丙泊酚对大鼠局灶性脑缺血-再灌注时脑组织热休克蛋白70基因和蛋白表达的影响

目的观察丙泊酚对大鼠局灶性脑缺血-再灌注时脑组织热休克蛋白(HSP)70 mRNA和HSP70蛋白表达的影响,以探讨其脑保护的机制。方法采用大脑中动脉线栓法建立大鼠局灶性脑缺血-再灌注模型。60只雄性Wistar大鼠,随机分为假手术组(Sham组)、缺血-再灌注组(I-R组)和丙泊酚组(P组),每组20只。大鼠脑缺血2 h,然后进行再灌注。在再灌注3、6、24、72 h断头取脑组织,采用原位杂交法和免疫组织化学染色检测大鼠脑组织HSP70 mRNA和HSP70蛋白的表达。结果局灶性脑缺血-再灌注后,HSP70 mRNA和HSP70蛋白的表达增加(P<0.01),但HSP70 mRNA表达较早,分布范围较广泛,而HSP70蛋白表达以半暗带区为主。应用丙泊酚能显著地促进脑缺血-再灌注后脑组织中HSP70 mRNA和HSP70蛋白的表达(P<0.01),与脑缺血-再灌注组相比较,HSP70 mRNA和HSP70蛋白不仅表达增多、范围增加,而且还能延缓下降(P<0.05)。结论丙泊酚能促进大鼠局灶性脑缺血-再灌注时HSP70的表达,这可能是其脑保护作用的部分机制。     

重复电针预处理对脑缺血再灌注大鼠脑皮层热休克蛋白70表达的影响

目的 探讨重复电针刺激百会穴预处理对脑缺血再灌注大鼠脑皮质热休克蛋白70表达的影响。方法 20只雄性SD大鼠,随机分为对照组(n=10)和电针组(n=10)。对照组未行任何预处理;电针组予电针刺激百会穴,30 min·d-1,连续5d。最后一次预处理后24 h时,两组均采用颈内动脉尼龙线线栓法致大脑中动脉阻闭(120 min)。缺血再灌注后24 h将大鼠灌注固定,取脑组织行病理学观察及免疫组织化学染色,并按照McCarthy等的方法对免疫组化染色结果进行半定量分析。结果 半定量分析结果,电针组皮层HSP70表达(1.9±0.6)明显强于对照组(0.9±0.4)(P<0.05)。电针组组织病理学损害轻于对照组。结论 重复电针刺激百会穴预处理减轻脑缺血再灌注损伤与皮层HSP70表达增强有关。     

肝脏缺血-再灌注2相损伤对胰岛β细胞分泌功能的影响

目的 研究大鼠肝脏缺血-再灌注2相损伤对胰岛β细胞分泌功能的影响.方法 36只SD大鼠随机分为假手术组(C组)和IR组,每组18只.IR组制成肝脏缺血1 h再灌注12 h的70%肝脏I/R损伤模型.每组取10只大鼠,再灌注12 h后采血,测定血糖及血胰岛素浓度并计算胰岛素分泌指数.每组另各取8只大鼠,进行高血糖钳夹试验.结果 IR组血糖水平明显高于C组[(9.02±1.37)mmol/L vs.(5.52±0.95)mmol/L](P<0.01),胰岛素浓度也显著高于C组[(116.45±10.87)mU/L vs.(73.65±22.87)mU/L](P<0.01),但IR组胰岛素分泌指数显著低于C组[(462.80±46.28) vs.(1 046.54±30.21)](P<0.01).高血糖钳夹试验显示,IR组2相胰岛素分泌量显著低于C组[(83.17±6.01)mU/L vs.(99.80±10.81)mU/L](P<0.05),葡萄槠代谢率也明显低于C组[(29.68±4.92)mg·kg-1·min-1 vs.(53.16±3.45)mg·kg-1·min-1](P<0.01),胰岛素敏感性指数显著低于C组[(30.97±8.11) vs.(64.34±7.21)](P<0.01).结论 肝脏缺血-再灌注损伤12 h后,大鼠胰岛β细胞分泌功能受损,外周组织的胰岛素敏感性明显下降.     

