NADPH氧化酶2活化介导脑内微清蛋白表达下降在小鼠脓毒症相关性脑病中的作用

目的观察脑内NADPH氧化酶2(NOX2)介导微清蛋白(PV)表达变化在脓毒症相关性脑病(SAE)中的作用,并探讨其可能机制。方法采用盲肠结扎穿孔法(CLP)建立SAE模型。60只成年雄性C57BL/6小鼠随机分为四组:对照+溶剂组(A组,n=10)、对照+夹竹桃麻素(apocynin,APO)组(B组,n=10)、CLP+溶剂组(C组,n=20)及CLP+夹竹桃麻素组(D组,n=20)。B、D组术后1h腹腔注射APO 5mg/kg,以后每天注射一次直至术后第10天;其余两组注射等容溶剂。术后第13天行旷场实验测试及条件性恐惧实验训练,第14天行条件性恐惧测试。行为学测试后2h取小鼠皮层及海马,采用Western blot检测其PV和NOX2两个亚基gp91phox、p22phox的表达。结果旷场实验中,四组小鼠总探索路程差异无统计学意义。与A、B组比较,C组中央格停留时间和僵直反应时间明显缩短,并伴有皮层及海马PV表达明显下降,而gp91phox及p22phox表达明显增高(P0.05);与C组比较,D组中央格停留时间和僵直反应时间明显延长,并伴有皮层及海马PV表达明显上升,而gp91phox及p22phox表达明显降低(P0.05)。结论脑内NOX2活化介导PV表达降低可能参与SAE发病。     

神经病理性疼痛致抑郁大鼠海马促炎细胞因子含量的变化

目的观察神经病理性疼痛致抑郁大鼠海马促炎细胞因子含量的变化,并探讨其与抑郁严重程度的关系。方法成年雄性SD大鼠28只,随机分为两组:假手术组(Sham组)和保留性坐骨神经损伤组(SNI组),每组14只。于术前1d及术后7、14、21d测试机械痛阈值(MWT);于术前1~3d测试糖水偏好、强迫游泳实验基础值;术后21~23d进行糖水偏好实验、强迫游泳实验;行为学测试结束后取海马组织,采用ELISA法检测海马中IL-1β、IL-6、TNF-α的含量,并对其与糖水偏好的相关性进行分析。结果与Sham组比较,SNI组大鼠在术后7、14、21d MWT明显降低(P0.001),术后21d糖水偏好明显降低(P0.01),术后23d强迫游泳不动时间明显延长(P0.05);术后21d海马组织IL-6、TNF-α含量明显升高(P0.05或P0.01),而IL-1β含量差异无统计学意义;SNI组海马组织IL-1β(r2=0.60,P0.01)、IL-6(r2=0.55,P0.01)、TNF-α(r2=0.60,P0.01)含量与糖水偏好呈明显负相关。结论神经病理性疼痛致抑郁大鼠海马促炎细胞因子IL-6、TNF-α含量升高,且与抑郁严重程度呈负相关。     

NADPH氧化酶下调海马内微清蛋白表达在老年小鼠术后认知功能障碍中的作用

目的观察海马内微清蛋白(PV)表达变化在老年小鼠术后认知功能障碍(POCD)中的作用,并探讨磷酸酰胺腺嘌呤二核苷酸(NADPH)氧化酶是否参与其中。方法采用异氟醚麻醉+剖腹探查术建立POCD动物模型。雄性16月龄C57BL/6小鼠21只,随机均分为三组(n=7):对照+溶剂组(C+V组)、异氟醚麻醉+剖腹探查+溶剂组(POCD+V组)、异氟醚麻醉+剖腹探查+夹竹桃麻素组(POCD+A组)。POCD+A组术后1h腹腔注射夹竹桃麻素5mg/kg,其后每天注射一次直至术后第7天;其余两组注射等容量的溶剂。术后第6天行旷场实验和条件性恐惧实验训练,第7天在给予药物或溶剂后2h行条件性恐惧测试。行为学测试后2h取小鼠海马组织,采用Western blot检测PV、gp91phox、p22phox及羟基壬烯酸(4-HNE)的表达变化。结果三组小鼠总探索路程和中央格停留时间差异无统计学意义。与C+V组比较,POCD+V组在场景性条件恐惧测试中僵直时间明显缩短,海马PV表达明显下降,而gp91phox、p22phox和4-HNE表达明显升高(P0.05);与POCD+V组比较,POCD+A组在场景性条件恐惧测试中僵直时间明显延长,海马PV表达明显升高,gp91phox、p22phox和4-HNE表达明显降低(P0.05)。结论海马NADPH氧化酶活化介导PV表达降低可能参与老年小鼠POCD。     

