慢性间歇低氧大鼠的血压变化及其颈动脉体中还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶的表达

目的 探讨慢性间歇低氧(chronic intermittent hypoxia,CIH)大鼠的血压变化及其颈动脉体中还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶的表达情况,以明确CIH致血压升高的可能机制.方法 清洁级雄性SD大鼠30只按随机数字表法分为CIH组、慢性持续缺氧组及对照组.尾袖法测量大鼠尾动脉收缩压,采用RT-PCR法检测大鼠颈动脉体中NADPH氧化酶各亚基gp91 phox、p22phox及p47phox mRNA的表达,对颈动脉体行免疫组织化学染色,并对p22phox的表达情况行半定量分析.结果 CIH组大鼠的尾动脉收缩压为[(145±11)mm Hg,1 mm Hg=0.133kPa],较CIH组(129±9)mm Hg及对照组(124±7)mm Hg显著升高(F值为19.895,P＜0.01),CIH组gp91phox、p22phox及p47phox mRNA的表达(分别为2.82±0.51、2.74±0.45和2.88±0.47)较慢性持续缺氧组(分别为2.35±0.42、2.25±0.38和2.41±0.43)及对照组(分别为2.23±0.35、2.16±0.30和2.30±0.36)显著升高(F值分别为5.794、6.854和7.163,P＜0.01)免疫组织化学检查结果显示CIH组p22phox蛋白相对表达量(99±12)较其他两组(分别为38±7和34±8)增多.结论 CIH可刺激大鼠颈动脉体中NADPH氧化酶的表达上调并使血压升高,NADPH氧化酶在颈动脉体中的过度表达可能与OSAHS患者发生高血压病存在相关联系.

Abstract：

Objective Chronic intermittent hypoxia (CIH) occurs in patients with obstructive sleep apnea-hyponea syndrome (OSAHS) and has adverse effects on multiple physiological functions. Previous studies have shown that reflexes arising from carotid bodies mediate CIH evoked circulation-respiratory responses, and reactive oxygen species (ROS) play important roles in eliciting systemic responses to CIH.But very little is known about the molecular mechanisms underlying CIH. NADPH oxidase is the most important sources of ROS. In the present study we examined changes of blood pressure and expression of NADPH oxidase in carotid body in rats exposed to intermittent hypoxia. Methods Thirty healthy male SD rats were randomly divided into 3 groups, a CIH group, a chronic continuous hypoxia group and a control group. The systolic blood pressure (SBP) was measured with tail-cuff method. RT-PCR was used to examine mRNA expressions of NADPH oxidase subunit gp91phox, p22phox, p47phox. Immunohistochemistry and semiquantitative analysis of NADPH oxidase subunits p22phox were done in the carotid body sections of all rats. Results Compared with normal group [ ( 124 ± 7 ) mm Hg, 1 mm Hg = 0. 133 kPa ] and chronic continuous hvpoxia group[ (129 ± 9) mm Hg], the SBP in CIH group [( 145 ± 11 ) mm Hg] was significantly higher( F = 19. 895, P ＜0. 01 ＝, and the expression of NADPH oxidase subunits gp91phox,p22phox,p47phox mRNA in CIH group ( 2. 82 ± 0. 51, 2. 74 ± 0. 45, 2. 88 ± 0. 47, respectively ) were significantly higher than those in chronic continuous hypoxia group ( 2. 35 ± 0. 42, 2. 25 ± 0. 38, 2. 41 ±0. 43, respectively)and normal group(2. 23 ±0. 35, 2. 16 ±0. 30, 2. 30 ±0. 36, respectively) ( F =5.794,6. 854, 7. 163, respectively, P ＜ 0. 01 ). The Immunohistochemistry and semiquantitative analysis showed that the expression of NADPH oxidase subunit p22phox in the carotid body in CIH group ( 99 ± 12 ) were more than those in chronic continuous hypoxia and control groups ( 38 ± 7 and 34 ± 8, P ＜ 0. 05 ).Conclusion CIH upregulates the expression of NADPH oxidase in rat carotid body and elevates the rat SBP. These results indicate that NADPH oxidase up-expression is closely associated with OSAHS patients with hypertension.     

