NADPH氧化酶Nox-4在不同亚型GERD患者食管上皮中的表达及DIS发生中的作用
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摘要
目的探讨烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶4(Nox-4)在不同亚型胃食管反流病(GERD)患者食管上皮中的表达及其在食管上皮细胞间隙增宽(DIS)发生中的意义。方法选取2017年10月至2018年8月在新疆维吾尔自治区人民医院住院的36例GERD患者设为研究组,同期完成体检的健康受试者9例设为对照组。GERD组行24 h pH监测、高分辨率食管测压监测并内镜下取食管齿状线上3 cm处的局部黏膜作为标本,后续采用HE染色、免疫组化、实时荧光定量反转录-聚合酶链式反应(real-time RT-PCR)、酶联免疫吸附测定(ELISA)等方法检测细胞间隙、氧化应激(Nox-4)、核因子红系相关因子2(Nrf-2)、血红素加氧酶-1(HO-1)以及紧密连接蛋白(TJ蛋白)[咬合蛋白(Occludin)、闭合蛋白-1(Claudin-1)、闭合蛋白-4(Claudin-4)及带状闭合蛋白-1(ZO-1)]的相对表达量。结果食管24 h pH监测显示,RE组酸反流(pH≤4)次数以及DeMeester评分明显高于NERD组,差异有统计学意义(P<0.05)。RE组食管下段括约肌(LES)长度、LES静息压、LES残余压及食管远端收缩积分(DCI)等指标略高于NERD组,但两组比较无统计学差异(P>0.05)。对照组、NERD、RE组GERD患者光镜下测量的细胞间隙分别为(0.64±0.08)μm、(1.14±0.11)μm和(1.27±0.10)μm,两两相比均有统计学差异(P<0.01)。对照组、NERD、RE组三组间Nox-4 mRNA和血清Nox-4浓度比较有统计学差异(P<0.01),RE组Nox-4 mRNA和血清Nox-4浓度最高,对照组最低。Nrf-2、HO-1、Occludin、Claudin-1、Claudin-4及ZO-1mRNA表达水平在NERD组、RE组分别低于对照组,差异有统计学意义(P<0.01)。结论 TJ蛋白在不同亚型GERD患者中表达量明显降低,而这些蛋白的低表达可能与酸反流引起食管Nox-4的过量产生有关。
Objective To investigate the expression of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase 4(Nox-4) in the esophageal epithelium of patients with different subtypes of gastroesophageal reflux disease(GERD) and the meaning of Nox-4 in the occurrence of dilated intercellular space(DIS).Methods Thirty-six patients with GERD admitted to the People′s Hospital of Xinjiang Uygur Autonomous Region from October 2017 to August 2018 were enrolled as study group.Nine healthy subjects who completed the physical examination at the same time were set as the control group.In the GERD group,24 h pH monitoring,high-resolution esophageal manometry was used,and endoscopic local mucosa specimen was taken from 3 cm above the dentate line of the esophagus.Then HE staining,immunohistochemistry,and reverse transcription-polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA) were used to detect intercellular space,and the relative expression of oxidative stress(Nox-4),nuclear factor erythroid-derived factor 2-related factor 2(Nrf-2),heme oxygenase-1(HO-1) and tight junction proteins(TJ proteins)[Occludin,Claudin-1,Claudin-4,and zonula occludens-1(ZO-1)].Results Esophageal 24 h pH monitoring showed that the number of acid reflux(pH≤4) and DeMeester score in RE group was significantly higher than that in NERD group(P<0.05).The lower esophageal sphincter(LES) length,LES resting pressure,LES residual pressure and distal contractile integral(DCI) in the RE group were slightly higher than those in the NERD group,but there was no significant difference between the two groups(P>0.05).The intercellular space measured by light microscopy in the control group,NERD group,and RE group were(0.64±0.08)μm,(1.14±0.11)μm,and(1.27±0.10)μm,respectively,and the differences among the pairuise comparisons were statistical significant(P<0.01).There were significant differences in the concentrations of Nox-4 mRNA and serum Nox-4 among the control group,NERD group and RE groups(P<0.01).The concentration of Nox-4 mRNA and serum Nox-4 was the highest in the RE group and the lowest in the control group.The mRNA expression levels of Nrf-2,HO-1,Occludin,Claudin-1,Claudin-4 and ZO-1 in the NERD group and the RE group were significantly lower than those in the control group(P<0.01),respectively.Conclusion The expression of TJ protein was significantly decreased in patients with different subtypes of GERD,and the low expression may be related to the overproduction of esophageal Nox-4 caused by acid reflux.
