expressed in colonic macrophages contributes to the pathogenesis of colonic inflammation in trinitrobenzene sulfonic acid-induced murine colitis[J].J Pharmacol Exp Ther,2017,360(1):192-200.
[19]Szanto I,Rubbia-Brandt L,Kiss P,et al.Expression of NOX1,a superoxide-generating NADPH oxidase,in colon cancer and inflammatory bowel disease[J].J Pathol,2005,207(2):164-176.
[20]Chen L,Xu B,Liu L,et al.Cadmium induction of reactive oxygen species activates the m TOR pathway,leading to neuronal cell death[J].Free Radic Biol Med,2011,50(5):624-632.
[21]Singel K L,Segal B H.NOX2-dependent regulation of inflammation[J].Clin Sci,2016,130(7):479-490.
[22]Hayes P,Dhillon S,O'Neill K,et al.Defects in NADPH oxidase genes NOX1 and DUOX2 in very early onset inflammatory bowel disease[J].Cell Mol Gastroenterol Hepatol,2015,1(5):489-502.
[23]Dhillon S S,Fattouh R,Elkadri A,et al.Variants in nicotinamide adenine dinucleotide phosphate oxidase complex components determine susceptibility to very early onset inflammatory bowel disease[J].Gastroenterol,2014,147(3):680-689.e2.
[24]Nauseef W M.Biological roles for the NOX family NADPH oxidases[J].J Biol Chem,2008,283(25):16961-16965.
[25]Brown D I,Griendling K K.Nox proteins in signal transduction[J].Free Radic Biol Med,2009,47(9):1239-1253.
[26]Lipinski S,Till A,Sina C,et al.DUOX2-derived reactive oxygen species are effectors of NOD2-mediated antibacterial responses[J].J Cell Sci,2009,122(Pt 19):3522-3530.
[27]Achitei D,Ciobica A,Balan G,et al.Different profile of peripheral antioxidant enzymes and lipid peroxidation in active and non-active inflammatory bowel disease patients[J].Dig Dis Sci,2013,58(5):1244-1249.
[28]杜莉莉,吕润潇,杨晓漪,等.胎盘间充质干细胞低氧培养液对肠黏膜上皮细胞氧化应激损伤的保护作用[J].中国医科大学学报,2016,45(2):131-135,140.
[29]Chu F F,Esworthy R S,Doroshow J H,et al.Deficiency in Duox2 activity alleviates ileitis in GPx1-and GPx2-knockout mice without affecting apoptosis incidence in the crypt epithelium[J].Redox Biol,2016(11):144-156.
[2]Maloy K J,Powrie F.Intestinal homeostasis and its breakdown in inflammatory bowel disease[J].Nature,2011,474(7351):298-306.
[3]Biasi F,Leonarduzzi G,Oteiza P I,et al.Inflammatory bowel disease:mechanisms,redox considerations,and therapeutic targets[J].Antioxid Redox Signal,2013,19(14):1711-1747.
[4]Circu M L,Aw T Y.Intestinal redox biology and oxidative stress[J].Semin Cell Dev Biol,2012,23(7):729-737.
[5]Rada B,Leto T L.Oxidative innate immune defenses by Nox/Duox family NADPH oxidases[J].Contrib Microbiol,2008,15:164-187.
[6]Bhattacharyya A,Chattopadhyay R,Mitra S,et al.Oxidative stress:an essential factor in the pathogenesis of gastrointestinal mucosal diseases[J].Physiol Rev,2014,94(2):329-354.
[7]Cifuentes-Pagano E,Csanyi G,Pagano P J.NADPH oxidase inhibitors:a decade of discovery from Nox2ds to HTS[J].Cell Mol Life Sci:CMLS,2012,69(14):2315-2325.
[8]Katsuyama M,Matsuno K,Yabe-Nishimura C.Physiological roles of NOX/NADPH oxidase,the superoxide-generating enzyme[J].J Clin Biochem Nutr,2012,50(1):9-22.
[9]Lam G,Apostolopoulos V,Zulli A,et al.NADPH oxidases and inflammatory bowel disease[J].Curr Med Chem,2015,22(17):2100-2109.