阻塞性睡眠呼吸暂停低通气综合征患者诱导痰中烟酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox水平与病情的关系

目的 观察OSAHS患者诱导痰中烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶p22phox的水平,探讨OSAHS患者气道局部氧化应激与病情严重程度的关系.方法 对2007年9月至2008年6月华中科技大学附属同济医院呼吸科就诊并进行睡眠监测的30例OSAHS患者和23名健康体检者分别行痰诱导,睡前和晨起各1次.其中OSAHS组男27例,女3例,平均年龄(43±9)岁;健康对照组男21名,女2名,平均年龄(45±10)岁.痰细胞涂片并行细胞分类计数,以逆转录PCR法检测诱导痰细胞中NADPH氧化酶p22phox mRNA水平,以免疫细胞化学法检测诱导痰细胞中NADPH氧化酶p22phox蛋白的表达水平.结果 OSAHS组诱导痰细胞中巨噬细胞百分比为(34±10)%,低于对照组的(66±5)%;而中性粒细胞百分比为(65±10)%,高于对照组的(33±5)%,两组比较,差异均有统计学意义(t=15.051、14.359,P<0.05).诱导痰细胞中NADPH氧化酶p22phox阳性细胞主要为中性粒细胞和巨噬细胞.OSAHS组2次诱导痰细胞中NADPH氧化酶p22phox mRNA水平及中性粒细胞和巨噬细胞阳性率均明显高于对照组.OSAHS组晨起时NADPH氧化酶p22phoxmRNA的表达水平及中性粒细胞和巨噬细胞阳性率高于睡前,并与最低脉搏氧饱和度呈负相关,与呼吸暂停低通气指数呈正相关.结论 OSAHS患者存在气道局部NADPH氧化酶p22phox表达水平的变化,并与病情严重程度相关.     

NADPH氧化酶对门静脉海绵样变大鼠体内氧化应激的影响

目的: 考察NADPH氧化酶对门静脉海绵样变(cavernous transformation of portal vein, CTPV)大鼠体内氧化应激的影响.方法: 将大鼠按随机抓取的方式分为假手术组、CTPV(门静脉海绵样变性)模型组. 采用门脉部分结扎法复制CTPV大鼠动物模型;测定门静脉内血清超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活力及丙二醛(MDA)、门静脉组织一氧化氮(NO)、内皮型一氧化氮合酶(eNOS)的含量;采用实时荧光定量RT-PCR法测定门静脉组织NADPH氧化酶p22phox以及gp91phox亚基的mRNA表达.结果: 与S h am组大鼠相比, CT PV组大鼠SOD、GSH-Px活性(酶活力单位)降低(93.79±8.87 μU/L vs 103.05±8.07 μU/L, 157.44±26.46 vs 709.09+83.21, 均P<0.05), 而MDA含量增加(5.33±0.35 μmol/L vs 3.59±0.44μmol/L, P<0.01);实时荧光定量RT-PCR显示门静脉NADPH氧化酶亚基gp91phox以及p22phox的mRNA表达增强(16.77±3.27 vs1.31±0.95, 11.64±7.34 vs 1.93±0.86, 均P<0.01);门静脉组织NO含量及eNOS活性均降低(2.33±0.82 μmol/L vs 85.00±3.16μmol/L, 0.24±0.11 U/mg prot vs 1.76±0.78U/mg prot, 均P<0.01).结论: C T P V大鼠体内氧化应激状态与NADPH氧化酶亚基gp91phox以及p22phoxmRNA过表达有关;NADPH氧化酶依赖的氧化应激可能与CTPV大鼠门静脉内皮功能障碍的发生发展密切相关.     

晚期糖基化终产物对大鼠血管外膜成纤维细胞中烟酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox亚基及活性氧表达的影响

