缺血预处理对肾脏的保护作用及其机制的实验研究

目的：在肾脏缺血预处理的动物实验模型中，探讨缺血预处理对肾脏的保护作用及其机制。方法：将36只小白兔随机分为4组，即对照组，缺血预处理组（IP），缺血再灌注组（I／R）和缺血预处理后再灌注组（IP＋I／R），分别检测各组动物血液中肌酐（Cr)和一氧化氮（NO）浓度，肾组织中一氧化氮合酶（NOS,eNOS，iNOS,nNOS)在肾组织中的表达及组织学变化。结果：急性肾缺血60min再灌注60min时，发现IP组及IP＋I／R组血液中Cr浓度变化与对照组之间差异无显著性意义（P＞0．05），但明显低于I／R组（P＜0．05），在IP组和IP＋I／R组血液中，NO浓度明显高于对照组和I／R组，eNOS阳性表达在IP组和IP＋I／R组明显高于I／R组和对照组（P＜0．01），iNOS和nNOS在肾组织中未见明显表达，组织学变化发现I／R组肾细胞发生明显变性坏死，而IP组和对照组未见明显改变，结论：缺血预处理在肾脏缺血再灌注中具有保护作用。而NOS可能参与了该作用机制。     

缺血预处理对大鼠部分肝脏切除术后肝脏一氧化氮合酶的影响及意义

目的 探讨缺血预处理（IPC）对大鼠部分肝脏切除术后肝脏一氧化氮合酶（NOS）的影响及意义。方法 20只SD大鼠随机分为2组：单纯热缺血组（WI）和缺血预处理组（IPC）。WI组于缺血前、再灌注后0．5、1、2、3h，IPC组于IPC前、IPC结束时、再灌注后0．5、1、2及3h分别切取肝脏组织约0．1g，用荧光定量PCR法检测其内皮型NOS（eNOS） mRNA和诱导型NOS（iNOS） mRNA。结果 两组肝脏热缺血再灌注后早期eNOS mRNA表达均增强，IPC组的表达高于WI组（P〈0、01或P〈0．05）。两组肝脏热缺血再灌注1h后iNOS mRNA开始表达，IPC组的表达低于WI组（P〈0．05）。结论 IPC可能通过促进肝脏热缺血再灌注早期eNOS mRNA的表达和抑制其稍后iNOS mRNA的表达，而发挥其对大鼠肝脏切除术中肝脏热缺血再灌注损伤的保护作用。     

中介素减轻大鼠肾脏缺血再灌注损伤

目的 探讨中介素(IMD)对大鼠肾脏缺血再灌注损伤(IRI)的影响,及其过程中一氧化氮合酶(NOS)的作用和机制.方法 将健康雄性Wistar大鼠分为4组:假手术组,行右肾切除术,1周后单纯分离左侧肾蒂及肾动脉,而不夹闭肾动脉；肾脏缺血再灌注(IR)组,行右肾切除术,1周后行左肾缺血再灌注手术；IMD基因转染组,右肾切除后左肾行超声微泡介导的IMD-pCDNA3.1(+)质粒转染术,饲养1周,再行左肾缺血再灌注手术；空质粒转染组,右肾切除后左肾行超声微泡介导的pCDNA3.1(+)质粒转染术,饲养1周,再行左肾缺血再灌注手术.大鼠于缺血再灌注术后24 h处死,用免疫组织化学方法检测肾组织IMD表达,取肾组织进行病理学观察,取血清测定尿素氮(BUN)和肌酐(Cr)浓度,检测肾组织中内皮型NOS(eNOS)、诱导型NOS(iNOS)以及神经型NOS(nNOS) mRNA及其蛋白的表达.结果 假手术组大鼠肾组织中IMD位于肾小管及间质细胞胞浆内,其表达灰度值为66±35;肾脏IR组大鼠肾小管上皮细胞和间质中IMD表达灰度值为176±48,高于假手术组(P＜0.01)；IMD基因转染组肾组织中IMD表达灰度值为262±68,高于肾脏IR组(P＜0.01)；空质粒转染组IMD表达灰度值为180±51,和肾脏IR组间表达的差异无统计学意义(P＞0.05).与肾脏IR组相比较,IMD基因转染组大鼠肾脏组织病理损伤程度较轻,血清BUN和Cr较低(P＜0.05),eNOS mRNA及eNOS表达升高(P＜0.05),iNOS mRNA及iNOS表达降低(P＜0.05),而两组间nNOSmRNA及nNOS表达的差异无统计学意义(P＞0.05).结论 中介素可能通过促进eNOS表达、抑制iNOS表达从而减轻大鼠肾脏IRI.     