兔髂动脉再狭窄与NADPH氧化酶的关系

目的研究NADPH氧化酶与血管壁活性氧(ROS)生成及再狭窄的关系。方法构建兔髂动脉2次损伤后再狭窄模型,逆转录-聚合酶链反应(RT-PCR)检测2次损伤后不同时段兔髂动脉NADPH氧化酶催化亚基gp91phox和p22phox mRNA表达的变化;原位杂交观察gp91phox和p22phox mRNA表达在血管壁的定位。结果gp91phox mRNA的表达在2次损伤后逐渐上升,在14 d(1．554±0．105)、28 d(1．444±0．360)达到峰值,与术后即刻组(0．572±0．018)比较,差异有统计学意义(P<0．01);p22phox mRNA的表达在2次损伤即刻(1．514±0．036)即处于高水平,术后1 d(0．832±0．059)略有下降后又逐渐升高,并在14 d(1．714±0．249)、28 d(1．564±0．151)达峰值。在2次损伤后,各时段血管壁gp91phox和p22phox mRNA均可见阳性表达,其表达主要位于新生内膜和外膜。结论NADPH氧化酶各个亚基在再狭窄过程中可能行使不同功能;氧化酶主要在新生内膜与外膜发挥作用。     

NMDA受体2B亚基模拟表位对慢性神经病理性痛大鼠的镇痛效应

目的 评价N-甲基-D-天冬氨酸受体2B亚基(NR2B)模拟表位对慢性神经病理性痛大鼠的镇痛效应.方法 成年雄性SD大鼠25只,体重200-250 g,随机分为5组(n=5),C组不进行任何处理;神经损伤(SNI)组行左后肢保留性SNI术;NR2B单纯免疫组(pNR2B组)于皮下两点注射携带NR2B模拟表位的特异性噬菌体(pNR2B)4×1012 pfμ/ml,每点100 μl,间隔1周后重复注射,共重复2次;无关噬菌体+SNI组(pSNI组)和pNR2B+SNI组(pNR2B/SNI组)于相应时点分别注射无关噬菌体或携带NR2B模拟表位的pNB2B,于首次注射后1周时行左后肢SNI术.持续观察大鼠痛行为学,分别于SNI术前1 d、术后1 d、1、2和3周测定左后爪机械刺激缩足反应阈值(MWT).首次注射后4周,采用间接ELISA法测定血清NR2B抗体和噬菌体抗体水平.结果 与C组和pNR2B组比较,SNI组和pSNI组术后1 d出现痛行为学症状,MWT降低,并持续至术后3周;pNR2B/SNI组术后1 d、1、2周痛行为学症状无明显缓解,术后3周痛行为学症状明显缓解,MWT差异无统计学意义(P＞0.05).与SNI组和pSNI组比较,pNR2B/SNI组术后1周痛行为学症状明显缓解,MWT升高,持续至术后3周.pNR2B组、pSNI组和pNR2B/SNI组血清噬菌体抗体水平高于C组和SNI组(P＜0.05),pNR2B组和pNR2B/SNI组血清NR2B抗体水平明显高于其他3组(P＜0.05).结论 NR2B模拟表位通过诱导SNI致慢性神经病理性痛大鼠产生NR2B抗体,产生明显的镇痛效应.     