目的:探讨晚期糖基化终产物(AGE)对大鼠血管外膜成纤维细胞(AF)中烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶p22phox亚基及活性氧表达的影响。方法:用组织贴块法培养SD大鼠的血管外膜成纤维细胞,用逆转录—聚合酶链反应、蛋白质印迹检测不同浓度的糖基化人血清白蛋白(AGE-HSA,分为对照组、100μg/ml AGE-HSA组,200μg/ml AGE-HSA组、300μg/ml AGE-HSA组)对NADPH氧化酶p22phox亚基信使核糖核酸(mRNA)及蛋白的表达的影响,并观察不同干预因素[分为对照组1、200μg/ml AGE-HSA组(200μg/ml处理组1)、200μg/ml AGE-HSA+50μg/ml抗RAGE中和抗体组(抗RAGE中和抗体组1)及200μg/ml AGE-HSA+30 nmol/L坎地沙坦组(坎地沙坦组1)对AGE-HSA上调NADPH氧化酶p22phox亚基mRNA和蛋白表达的影响。用2’,7’-二氯荧光黄双乙酸盐检测不同干预因素[空白对照组、AGE-HSA 200μg/ml组(200μg/ml处理组2)、200μg/ml AGE-HSA+50μg/ml抗RAGE中和抗体组(抗RAGE中和抗体组2)、200μg/ml AGE-HSA+30μmol/L夹竹桃素组(夹竹桃组),200μg/ml AGE-HSA+30 nmol/L坎地沙坦组(坎地沙坦组2)对血管外膜成纤维细胞内活性氧表达的影响。结果:3个浓度(100μg/ml、200μg/ml和300μg/ml)的AGE-HSA组均与对照组比较,血管外膜成纤维细胞NADPH氧化酶p22phox亚基mRNA及蛋白的表达随AGE-HSA增加呈浓度依赖性上调;抗RAGE中和抗体组1、坎地沙坦组1比200μg/ml处理组1的p22phox mRNA及蛋白表达降低;抗RAGE中和抗体组2、夹竹桃素组2及坎地沙坦组2比200μg/mlAGE-HSA处理组2血管外膜成纤维细胞内活性氧相对荧光强度降低,上述比较差异均有统计学意义(P<0.05)。结论:AGE-HSA经由RAGE影响活性氧与NADPH氧化酶p22phox亚基的表达上调,活性氧的上调与NADPH氧化酶p22phox亚基表达相关。NADPH氧化酶抑制剂及坎地沙坦可通过下调NADPH氧化酶p22phox亚基的表达减少血管外膜成纤维细胞内活性氧的产生。     

辛伐他汀对高糖损伤乳鼠心肌细胞NADPH氧化酶亚基基因表达的影响

取原代培养的SD雄性乳鼠心肌细胞在高糖刺激下加入10-7、10-6和10-5mol/L辛伐他汀作用72 h.结果显示,与对照组比较,高糖组心肌细胞活力明显降低(P＜0.01),乳酸脱氢酶(LDH)活性显著增加(P＜0.01),NADPH氧化酶亚基p22phox、p47phox mRNA表达和活性氧簇(ROS)水平明显增高(均P＜0.05);与高糖组比较,辛伐他汀各组心肌细胞活力明显增加(P＜0.05),LDH活性显著降低(P＜0.05),p22phox、p47phox mRNA表达和ROS水平明显降低,且辛伐他汀浓度对心肌细胞活力的影响呈剂量依赖效应.这些结果提示辛伐他汀能够抑制NADPH氧化酶亚基的基因表达,减轻高糖引起的心肌损伤.     

NADPH氧化酶在肿瘤坏死因子-α致乳鼠心肌细胞肥大中的作用

目的 探讨NADPH氧化酶来源的活性氧(ROS)在肿瘤坏死因子(TNF)-α诱导乳鼠心肌细胞肥大中的作用.方法 原代培养SD乳鼠心肌细胞,分为正常对照组,TNF-α组(80ng/ml),APO(100 μmol/L)组和TNF-α+APO组.BCA法测定心肌细胞蛋白合成总量.荧光染料(DCFH-DA)及激光共聚焦显微镜检测心肌细胞活性氧水平.RT-PCR检测p22phox mRNA的表达.免疫细胞化学染色法检测心肌细胞p22phox的表达.结果 TNF-α可显著增加心肌细胞ROS生成,APO显著抑制TNF-α刺激后ROS的产生.TNF-α促进心肌细胞p22phox mRNA和蛋白的表达,APO可抑制TNF-α的作用.结论 TNF-α可能通过上调NADPH氧化酶亚基p22phox的表达,促进心肌产生ROS增加,导致了心肌细胞肥大的发生.     