炎症中诱导型氧化氮合酶与环加氧酶2相互作用及胰岛素抗炎效应

应激反应、缺血缺氧、炎症感染是烧伤后全身反应的主要环节，诱导型氧化氮合酶（iNOS）及环加氧酶2（COX-2）是介导炎性反应的重要递质。氧化氮合酶（NOS）分两种类型，即组成型NOS（cNOS）和iNOS（又称NOSⅡ）。cNOS又分为神经元NOS（nNOS，NOSⅠ）及内皮NOS（eNOS，NOSⅢ），它们的生成量通常以pmol为单位，iNOS的生成量通常以nmol为单位。COX是花生四烯酸转化为前列腺素（PGs）的关键酶，也分为两型：COX-1、     

大鼠脊髓损伤后一氧化氮合酶基因表达的变化

目的　探讨大鼠脊髓损伤后3种类型一氧化氮合酶（NOS）mRNA表达的变化规律。方法　成年SD大鼠36只,随机分为种类6组,每组6只大鼠。建立大鼠脊髓压迫伤模型,以逆转录-聚合酶链反应（RT-PCR）法测定伤段脊髓组织神经型（nNOS）、诱导型（iNOS）及内皮型（eNOS）一氧化氮合酶的mRNA表达情况。结果　脊髓压迫伤后nNOSmRNA及NOSRNA表达增强,伤后6h达到高峰0.633±0.012、1.236±0.207;iNOSmRNA表达亦增高,但在伤后24h才达到高峰1.043±0.049。结论　脊髓损伤后NOSmRNA的表达增强,但不同类型的NOSmRNA变化规律不同,增强或抑制不同NOSmRNA的表达可能减轻脊髓继发性损伤。     

甘氨酸对肾缺血-再灌注损伤的保护作用及对iNOS表达的影响

目的探讨甘氨酸对急性肾缺血-再灌注损伤的保护作用及作用机制。方法将24只Wistar大鼠随机分为假手术组（A组）、对照组（B组）、甘氨酸治疗组（C组），通过夹闭大鼠双侧肾蒂30min，再灌注30min制成肾缺血-再灌注损伤动物模型，检测尿量、血尿素氮（BUN）、血肌酐（Cr），肾组织光镜及电镜检查并使用免疫组化法检测iNOS表达。结果B组比A组尿量增加，血BUN、Cr升高，肾小管上皮细胞变性坏死明显，iNOS表达明显增强（P〈0．05）。C组比B组尿量明显增加，血BUN、Cr明显下降，肾小管上皮细胞变性坏死程度减轻，iNOS表达明显减少（P〈0．05）。结论甘氨酸对大鼠肾脏缺血-再灌注损伤有明显的保护作用，其机制可能与抑制大鼠缺血-再灌注后肾脏iNOS蛋白表达有关。     

