热休克预处理对严重烧伤大鼠胃黏膜的保护作用及机制

目的观察热休克预处理（HS）对严重烧伤大鼠胃黏膜热休克蛋白（HSP）60、HSP70表达及线粒体超氧化物歧化酶（SOD）、细胞色素氧化酶（CCO）活性的影响，探讨HS对严重烧伤大鼠胃黏膜的保护作用及机制。方法将Wistar大鼠随机分为烧伤组（40只）：烧伤后即制作急性胃黏膜损伤模型；另取8只大鼠不致伤作为空白对照。HS组（40只）：于烧伤前20h行HS，另取8只大鼠仅行HS不烧伤，作为实验对照。放线菌素D组（40只）：在HS前30min静脉注射放线菌素D0．1mg／kg，随后的处理同HS组；另取8只大鼠只注射放线菌素D不烧伤，作为实验对照。于伤后3、6、12、24、48h处死各组大鼠（每组每时相点8只）。取胃黏膜组织检测胃黏膜损伤指数（UI）、HSP70 mRNA、HSP60、HSP70的表达及SOD、CCO的活性。结果大鼠烧伤后uI呈时间依赖性增加，烧伤组伤后24h胃黏膜损伤程度最严重，UI为12．8±1．9。除伤后3h外，HS组大鼠各时相点uI均低于烧伤组（P＜0．05或0．01）。放线菌素D组大鼠胃黏膜损伤程度明显重于烧伤组及HS组（P＜0．05）。烧伤组、HS组除伤后24、48h外其余各时相点HSP70 mRNA均增加，而放线菌素D组伤后24、48h有所增加。与烧伤组比较，HS组大鼠胃黏膜HSP60及HSP70表达显著增加，除伤后48h外其余各时相点比较，差异均有统计学意义（P＜0．05或0．01）。放线菌素D组大鼠HSP60和HSP70表达明显受抑（P＜0．05）。大鼠烧伤后胃黏膜CCO、SOD活性不断降低，经HS后CCO及SOD下降不明显，在伤后6、12、24h均高于同时相点烧伤组（P＜0．05或0．01）。结论HS对大鼠严重烫伤后急性胃黏膜损害具有保护作用，其机制可能与HSP60、HSPT0诱导表达增强，线粒体SOD及CCO活性增加等有关。     

Induction of Heat Shock Response: Effect on the Rat Liver with Carbon Tetrachloride-induced Fibrosis from Ischemia-reperfusion Injury

n = 56) received no pretreatment except anesthesia, and the heat shock group (group HS, n = 56) were exposed to heat shock (42°C) for 15 minutes. After a 48-hour recovery all rats were subjected to 30 minutes of warm ischemia. Western blotting analysis was employed for heat shock protein (HSP) 72 detection. The adenine nucleotide levels in liver tissue and the liver enzyme levels in serum were measured before and after ischemic intervention (seven animals were used at each of six time point measurements in both groups). HSP72 was induced in group HS at greater intensity than in group C. The survival rate on postoperative day 7 in group C (3/14) was significantly poorer than that in group HS (14/14) ( p < 0.01). The higher survival rate in group HS was accompanied by more rapid recovery of the adenosine triphosphate level and lower serum levels of liver enzymes after reperfusion ( p < 0.01 vs. group C). Heat shock preconditioning induces HSP72 in the rat liver with fibrosis and provides significantly increased tolerance of warm-ischemia reperfusion injury.     