肝缺血预处理对肝再灌注期间热休克蛋白70的表达及肝脏病理改变的影响

评价缺血预处理对大鼠肝缺血再灌注后肝细胞热休克蛋白70(HSP70)的表达和肝组织形态学的影响。方法:大鼠78只分为3组:缺血预处理组、肝血流持续阻断组及假处理组。用免疫金银法动态观察再灌注24h内HSP70蛋白的表达,同时观察肝脏的病理改变。结果:肝缺血预处理能较早诱导HSP70的表达,肝细胞损伤也相应较轻。结论:肝缺血预处理可增加组织对缺血再灌注损伤的耐受性,该保护作用可能与早期诱导HSP70蛋白的产生有关。     

浅低温对犬全脑缺血再灌注后脑组织诱导型热休克蛋白70表达的影响

目的:研究犬全脑缺血再灌时浅低温对热休克蛋白70(HSP70)表达的影响,探讨热休克蛋白在低温脑复苏中的作用。方法:采用犬心脏停跳再复苏模型。14只犬随机分成三组:非缺血对照组(n=4)、常规治疗组(n=5)和浅低温组(n=5)。用免疫组化测定脑组织内HSP70表达阳性细胞数密度和反应产物灰度值。结果:犬全脑缺血再灌后HSP70表达明显增强(P<0.01)。浅低温组HSP70表达增强比常规治疗组更明显(P<0.01)。病理损伤明显减轻。结论:浅低温能增加再灌脑组织HSP70表达,并可能是低温脑复苏机制之一。     

缺血后处理对大鼠局灶性脑缺血再灌注损伤的影响

目的探讨缺血后处理对大鼠局灶性脑缺血再灌注损伤的影响。方法36只雄性SD 大鼠随机分为3组(n=12),对照组:即单纯缺血再灌注组;缺血后处理15s组(I-15 s组):大脑中动脉线栓阻闭(MCAO)90 min后,再灌注15 s,缺血15 s,反复3次;缺血后处理30 s组(I-30 s组):MCAO 90 min后,再灌注30 s,缺血30 s,反复3次。再灌注24 h后对所有动物行神经功能障碍评分(NDS),然后取大脑测定脑梗死容积。结果再灌注24 h后I-15 s和I-30 s组NDS与对照组比较差异无统计学意义(P>0.05)。再灌注24 h后脑梗死容积I-15s组为(271±97)mm3,I-30 s组为(217±85)mm3,小于对照组[(378±103)mm3](P<0.01),I-15 s和I-30 s组比较差异无统计学意义(P>0.05)。结论缺血后处理可减轻局灶性脑缺血再灌注大鼠脑的病理性损伤。     

丙泊酚对大鼠缺血-再灌注脑TNF-α、IL-10、NF-κB的影响

目的 研究丙泊酚对缺血性脑炎性因子表达的影响.方法 SD大鼠30只随机分为假手术对照(S)组、脑缺血-再灌注(IR)组(双侧颈总动脉夹闭造成暂时性脑缺血)、丙泊酚预处理(PP)组(缺血前60 min输注丙泊酚50 mg·kg-1·h-1),丙泊酚后处理(PA)组(再灌注后10 min给予丙泊酚50 mg·kg-1·h-1)以及不行脑缺血丙泊酚(P)组(输注丙泊酚50 mg·kg-1·h-1),每组6只.用酶联免疫吸附法(ELISA)测定脑组织肿瘤坏死因子α(TNF-α)和白细胞介素-10(IL-10),用同位素([32P]-ATP)方法测定脑内核因子-κB(NF-κB)的变化.结果 与S组比较,IR组TNF-α明显增高[(2.57±0.19)Pg/g vs.(1.60±0.15)pg/g](P<0.05),同时IL-10明显增高[(11.59±1.32)pg/gvs.(7.97±1.96)pg/g](P<0.05).与IR组比较,PP组TNF-α明显减低[(1.88±0.26)pg/g vs.(2.57±0.19)pg/g](P<0.05),IL-10水平明显降低[(8.35±1.00)pg/g vs.(11.59±1.32)Pg/g](P<0.05),NF-κB活性明显减低.PA组TNF-α、IL-10、NF-κB与IR组差异无统计学意义.IL-10和NF-κB水平与TNF-α活性呈现平行变化关系.结论 脑缺血前丙泊酚预处理可抑制脑的炎性介质TNF-α、IL-10和NF-κB的增高,但脑缺血-再灌注后应用丙泊酚对缺血性炎性介质的增高没有抑制作用;丙泊酚对缺血性炎性介质TNF-α的作用可能与抑制NF-κB转导途径有关.     