臭氧氧化预处理在肾缺血再灌注损伤中对内皮型一氧化氮合酶表达的影响

目的 观察臭氧氧化预处理对大鼠急性肾缺血再灌注损伤的保护作用及其对内皮型一氧化氮合酶（eNOS）表达的影响.方法 38只8周龄雄性Wistar大鼠按随机数字表法随机分为4组.（1）假手术组（n＝8）：仅切除右肾;（2）缺血再灌注组（n＝10）：切除右肾、游离左肾后夹闭左肾动、静脉45 min再开通;（3）臭氧氧化预处理组（n＝10）：与缺血再灌注组建模方法一致,但在建模前15 d起每天1次经直肠灌注法吹入氧气和臭氧的混合气体5~5.5 mL;（4）氧气预处理组（n＝10）：与臭氧氧化预处理组建模方法一致,但经直肠仅吹入氧气（13 mg/kg）.建模后24 h检测各组大鼠血清肌酐、血尿素氮和血清一氧化氮浓度;48 h后采用免疫组织化学法和逆转录PCR法检测各组大鼠肾组织eNOS表达;蛋白质印迹法检测肾组织胞浆eNOS含量.结果 建模后24 h,缺血再灌注组大鼠血清肌酐为（117±20）μmol/L,血尿素氮为（32.8±7.6）mmol/L,血清一氧化氮为（58±12）μmol/L,均高于假手术组,差异有统计学意义（均P〈0.05）.臭氧氧化预处理组血清肌酐为（63±16）μmol/L,血尿素氮为（17.4±5.2）mmol/L,低于缺血再灌注组和氧气预处理组,差异有统计学意义（均P〈0.05）;血清一氧化氮为（84±14）μmol/L,均高于缺血再灌注组和氧气预处理组,差异有统计学意义（均P〈0.05）.臭氧氧化预处理组肾组织学Paller评分（41±6）低于缺血再灌注组（62±9）,eNOS灰度值（163±15）高于缺血再灌注组（126±18）,差异有统计学意义（均P〈0.05）.假手术组大鼠肾组织内有微量eNOS mRNA表达,缺血再灌注组、臭氧氧化预处理组和氧气预处理组eNOS mRNA表达增强,均高于假手术组（均P〈0.05）,臭氧氧化预处理组eNOS mRNA表达高于缺血再灌注组和氧气预处理组（均P〈0.05）.假手术组无明显eNOS蛋白表达,缺血再灌注组、臭氧氧化预处理组和氧气预处理组eNOS蛋白表达增强,均高于假手术组（均P〈0.05）,臭氧氧化预处理组eNOS蛋白表达高于缺血再灌注组和氧气预处理组（均P〈0.05）.结论 臭氧氧化预处理对大鼠肾缺血再灌注损伤有保护作用,作用机制可能与其诱导eNOS合成增多、一氧化氮产生增多有关.     

缺血后处理对大鼠肾缺血再灌注损伤的影响

目的探讨缺血后处理对大鼠肾缺血再灌注（I／R）损伤的影响及其机制。方法18只雄性SD大鼠随机分为3组（n=6）：假手术组（S组）、缺血再灌注组（I／R组）和缺血后处理组（IPo组）。采用夹闭双侧肾蒂45min-再灌注6h制备肾脏缺血再灌注损伤模型。IPo组在夹闭双侧肾蒂45min后，再灌注10s，缺血10s，重复3次后，完全恢复肾血流。再灌注6h时开胸，取心脏血后处死大鼠，取肾组织。测定血清肌酐（Cr）、尿素氮（BUN）和尿酸（UA）浓度，肾组织中丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性；光镜下观察肾组织病理学改变；采用原位末端脱氧核苷酸转移酶标记（TUNEL）法检测肾组织中凋亡细胞，光镜下计数凋亡细胞，并计算肾小管上皮细胞凋亡指数（AI）。结果与S组比较，I／R组和IPo组Cr和BUN浓度升高（P〈0．05），UA浓度差异无统计学意义（P〉0．05），肾组织SOD活性降低，MDA含量升高，肾小管上皮细胞凋亡指数增加（P〈0．05），病理损伤明显。与I／R组比较，IPo组Cr和BUN浓度降低（P〈0．05），UA浓度差异无统计学意义（P〉0．05），SOD活性升高，MDA含量降低，肾小管上皮细胞凋亡指数减少（P〈0．05），病理损伤减轻。结论缺血后处理能减轻大鼠肾缺血再灌注损伤，其机制与增强肾脏抗氧化能力和抑制肾组织细胞凋亡有关。     