Hypertonic saline resuscitation after mesenteric ischemia/reperfusion induces ileal apoptosis

BACKGROUND: We have previously demonstrated that hypertonic saline (HS) resuscitation decreased inflammation and mucosal injury after mesenteric ischemia/reperfusion (I/R). In contrast to I/R cell necrosis, apoptosis provides controlled cell death that minimizes inflammation. We therefore hypothesized that HS resuscitation after mesenteric I/R would induce apoptosis and decrease mucosal injury. METHODS: Rats underwent 60 minutes of superior mesenteric artery occlusion (SMAO) and then received no resuscitation or resuscitation with 4 mL/kg of HS, 4 mL/kg of lactated Ringer's (LR) solution (equal volume), or 32 mL/kg of LR solution (equal salt load). Rats were killed at 6 hours of reperfusion, and ileum was harvested for analysis. DNA fragmentation (apoptosis) was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and mucosal injury by histology (Chiu score 0-5). Caspase-3 (proapoptotic mediator) and Bcl-xL (antiapoptotic mediator) protein expression were analyzed by Western immunoblot. RESULTS: SMAO with no resuscitation, SMAO with 4 mL/kg of LR, and SMAO with 32 mL/kg of LR increased apoptosis (quantitated by TUNEL) and I/R-induced mucosal injury (quantitated by Chiu score). This was associated with an increase to similar levels in both proapoptotic caspase-3 and antiapoptotic Bcl-xL protein expression. Moreover, SMAO with 4 mL/kg of HS further increased apoptosis but decreased mucosal injury. This was associated with a differential expression of proapoptotic caspase-3 over antiapoptotic Bcl-xL. CONCLUSION: HS resuscitation after mesenteric I/R significantly increased ileal mucosal apoptosis while decreasing mucosal injury and may represent a novel mechanism by which HS resuscitation after mesenteric I/R reduces inflammation and imparts protection to the gut.     

Chronic COX-2 inhibition reduces medullary HSP70 expression and induces papillary apoptosis in dehydrated rats

BACKGROUND: Papillary cells adapt to their hyperosmotic environment by accumulating organic osmolytes and by enhanced synthesis of heat shock protein 70 (HSP70), which protect against high-solute concentrations. Because cyclooxygenase-2 (COX-2) is expressed abundantly in the renal papilla and is induced by dehydration, and because HSP70 expression is stimulated by specific prostaglandins, COX-2 inhibition may interfere with cellular osmoadaptation. METHODS: In vivo, rats received rofecoxib before water deprivation. Medullary expression of several tonicity-responsive genes was analyzed and apoptosis was monitored by transferase-mediated dUTP nick-end labeled (TUNEL) staining and determination of papillary caspase-3 activity. In vitro, inner medullary collecting duct 3 (IMCD3) cells were exposed to hypertonic medium containing a COX-2-specific inhibitor. Thereafter, expression of tonicity-responsive genes was analyzed and resistance to high-solute concentrations was examined. Further, the effect of Delta 12-PGJ2, a urinary prostaglandin, and of HSP70 overexpression on resistance against high urea concentration, was evaluated. RESULTS: Rofecoxib treatment significantly increased urine osmolality due to higher urea concentrations, but reduced papillary HSP70 abundance by 50%. TUNEL staining showed numerous apoptotic cells in the papilla, associated with increased caspase-3 activity. These in vivo results were confirmed by experiments on cultured IMCD3 cells, in which COX-2 inhibition impaired the tonicity-induced up-regulation of HSP70 expression and rendered the cells susceptible to high urea concentrations. Furthermore, Delta 12-PGJ2 increased both HSP70 expression and resistance against high urea, which was causally linked to higher HSP70 levels. CONCLUSION: These observations support the view that chronic COX-2 inhibition reduces medullary HSP70 expression, thus rendering papillary cells susceptible to damage by high urea concentrations, especially when accompanied by dehydration.     

Protective effect of heat preconditioning of rat liver graft resulting in improved transplant survival