TMEM16A下调缓解坐骨神经分支选择性损伤大鼠的神经病理性痛

目的探讨TMEM16A在坐骨神经分支选择性损伤(SNI)神经病理性痛模型大鼠中的作用。方法成年雄性SD大鼠,体重180～220 g,随机分为假手术组(sham组,n=6)和SNI组(n=12),sham组大鼠仅暴露左侧坐骨神经分支;SNI组行SNI。在术前1 d、术后3、7、10、14 d测定大鼠热缩足潜伏期(TWL)、冷缩足潜伏期(CWL)和机械缩足阈值(MWT)。采用Western blot测定术前1 d和术后7、14 d术侧背根神经节(DRG)中TMEM16A蛋白含量。另取72只雄性SD大鼠随机分为四组:sham+生理盐水组(CS组)、SNI+生理盐水组(SS组)、SNI+CaCCinh-A01组(SC组)和SNI+T16Ainh-A01组(ST组),每组18只。在给药前3 d行鞘内置管术,CS、SS组大鼠术后14 d鞘内单次注射生理盐水10μl,SC、ST组大鼠相同时点鞘内单次注射10μl浓度为1 mg/ml的特异性钙激活氯通道(CaCCs)抑制剂CaCCinh-A01或T16Ainh-A01,在给药后的8 h内每隔1 h测定TWL、CWL和MWT。另设相同四组大鼠于术后第12天开始每隔6 h分别鞘内注射10μl的生理盐水、CaCCinh-A01或T16Ainh-A01,共注射5次,于术后第14天提取术侧DRG进行Western blot和免疫荧光实验,观察TMEM16A蛋白含量及TMEM16A分布特点。结果与sham组比较,SNI组术后3、7、10、14 d CWL明显延长(P0.05),MWT明显降低(P0.05),TWL差异无统计学意义。与术前1 d比较,SNI组TMEM16A蛋白含量在术后7、14 d明显增高,且术后14 d明显高于术后7 d(P0.05)。与SS组比较,SC组和ST组CWL从给药后1 h开始降低,3 h达到最低,且在给药后的1～4 h内SC组CWL明显小于ST组(P0.05);MWT从给药后1 h开始升高,分别在2 h和3 h达到最高且在给药后的1、2、4、7和8 h内SC组MWT明显高于ST组(P0.05);TWL在各时点差异均无统计学意义。与SS组比较,SC组和ST组TMEM16A蛋白含量明显降低(P0.05),且ST组明显低于SC组(P0.05)。免疫荧光结果显示TMEM16A主要表达在与伤害感受相关的中小神经元上。结论 TMEM16A可能在SNI诱导的持续性痛觉过敏中起关键作用。TMEM16A可为神经病理性痛提供新的药物靶点。     

疼痛调控相关miRNAs的筛选及脊髓miR-29c对神经病理性痛大鼠痛阈的影响

目的通过microRNAs(miRNAs)芯片技术筛选疼痛调控相关的miRNAs,观察microRNA-29c(miR-29c)对神经病理性痛大鼠痛阈的影响。方法实验一:雌性SD大鼠,随机分为神经病理性痛组(SNI组)及妊娠神经病理性痛组(PSNI组),测定机械缩足阈值(MWT),观察分娩对疼痛的影响。在SNI组及PSNI组大鼠分娩后MWT差异显著时,取脊髓腰膨大组织,筛选差异表达的miRNAs。实验二:选取目的miRNAs,包被过表达或沉默慢病毒,脊髓内注射干预后观察SNI组大鼠MWT变化。结果与SNI组大鼠比较,PSNI组大鼠分娩后第3天MWT明显升高,脊髓c-Fos蛋白含量明显降低(P0.05);miRNAs芯片分析共发现71条差异表达的miRNAs,在PSNI大鼠显著上调37条,显著下调34条;选取并包被miR-124a过表达、miR-29c沉默慢病毒注射,miR-29c表达沉默可明显提高SNI组大鼠MWT。结论 miR-29c是通过基因芯片筛选获得的miRNA,下调SNI大鼠脊髓miR-29c表达,能够显著改善病理性痛状态。     

艾司氯胺酮对小鼠抗抑郁作用的机制与海马GABAB受体的关系

目的评价艾司氯胺酮对小鼠抗抑郁作用的机制与海马γ-氨基丁酸B型受体(GABABR)的关系。方法雄性C57BL/6J(B6)小鼠54只, 8周龄, 体质量25～30 g, 取40只小鼠采用慢性社会挫败应激法建立抑郁模型, 造模后第11天时采用社交回避实验筛选出26只抑郁易感小鼠, 采用随机数字表法分为2组(n=13):抑郁易感组(Sus组)和抑郁易感+艾司氯胺酮组(Sus+S-ket组);剩余的14只小鼠作为对照组(C组)。造模后第12天开始, Sus+S-ket组连续3 d每天腹腔注射艾司氯胺酮10 mg/kg, C组和Sus组连续3 d每天腹腔注射等容量生理盐水。最后一次腹腔注射后1 h时行旷场实验, 记录运动总距离;旷场实验结束后1 d时行强迫游泳实验, 记录不动时间;强迫游泳实验结束后1 d时行糖水偏好实验, 计算糖水消耗比例。行为学测试结束后1 h时深麻醉下处死小鼠, 取海马组织, 采用Western blot法检测GABABR1、GABABR2、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)、脑源性神经营养因子(BDNF)、酪氨酸激酶受体B(TrkB)、磷...     