黄芪当归合剂对大鼠急性缺血/再灌注肾损伤的保护及加速修复作用

目的 研究黄芪当归合剂在急性缺血/再灌注肾损伤修复过程中的作用及细胞内信号转导机制。方法 无创动脉夹夹闭双侧肾蒂造成急性缺血/再灌注肾损伤大鼠模型,以黄芪当归合剂灌胃,采用苦味酸法测定血清肌酐,免疫组织化学检测肾组织增殖细胞核抗原（PCNA）的表达及分布,特异性底物磷酸化结合免疫沉淀法测定肾组织细胞外信号调节激酶（ERK）、氨基末端激酶（JNK）活性。结果 与对照组相比,再灌注24h给药组大鼠血肌酐升高幅度降低,肾小管上皮细胞坏死减少,PCNA阳性细胞明显增多。单纯缺血使肾组织ERK活性降低,再灌注5min其活性恢复,给药组与对照组无差异;单纯缺血对JNK活性无明显影响,再灌注5min、20min给药组JNK活性明显增高。结论 黄芪当归合剂可减轻急性缺血/再灌注肾损伤,促进损伤修复,这一作用可能与其影响缺血再灌注后JNK信号通路的变化有关。     

大鼠肝缺血／再灌注后肝组织一氧化氮和不同亚型一氧化氮合酶的变化

目的探讨一氧化氮（NO）和一氧化氮合成酶（NOS）在肝缺血／再灌注（I／R）过程中的变化和作用。方法健康雄性SD大鼠24只，随机分为3组（每组8只）：①正常对照组，术中只分离肝周围韧带，不做肝门阻断及再灌注。②I/R组，进行45min的部分肝门阻断及60min的再灌注。③L-精氨酸（L—Arg）组，缺血前20min经阴茎背静脉注射L—Arg（300mg／kg），余同②组。实验结束后，取下腔静脉血2ml，并迅速切取缺血肝组织。检测血清丙氨酸转氨酶（ALT）、门冬氨酸转氨酶（AST）、乳酸脱氢酶（LDH）；测定肝组织中超氧化物歧化酶（SOD）、丙二醛（MDA）、黄嘌呤氧化酶（XOD）、一氧化氮（NO）和一氧化氯合成酶（NOS）等指标；观察光镜和电镜下肝组织学变化。结果与正常对照组相比，I／R组iNOS升高，NO降低；L-Arg组NO、eNOS均高于I／R组。2、3组比1组大鼠的肝组织病理损害重、肝功能差，L—Arg组病理损害较I／R组明显减轻、肝功能改善。结论NO对大鼠肝I／R损伤具有保护作用．不同亚型NOS的变化参与其中。     

Reperfusion liver injury induces down-regulation of eNOS and up-regulation of iNOS in lung tissues

OBJECTIVES: Acute lung injury and inflammation can occur after hepatic ischemia/reperfusion (I/R). Little is known regarding the possible role of nitric oxide synthase expression in this complex type of lung injury. METHODS: Real-time polymerase chain reactions and immunohistochemistry were used to assess the mRNA and protein expression of eNOS and iNOS in lung tissue after I/R challenge to the liver. Ischemia was induced by clamping the hepatic artery and portal vein for 40 minutes. After flow was restored, the liver was reperfused for 300 minutes. Blood samples were collected to assay three inflammatory parameters: tumor necrosis factor (TNF)-alpha, hydroxyl radicals, and NO. Lung lavage samples were assayed for protein and myeloperoxidase. The expression of eNOS and iNOS in lung tissues (n = 3) was also evaluated after I/R challenge to the liver. The iNOS inhibitor aminoguanidine was also tested in this I/R model. RESULTS: Reperfusion of the liver produced increased blood concentrations of TNF, hydroxyl radicals, and NO (P < .001; n = 8). Bronchial lavage fluids showed higher levels of protein and myeloperoxidase in the I/R than in the sham-treated group (P < .01). eNOS expression was down-regulated and iNOS expression up-regulated in I/R lung tissues (n = 3). The iNOS inhibitor aminoguanidine (10 mg/kg) significantly attenuated the lung injury. CONCLUSIONS: I/R injury to the liver induced lung injury involving systemic inflammatory responses and iNOS expression. Administration of aminoguanidine significantly attenuated the injury, suggesting that iNOS expression may play a critical role in lung injury induced by I/R of the liver.     