BACKGROUND: The protective effect of heat preconditioning of the liver against ischemia-reperfusion injury has been reported mostly in models of transient ischemia in relation to heat shock protein 70 (HSP70). We estimated the effect of heat preconditioning of liver grafts on the transplant survival rate and on apoptosis of sinusoidal endothelial cells (SEC) as well as hepatocytes in a rat model of liver transplantation. METHODS: Donor rats of the heat shock (HS) group were subjected to heat preconditioning 48 hr before graft harvest, and HSP70 levels were estimated by. Western blot analysis and immunohistochemistry. The liver isografts from the HS group and control (C) group were preserved in Euro-Collins solution for 6 or 8 hr and transplanted orthotopically. Serum hyaluronic acid and alanine aminotransferase were measured, and apoptosis of the SEC and hepatocytes was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining and electron microscopy. RESULTS: HSP70 expression was detected not only in hepatocytes but also in SEC. In the 8-hr preservation model, the 1-week survival rate was 60% in the HS group and 0% in the C group. Serum hyaluronic acid and alanine aminotransferase levels in the HS group were significantly lower than those in the C group at 3 hr after reperfusion, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling-positive SEC in the C group (35.2%) was markedly increased compared with the HS group (10.1%). Electron microscopic examination confirmed the features of apoptosis of SEC. CONCLUSIONS: Heat preconditioning of the graft improved the survival rate of the liver transplants. Induction of HSP70 in SEC as well as in hepatocytes might attenuate preservation-reperfusion injury by inhibiting apoptosis of SEC.     

Hyperbaric oxygen treatment prevents nitric oxide‐induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70

Heat shock proteins (HSPs), inflammatory cytokines, nitric oxide (NO), and localized hypoxia‐induced apoptosis are thought to be correlated to the degree of cartilage injury. We investigated the effect of hyperbaric oxygen (HBO) on (1) interleukin‐1β (IL‐1β)‐induced NO production and apoptosis of rabbit chondrocytes and (2) healing of articular cartilage defects. For the in vitro study, RT‐PCR and Western blotting were performed to detect mRNA and protein expressions of HSP70, inducible NO synthase (iNOS), and caspase 3 in IL‐1β‐treated chondrocytes. To clarify that the HSP70 was necessary for anti‐iNOS and anti‐apoptotic activity by HBO, we treated the cells with an HSP70 inhibitor, KNK437. For the in vivo study, cartilage defects were created in rabbits. The HBO group was exposed to 100% oxygen at 2.5 ATA for 1.5 h a day for 10 weeks. The control group was exposed to normal air. After sacrifice, specimen sections were sent for examination using a scoring system. Immunohistochemical analyses were performed to detect the expressions of iNOS, HSP70, and caspase 3. Our results suggested that HBO upregulated the mRNA and protein expressions of HSP70 and suppressed those of iNOS and caspase 3 in chondrocytes. KNK437 inhibited the HBO‐induced downregulation of iNOS and casapase 3 activities. The histological scores showed that HBO markedly enhanced cartilage repair. Immunohistostaining showed that HBO enhanced HSP70 expression and suppressed iNOS and caspase 3 expressions in chondrocytes. Accordingly, HBO treatment prevents NO‐induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 376–384, 2013     

Effects of dihydroartemisinin on HSP70 expression in human prostate cancer PC‐3 cells

We aimed to evaluate the effects of dihydroartemisinin (DHA) on heat‐shock protein 70 (HSP70) expression in human prostate cancer PC‐3 cells and to examine the molecular mechanism. The viability of PC‐3 cells following treatment with 25, 50, 100 and 200 μmol/L DHA for 48 hr was detected by flow cytometry and MTT assay. The expression of HSP70 mRNA was detected by RT‐qPCR. The expression levels and locations of HSP70, caspase‐3 and apoptosis‐inducing factor (AIF) were detected with immunofluorescence assay. With 100 μmol/L HSP70 inhibitor quercetin as positive control and dimethyl sulphoxide (DMSO) as solvent control, the protein expressions of HSP70, apoptotic protease activating factor‐1 (Apaf‐1) and AIF were detected by Western blot. DHA promoted PC‐3 cell apoptosis dose‐dependently. With increasing DHA dose, the expression of HSP70 mRNA significantly decreased (p < 0.05). DHA did not change the location of HSP70 or AIF. Compared with control and DMSO groups, the expression of HSP70 protein significantly decreased, and those of Apaf‐1, caspase‐3 and AIF significantly increased following treatment with DHA and quercetin for 48 hr. In conclusion, DHA inhibits the expression of HSP70 and induces the apoptosis of PC‐3 cells. The results provide valuable experimental evidence for prostate cancer therapies using DHA.     