鞘内注射舒芬太尼对神经病理性痛大鼠脊髓背角NMDA受体及CGRP表达的影响

目的 评价鞘内注射舒芬太尼对神经病理性痛大鼠脊髓背角N-甲基-D-天冬氨酸(NMDA)受体及降钙素相关基因肽(CGRP)表达的影响.方法 雄性SD大鼠36只,体重220～280 g,随机分为4组(n=9):正常对照组(C组)、假手术组(S组)、坐骨神经分支选择性损伤组(SNI组)和舒芬太尼+坐骨神经分支选择性损伤组(S+SNI组).SNI组和S+SNI组制备SNI模型,S+SNI组在SNI术后14 d内每天鞘内注射舒芬太尼1 μg(用生理盐水稀释至10 μl),其余各组给予等容量生理盐水.于SNI给药前2 d(基础状态)及给药1、2、7、14 d测定机械痛阈和热缩足潜伏期,分别于给药2、7、14 d测定痛阈后立即处死3只大鼠,采用免疫组化法测定L5节段脊髓背角NMDA受体和CGRP表达水平.结果 与C组和S组比较,SNI组机械痛阚降低,NMDA受体和CGRP表达上调(P<0.01),热缩足潜伏期差异无统计学意义(P>0.05).与SNI组比较,S+SNI组机械痛阈升高,热缩足潜伏期延长,NMDA受体和CGRP表达下调(P<0.01).结论 鞘内注射舒芬太尼可抑制脊髓背角NMDA受体和CGRP表达上调,从而减轻大鼠神经病理性痛.     

乳酸对慢性神经病理性痛小鼠认知功能的影响

目的 探索乳酸对慢性神经病理性痛小鼠认知功能的影响。
方法 选择C57BL/6J雄性小鼠36只，8～10周龄，体重25～30 g。采用坐骨神经损伤（SNI）制备慢性神经病理性痛模型。将小鼠随机分为三组:假手术组（Sham组）、坐骨神经损伤组（SNI组）和坐骨神经损伤+乳酸处理组（SNIL组），每组12只。Sham组仅暴露神经，不进行结扎处理。SNI组和SNIL组制备慢性神经病理性痛模型。乳酸通过生理盐水稀释至终浓度100 mg/ml。SNIL组于手术当天至术后第20天每天腹腔注射乳酸0.25 ml。Sham组和SNI组在相同时点给予等体积生理盐水。于手术当天和术后第21天测量MWT，于术后第20天采用旷场实验测量运动距离，采用新物体识别实验计算新物体探索时间百分比。于术后第21天采用Y迷宫实验计算轮替率。术后第22天处死小鼠，采用Western blot法检测脑源性神经营养因子（BDNF）、早期生长反应因子-1（Egr-1）和细胞骨架活性调节蛋白（Arc）的相对含量。
结果 与Sham组比较，SNI组和SNIL组术后第21天MWT明显降低，SNI组新物体探索时间百分比明显降低，轮替率、海马组织BDNF、Egr-1、Arc相对含量明显降低（P<0.05）。与SNI组比较，SNIL组新物体探索时间明显延长，轮替率、海马组织BDNF、Egr-1、Arc相对含量明显升高（P<0.05）。
结论 乳酸可以通过增加慢性神经病理性痛小鼠海马突触可塑性相关蛋白的表达，减轻认知功能损伤。     

Nicotinamide Adenine Dinucleotide Phosphate Oxidase in the Formation of Superoxide in Osteoclasts

Osteoclasts use a variety of chemical agents to degrade bone. One important component of this process is the generation of superoxide. It has been reported that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is the enzyme responsible for superoxide production in phagocyte; however, the NADPH oxidase present in osteoclasts has not been studied in detail. One of the membrane-bound subunits of the NADPH oxidase is gp91^phox which represents the rate-limiting component for the formation of the NADPH oxidase complex. This study was designed to demonstrate the presence of gp91^phox in individual osteoclasts using the RT-PCR technique developed for limited numbers of cells. Compared with white cells, 1.8 times the amount of gp91^phox mRNA was found in osteoclasts. This difference may be related to the size of the osteoclast and the multiple nuclei present. The presence of gp91^phox in osteoclasts was confirmed at protein level by immunocytochemistry. Osteoclastic superoxide generation is inhibited by diphenylene iodonium, a specific inhibitor of the NADPH oxidase. These studies suggest that superoxide generation by osteoclasts correlates with the activity of NADPH oxidase. Received: 8 September 1997 / Accepted: 8 April 1998     