Modulation of nitric oxide synthase isoenzymes inreperfused skeletal muscle

Objective:To investigate the modulation of nitric oxide synthase(NOS)isoenzymes in skeletal muscle during 3h ischemia/reperfusion (I/R,3h ischemia followed by 3h reperfusion).Methods:The extensor digitorum longuses(EDLs) from 20 adult rats were divided into 4 groups:the normal,the sham operation,the ischemia(3h),and the ischemia/reperfusion group.One normal EDL from each rat was used as the non-operated control,and the opposite ones are distributed into the 3 remaining groups.All the samples were studied with Western blotting technique and immunohistochemistry staining.Results:Three sizes or protein bands verified with the proteins of relative molecule to be of 155000,140000 and 135000,were detected in the EDL homogenate by Western blotting,which were comparable with the positive controls for nNOS,eNOS and iNOS,respectively.Immunostaining demonstrated that nNOS was present in the muscle fiber,with a similar location of the muscle stria,eNOS was found apparently in microvascular endothelia,but not found in muscle fibers,and iNOS was found in the leukocytes around the muscle fiber and some endothelia cells.Immunostaining paralleled the Western blotting results.Conclusions:It suggests that the constitutive nNOS and eNOS protein can be regulated by I/R,and I/R results in a down regulation of nNOW and up-regulation of eNOS and iNOS in reperfused skeletal muscle.The fact that nNOS is present around stria suggests that nNOS may have a close relationship with muscle function.The localization of eNOS in endothelial cell indicates its role in regulating blood supply of the muscle.Based on these findings,it is possible that No produced by distinct NOS may play a different role in I/R injury.     

Limitations of ischemic tolerance in oxidative skeletal muscle: perfusion vs tissue protection

OBJECTIVES: This study determined if ischemic tolerance occurs in oxidative skeletal muscle following a severe ischemia/reperfusion (I/R) insult and if such protection involves the induction of nitric oxide synthase (NOS). METHODS: The soleus muscle of male Wistar rats (250-350 g) was preconditioned (PC + I/R) using five cycles of ischemia (10 min) and reperfusion (10 min) or had no PC (I/R) and 24 h later 2 h no-flow ischemia was induced. Calcium dependent (cNOS) and independent (iNOS) NOS activities were determined from PC (n = 5), or sham (n = 5) and the role of iNOS was tested by application of aminoguanidine (AMG) (100 microM; n = 4) to the muscle bath. Direct measures of the number of perfused capillaries (Npc; #/mm) during 90-min reperfusion were obtained using intravital microscopy. Tissue injury was estimated using the fluorescent vital dyes ethidium bromide (E; labels injured cells) and bisbenzimide (B; labels all cells) and expressed as the ratio E/B. RESULTS: PC prevented microvascular flow deficits (Npc:I/R = 23.4 +/- 1.3 vs PC + I/R = 29.9 +/- 1.1) and resulted in a modest, but significant reduction (21%) in tissue injury (I/R = 0.82 +/- 0.03 vs PC + I/R = 0.64 +/- 0.04). PC led to a nine fold increase in iNOS activity, but decreased cNOS activity by 94% compared to sham. AMG prevented the parenchymal protection following PC, but had no effect on microvascular perfusion. CONCLUSIONS: Ischemic tolerance, 24 h following PC, preserved microvascular perfusion, but only modestly improved tissue viability in the soleus muscle.     