[10]Ouyang Q,Xue L Y.Inflammatory bowel disease in the 21(st)century in China:turning challenges into opportunities[J].J Dig Dis,2012,13(4):195-199.
[11]Jemal A,Bray F,Center M M,et al.Global cancer statistics[J].CA:Cancer J Clin,2011,61(2):69-90.
[12]Bedard K,Krause K H.The NOX family of ROS-generating NADPH oxidases:physiology and pathophysiology[J].Physiol Rev,2007,87(1):245-313.
[13]韩晓燕,高丽萍,刘箐.NOX家族蛋白的研究进展[J].生命科学,2012,24(6):568-577.
[14]Reuter S,Gupta S C,Chaturvedi M M,et al.Oxidative stress,inflammation,and cancer:how are they linked?[J].Free Radic Biol Med,2010,49(11):1603-1616.
[15]Jones R M,Luo L,Ardita C S,et al.Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species[J].EMBO J,2013,32(23):3017-3028.
[16]杨茉莉,李宏谦,李小珍,等.NADPH氧化酶Nox1和Duox2在小鼠肠炎中的表达和意义[J].世界华人消化杂志,2015,23(10):1560-1567.
[17]Kato M,Marumo M,Nakayama J,et al.The ROS-generating oxidase Nox1 is required for epithelial restitution following colitis[J].Exp Anim,2016,65(3):197-205.
[18]Yokota H,Tsuzuki A,Shimada Y,et al.NOX1/NADPH oxidase expressed in colonic macrophages contributes to the pathogenesis of colonic inflammation in trinitrobenzene sulfonic acid-induced murine colitis[J].J Pharmacol Exp Ther,2017,360(1):192-200.
[19]Szanto I,Rubbia-Brandt L,Kiss P,et al.Expression of NOX1,a superoxide-generating NADPH oxidase,in colon cancer and inflammatory bowel disease[J].J Pathol,2005,207(2):164-176.
[20]Chen L,Xu B,Liu L,et al.Cadmium induction of reactive oxygen species activates the m TOR pathway,leading to neuronal cell death[J].Free Radic Biol Med,2011,50(5):624-632.
[21]Singel K L,Segal B H.NOX2-dependent regulation of inflammation[J].Clin Sci,2016,130(7):479-490.
[22]Hayes P,Dhillon S,O'Neill K,et al.Defects in NADPH oxidase genes NOX1 and DUOX2 in very early onset inflammatory bowel disease[J].Cell Mol Gastroenterol Hepatol,2015,1(5):489-502.
[23]Dhillon S S,Fattouh R,Elkadri A,et al.Variants in nicotinamide adenine dinucleotide phosphate oxidase complex components determine susceptibility to very early onset inflammatory bowel disease[J].Gastroenterol,2014,147(3):680-689.e2.
[24]Nauseef W M.Biological roles for the NOX family NADPH oxidases[J].J Biol Chem,2008,283(25):16961-16965.
[25]Brown D I,Griendling K K.Nox proteins in signal transduction[J].Free Radic Biol Med,2009,47(9):1239-1253.
[26]Lipinski S,Till A,Sina C,et al.DUOX2-derived reactive oxygen species are effectors of NOD2-mediated antibacterial responses[J].J Cell Sci,2009,122(Pt 19):3522-3530.
[27]Achitei D,Ciobica A,Balan G,et al.Different profile of peripheral antioxidant enzymes and lipid peroxidation in active and non-active inflammatory bowel disease patients[J].Dig Dis Sci,2013,58(5):1244-1249.
[28]杜莉莉,吕润潇,杨晓漪,等.胎盘间充质干细胞低氧培养液对肠黏膜上皮细胞氧化应激损伤的保护作用[J].中国医科大学学报,2016,45(2):131-135,140.
[29]Chu F F,Esworthy R S,Doroshow J H,et al.Deficiency in Duox2 activity alleviates ileitis in GPx1-and GPx2-knockout mice without affecting apoptosis incidence in the crypt epithelium[J].Redox Biol,2016(11):144-156.