Evaluation of Ischemia-Reperfusion Liver Injury by Near-Infrared Spectroscopy in an Experimental Swine Model: The Effect of Desferoxamine

ABSTRACT

Introduction: Ischemia-reperfusion (I-R) injury has long been regarded a primary factor for the physiological dysfunction that can occur following major liver resection performed under vascular control. The aim of our study was to assess the effect of treatment with desferoxamine (DFO), a potent antioxidative agent, monitoring the I-R injury on a porcine model of major hepatectomy. Materials and Methods: Twelve female pigs were allocated to control (n = 6) and DFO groups (n = 6) and underwent 30 min of liver ischemia, during which a ≥30% hepatectomy was performed, followed by six hours of postoperative monitoring. The DFO group animals were preconditioned with a continuous iv solution of DFO to a total dose of 100 mg/kg during their postoperative period. Liver remnants (≈70% of initial liver volume) were evaluated by means of infrared spectroscopy, serum lactate measurement of the systemic, portal and hepatic vein blood, and by immunohistochemical assessment of apoptosis in consecutive liver biopsies. Results: DFO group demonstrated considerably faster restoration of tissue oxygenation (92.33% vs. 80%, p < .05) and serum lactate values (1.23 mmol/l vs. 2.27 mmol/l, p < .05). Moreover, apoptosis as estimated by TUNEL and caspase-3 staining was significantly lower in the DFO group (0.06% vs. 1.17% and 1.17% vs. 2%, respectively, p < .05). The severity of the I-R injury showed a linear correlation to the restoration of tissue oxygenation, as estimated by infrared-spectroscopy (r² = 0.81, p < .01). Conclusion: Iron chelation with DFO appears to attenuate I-R injury of the liver remnant following hepatectomy, as reflected by faster restoration of tissue oxygenation and lower apoptotic activity.     

Clotrimazole Protects the Liver Against Normothermic Ischemia-Reperfusion Injury in Rats

Objective

To investigate the possible antiapoptotic prosurvival role of the pregnane X receptor (PXR) in hepatic ischemia-reperfusion injury in rats using clotrimazole (CTZ), a strong PXR transactivator.

Materials and Methods

Male Sprague-Dawley rats were divided into 3 groups of 6 each: sham-treated, control, and CTZ-treated animals. Control and CTZ-treated animals were subjected to 30 minutes of normothermic ischemia of the whole liver followed by 6 hours of reperfusion. The animals were then killed, and the liver was excised and blood samples collected.

Results

Clotrimazole induced a significant increase in expression of the CYP3A gene, indicating PXR transactivation, whereas expression of the antiapoptotic Bcl-xL gene was not increased. Serum concentrations of aspartate aminotransaminase and alanine aminotransaminase were lower in CTZ-treated animals than in control animals (difference not significant). Levels of poly(adenosine diphosphate–ribose) polymerase, a caspase-3 substrate, remained significantly higher in the CTZ-treated group compared with controls (P < .05). Clotrimazole increased the expression of phospho-p 44/42 extracellular signal-regulated kinase 1,2 (P < .05). The gene expression of the heat shock proteins 27, 70 and 90 was significantly lower in CTZ-treated animals than in controls (P < .05).

Conclusion

Clotrimazole-mediated PXR transactivation protects the liver against ischemia-reperfusion apoptosis in rats. Phospho-p 44/42 extracellular signal-regulated kinase 1,2 is activated, whereas gene expression of heat shock proteins 27, 70, and 90 is downregulated by induction of PXR.     