辛伐他汀对高糖损伤乳鼠心肌细胞氧化应激的影响

目的探讨辛伐他汀对高糖损伤乳鼠心肌细胞内还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性氧通路的干预作用。方法培养新生1～3 d SD雄性大鼠心肌细胞,随机分为对照组、高浓度葡萄糖刺激组(GS组)、不同浓度辛伐他汀干预组[分别为GS+10~(-7) mol/L Sim组(GS+10~(-7) Msim组)、GS+10~(-6) mol/L Sim组(GS+10~(-6) MSim组)、GS+10~(-5) mol/L Sim组(GS++10~(-5) MSim组),以MTT比色法测定各组心肌细胞活力;化学比色法测定心肌细胞丙二醛含量、超氧化物歧化酶活力及活性氧水平:RT-PCR检测细胞内NADPH氧化酶p22phox mRNA、p47phox mRNA的表达水平。结果与GS组比较,GS+10~(-7) MSim组、GS+10~(-6) MSim组、GS+10~(-5) MSim组丙二醛含量、活性氧水平明显降低.p22phox mRNA、p47phox mRNA表达明显下调,超氧化物歧化酶活力明显升高(P<0.05)。结论心肌细胞内NADPH氧化酶源性的活性氧升高是介导高糖损伤心肌细胞的重要机制,辛伐他汀可能通过抑制NADPH氧化酶-活性氧通路减轻心肌细胞损伤。     

1,6-二磷酸果糖对病毒性心肌炎小鼠心肌烟酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox亚基蛋白表达的影响

目的 观察1,6-二磷酸果糖(FDP)对病毒性心肌炎小鼠心肌还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶p22phox亚基蛋白表达的影响,探讨FDP对病毒性心肌炎的保护作用.方法 4周龄雌性BALB/c小鼠30只,体质量(12±2)g.按体质量将小鼠随机分为病毒组和治疗组,每组15只.两组小鼠同时1次性腹腔注射柯萨奇B3病毒(CVB3) 0.1 ml,治疗组在注射CVB3后的第1天,每日腹腔注射1次FDP,连续注射7d,注射剂量为300 mg/kg.在注射结束后的第4、8、21天,两组分别各处死5只小鼠,取心脏做心肌病理检查,Western免疫印迹法检测病毒性心肌炎小鼠心肌细胞内NADPH氧化酶p22phox亚基蛋白表达,图像分析系统测量p22phox亚基蛋白阳性表达区域平均吸光度(A)值,并进行定量分析.结果 感染后第4天,光镜下病毒组小鼠心肌间可见少量炎症细胞浸润,心肌细胞肿胀;治疗组小鼠心肌间仅有少量炎性细胞浸润.感染后第8天,病毒组小鼠心肌出现坏死性崩解,大量炎症细胞浸润;治疗组小鼠心肌间可见稀疏的散在炎性细胞浸润.感染后第21天,病毒组小鼠心肌坏死灶中有慢性炎症细胞浸润,出现结缔组织增生;治疗组小鼠心肌可见少量慢性炎症细胞浸润.病毒组在第8天炎症浸润最严重.在病毒感染后第4、8、21天,治疗组心肌病变积分[(0.88±0.23)、(2.20±0.24)、(1.56±0.17)分]低于病毒组[(1.32±0.12)、(3.0±0.25)、(2.04±0.17)分,t值分别为3.793、5.1645、4.457,P均＜0.01].Western免疫印迹法分析结果显示,在病毒感染后第4、8、21天,治疗组NADPH氧化酶p22phox亚基蛋白表达(0.776±0.017、0.751±0.018、0.689±0.034)明显低于病毒组(1.052±0.015、0.952±0.019、0.907±0.025,t值分别为3.391、6.716、2.750,P均＜0.01或＜0.05).结论 FDP能下调NADPH氧化酶p22phox亚基蛋白表达,FDP可能通过改变NADPH酶的表达对心肌发挥保护性作用.     