臭氧氧化预处理对大鼠肾缺血再灌注损伤的热休克蛋白表达的影响

目的 探讨臭氧氧化预处理通过诱导热休克蛋白70(HSP70)的合成,保护大鼠肾脏缺血再灌注损伤的作用与机制.方法 建立原位大鼠单侧肾缺血再灌注动物模型,I/R前15 d经直肠吹入氧气和臭氧的混合气体5.0～5.5 ml(臭氧浓度50 mg/L,1 mg/kg体蕈,每日 1次).全自动生化分析仪检测尿素氮(BUN)、肌酐(Cr),比色法测定血清的脂质过氧化产物丙二醛(MDA)、超氧化物歧化酶(SOD).Western blot检测HSP70蛋白的含量;逆转录聚合酶链反应(RT-PCR)方法检测HSP70的表达.结果 肾缺血再灌注24 h后,血清中BUN、Cr、MDA明显增高,肾组织内HSPT0表达明显增强(P<0.05),经臭氧氧化预处理后,血清中的BUN、Cr、MDA均降低,SOD升高;HSP70表达升高更加明显(P<0.05).结论 臭氧氧化预处理可以诱导大鼠肾缺血再灌注组织中HSP70表达,减轻大鼠肾脏缺血再灌注损伤.     

内源性及外源性碱性成纤维细胞生长因子与脑缺血后神经元凋亡及其相关基因关系

目的　研究内源性及外源性碱性成纤维细胞生长因子 (bFGF)与脑缺血再灌注后HSP70蛋白、p53基因表达及细胞凋亡的关系。方法　线栓法制成大鼠大脑中动脉闭塞及再灌注模型 ,用原位杂交 ,免疫组织化学及原位末端标记的方法观察大鼠大脑中动脉闭塞 2h后再通 0～72h脑组织内源性bFGF基因与HSP70蛋白、p53基因的表达、凋亡细胞的分布及侧脑室注射外源性bFGF对后者的影响。结果　缺血组于缺血 2h再灌注 1h可见bFGF表达增高 (P <0 .0 5) ,bFGFmRNA表达于 1 2h达高峰 ,bFGF蛋白于 2 4h达高峰 ;bFGF组与缺血组相比 ,在 6～ 48h各时间点的HSP70蛋白表达增强 ,p53表达减弱 ,凋亡细胞数明显减少 (P <0 .0 5 ,P <0 .0 1 )。结论内源性及外源性bFGF可能抑制脑缺血再灌注后细胞凋亡并调控其相关基因的表达     