Effect of uric acid on liver injury during hemorrhagic shock

BACKGROUND: It remains unproven whether nitric oxide (NO) exerts a toxic effect on hepatocytes directly or through the formation of a more toxic compound during hemorrhagic shock (HS). NO reacts at a very high rate constant with superoxide to give peroxynitrite, a potentially toxic molecule. In this study, we investigated whether or not peroxynitrite contributed to tissue injury in the liver during HS. METHODS: Male Sprague-Dawley rats were subjected to decompensated HS followed by resuscitation. In addition to the time course of tissue injury and inducible NO synthase (iNOS) messenger RNA (mRNA) expression in the liver during HS, we investigated the effect of N6-(iminoethyl)-L-lysine(LNIL) (a specific inhibitor of iNOS) and also that of uric acid (a natural scavenger of peroxynitrite) on tissue injury and nitrotyrosine formation (a footprint of peroxynitrite) in the liver. RESULTS: The liver injury, evaluated by plasma aminotransferase levels and histology, became evident at the end of the shock period and had significantly increased 1 hour after the start of resuscitation (Shock-1 h). There was no iNOS mRNA expression in the liver at baseline, and it had clearly increased by Shock-1 h. Treatment with LNIL or uric acid significantly attenuated the tissue injury with a prominent reduction in nitrotyrosine formation in the liver. CONCLUSIONS: These lines of evidence suggest that one of the mechanisms by which NO production causes liver injury during HS may be its reaction with superoxide to form peroxynitrite.     

L-selectin blockade and liver function in rats after uncontrolled hemorrhagic shock.

Hemorrhagic shock (HS) and resuscitation can be seen as a global body ischemia-reperfusion (I/R) injury characterized by neutrophil infiltration and organ damage. Liver dysfunction occurs early after HS. Adhesion molecules are needed for the first steps ofneutrophil migration. Thus, the purpose of this study was to investigate the role of L-selectin in the liver after uncontrolled HS and resuscitation. Forty-eight Sprague Dawley rats were subjected to uncontrolled HS and resuscitation. Animals were divided into three groups: sham, uncontrolled HS and resuscitation, and uncontrolled HS and resuscitation with anti-L-selectin treatment. At 6 we evaluated liver injury tests, liver tissue myeloperoxidase (MPO), and liver histology. Survival was followed for 3 days and compared between groups. Statistical analysis included Fisher's exact test and one-way analysis of variance. Survival significantly increased from 30% in the control group to 60% in the treated group (p < .05). Hepatocellular and structural injury as well as neutrophil infiltration was significantly decreased in treated animals (p < .05). Thus, blockade of L-selectin resulted in decreased hepatocellular injury and increased survival in our model of uncontrolled HS. Selectins may be important therapeutic targets for blockade in the treatment of HS.     

Glutamine protects articular chondrocytes from heat stress and NO-induced apoptosis with HSP70 expression

OBJECTIVE: To investigate the effect of l-glutamine (Gln) on stress responses of chondrocytes exposed to heat stress or nitric oxide (NO). METHODS: Cultures of articular chondrocytes were established from rabbit joints, and treated for 12h with various concentrations of Gln (0-20 mM). In some experiments, cells were also treated with quercetin (Que), a heat shock protein 70 (HSP70) inhibitor. Heat stress (43 degrees C) was applied to the cells for 0-120 min. Apoptosis was induced by 0.5mM sodium nitroprusside (SNP) dihydrate that produces NO. After stress loading, HSP70 expression was detected by Western blot analysis. Cell viability was assessed by lactate dehydrogenase (LDH) release and tetrazolium salt-based assays, while apoptosis was evaluated by Hoechst 33342 staining, TUNEL methods and active caspase-3 determination. RESULTS: Gln demonstrated dose-dependent enhancing effect on stress-mediated induction of HSP70, while in the absence of any stress HSP70 was not induced by Gln alone. After heating or SNP loading, chondrocytes showed severe reduction in viability, while the cytotoxic outcome was almost completely abrogated by conditioning with Gln. The protective effect of Gln was significantly blocked by Que that effectively suppressed stress-induced HSP70 expression in chondrocytes. The Gln also rendered chondrocytes unsusceptible to NO-induced apoptosis that was frequently seen in SNP-treated culture. CONCLUSION: This study demonstrated that the treatment of chondrocytes with Gln protected the cells from heat stress and NO-induced apoptosis. These chondroprotective effects of Gln may be mediated by HSP70.     