Objective To investigate the expression of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase 4(Nox-4) in the esophageal epithelium of patients with different subtypes of gastroesophageal reflux disease(GERD) and the meaning of Nox-4 in the occurrence of dilated intercellular space(DIS).Methods Thirty-six patients with GERD admitted to the People′s Hospital of Xinjiang Uygur Autonomous Region from October 2017 to August 2018 were enrolled as study group.Nine healthy subjects who completed the physical examination at the same time were set as the control group.In the GERD group,24 h pH monitoring,high-resolution esophageal manometry was used,and endoscopic local mucosa specimen was taken from 3 cm above the dentate line of the esophagus.Then HE staining,immunohistochemistry,and reverse transcription-polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA) were used to detect intercellular space,and the relative expression of oxidative stress(Nox-4),nuclear factor erythroid-derived factor 2-related factor 2(Nrf-2),heme oxygenase-1(HO-1) and tight junction proteins(TJ proteins)[Occludin,Claudin-1,Claudin-4,and zonula occludens-1(ZO-1)].Results Esophageal 24 h pH monitoring showed that the number of acid reflux(pH≤4) and DeMeester score in RE group was significantly higher than that in NERD group(P<0.05).The lower esophageal sphincter(LES) length,LES resting pressure,LES residual pressure and distal contractile integral(DCI) in the RE group were slightly higher than those in the NERD group,but there was no significant difference between the two groups(P>0.05).The intercellular space measured by light microscopy in the control group,NERD group,and RE group were(0.64±0.08)μm,(1.14±0.11)μm,and(1.27±0.10)μm,respectively,and the differences among the pairuise comparisons were statistical significant(P<0.01).There were significant differences in the concentrations of Nox-4 mRNA and serum Nox-4 among the control group,NERD group and RE groups(P<0.01).The concentration of Nox-4 mRNA and serum Nox-4 was the highest in the RE group and the lowest in the control group.The mRNA expression levels of Nrf-2,HO-1,Occludin,Claudin-1,Claudin-4 and ZO-1 in the NERD group and the RE group were significantly lower than those in the control group(P<0.01),respectively.Conclusion The expression of TJ protein was significantly decreased in patients with different subtypes of GERD,and the low expression may be related to the overproduction of esophageal Nox-4 caused by acid reflux.
引文
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[11] 买买提·依斯热依力,吾布力卡斯木·吾拉木,克力木·阿不都热依木.TRPV-1、PAR-2在GERD患者食管粘膜中的表达及其临床意义[J].健康世界,2018,26(14):17-19.
[12] 刘贝妮,姚树坤.食管黏膜屏障功能及其检测方法研究进展[J].胃肠病学和肝病学杂志,2014,23(12):1373-1375.
[13] Oh TY,Lee JS,Ahn BO,et al.Oxidative damages are critical in pathogenesis of reflux esophagitis:implication of antioxidants in its treatment[J].Free Radic Biol Med,2001,30(8):905-915.
[14] 刘荣泉,那吉,陶家丽,等.贲门松弛与胃食管反流病患者的食管下段DIS改变[J].胃肠病学和肝病学杂志,2016,25(1):62-65.
[15] Chen H,Hu Y,Fang Y,et al.Nrf2 deficiency impairs the barrier function of mouse esophageal epithelium[J].Gut,2014,63(5):711-719.
[16] Souza RF,Huo XF,Mittal V,et al.Gastroesophageal reflux might cause esophagitis through a cytokine-mediated mechanism rather than caustic acid injury[J].Gastroenterology,2009,137(5):1776-1784.
[2] 克力木·阿不都热依木,张成,汪忠镐.胃食管反流病与食管裂孔疝外科临床研究现状与争议[J].中华胃食管反流病电子杂志,2014,1(1):4-6.
[3] 杜智,张成,克力木,等.高分辨率食管测压与食管24h pH监测在胃食管反流病中的应用价值[J].中华胃食管反流病电子杂志,2015,2(3):147-151.
[4] Farré R,De Vos R,Geboes K,et al.Critical role of stress in increased oesophageal mucosa permeability and dilated intercellular spaces[J].Gut,2007,56(9):1191-1197.
[5] Knowles CH,Aziz Q.Visceral hypersensitivity in non-erosive reflux disease[J].Gut,2008,57(5):674-683.
[6] 刘荣泉,朱海杭.非糜烂性反流病食管下段鳞状上皮细胞间隙增宽的研究进展[J].国际消化病杂志,2015,35(2):106-108.
[7] Johannessen R,Skogaker N,Halgunset J,et al.A standardized method for measuring intercellular spaces in esophageal biopsies in patients with suspected gastroesophageal reflux disease (the intercellular space ratio)[J].Scand J Gastroenterol,2013,48(11):1235-1241.
[8] 谭佳成,叶必星,林琳,等.胃食管反流病相关的细胞间隙增宽和紧密连接蛋白[J].胃肠病学,2013,18(10):636-638.
[9] Van Itallie CM,Anderson JM.Architecture of tight junctions and principles of molecular composition[J].Semin Cell Dev Biol,2014,36:157-165.
[10] 刘荣泉,傅奕,郑英,等.非糜烂性反流病患者食管上皮细胞钙黏蛋白的表达及细胞间隙的改变[J].山东医药,2015,55(21):88-89.
[11] 买买提·依斯热依力,吾布力卡斯木·吾拉木,克力木·阿不都热依木.TRPV-1、PAR-2在GERD患者食管粘膜中的表达及其临床意义[J].健康世界,2018,26(14):17-19.
[12] 刘贝妮,姚树坤.食管黏膜屏障功能及其检测方法研究进展[J].胃肠病学和肝病学杂志,2014,23(12):1373-1375.
[13] Oh TY,Lee JS,Ahn BO,et al.Oxidative damages are critical in pathogenesis of reflux esophagitis:implication of antioxidants in its treatment[J].Free Radic Biol Med,2001,30(8):905-915.
[14] 刘荣泉,那吉,陶家丽,等.贲门松弛与胃食管反流病患者的食管下段DIS改变[J].胃肠病学和肝病学杂志,2016,25(1):62-65.
[15] Chen H,Hu Y,Fang Y,et al.Nrf2 deficiency impairs the barrier function of mouse esophageal epithelium[J].Gut,2014,63(5):711-719.
[16] Souza RF,Huo XF,Mittal V,et al.Gastroesophageal reflux might cause esophagitis through a cytokine-mediated mechanism rather than caustic acid injury[J].Gastroenterology,2009,137(5):1776-1784.