血管紧张素Ⅱ和NADPH氧化酶与血管衰老的相关性研究

目的探讨血管紧张素Ⅱ（AngⅡ）和NADPH氧化酶在血管衰老中的地位及作用机理.方法健康Wistar大鼠分为青年组、老龄组、Valsantan组,分析各组大鼠主动脉形态结构及功能;测定血浆和主动脉AngⅡ水平、主动脉活性氧水平;分别应用RT-PCR和Western bolt检测各组大鼠AngⅡ1型和2型受体（AT1R和AT2R）、NADPH氧化酶p22phox的mRNA及蛋白表达.结果随增龄大鼠主动脉管壁增厚,纤维化程度增高,内皮功能受损,活性氧产生增加;主动脉AngⅡ含量增高,AT2R、p22phox的mRNA及蛋白表达上调,AT1R表达下降;Valsantan（AngⅡ1型受体特异性阻断剂）干预后,p22phox表达下降,活性氧水平降低,衰老血管形态结构和功能异常有所改善.结论衰老血管有其特征性结构和功能改变;AngⅡ经由AT1R上调NADPH氧化酶的基因表达可能是血管衰老的重要机制之一.     

辛伐他汀对心肌梗死后心力衰竭大鼠心肌氧化负荷和心室重构的影响

目的了解辛伐他汀对急性心肌梗死（AMI）后心衰大鼠心室重构和心功能的影响,并研究NADPH氧化酶亚基p47phox的变化以研究辛伐他汀在CHF中可能的作用机制。方法随机选取雄性Sprague Dawly,通过结扎大鼠左冠状动脉前降支制作大鼠AMI模型致CHF,并随机选取大鼠做假手术对照。AMI术后大鼠被随机分为大剂量辛伐他汀组[hSIM,4mg/（kg·d）],小剂量辛伐他汀组[lSIM,0.4mg/（c）]与单纯模型组。干预12周后测体质量（BM）,左室质量（LVM）等,并行血流动力学测定。选取左室非梗死区心肌检测p47phox mRNA相对表达率及蛋白含量。结果单纯模型组、hSIM和lSIM组LVM均较假手术组增加;hSIM和lSIM组平均最大左室压力变化（m±dp/dtmax）均较单纯模型组升高,p47phox mRNA表达、p47phox含量均降低;p47phox含量hSIM和lSIM组组有显著性差异。结论在AMI后CHF大鼠,大体检查和血流动力学检测可证实辛伐他汀减轻CHF的发展。其机制与NADPH氧化酶亚单位p47phox介导产生的反应性氧簇有关。     

手把区域多肽及氯沙坦抑制左旋谷氨酸钠大鼠腹部脂肪组织还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶亚单位的表达

目的 探讨手把区域多肽（HRP）及氯沙坦对左旋谷氨酸钠（MSG）大鼠胰岛素敏感性的影响及探讨其对腹腔脂肪组织局部肾素血管紧张素系统（RAS）和还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶亚单位基因表达的影响.方法 将8周龄体重在250～300 g的MSG大鼠共24只采用完全随机法分为MSG对照组（MSG组,n=6）、HRP干预组（MSG-HRP组,n=6,1.0 mg·d-1·kg-1）、氯沙坦干预组（MSG-L组,n=6,450 mg/L于饮用水中）、HRP及氯沙坦联合干预组（MSG-HRP-L组,n=6）,干预4周,正常SD大鼠为对照组（Con组,n=6）.12周龄时予以行胰岛素耐量试验评估大鼠胰岛素敏感性,计算胰岛素注射后30 min与0 rain时血糖比值.测定单位质量腹腔脂肪组织（前）肾素、（前）肾素受体[（P）RR]和选择性血管紧张素Ⅱ1型受体（AT1R） mRNA的表达及血管紧张素-Ⅱ（Ang-Ⅱ）蛋白水平,测定NADPH氧化酶亚单位P47phox和P22phoxmRNA的表达情况.采用方差分析及LSD两两比较法进行统计学分析.结果 外源性胰岛素注射后计算30 min血糖与基础血糖的比值,MSG组最高（92％±12％）,与Con组（66％±8％）、MSG-HRP组（76％±5％）、MSG-L组（78％±5％）、MSG-HRP-L组（75％±10％）比较均有统计学差异（F=6.875,P＜0.05）.本研究未能检测到腹腔脂肪组织中（前）肾素mRNA的表达.MSG-HRP组、MSG-L组、MSG-HRP-L组（P） RR mRNA表达量分别是MSG组大鼠的1.92、3.19和1.90倍（F=9.805,P＜0.05）.MSG-HRP组、MSG-L组、MSG-HRP-L组AT1 R mRNA分别是MSG大鼠组表达量的72％、45％和53％（F=14.508,P＜0.05）.与MSG组比较,MSG-HRP组脂肪局部Ang-Ⅱ水平下调[分别为（36±8）比（56±4） ng/g蛋白,P＜0.05],但是MSG-L组和MSG-HRP-L组水平均明显升高[分别为（79±14）比（70±16）比（56±4） ng/g蛋白,F=14.864,均P＜0.05].MSG-HRP组、MSG-L组、MSG-HRP-L组腹腔脂肪组织中P47phox mRNA分别是MSG大鼠组表达量的65％、51％和43％（F=7.082,均P＜0.05）.p22pho mRNA分别是MSG大鼠组表达量的57％、40％和41％（F=9.810,均P＜0.05）.结论 HRP和氯沙坦均可改善MSG大鼠胰岛素敏感性,其机制可能是其减少脂肪组织局部Ang-Ⅱ的数量或抑制脂肪组织中局部Ang-Ⅱ的效应,抑制氧化应激,从而改善脂肪胰岛素抵抗.     