膝关节骨关节炎发病过程中HSP70对软骨细胞凋亡的影响

目的:观察热休克蛋白70(HSP70)和半胱氨酸天冬氨酸蛋白酶-3蛋白(caspase-3)在膝关节骨关节炎(knee osteoarthritis,KOA)发病过程中的表达,探讨其相关性。方法:40只3月龄SD大鼠分为两组,实验组30只,对照组10只。实验组采用Hulth法复制膝骨性关节炎模型,作前交叉韧带切断加内侧1/3半月板切除术,对照组不作特殊处理。造模后,不采取任何措施,自由饲养。分别在术后1、2、4周取材股骨端和胫骨端关节,对标本组织进行肉眼、免疫组化及光镜观察,采用Mankin改良的关节软骨病理评分标准进行组织学评分。结果:肉眼及光镜观察,实验组均可见KOA的改变,如滑膜增生、表层软骨糜烂等;同时,免疫组化中,术后1、2、4周HSP70的表达逐渐增加,caspase-3的表达先增加后降低,而对照组并无类似改变。Mankin评分显示1周分别和2、4周比较,差异均有统计学意义(P<0.01)。结论:膝关节骨关节炎发病过程中,热休克蛋白抑制软骨细胞凋亡,并对软骨细胞具有保护作用,这将为临床上诸保守治疗提供一定程度的客观科学依据。     

Oncogenic ras results in increased cell kill due to defective thermoprotection in lung cancer cells

Background. The survival response of normal cells to heat stress is an upregulation of heat shock proteins and ras protein activation. We hypothesized that in lung cancer cells the presence of oncogenic ras interferes with thermoprotective mechanisms resulting in cell death.Methods. An equal number of lung tissue culture cells (normal and cancerous) were subjected to either heat stress and then recovery (43°C for 180 minutes, 37°C for 180 minutes) or recovery alone (37°C for 360 minutes). End points were surviving number of cells, cell-death time course, heat shock protein (HSP70, HSC70, HSP27) expression before and after heat stress, and time course for HSP70 expression during heat stress and recovery. Heated cells were compared with unheated control cells, then this difference was compared between cell types.Results. Heat stress in normal cells caused an 8% decrease in cell number versus a 78% ± 5% decrease in cancer cells (p < 0.05). In normal cells, heat stress caused a 4.4-fold increase in HSP70, no change in HSC70, and a 1.7-fold increase in HSP27. In contrast, cancer cells initially contained significantly less HSP70 (p < 0.05), and there was a 27-fold increase in HSP70 and a 2-fold increase in HSC70 with no HSP27 detected (comparison significant, p < 0.05). HSP70 time course in normal cells showed that HSP70 increased 100-fold, reaching a vertex at 2 hours and remaining elevated for 24 hours; in cancer cells, HSP70 maximum expression (100-fold) peaked at 5 hours, then decreased to slightly elevated at 24 hours.Conclusions. Cancer cells with oncogenic ras have defective thermoprotective mechanism(s) causing increased in vitro cell death, which provides an opportunity for thermal treatment of lung cancer.     