N-myc下游调节基因2在七氟醚预处理减轻大鼠局灶性脑缺血再灌注损伤中的作用

目的 评价N-myc下游调节基因2(NDRG2)在七氟醚预处理减轻大鼠局灶性脑缺血再灌注损伤中的作用.方法 健康雄性成年SD大鼠48只,体重280～320 g,采用随机数字表,将大鼠随机分为3组(n=16):假手术组(S组)、脑缺血再灌注组(I/R组)和七氟醚预处理组(Sev组).采用大脑中动脉阻断法制备大鼠局灶性脑缺血再灌注模型.大鼠吸入2％七氟醚lh,每天1次,连续5d行七氟醚预处理.Sev组于七氟醚预处理结束后24h制备局灶性脑缺血再灌注模型.再灌注24h时行神经功能评分,随后处死大鼠,取脑组织,测定脑梗死体积百分比,采用Western Blot法测定缺血半暗带区NDRG2和活化的Caspase-3的表达,采用免疫组织荧光测定缺血半暗带区NDRG2的表达及定位.结果 与S组比较,I/R组和Sev组脑梗死体积百分比升高,神经功能评分降低,脑组织缺血区半暗带NDRG2和活化的Caspase-3表达上调(P＜0.05);与I/R组比较,Sev组脑梗死体积百分比降低,神经功能评分升高,脑组织缺血区半暗带NDRG2和活化的Caspase-3表达下调(P＜0.05).Sev组核内NDRG2阳性染色较I/R组减浅.结论 七氟醚预处理可能通过抑制脑组织NDRG2的表达、活性和细胞凋亡,从而减轻大鼠局灶性脑缺血再灌注损伤.     

Diazoxide, as a postconditioning and delayed preconditioning trigger, increases HSP25 and HSP70 in the central nervous system following combined cerebral stroke and hemorrhagic shock

Combined hemorrhagic shock (Shock) and unilateral common carotid artery occlusion (Stroke) results in a decrease of oxygen availability to peripheral tissues and organs and the central nervous system (CNS). A variety of biochemical processes ensue, including organ failure, cellular apoptosis, and necrosis. The present study used male, Sprague-Dawley rats to assess the impact of cerebral insult. Using heat-shock protein 25 and 70 (HSP25, HSP70) as biomarkers, measured 24 h after injury, we tested the hypothesis that pharmacological induction of preconditioning can offer cytoprotection from combined Stroke and Shock. The compound, diazoxide (DZ), is known to induce preconditioning through its effect as a mitochondrial potassium ATP (mK(ATP)) channel opener and succinate dehydrogenase inhibitor. When administered 24 h prior to Stroke and Shock (delayed preconditioning), DZ increased cerebral cortical and hippocampal levels of HSP25 and HSP70. A more clinically relevant treatment paradigm was tested, where DZ was administered after the induction of Stroke and Shock (postconditioning). When administered 60 min (but not 10 min) after the induction of Stroke and Shock, DZ significantly increased HSP25 and HSP70 expression in the ipsilateral cerebral cortex and hippocampus. Taken together, these results suggest that DZ treatment may be efficacious for CNS injury resulting from blood loss and anoxia from combined cerebral ischemia and hemorrhagic shock. "Postconditioning" triggered by DZ, immediately before resuscitation, is a potentially effective treatment for ischemia-reperfusion injury from combined Stroke and Shock.     

乙醇预处理与HSP70的诱导及对肝脏缺血再灌流损伤的影响

目的探讨乙醇预处理对肝脏缺血再灌流损伤的影响以及与热休克蛋白70诱导的关系。方法雄性成年Wistar大鼠232只,胃饲乙醇浓度40%,剂量为5 g/kg体重,随机分为5组:正常对照组(N组)、胃饲乙醇组(E组)、缺血组(IR组)、生理盐水预处理组(NPC组)、胃饲乙醇预处理组(EPC组)。动物手术采用门静脉转流下的肝脏缺血模式,肝门阻断时限为90 min,于再灌流0、13、、6、12、24、72 h活杀留取血液及肝脏标本。结果EPC组3、6 h血清ALT(1 230.88±132.50、888.88±126.67)IU/L、AST(1 866.38±61.77)(、1 433.88±42.74)IU/L均明显低于IR组及NPC组,肝脏病理改变较轻,而肝组织HSP70含量高于后者。结论乙醇预处理可以减轻大鼠肝脏90 min的缺血再灌流损伤,其肝脏保护作用与肝组织HSP70升高相一致,HSP70可能是其发挥肝脏保护的物质基础之一。     