Role of extracellular superoxide dismutase in the mouse angiotensin slow pressor response

Low rates of angiotensin II (Ang II) infusion raise blood pressure, renal vascular resistance (RVR), NADPH oxidase activity, and superoxide. We tested the hypothesis that these effects are ameliorated by extracellular superoxide dismutase (EC-SOD). EC-SOD knockout (-/-) and wild type (+/+) mice were equipped with blood pressure telemeters and infused subcutaneously with Ang II (400 ng/kg per minute) or vehicle for 2 weeks. During vehicle infusion, EC-SOD -/- mice had significantly (P<0.05) higher MAP (+/+: 107+/-3 mm Hg versus -/-: 114+/-2 mm Hg; n=11 to 14), RVR, lipid peroxidation, renal cortical p22(phox) expression, and NADPH oxidase activity. Ang II infusion in EC-SOD +/+ mice significantly (P<0.05) increased MAP, RVR, p22(phox), NADPH oxidase activity, and lipid peroxidation. Ang II reduced SOD activity in plasma, aorta, and kidney accompanied by reduced renal EC-SOD expression. During Ang II infusion, both groups had similar values for MAP (+/+ Ang II: 125+/-3 versus -/- Ang II: 124+/-3 mmHg; P value not significant), RVR, NADPH oxidase activity, and lipid peroxidation. SOD activity in the kidneys of Ang II-infused mice was paradoxically higher in EC-SOD -/- mice (+/+: 8.8+/-1.2 U/mg protein(-1) versus -/-: 13.7+/-1.6 U/mg protein(-1); P<0.05) accompanied by a significant upregulation of mRNA and protein for Cu/Zn-SOD. In conclusion, EC-SOD protects normal mice against oxidative stress by attenuating renal p22(phox) expression, NADPH oxidase activation, and the accompanying renal vasoconstriction and hypertension. However, during an Ang II slow pressor response, renal EC-SOD expression is reduced and, in its absence, renal Cu/Zn-SOD is upregulated and may prevent excessive Ang II-induced renal oxidative stress, renal vasoconstriction, and hypertension.     

上海地区汉族人群NADPH氧化酶p22phox编码基因CYBA—A930G多态性与高血压性脑出血的相关性

目的研究上海汉族人群中NADPH氧化酶p22phox亚基-A930G基因多态性与高血压性脑出血的相关性。方法纳入高血压性脑出血患者和正常对照者,采用聚合酶链反应和限制性片段长度多态性技术检测NADPH氧化酶p22pb“亚基-A930G基因型和等位基因。结果共纳入高血压脑出血患者128例和对照组151例。高血压脑出血组收缩压、舒张压、血糖和三酰甘油水平以及吸烟和饮酒的患者构成比显著性高于对照组（P均〈0．05）,AA、AG和GG基因型（42．2％、44．5％、13．3％对63．6％、27．8％、8．6％;x212．757,P=0．002）以及A、G等位基因（64．5％、35．5％对77．5％、22．5％;X2：8．734,P=0．001）频率与对照组均存在显著性差异。多变量logistic回归分析显示,收缩压≥140mmHg（1mmHg=0．133kPa）[优势比（oddsratio,OR）13．952,95％可信区间（confidence interval,CI．242～26．879;P〈0．001]、载脂蛋白A≥0．99mmol／L（OR 3．139,95％CI 1．012～9．733;P=0．048）和AG＋GG基因型（OR2．333,95％CI 1．253～4．342;P=0．008）为高血压性脑出血的独立危险因素。结论在中国上海地区汉族人群中,NADPH氧化酶D22phox亚基～A930G多态性是高血压性脑出血的独立危险因素。     