热休克蛋白70和血红素加氧酶-1表达在肾缺血后处理减轻肾缺血再灌注损伤中的作用

目的 评价热休克蛋白70(HSP70)和血红素加氧酶-1(HO-1)表达在肾缺血后处理减轻肾缺血再灌注损伤中的作用.方法健康雄性SD大鼠140只,体重250～280 g,采用随机数字表法,将大鼠随机分为4组(n=35):假手术组(S组)仅开腹,游离双侧肾脏,分离双侧肾蒂不夹团;肾缺血再灌注组(I/R组)夹闭双侧肾蒂缺血45 min,恢复灌注;缺血后处理组(IPo组)夹闭双侧肾蒂45 min,再灌注10 s,缺血10 s,反复3次,恢复灌注;HSP抑制剂槲皮黄酮+缺血后处理组(Q+IPo组)缺血前1 h 腹腔注射槲皮黄酮100 mg/kg,余操作同IPo组.于再灌注即刻(T0)、1、3、6、12、24、48 h(T1～6)时各组随机取5只大鼠抽心脏血后取肾,检测肾组织HSP70、HO-1的mRNA和蛋白表达,T3时抽心脏血,测定血清肌酐(Cr)和尿素氮(BUN)浓度、caspase-3 mRNA的表达,TUNNEL法检测肾组织凋亡细胞,计算凋亡指数(AI),光镜下观察肾组织病理学结果.结果 与S组比较,其余组T3时血清Cr和BUN浓度和AJ升高,caspase-3 mRNA表达上调,各时点HSF70、BO-1的mRNA和蛋白表达上调(P＜0.05);与I/R组比较,IPo组T3时血清Cr和BUN浓度和AI降低,caspase-3 mRNA表达下调,T1～5时HSP70、HO-1的mRNA和蛋白表达上调(P＜0.05);与IPo组比较,Q+IPo组T3时血清Cr和BUN浓度和AJ升高,caspase-3mRNA表达上调,T1～5时HSP70、HO-1的mRNA和蛋白表达下调(P＜0.05).IPo组肾组织病理学损伤较I/R组减轻,Q+IPo组肾组织病理学损伤程度与I/R组相似.结论 HSP70和H0-1表达参与了肾缺血后处理减轻肾缺血再灌注损伤的过程.

Abstract：

Objective To evaluate the role of the expression of heat shock protein 70 (HSP70) and heme oxygenase-1 (HO-1) in the reduction of renal ischemia-reperfusion (I/R) injury by ischemic postconditioning in tats.Methods One hundred and forty healthy male SD rats weighing 250-280 g were randomized into 4 groups ( n = 35 each) : sham operation group (S group) ; I/R group; ischemic postconditioning group (IPo group); quercetin (an inhibitor of HSP) + ischemic postconditioning group (Q + IPo group). Renal I/R was produced by clamping bilateral renal pedicels for 45 min followed by reperfusion. In group S, bilateral kidneys were only exposed through a midline incision but their- pedicels were not clamped. In IPo and Q + IPo groups, 45 min ischemia was followed by three 10 s episodes of ischemia at 10 s intervals for reperfusion and in addition intraperitoneal quercetin 100 mg/kg was injected at 1 h before ischemia in group Q + IPo. Blood samples from hearts were obtained at 0, 1, 3, 6, 12, 24 and 48 h of reperfusion (T0-6) and the rats were then sacrificed and kidneys removed to detect the expression of HSP70 and HO-1 mRNA and protein in renal tissues. The blood samples obtained at T3 were used to determine serum creatinine (Cr) and urea nitrogen (BUN) concentrations and the expression of caspase-3 mRNA . The apoptosis in the renal tissues was detected using TUNEL and apoptotic index ( AI) was calculated. Microscopic examination was performed with light microscope. Results Compared with group S, the serum Cr and BUN concentrations and AI were significantly increased at T3,the expression of caspase-3 mRNA was up-regulated at T3, and the expression of HSP70 and HO-1 mRNA and protein was up-regulated at T0-6 in the other groups (P ＜ 0.05) . Compared with group I/R, the serum Cr and BUN concentrations and AI were significantly decreased at T3, the expression of caspase-3 mRNA was down-regulated at T3, and the expression of HSP70 and HO-1 mRNA and protein was up-regulated at T1-5 in group IPo ( P ＜ 0.05) . Compared with group IPo, the serum Cr and BUN concentrations and AI were significantly increased at T3, the expression of caspase-3 mRNA was up-regulated at T3, and the expression of HSP70 and HO-1 mRNA and protein was down-regulated at T1-5, in group Q + IPo ( P ＜ 0.05) . The microscopic examination showed that the renal I/R injury was significantly attenuated by ischemic postconditioning and the degree of injury in group IPo was similar to that in group I/R. Conclusion The expression of HSP70 and HO-1 is involved in the reduction of renal I/R injury by ischemic postconditioning in rats.     