Phagocyte NADPH Oxidase Restrains the Inflammasome in ANCA-Induced GN

ANCA-activated phagocytes cause vasculitis and necrotizing crescentic GN (NCGN). ANCA-induced phagocyte NADPH oxidase (Phox) may contribute by generating tissue-damaging reactive oxygen species. We tested an alternative hypothesis, in which Phox restrains inflammation by downregulating caspase-1, thereby reducing IL-1β generation and limiting NCGN. In an antimyeloperoxidase (anti-MPO) antibody-mediated disease model, mice transplanted with either gp91^phox-deficient or p47^phox-deficient bone marrow showed accelerated disease with increased crescents, necrosis, glomerular monocytes, and renal IL-1β levels compared with mice transplanted with wild-type bone marrow. IL-1β receptor blockade abrogated aggravated NCGN in gp91^phox-deficient mice. In vitro, challenge with anti-MPO antibody strongly enhanced caspase-1 activity and IL-1β generation in gp91^phox-deficient and p47^phox-deficient monocytes compared with wild-type monocytes. This enhanced IL-1β generation was abrogated when caspase-1 was blocked. ANCA-induced superoxide and IL-1β generation were inversely related in human monocytes. Furthermore, transplantation of gp91^phox/caspase-1 double-deficient bone marrow rescued the accelerated NCGN phenotype in gp91^phox bone marrow-deficient mice. These results suggest that Phox-generated reactive oxygen species downregulate caspase-1, thereby keeping the inflammasome in check and limiting ANCA-induced inflammation. IL-1 receptor blockade may provide a promising strategy in NCGN, whereas our data question the benefit of antioxidants.     

Rac1 Is Required for Cardiomyocyte Apoptosis During Hyperglycemia

OBJECTIVE

Hyperglycemia induces reactive oxygen species (ROS) and apoptosis in cardiomyocytes, which contributes to diabetic cardiomyopathy. The present study was to investigate the role of Rac1 in ROS production and cardiomyocyte apoptosis during hyperglycemia.

RESEARCH DESIGN AND METHODS

Mice with cardiomyocyte-specific Rac1 knockout (Rac1-ko) were generated. Hyperglycemia was induced in Rac1-ko mice and their wild-type littermates by injection of streptozotocin (STZ). In cultured adult rat cardiomyocytes, apoptosis was induced by high glucose.

RESULTS

The results showed a mouse model of STZ-induced diabetes, 7 days of hyperglycemia-upregulated Rac1 and NADPH oxidase activation, elevated ROS production, and induced apoptosis in the heart. These effects of hyperglycemia were significantly decreased in Rac1-ko mice or wild-type mice treated with apocynin. Interestingly, deficiency of Rac1 or apocynin treatment significantly reduced hyperglycemia-induced mitochondrial ROS production in the heart. Deficiency of Rac1 also attenuated myocardial dysfunction after 2 months of STZ injection. In cultured cardiomyocytes, high glucose upregulated Rac1 and NADPH oxidase activity and induced apoptotic cell death, which were blocked by overexpression of a dominant negative mutant of Rac1, knockdown of gp91^phox or p47^phox, or NADPH oxidase inhibitor. In type 2 diabetic db/db mice, administration of Rac1 inhibitor, NSC23766, significantly inhibited NADPH oxidase activity and apoptosis and slightly improved myocardial function.