Induction of heat shock protein 70 (HSP70) by zinc bis (dl-hydrogen aspartate) reduces ischemic small-bowel tissue damage in rats

The aim of the study was to determine whether the induction of HSP70 by Zn²⁺ is able to protect the small bowel of rats against ischemia. Twenty-four male Wistar rats (weight 200–300 g) were divided into four groups: (1) saline treatment for 24 h (n=4); (2) Zn²⁺ treatment for 24 h (n=4); (3) Saline pretreatment for 24 h and ischemia (n=8); (4) Zn²⁺ pretreatment for 24h and ischemia (n=8). Pretreatment with Zn²⁺ was carried out by intraperitoneal administration of 50 mg/kg zinc bis (dl-hydrogen aspartate)=10 mg/kg Zn²⁺. Ischemia in a defined segment of the small bowel was produced by ligation of the mesenteric vein and artery and ligation of both ends of the segment. Tissue samples were collected before and 2, 4 and 6 h after ligation and investigated by histology, immunohistochemistry and Western blotting. Twenty-four h after i.p. Zn²⁺ injection, the small bowel expressed increased HSP70 tissue levels. Histology with subsequent grading of ischemic tissue injury showed significantly decreased tissue necrosis after Zn²⁺ pretreatment and HSP70 induction compared with saline pretreated controls. In conclusion, this study proves that Zn²⁺ is inducing HSP70 in the small bowel in vivo and hereby able to protect the small bowel against ischemia.     

The time course of acquisition of ischemic tolerance and induction of heat shock protein 70 after a brief period of ischemia in the spinal cord in rabbits

We examined the time course of development of ischemic tolerance in the spinal cord and sought its mechanism exploring the expression of heat shock protein 70 (HSP70). Spinal cord ischemia was produced in rabbits by occlusion of the abdominal aorta. In Experiment 1, neurologic and histopathologic outcome was evaluated 48 h after prolonged ischemia (20 min) that was given 2 days, 4 days, or 7 days after a short period of ischemia (ischemic pretreatment) sufficient to abolish postsynaptic component of spinal cord evoked potentials. Control animals were given prolonged ischemia 4 days after sham operation. In Experiment 2, HSP70 expression in motor neurons after pretreatment without exposure to prolonged ischemia was examined by immunohistochemical staining. Ischemic pretreatment 4 days (but not 2 days or 7 days) before 20 min ischemia exhibited protective effects against spinal cord injury. In the cytoplasm, HSP70 immunoreactivity was mildly increased after 2, 4, and 7 days of ischemic pretreatment. However, the incidence of nuclear HSP70 immunoreactivity 2 days, 4 days, and 7 days after ischemic pretreatment was 2 of 6 animals, 4 of 6 animals, and 1 of 6 animals, respectively (none in the control group). These results suggest that ischemic tolerance is apparent 4 days after ischemic pretreatment and that HSP70 immunoreactivity in the nucleus may provide some insight into the mechanisms of ischemic tolerance in the spinal cord.     

Hyperthermia-induced HSP expression correlates with improved rat renal isograft viability and survival in kidneys harvested from non-heart-beating donors