NAD(P)H oxidase inhibition improves endothelial function in rat and human blood vessels

The NO/superoxide (O2-) balance is a key regulator of endothelial function. O2- levels are elevated in many forms of cardiovascular disease; therefore, decreasing O2- should improve endothelial function. To explore this hypothesis, internal mammary arteries and saphenous veins, obtained from patients undergoing coronary artery revascularization, and aortic and carotid arteries from Wistar-Kyoto and spontaneously hypertensive stroke-prone rats were incubated with O2- dismutase or NAD(P)H oxidase inhibitors. O2- levels were measured using lucigenin chemiluminescence; NO bioavailability was assessed in organ chambers; and mRNA expression of NAD(P)H oxidase components was quantified by use of a Light Cycler. In rat arteries, phenylarsine oxide, 4-(2-aminoethyl)-benzenesulfanyl fluoride, and apocynin all decreased NADH-stimulated O2- production, but only apocynin increased NO bioavailability. In human internal mammary arteries and saphenous veins, apocynin decreased NAD(P)H-stimulated O2- generation and caused vasorelaxation that was endothelium dependent and reversed on addition of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester. In addition, it increased NO production from cultured human endothelial saphenous vein cells. Polyethylene-glycolated O2- dismutase also increased NO bioavailability in rat carotid arteries and human blood vessels, but the effects were smaller than those observed with apocynin. NADH-generated O2- and mRNA expression of p22(phox), gp91(phox), and nox-1 were comparable between the 2 strains of rat. This is the first study to demonstrate pharmacological effects of apocynin in human blood vessels. The increases in NO bioavailability shown here suggest that the NAD(P)H oxidase pathway may be a novel target for drug intervention in cardiovascular disease.     

Rosuvastatin, a 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor, decreases cardiac oxidative stress and remodeling in Ren2 transgenic rats

Angiotensin-II (Ang-II)-stimulated increases in nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity and oxidative stress are known to play a key role in cardiac remodeling. Inhibition of isoprenylation and activation of small G proteins, such as Rac1, a component of NADPH oxidase, may mediate the antioxidant actions of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins). In this study, we investigated the effects of rosuvastatin on cardiac oxidative stress and remodeling in transgenic rats (Ren2) overexpressing the mouse renin gene with elevated cardiac levels of Ang-II. We treated 6- to 7-wk-old Ren2 rats and age-matched Sprague-Dawley (SD) rats with rosuvastatin (10 mg/kg.d) or vehicle for 3 wk. At the end of the treatment period, left ventricular mass, wall thickness, ejection fraction (by echocardiography), and cardiac remodeling (by light microscopy and immunohistochemistry) were assessed. In addition, myocardial content of nitrotyrosine, malondialdehyde, NADPH-oxidase subunits (gp91(phox), p40(phox), and p22(phox)), and Rac1 were analyzed by immunochemistry. Systolic blood pressure was significantly higher in Ren2 rats, compared with SD rats (P < 0.05); rosuvastatin had no significant effect on systolic blood pressure in either group. In Ren2, but not SD rats, rosuvastatin significantly improved the ventricular ejection fraction, cardiac hypertrophy, and perivascular fibrosis (P < 0.05). In addition, rosuvastatin administration significantly decreased the accentuated myocardial gp91(phox), p40(phox), p22(phox), and Rac1 expression. These changes were accompanied by a parallel reduction in myocardial lipid peroxidation (nitrotyrosine and malondialdehyde content) (P < 0.05). These results suggest that in vivo statin treatment through its direct actions on the heart reduces oxidative stress and remodeling including ventricular mass regression in the Ang-II-dependent Ren2 model.     