CONCLUSIONS

Rac1 is pivotal in hyperglycemia-induced apoptosis in cardiomyocytes. The role of Rac1 is mediated through NADPH oxidase activation and associated with mitochondrial ROS generation. Our study suggests that Rac1 may serve as a potential therapeutic target for cardiac complications of diabetes.Diabetic cardiomyopathy has been defined as ventricular dysfunction that occurs in the absence of changes in blood pressure and coronary artery disease (1,2). Cell death by apoptosis is the predominant damage in diabetic cardiomyopathy (3,4). Diabetes increases cardiac apoptosis in animals and patients (3–7). Cardiomyocyte death causes a loss of contractile tissue, which initiates a cardiac remodeling (8). Loss of cardiomyocytes and hypertrophy of the remaining cells characterize the diabetic cardiomyopathy (9,10). Thus, suppression of cardiomyocyte apoptosis results in a significant prevention of the development of diabetic cardiomyopathy (4). However, the underlying mechanisms by which diabetes induce apoptosis remain not fully understood.All forms of diabetes are characterized by chronic hyperglycemia. Hyperglycemia induces reactive oxygen species (ROS) production in cardiomyocytes (6,11), which plays a crucial role in cardiomyocyte apoptosis in diabetes because the administration of antioxidant agents are able to rescue hyperglycemia-induced cardiomyocytes (4,6). The mechanisms activated by hyperglycemia, leading to myocardial oxidative stress and apoptosis, are not completely clarified.Although multiple sources of ROS have been demonstrated, NADPH oxidase is a critical determinant of the redox state of the myocardium (12–15). Higher myocardial NADPH oxidase activity has been detected in diabetes (16,17); more importantly, NADPH oxidase activity is markedly increased by high glucose levels (18). The NADPH oxidase is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b558, which contains gp91^phox and p22^phox, the cytosolic regulatory subunits p47^phox and p67^phox, and the small guanosine triphosphate-binding protein Rac. An important step for the assembly and function of this multicomponent NADPH oxidase complex is the heterodimerization of gp91^phox with p67^phox, which is mediated by Rac (19). Three isoforms of Rac (Rac1, Rac2, and Rac3) have been identified (20), and Rac1 is the predominant isoform expressed in cardiomyocytes (21). Thus, Rac1 activation may lead to myocardial oxidative stress and apoptosis during hyperglycemia. A recent study showed that Rac1 contributes to vascular injury in diabetes (22). However, no direct evidence is available on Rac1 and NADPH oxidase activation in cardiomyocyte apoptosis in diabetes.In this study, we generated cardiomyocyte-specific Rac1 knockout (Rac1-ko) mice; analyzed the impact of Rac1 on NADPH oxidase activation, mitochondrial ROS generation, and intracellular ROS production; and investigated the role of Rac1 and NADPH oxidase activation in cardiomyocyte apoptosis during hyperglycemia.     

COMPUTER‐AIDED ULTRASONOGRAPHY (HISTOSCANNING): A NOVEL TECHNOLOGY FOR LOCATING AND CHARACTERIZING PROSTATE CANCER

OBJECTIVE

To test the hypothesis that exposure of a renal epithelial cell line, NRK52E, to calcium oxalate monohydrate crystals (COM) would up‐regulate NADPH oxidase subunit p47^phox, enhance superoxide production and increase monocyte chemoattractant protein‐1 (MCP‐1) and osteopontin mRNA levels.

MATERIALS AND METHODS

Confluent cultures of NRK52E cells were exposed to COM (66.7 µg/cm²) with or with no pretreatment with diphenileneiodium chloride (DPI, 10 × 10^?6m ) an inhibitor for NADPH oxidase, under serum‐free conditions. The conditioned medium was collected and total cellular RNA isolated from the cells, and subjected to enzyme‐linked immunosorbent assay and real‐time polymerase chain reaction (PCR). Production of reactive oxygen species (ROS) was estimated by dihydroethidium (DHE) staining using a fluorescence microscope. Immunohistochemistry and real‐time PCR were used to analyse p47^phox in NRK52E cells.

RESULTS

In COM treated NRK52E cells there was enhanced expression of p47^phox and production of superoxide. COM‐induced production of MCP‐1 and osteopontin was significantly reduced after treatment with DPI.

CONCLUSIONS

While the generation of a lot of ROS might play a major role in tissue injury or death, the regulated generation of low concentration of ROS, possibly by NADPH oxidase, may represent a second messenger system for generation of COM‐induced MCP‐1 and osteopontin production in the renal tubules.     

rAd5/NR2B镇痛疫苗对神经病理性痛大鼠认知功能的影响

目的 评价N-甲基-D天冬氨酸受体2B亚基(NR2B)基因重组腺病毒(rAd5/NR2B)对神经病理性痛大鼠认知功能的影响.方法 雌性SD大鼠40只,体重180～200 g,随机分为4组(n=10):对照组(C组)、rAd5/NR2B镇痛免疫组(rAd5/NR2B组)、神经病理性痛组(SP组)和rAd5/NR2B镇痛疫苗+神经病理性痛组(rAd5/NR2B+SP组).C组和rAd5/NR2B组分别胃内灌注生理盐水0.1 ml和rAd5/NR2B镇痛疫苗1×10~8 PFU,2周后重复灌注.SP组制备神经病理性痛模型,1周后腹腔注射生理盐水0.1 ml.rAd5/NR2B+SP组胃内灌注rAd5/NR2B镇痛疫苗1×10~8 PFU,2周后重复灌注,再1周后制备神经病理性痛模型.测定大鼠机械缩爪阈值(MWT).行Morris水迷宫实验测定大鼠认知功能,记录潜伏期和游泳速度.采用免疫组织化学法检测大鼠海马NR2B的表达水平.结果 (1)与C组比较,rAd5/NR2B组MWT差异无统计学意义(P＞0.05),SP组和rAd5/NR2B+SP组MWT降低(P＜0.05);与SP组比较,rAd5/NR2B+SP组MWT升高(P＜0.05).(2)各组游泳速度比较差异无统计学意义(P＞0.05);与C组比较,rAd5/NR2B组潜伏期差异无统计学意义(P＞0.05),SP组和rAd5/NR2B+SP组潜伏期延长(P＜0.05);与SP组比较,rAd5/NR2B+SP组潜伏期差异无统计学意义(P＞0.05).(3)与C组比较,rAd5/NR2B组和rAd5/NR2B+SP组海马NR2B表达差异无统计学意义(P＞0.05),SP组海马NR2B表达上调(P＜0.05);与SP组比较,rAd5/NR2B+SP组海马NR2B表达下调(P＜0.05).结论 rAd5/NR2B疼痛疫苗对神经病理性痛大鼠认知功能无明显影响.     