Transient sublethal hyperthermia followed by recovery from heat stress, referred to as heat shock preconditioning, exerts a protective effect on ischemia/reperfusion-induced injury in many systems. This effect is considered to be correlated to heat shock proteins (HSPs) and might be a critical factor in kidney graft function and survival. This study was designed to examine the impact of heat shock preconditioning on kidney isograft function and survival in a model utilizing non-heart-beating (NHB) donors. Four groups of male Lewis rats (n = 10/group) subjected either to whole body hyperthermia (groups A and C) or to sham anesthesia (groups B and D) were allowed 24 h recovery. Thereafter, 20 min of warm ischemia (A/B), and in a separate set of experiments 40 min of warm ischemia (C/D), were induced by suprarenal aortic cross clamping before renal procurement. After 24-h preservation with University of Wisconsin solution at 4 °C, orthotopic kidney transplantations were performed to syngeneic bilaterally nephrectomized recipients. Tissue specimens were taken to determine HO-1/HSP32, 72, and 90 induction by Western blot analysis. Renal function was measured by means of serum creatinine and creatinine clearance on days 0, 3, and 7 as well as urine volume, protein content, and creatinine levels daily. HO-1/HSP32 and HSP72 were found to be expressed constitutively. Moreover, heat shock strongly induced renal HSP72 and HSP32/HO-1, and to a lesser extent HSP90, expression. For recipients of group A grafts, the graft survival rate was 10/10, whereas it was 7/10 (70 %) in recipients of group B grafts (log rank p < 0.05). Following 40 min of warm ischemia, 6/10 (60 %) recipients survived, whereas all sham treated animals died with anuria within 6 days (log rank p = 0.01). Heat shock preconditioning strongly improved graft viability and reduced functional impairment. Creatinine clearance (CRC) on day 3 post Tx was 0.43 ± 0.24 ml/min in preconditioned animals (group A) and 0.07 ± 0.09 ml/min (p < 0.001) in sham preconditioned (group B), whereas it was 0.91 ± 0.33 ml/min and 0.03 ± 0.02 ml/min (p < 0.00 001) on day 7 post Tx. Following 40 min NHB time, CRC in survivors of preconditioned graft recipients (group C) was 0.32 ± 0.2 ml/min (day 3 post Tx) and 0.23 ± 0.08 ml/min (day 7 post Tx) and was significantly better than CRC of group B (p < 0.01 and p < 0.00001, respectively). CRCs prior to NHB procedures were comparable in all animals ranging between 1.31 and 1.72 ml/min. Serum creatinine as well as proteinuria were significantly increased after transplantation in both groups but recovered within 5 days in recipients of preconditioned grafts, whereas kidneys from donors without HP did not recover function. Histological alterations were also diminished following HP. Hyperthermic preconditioning induces strong and long lasting HO-1/HSP32, HSP72, and HSP90 expression in rat kidneys. HP increases survival following transplantation and improves renal graft function including proteinuria, volume output, and creatinine clearance. HSP induction might be used to develop novel approaches in clinical transplantation. Received: 3 November 2000 Revised: 27 February 2001 Accepted: 29 May 2001     

Prior induction of heat shock proteins by a nitric oxide donor attenuates cardiac ischemia/reperfusion injury in the rat

BACKGROUND: Recent studies have demonstrated that nitric oxide (NO) releasers considerably increase heat shock proteins (HSPs) in the in vitro cell system, providing resistance to oxidant damage. This study was designed to examine the cellular responses of HSPs induced by prior administration of an NO releaser, FK409 (FK), in an in vivo transplantation model. METHODS: Lewis rats received either saline or FK solution intravenously administered at different time points before graft harvesting (10 micromol/kg) or for 15 min during reperfusion (0.66 micromol/kg/min). Tissue specimens were taken to determine HSP70 and heme oxygenase-1/HSP32 (HO-1) expression, and glutathione content. After 24-hr preservation with University of Wisconsin solution, heterotopic cardiac transplantations were performed, and graft survival was determined at 14 days. Tissue samples for end labeling of nuclear DNA fragments (TdT-mediated d-uridine triphosphate biotin nick end labeling; TUNEL) and propidium iodide staining were taken 15 min after reperfusion. RESULTS: The gene and protein expression of HSP70 after FK administration peaked at 12 min and 60-90 min, whereas those of HO-1 peaked at 6 min and 90 min, respectively. Then, representative cardiac grafts taken 60 min after FK treatment were examined for further assay. Localization of induced HSP70 and HO-1 molecules were observed in the myocardium and vascular endothelium, respectively. Prior treatment of FK was effective in preventing the reduction of tissue glutathione contents compared with control (P<0.05). Fewer TUNEL and propidium iodide-positive cells were also observed in the FK group (P<0.0005, vs. control). The graft survival rate was higher in the FK group (9/10 vs. 1/10 of control; P<0.001), whereas the groups either harvested 10 min after FK pretreatment or continuously infused for 15 min during reperfusion were inferior, similar to that of control. CONCLUSION: Prior induction of HSP70 and HO-1 with a relatively low dose of FK administration attenuates ischemia and reperfusion injury, which was due to antioxidant and antiapoptotic activities augmented by such stress proteins. Thus, NO releasers as a pharmacological maneuver may provide an innovative approach for the prevention of ischemia and reperfusion injury.     