热休克蛋白70抑制大鼠颅脑损伤诱生型一氧化氮合酶mRNA的表达

目的 探讨热休克蛋白７０（ＨＳＰ７０）对大鼠感染性脑损伤（感脑）诱生型一氧化氮合酶（ｉＮＯＳ）的表达及一氧化氮（ＮＯ）合成的影响。方法 将大鼠７２只随机分为正常对照组、感染性脑损伤组和热休克处理组,每组又ｈ共３个时间点。采用百日咳菌液通过左颈内动脉注入制成大鼠感染性脑损伤模型,用Ｗｅｓｔｅｒｎ印迹杂交技术检测各组各时间点的ＨＳＰ７０的表达,同时用原位杂交方法检测各组ｉＮＯＳｍＲＮＡ的表达及Ｇｒｉｅｓｓ法测定各组的ＮＯ含量的变化。结果 Ｗｅｓｔｅｒｎ印迹杂交分析结果表明,大鼠感脑各组及正常组有一定量的ＨＳＰ７０表达,而热休克处理组的ＨＳＰ７０的量明显高于感脑组（Ｐ〈０．０１）。原位杂交结果提示ｉＮＯＳ在感脑的大脑皮质神经细胞４、８、２４ｈ开始表达,可见明显的杂交信号,而休克处理组仅有少量的阳性颗粒。ＮＯ含量在感脑     

Effect of Caspase Inhibition on Thymic Apoptosis in Hemorrhagic Shock

In hemorrhagic shock (HS) an increased thymic apoptosis (TA) was described. The aim of this study was to evaluate the effect of administration of the caspase inhibitor N-benzyloxy-carbonil-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) during the resuscitation phase on TA, organ dysfunctions, and tumor necrosis factor (TNF)-α release in HS. Forty rats were randomly assigned to four groups: no HS/resuscitation (sham); HS/resuscitation with shed blood and normal saline (control); HS/resuscitation with shed blood and phosphate-buffered solution (PBS) (vehicle); and HS/resuscitation with shed blood and Z-VAD-FMK (inhibitor). Rats were subjected to HS by blood removal to a MAP of 35–40 mmHg. After a 1-h shock period, the animals were resuscitated according to the protocol. At 1 and 3 h after resuscitation, transaminases, creatinine, urea, lipase, TNF-α, and TA were evaluated. Our study showed that a nonlethal HS is early able to induce organ dysfunctions and increased TA. Administration of Z-VAD-FMK did not significantly decrease organ dysfunctions, while it induced a significant TNF-α release. TA was significantly reduced by Z-VAD-FMK after 1 h, but not after 3 h. Our results suggest that postinjury caspase inhibition does not attenuate organ dysfunctions, and also does not permanently reduce TA induced by HS and resuscitation in rats.     

Cyclosporin A Has a Protective Effect with Induced Upregulation of Hsp70 and nNOS on Severe Spinal Cord Ischemic Injury in Rabbits

The purpose of this study is to ascertain the neuroprotective effect of cyclosporin A on the 25-min surgical ischemia model in the spinal cords of rabbits with neuropathological correlation and histoimmunochemical analyses measuring HSP70 and neuronal NOS (nNOS). New Zealand white rabbits were randomly divided into four groups (each n = 8): the C2, C7, Cs2, and Cs7 groups. The C2 and C7 groups underwent a 25-min surgical aortic cross-clamp without intervention and were sacrificed respectively on day 2 and on day 7 postoperatively. The Cs2 and Cs7 groups received cyclosporin A (25 mg/kg) intravenously 15 min after the 25-min cross-clamp and were sacrificed respectively on day 2 and day 7 postoperatively. Neurologic functions were evaluated on postoperative days 2 and 7 using the Tarlov scoring system. Then the rabbits were sacrificed for histopathologic observation. HSP 70 and nNOS stains with TUNEL assay were done for the C2 and Cs2 groups. All rabbits survived the experimental procedure. Tarlov's score for the Cs7 group (2.75 ± 0.89) was significantly higher than that of the C7 group (1.25 ± 1.39) (p <. 05). Tarlov's score of the Cs7 group was also statistically higher on day 7 than on day 2 (p <. 05). Strong correlation between the neurological and histological scorings was found. The TUNEL assay showed that the mean number of positive cells in the C2 group was 17.5 ± 22.6 and in the Cs2 group was 12.5 ± 11.1, but there was no statistically significant difference between the groups. There was more expression of HSP70 and nNOS in the cyclosporin groups than in the ischemia groups. In conclusion, this study demonstrates that cyclosporin A reduces neurological injury in the rabbit model of 25-min spinal cord ischemia. The neuroprotective effect of cyclosporin A against ischemia seems to be related to overexpressions of nNOS and HSP70.     