胰高血糖素样肽1对1型糖尿病大鼠心肌烟酰胺腺嘌呤二核苷酸氧化酶亚单位表达的影响

目的 探讨胰高血糖素样肽1 （GLP-1）对1型糖尿病大鼠心肌组织烟酰胺腺嘌呤二核苷酸（NADPH）氧化酶亚单位p22phox和Nox4表达的影响.方法 将42只雄性SD大鼠用随机数字表法分为正常对照组（NC组,n=7）和糖尿病造模组（n=35）.采用链脲佐菌素（STZ）对糖尿病造模组制备1型糖尿病大鼠模型.将造模成功的29只1型糖尿病大鼠用随机数字表法分为糖尿病组（DM组,n=10）,糖尿病GLP-1低剂量治疗组（DL组,n=10）和糖尿病GLP-1高剂量治疗组（DH组,n=9）.DL组予以艾塞那肽1μg/kg,2次/d皮下注射,DH组予以艾塞那肽5μg/kg,2次/d皮下注射.艾塞那肽治疗8周后处死动物.用实时荧光定量聚合酶链反应测定4组大鼠心肌p22phox和Nox4 mRNA的表达,免疫组化法检测心肌铜-锌-超氧化物歧化酶（Cu-Zn-SOD）蛋白表达.多组比较采用单因素方差分析.结果 与NC组比较,DM组大鼠心肌p22phox和Nox4 mRNA表达显著升高（t=5.77、5.36,均P＜0.05）,心肌Cu-Zn-SOD蛋白表达显著升高（t=59.91,P＜0.05）.艾塞那肽治疗8周后,与DM组比较,DL组和DH组大鼠心肌p22phox和Nox4 mRNA表达均显著降低（t=16.86、7.66和16.11、7.59,均P＜0.05）,心肌Cu-Zn-SOD蛋白表达均显著降低（t=56.00、47.05,均P＜0.05）.与DL组比较,DH组大鼠心肌p22phox和Nox4 mRNA表达显著降低（t=10.14、8.67,均P ＜0.05）,而心肌Cu-Zn-SOD表达无明显变化（=81.91,P＞0.05）.结论 GLP-1可剂量依赖性地下调1型糖尿病大鼠心肌p22phox和Nox4的表达,非剂量依赖性地上调心肌Cu-Zn-SOD表达,减轻氧化应激对心肌的损害,对糖尿病大鼠心肌产生保护作用.     

Leptin regulates cardiomyocyte contractile function through endothelin-1 receptor-NADPH oxidase pathway

Leptin, the obese gene product, plays an important role in the regulation of cardiac function. However, the mechanism behind leptin-induced cardiomyocyte contractile response is poorly understood. This study was designed to examine whether endothelin-1 receptor and NADPH oxidase play any role in leptin-induced cardiac contractile response. Isolated murine cardiomyocytes were exposed to leptin (5, 50, and 100 nmol/L) for 60 minutes in the absence or presence of the ETA receptor antagonist BQ123 (1 micromol/L), the ETB receptor antagonist BQ788 (1 micromol/L), or the NADPH oxidase inhibitor apocynin (100 micromol/L) before mechanical function was studied. Superoxide levels were measured by dihydroethidium fluorescent dye and the superoxide dismutase-inhibitable reduction of cytochrome c. NADPH oxidase subunit expression (p22phox, p47phox, p67phox, and gp91phox) was evaluated with Western blot. Leptin depressed peak shortening and maximal velocity of shortening/relengthening (+/-dL/dt), prolonged the duration of relengthening (TR90) without affecting the time-to-peak cell shortening. Consistent with the mechanical characteristics, myocytes treated with leptin displayed a reduced electrically stimulated rise in intracellular Ca2+ (change in fura-2 fluorescence intensity) associated with a prolonged intracellular Ca2+ decay rate. All of the abnormalities were significantly attenuated by apocynin, BQ123, or BQ788. Intracellular superoxide generation was enhanced after leptin treatment, which was partially blocked by apocynin, BQ123, or BQ788. Leptin had no effect on p22phox and gp91phox but upregulated protein expression of p67phox and p47phox, both of which were inhibited by apocynin, BQ123, or BQ788. These results suggest that leptin suppresses cardiac contractile function in ventricular myocytes through the endothelin-1 receptor and NADPH oxidase-mediated pathway.