Evaluation of warm ischemia-reperfusion injury using heat shock protein in the rat liver

We focused on heat shock protein 70 (HSP70) as a marker of viability in hepatic warm ischemia-reperfusion. Segmental hepatic warm ischemia was produced in rats for 15, 30, 60, 90, 120, or 180 min. Liver sections were evaluated at 30, 60, and 120 min of reperfusion. Expression of HSP70 and messenger RNA (mRNA), apoptosis, and apoptosis-associated genes such as Bcl-2 and Bax were studied. Expression of HSP70 and mRNA was augmented as warm ischemia was prolonged, but was markedly suppressed in livers with more than 120 min of ischemia. The highest accumulation of HSP70 was observed in the nucleus. In livers subjected to longer duration of warm ischemia, necrosis and apoptosis were evident and Bcl-2 mRNA expression and Bcl-2/Bax protein ratio were markedly diminished. Apoptosis may be related to the process of cellular injury induced by warm ischemia-reperfusion. Expression of HSP70 and the Bcl-2 family can be effective markers of viability in hepatic warm ischemia-reperfusion.     

Lipid peroxidation and the expressional regulation of the heat-shock response during ischemia-reperfusion of rat kidney

Because of the continuing shortage of donor organs, 'marginal kidneys' are increasingly being used. The purpose of our experiments was to characterize the extent of lipid peroxidation after ischemia-reperfusion (IR) injury in rat kidney, to analyze the expressional regulation of the heat-shock response and now to discuss the clinical application of these results. After ischemia, xanthine oxidase (XO) is thought to be the main oxygen radical-generating system and malondialdehyde (MDA) is considered to be a marker of LPO. In young rats (10 weeks) a unilateral warm ischemia of 40 and 60 min duration with subsequent reperfusion up to 1 h was conducted. Beside the 'footprints' of oxidative stress, the cytosolic antioxidative capacity, expressed as superoxide anion (SOA) scavenging capacity, was investigated. There was only a moderate and transient increase of renal MDA 5 and 10 min after the onset of reoxygenation (133.57/70.67 and 97.84/91.57 vs. 49.47 nmol/g wet weight (ww) in preischemic controls). ATP breakdown (to 83/65 from 2,947 nmol/g ww) with consecutive accumulation of hypoxanthine (up to 1,105 nmol/g ww) at the end of the ischemic period and the subsequent rapid decline of hypoxanthine by XO during reperfusion were used for an assessment of the SOA-generating capacity of these kidneys. Only 1/25-1/50 of the kidney cytosol was able to scavenge the whole amount of SOA generated by the total XO activity of rat kidney. Thus, it could be analytically and stoichiometrically shown that after IR there is only a moderate oxidative stress in kidneys of young rats; this is due to their high SOA-scavenging capacity compared to their SOA-generating ability. We investigated the time course of HSP70-1 and -2 mRNA expression and its relation to cellular ATP levels in renal cortex after different periods of unilateral warm renal ischemia (10-60 min) and reperfusion (up to 60 min) in 10-week-old male Wistar rats, since IR is known to cause induction of both genes. Immediately after ischemia there was a significant induction of both HSP70i genes. While HSP70-1 expression constantly increased (up to 4-fold) during reperfusion, even to a higher extent with prolongation of ischemia, HSP70-2 mRNA - generally being expressed on a far lower level than HSP70-1 mRNA - was strongly induced (3-fold) during reperfusion only after brief periods (10 min) of ischemia. Cellular ATP levels rapidly dropped down to 5% with ischemia and the pattern of recovery during reperfusion significantly depended on the duration of the ischemic period thus showing a good relation to the heat-shock (protein) gene expression. We conclude that the HSP70-2 is the more sensitive gene with a lower threshold activation by mild injury, while the HSP70-1 gene mediates the big response of HSP induction after severe injury. Thus, the measurement of the cytosolic antioxidative capacity and the differential expression of HSP70-1 and -2 mRNA could be promising clinical tools to assess the donor viability.     