L-Arginine transport is augmented through up-regulation of tubular CAT-2 mRNA in ischemic acute renal failure in rats

BACKGROUND: Ischemic acute renal failure (iARF) is associated with increased nitric oxide (NO) production during the reperfusion period, as endothelial nitric oxide synthase (eNOS) is maximally activated, and renal tubular inducible NOS (iNOS) is stimulated. Increased NO production leads to augmented tubular injury, probably through the formation of peroxynitrite. l-Arginine (l-Arg), the only precursor for NO, is transported into cells by cationic amino acid transporters, CAT-1 and CAT-2. We hypothesized that the increased NO production observed in iARF may result from increased l-Arg uptake, which would be reflected in the augmented expression of l-Arg transporter(s). METHODS: Ischemic acute renal failure was induced in rats by right nephrectomy + left renal artery clamping for 60 minutes. l-Arg uptake was examined in freshly harvested glomeruli and tubuli from control, sham operated, and animals subjected to 15, 30, and 60 minutes, and 24 hours of reperfusion, following 60 minutes of ischemia. Using RT-PCR, renal tissues were examined further for the expression of iNOS, CAT-1, CAT-2, arginase I and arginase II. RESULTS: Tubular expression of iNOS mRNA was initiated by ischemia, continued to increase after 60 minutes of reperfusion, and decreased after 24 hours. l-Arg transport into glomeruli was similar in all experimental groups. l-Arg uptake into tubuli was markedly augmented following the 60-minute reperfusion, while it moderately increased after 24 hours of reperfusion. This was accompanied by a parallel, preferential increase in tubular CAT-2 mRNA expression at 60 minutes of reperfusion. CAT-1 mRNA expression was unchanged, as detected by RT-PCR. In addition, the expression of arginase II and arginase I mRNA was attenuated by 30 minutes and one hour of reperfusion, and returned to baseline values after 24 hours of reperfusion. CONCLUSIONS: Ischemic ARF is associated with augmented tubular CAT-2 mRNA expression, which leads to enhanced l-Arg transport and increased NO production. This may contribute to the renal injury exhibited in iARF.     

Ischemia/reperfusion-induced low reactivity of the rat superior mesenteric vascular bed is associated with expression of nitric oxide synthases

Our objective was to investigate the mRNA and protein expressions of eNOS and iNOS in the mesenteric vascular bed after ischemia and reperfusion of the rat superior mesenteric artery (SMA) and the role of nitric oxide (NO) in the response of the vascular bed to vasoconstrictors following reperfusion of the SMA. METHODS: Real-time polymerase chain reaction and immunohistochemistry were used to monitor the mRNA and protein expression of eNOS and iNOS after I/R challenge to the rat SMA. Ischemia was induced by clamping the SMA for 40 minutes, after which the flow was restored and the vessels were reperfused for 300 minutes. Blood samples were collected for assays of lactic dehydrogenase, tumor necrosis factor (TNF), hydroxyl radical, and NO. After ischemia/reperfusion, the vascular beds were separated for analysis of the expression of eNOS and iNOS. The SMA with its associated intestinal tissue was isolated and perfused in vitro with Tyrode's solution (N = 8) then challenged with phenylephrine. RESULTS: Reperfusion of the SMA induced an increase in blood concentrations of lactic dehydrogenase (P < .001; N = 8), hydroxyl radical (P < .05), TNF (P < .001), and NO (P < .05). ENOS and iNOS mRNA expression increased 1.3 +/- 0.1-fold and 19.6 +/- 3.5-fold, respectively when compared to the sham-operated group. Protein expression increased 1.9 +/- 0.4-fold and 12.6 +/- 3.1-fold, respectively, after reperfusion (N = 3) when compared with sham-treated rats. In vitro challenge showed that administration of phenylephrine (10(-8) approximately 10(-4) nmol) produced vasoconstriction in a dose-related manner. Maximum contractile responses to phenylephrine were attenuated in reperfused SMA. Addition of the NOS inhibitor N(G)-nitro-L-arginine (L-NNA, 10(-4) M) resulted in full recovery of the response to phenylephrine. CONCLUSIONS: Ischemia/reperfusion of the SMA results in a decrease in vascular reactivity of the mesenteric vessels that is dependent on NOS expression by the intestinal vascular bed.     