夹竹桃麻素对高草酸尿症大鼠肾脏氧化应激损伤的保护作用

目的 观察NADPH氧化酶特异抑制剂夹竹桃麻素（apocynin）对高草酸尿症大鼠肾脏氧化应激（OS）损伤的保护作用。 方法 自由饮用含有0.8%乙二醇的水4周建立高草酸尿症SD大鼠模型。大鼠按随机数字表法分为4个组：空白组、高草酸尿症组、apocynin干预组、apocynin对照组。后两组给予apocynin（0.2 g&#8226;kg-1&#8226;d-1）灌胃,对照组给予正常饮水。4周后检测大鼠肾脏OS 指标（尿H2O2和8-异前列腺素）,以及Ccr及肾脏/体质量比值。免疫组化观察NADPH氧化酶亚基p47phox在肾脏中的表达位置。RT-PCR和免疫印迹法分别检测肾组织NADPH氧化酶亚基p47phox、gp91phox、Nox-1 mRNA以及p47phox蛋白的表达水平。 结果 p47phox在各组肾脏中均有广泛的表达,包括肾皮质区、内髓区、外髓区等。与空白组比较,高草酸尿症组大鼠尿H2O2和8-异前列腺素水平显著升高,Ccr降低,肾脏/体质量比值增高（均P < 0.05）;肾脏p47phox、gp91phox和Nox-1 的mRNA表达均显著增加（均P ＜ 0.05）, p47phox蛋白表达也增多（P ＜ 0.01）。apocynin干预治疗可抑制肾脏p47phox、Nox-1 mRNA及p47phox蛋白的表达,但gp91phox mRNA表达未明显减少,而大鼠尿H2O2和8-异前列腺素水平下降,Ccr增加,肾脏/体质量比值减少,但仍高于对照组水平。 结论 NADPH氧化酶是高草酸尿症诱导大鼠肾脏OS损伤过程中活性氧形成的来源之一。使用apocynin抑制NADPH氧化酶活性可部分减轻肾脏的OS损伤程度,保护肾功能。     

Production of N(epsilon)-(carboxymethyl)lysine is impaired in mice deficient in NADPH oxidase: a role for phagocyte-derived oxidants in the formation of advanced glycation end products during inflammation

Advanced glycation end products (AGEs) derived from glucose are implicated in the pathogenesis of diabetic vascular disease. However, many lines of evidence suggest that other pathways also promote AGE formation. One potential mechanism involves oxidants produced by the NADPH oxidase of neutrophils, monocytes, and macrophages. In vitro studies have demonstrated that glycolaldehyde, a product of serine oxidation, reacts with proteins to form N(epsilon)-(carboxymethyl)lysine (CML), a chemically well-characterized AGE. We used mice deficient in phagocyte NADPH oxidase (gp91-phox(-/-)) to explore the role of oxidants in AGE production in isolated neutrophils and intact animals. Activated neutrophils harvested from wild-type mice generated CML on ribonuclease A (RNase A), a model protein, by a pathway that required L-serine. CML formation by gp91-phox(-/-) neutrophils was impaired, suggesting that oxidants produced by phagocyte NADPH oxidase contribute to the cellular formation of AGEs. To determine whether these observations are physiologically relevant, we used isotope-dilution gas chromatography/mass spectrometry to quantify levels of protein-bound CML in mice suffering from acute peritoneal inflammation. Phagocytes from the gp91-phox(-/-) mice contained much lower levels of CML than those from the wild-type mice. Therefore, oxidants generated by phagocyte NADPH oxidase may play a role in AGE formation in vivo by a glucose-independent pathway.     