Ergothioneine pretreatment protects the liver from ischemia-reperfusion injury caused by increasing hepatic heat shock protein 70

BACKGROUND: Reperfusion of the liver after ischemia induces the expression of the heat shock genes and the synthesis of the heat shock proteins (HSP). We studied the effects of the natural antioxidant ergothioneine (EGT) treatment on the expression of HSP70 in ischemic-reperfused (IR) liver. METHODS: Adult male Wistar rats were randomly divided into three groups: Sham group given standard laboratory chow and water for 3 weeks followed by sham operation; Control group given standard laboratory chow and water for 3 weeks followed by liver IR injury; EGT group given standard laboratory chow supplementation l-ergothioneine (1.2 mg/kg/d body weight) administered by gavage and water for 3 weeks followed by liver IR injury. Ten rats from each group were killed to determine serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic dehydrogenase (LDH), tissue malondialdehyde (MDA), HSP70 levels, and histologic changes at 30, 60, and 120 min of reperfusion, respectively. Survival was followed for 1 week. RESULTS: IR caused significant increase in serum AST, ALT, LDH, and tissue MDA levels. As compared with the control group, animals treated with EGT experienced a significant decrease in serum AST, ALT, and LDH levels in all reperfusion periods. Tissue MDA levels in animals receiving EGT were significantly reduced as compared with control group at 30 min and 60 min after reperfusion. After ischemia, reperfusion caused a remarkable production of HSP70 in the control group. When the rats were pretreated with EGT, the levels of HSP70 increased significantly in their livers after reperfusion compared with the control group. Liver injury in the EGT-treated animals was lower to that in the control group. The 7-day survival rate was significantly improved (from 50% to 80%) by EGT pretreatment. CONCLUSION: HSP70 has been shown to induce tolerance against warm IR injury in rat livers. EGT pretreatment protects the liver from IR injury by over-expression of HSP and the subsequent suppression of lipid peroxidation.     

Differential expression of heat shock proteins 70-1 and 70-2 mRNA after ischemia-reperfusion injury of rat kidney

Ischemia-reperfusion injury in the kidney is known to cause induction of the inducible form of the 70 kDa heat shock protein HSP70i (or HSP72). However, knowledge of the expressional regulation of the two coding genes for HSP70i –HSP70-1 gene and HSP70-2 gene – is very limited. We investigated the time course of HSP70-1 and -2 mRNA expression and its relation to cellular ATP levels in the renal cortex after different periods of unilateral warm renal ischemia (10–60 min) and reperfusion (up to 60 min) in 10-week-old male Wistar rats. Immediately after ischemia there was a significant induction of both HSP70i genes. While HSP70-1 expression constantly increased (up to 4-fold) during reperfusion, even to a higher extent with prolongation of ischemia, HSP70-2 mRNA – which was generally expressed at a far lower level than HSP70-1 mRNA – was strongly induced (3-fold) during reperfusion only after brief periods (10 min) of ischemia. Cellular ATP levels rapidly dropped to 5% with ischemia and the pattern of recovery during reperfusion significantly depended on the duration of the ischemic period, thus showing a good relation with the heat shock (protein) gene expression. We conclude that HSP70-2 is the more sensitive gene with a lower activation threshold by mild injury, while the HSP70-1 gene mediates the major response of heat shock protein induction after severe injury. Received: 16 November 1998 / Accepted: 11 March 1999