Effects of Partial Liver Ischemia Followed by Global Liver Reperfusion on the Remote Tissue Expression of Nitric Oxide Synthase: Lungs and Kidneys

Hepatic ischemia followed by reperfusion (IR) results in mild to severe remote organ injury. Oxidative stress and nitric oxide (NO) seem to be involved in the IR injury. Our aim was to investigate the effects of liver I/R on hepatic function and lipid peroxidation, leukocyte infiltration and NO synthase (NOS) immunostaining in the lung and the kidney. We randomized 24 male Wistar rats into 3 groups: 1) control; 2) 60 minutes of partial (70%) liver I and 2 hours of global liver R; and 3) 60 minutes of partial (70%) liver I and 6 hours of global liver R. Groups 2 and 3 showed significant increases in plasma alanine and aspartate aminotransferase levels and in tissue malondialdehyde and myeloperoxidase contents. In the kidney, positive endothelial NOS (eNOS) staining was significantly decreased in group 3 compared with group 1. However, staining for inducible NOS (iNOS) and neuronal NOS (nNOS) did not differ among the groups. In the lung, the staining for eNOS and iNOS did not show significant differences among the groups; no positive nNOS staining was observed in any group. These results suggested that partial liver I followed by global liver R induced liver, kidney, and lung injuries characterized by neutrophil sequestration and increased oxidative stress. In addition, we supposed that the reduced NO formation via eNOS may be implicated in the moderate impairment of renal function, observed by others at 24 hours after liver I/R.     

卡托普利对缺血-再灌注鼠心肌一氧化氮及其合酶活性的影响

目的　研究一氧化氮 (NO)和一氧化氮合酶 (NOS)在心肌再灌注损伤中的作用 ,探讨卡托普利(captopril)对缺血 -再灌注鼠心肌保护的机制。　方法　采用 L angendorff离体鼠心灌流模型 ,将 18只 SD大白鼠随机分为 3组 (每组 6只 ) ,对照组、缺血 -再灌注组、卡托普利组。观察心肌 NOS同工酶活性、过氧化物歧化酶活性、丙二醛含量、肌酸激酶含量和冠脉流出液 NO的变化。　结果　缺血 -再灌注组与对照组比较心肌诱导型 NOS(i NOS)活性增高 (P<0 .0 0 1) ,而心肌原生型 NOS(c NOS)活性及总 NOS活性显著降低 (P<0 .0 0 1,0 .0 5 ) ,冠脉流出液 NO含量下降(P<0 .0 1)。卡托普利组再灌注 30分钟 ,心肌 i NOS活性低于缺血 -再灌注组 (P<0 .0 1) ,c NOS活性和总 NOS活性高于缺血 -再灌注组 (P<0 .0 1,0 .0 5 ) ,再灌注期间冠脉流出液 NO水平高于缺血 -再灌注组 (P<0 .0 1) ,心肌损伤较缺血 -再灌注组减轻。　结论　心肌 NOS同工酶活性及 NO产生的失常是心肌再灌注损伤的重要机制之一 ,卡托普利可通过调节心肌 NOS同工酶活性 ,维持正常的 NO水平 ,起到心肌保护作用。