NOX2 Deficiency Protects Against Streptozotocin-Induced ��-Cell Destruction and Development of Diabetes in Mice

OBJECTIVE

The role of NOX2-containing NADPH oxidase in the development of diabetes is not fully understood. We hypothesized that NOX2 deficiency decreases reactive oxygen species (ROS) production and immune response and protects against streptozotocin (STZ)-induced β-cell destruction and development of diabetes in mice.

RESEARCH DESIGN AND METHODS

Five groups of mice—wild-type (WT), NOX2^−/−, WT treated with apocynin, and WT adoptively transferred with NOX2^−/− or WT splenocytes—were treated with multiple-low-dose STZ. Blood glucose and insulin levels were monitored, and an intraperitoneal glucose tolerance test was performed. Isolated WT and NOX2^−/− pancreatic islets were treated with cytokines for 48 h.

RESULTS

Significantly lower blood glucose levels, higher insulin levels, and better glucose tolerance was observed in NOX2^−/− mice and in WT mice adoptively transferred with NOX2^−/− splenocytes compared with the respective control groups after STZ treatment. Compared with WT, β-cell apoptosis, as determined by TUNEL staining, and insulitis were significantly decreased, whereas β-cell mass was significantly increased in NOX2^−/− mice. In response to cytokine stimulation, ROS production was significantly decreased, and insulin secretion was preserved in NOX2^−/− compared with WT islets. Furthermore, proinflammatory cytokine release induced by concanavalin A was significantly decreased in NOX2^−/− compared with WT splenocytes.

CONCLUSIONS

NOX2 deficiency decreases β-cell destruction and preserves islet function in STZ-induced diabetes by reducing ROS production, immune response, and β-cell apoptosis.Type 1 diabetes is a T-cell–mediated autoimmune disease characterized by the selective destruction of insulin-secreting β-cells in the islets of Langerhans. It is a multifactorial process involving autoantigen presentation by macrophages, dentritic cells, and B-cells; activation of autoreactive CD4⁺ T-cells; and activation and recruitment of β-cell–specific CD8⁺ T-cells, leading to increased cytokine and reactive oxygen species (ROS) production and destruction of β-cells (1). The mechanisms of putative type 1 diabetes induced by multiple-low-dose streptozotocin (STZ) includes the direct β-cell destruction, which is mainly induced via DNA alkylation (2) and the indirect β-cell destruction from T-cell–dependent immune reaction (3). Furthermore, in response to cytokine stimulation including interleukin (IL)-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, β-cells also generate ROS and reactive nitrogen species, which may facilitate their destruction (4). Additionally, overexpression of antioxidant enzymes including superoxide dismutase (SOD), catalase, and glutathione peroxidase 1 (Gpx1) protects against the onset and development of diabetes and supports an important role of ROS in the pathogenesis of immune-mediated diabetes (5–12). NADPH oxidase is one of the main sources of superoxide radical formation in many cell types including phagocyte and β-cells (13). This ROS-producing enzyme consists of two membrane subunits (NOX2 and p22^phox) and at least four cytosolic components (p40^phox, p47^phox, p67^phox, and Rac1). NADPH oxidase is a highly regulated enzyme. In the resting cells, the cytosolic complex is separated from the membrane-bound catalytic core. Upon stimulation, the cytosolic component p47^phox becomes phosphorylated and the cytosolic complex migrates and binds to the membrane subunits to assemble into an active oxidase (14). It catalyzes the reduction of oxygen to superoxide anion using NADPH as a substrate and plays a major role in antimicrobial host defense as well as in tissue damage of autoimmune diseases (15,16). NOX2 is one of the critical subunits of NADPH oxidase. T-cells deficient in NOX2 exhibit an impaired ability to produce superoxide in response to anti-CD3 stimulation (17). Furthermore, glucose stimulates β-cell superoxide production, which can be inhibited by a selective NADPH oxidase inhibitor diphenylene iodonium, suggesting a functional NADPH oxidase in the islet (18). However, a definitive role of NADPH oxidase in the development of diabetes remains to be determined.In the present study, NOX2^−/− mice were used to investigate the role of NADPH oxidase in β-cell destruction induced by multiple-low-dose STZ. We demonstrated that NOX2 deficiency attenuates the severity of hyperglycemia and the loss of β-cell mass induced by STZ treatments via reduced ROS production and suppressed immune response.