Small interfering RNA targeting heat shock protein 70 enhances chemosensitivity in human bladder cancer cells

ObjectivesTo evaluate the expression levels of heat shock protein 70 (HSP70) in human urothelial cancer of the bladder and to assess the therapeutic effects of treatment with small interfering RNA (siRNA) targeting HSP70 on human bladder cancer KoTCC-1 cells.Materials and methodsHSP70 expression in bladder cancer specimens obtained from 235 patients were evaluated by immunohistochemical staining. We then analyzed changes in the growth and chemosensitivity of KoTCC-1 cells following treatment with HSP70 siRNA.ResultsExpression levels of HSP70 protein in bladder cancer specimens were significantly related to major prognostic indicators, including pathologic stage and tumor grade. Treatment of KoTCC-1 with HSP70 siRNA resulted in a dose-dependent inhibition of HSP70 expression. HSP70 siRNA significantly inhibited the growth of KoTCC-1 compared with that after treatment with scrambled control siRNA. Among several chemotherapeutic agents, the most powerful synergistic cytotoxic effect was observed when KoTCC-1 was treated with gemcitabine plus HSP70 siRNA, which induced more than 50% reduction in the IC₅₀ of gemcitabine. Furthermore, a significant increase in the subG0-G1 fraction of KoTCC-1 and the DNA fragmentation was observed only after combined treatment with HSP70 siRNA and sublethal doses of gemcitabine, but not after treatment with either agent alone. Similarly, caspase-3 and caspase-9, but not caspase-8, in KoTCC-1 were synergistically activated by combined treatment with gemcitabine and HSP70 siRNA.ConclusionsSilencing of HSP70 expression using siRNA could be an attractive therapeutic strategy for bladder cancer by inducing inhibition of tumor growth as well as enhancing chemosensitivity.     

Impact of fluid therapy on apoptosis and organ injury during haemorrhagic shock in an oxygen-debt-controlled pig model

Introduction

Apoptosis, or programmed cell death, seems to play a role in the physiology of shock. The influence of fluid resuscitation on the occurrence of apoptosis during haemorrhage is still unclear. Using an experimental randomised study, the goal of this investigation was to find a relation between different frequently used resuscitation fluids and evidence of apoptosis.

Materials and methods

Sixty female pigs with a mean body weight of 20 kg were randomised into six groups, each receiving a different resuscitation fluid therapy: malated Ringer, lactated Ringer, hypertonic saline, hypertonic saline solution/Dextran 60, carbonate/gelatine and a sham group (no shock, no resuscitation). A haemorrhagic shock with a predefined oxygen debt with high mortality expected was induced for a period of 60 min. Then, the resuscitation fluid therapy within each group was initiated. At the beginning, after 1 h of shock and 1 and 2 h after resuscitation, biopsies from the liver were taken, as one of the most important metabolism organs of shock. Three hours after the beginning of the resuscitation period, the animals were allowed to recover under observation for 3 days. At the end of this period, a state of narcosis was induced and another liver biopsy was taken. Finally, the animals were sacrificed and samples were taken from the liver, kidney, heart and hippocampus. The TUNEL method was used for identifying apoptosis. Impairment of liver function was indicated by the measurement of transaminase levels.

Results

There was no observed difference in the rate of apoptosis in all groups and a low number of apoptotic cells were found in all the organs sampled. The sham group also showed a low count of apoptosis. The hypoxia-sensitive neurons within the hippocampus did not show any signs of apoptosis. The high oxygen debt during haemorrhage led to a high mortality. The non-treated animals died very quickly, as an indicator for severe shock. Animals treated with hypertonic saline showed a significant increase in aspartate transaminase (AST) plasma levels on the first day after shock.

Conclusion

The different resuscitation fluids used in the treatment of haemorrhagic shock in this experimental model showed no evidence of a different apoptosis rate in the end organs.     

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