Glutamate-induced cell death of immortalized murine hippocampal neurons: neuroprotective activity of heme oxygenase-1, heat shock protein 70, and sodium selenite

HT22 immortalized hippocampal neurons serve as a cellular model system to study oxidative stress, an imbalance of cellular redox homeostasis. Glutamate induces HT22 cell death by inhibiting the uptake of cystine into the cells via the cystine/glutamate transport system xc-, thus leading to reduced levels of glutathione. Here, we show that glutamate-induced cell death is attenuated in HT22 cells overexpressing heat shock protein 70 or heme oxygenase-1. Moreover, supplementing the culture medium with sodium selenite completely protected HT22 against oxidative glutamate toxicity. In contrast, neither heat shock protein 70 nor heme oxygenase-1 expression or increased concentrations of sodium selenite protected HT22 cells against serum withdrawal-induced cell death. These data indicate that glutamate-induced cell death differs substantially from that induced by growth factor deprivation.     

Expression of heat shock protein 70 in leukocytes of patients with sepsis

The heat shock proteins are known to protect cells against diverse injuries such as cytotoxicity by TNFalpha acting mainly as chaperones for denatured proteins. Lipopolysaccharide stimulates the production and the release of numerous endogenous mediators of sepsis: tumor necrosis factor alpha, interleukin-1, and interleukin-6 that induce fever production. Moreover, temperature at 40 degrees C is sufficient to induce heat shock and attenuate both TNFalpha and IL-1 expression. We demonstrate a distinct profile in gene expression of HSP 70 family in leukocytes obtained from different phases of septic patients. Our findings strongly suggest that HSP 70 may play a role in the outcome of septic shock patients.     

一氧化碳中毒迟发性脑病模型小鼠脑内血红素加氧酶1 mRNA和蛋白的表达

背景：研究表明一氧化碳中毒迟发性脑病症状的出现与一氧化碳中毒后神经组织细胞凋亡持续发生关系较大。有关血红素加氧酶的细胞保护作用尤其在脑损伤中的细胞保护作用还存在争议。 目的：观察一氧化碳中毒后不同时间点小鼠脑内血红素加氧酶1 mRNA和蛋白的表达变化。 方法：雄性昆明小鼠随机分为2组,一氧化碳中毒组腹腔注射一氧化碳制备一氧化碳中毒迟发性脑病模型,空气对照组腹腔注射空气。应用原位杂交及Western blot法观察两组在不同时间点海马区血红素加氧酶1 mRNA及蛋白表达变化。 结果与结论：空气对照组血红素加氧酶1 mRNA表达阳性细胞较少,染色较浅;一氧化碳中毒组海马阳性细胞数较多,染色较深。血红素加氧酶1 mRNA在1 d表达增加(P < 0.01),3 d达高峰(P < 0.01),5 d时下降(P < 0.01),21 d时仍高于空气对照组(P < 0.01)。血红素加氧酶1蛋白表达与血红素加氧酶1 mRNA表达变化相一致。结果表明血红素加氧酶1 mRNA及其蛋白的表达增加可能在一氧化碳中毒所致迟发性脑病的发病机制中起重要作用。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

睡眠剥夺对HSP70表达及脑超微结构的影响

目的：观察睡眠剥夺（SD）后大鼠脑组织HSP70表达的变化及对超微结构的影响。方法：44只雄性SD大鼠随机分为11组，每组4只，免疫组织化学方法检测HSP70的表达，电镜观察海马超微结构的变化。结果：睡眠剥夺后12小时即可在大脑皮质及海马观察到HSP70阳性细胞，2—3天数量达到高峰，7天时明显下降。白天睡眠剥夺12小时（SDd12h）组HSP70阳性细胞数较夜晚睡眠剥夺12小时（SDn12h）组多（P〈0．05）。RS组大脑皮质HSP70阳性细胞数较白天睡眠剥夺1天（SDd1d）组减少（P〈0．05）。白天睡眠剥夺3天（SDd3d）海马出现超微结构改变，白天睡眠剥夺7天（SDd7d）后改变更加明显。结论：睡眠剥夺可影响大鼠脑组织HSP70表达及超微结构。     

远期缺血预适应激活热休克蛋白27、促红细胞生成素、血红素加氧酶1对肾脏缺血再灌注损伤的影响**

背景：缺血再灌注损伤是临床导致急性肾衰竭等其他疾病的重要原因,其机制为多因素、多途径的复杂的病理过程。 目的：观察肾脏进行预处理后激活热休克蛋白、促红细胞生成素和血红素加氧酶1对肾脏缺血再灌注损伤的影响。 方法：雄性C57BL/6小鼠90只随机分成3组。缺血再灌注组为右肾切除,左肾缺血25 min再灌注24 h;预适应组为双侧肾脏缺血20 min再灌注8 d后再进行缺血再灌注。假手术组开腹游离肾蒂。 结果与结论：血清肌酐和尿素氮检测预适应组和假手术组明显低于缺血再灌注组(P < 0.01);MPO染色发现缺血再灌注组大量中性粒细胞浸润(P < 0.01);PAS染色发现预适应组肾组织病理情况轻于缺血再灌注组(P < 0.05);TUNEL染色分析结果表明预适应组和假手术组细胞凋亡数明显少于缺血再灌注组(P < 0.01);预适应组热休克蛋白27 mRNA表达明显高于缺血再灌注和假手术组(P < 0.05),热休克蛋白27 mRNA于第8天时最强,促红细胞生成素、血红素加氧酶1 mRNA在24~    48 h达到峰值A,然后逐渐下降,第8天后达到峰值B,B>A,并且高于假手术组(P < 0.01)。提示远期缺血预适应激活热休克蛋白27、促红细胞生成素、血红素加氧酶1,能减少炎症因子浸润、促进肾小管细胞修复和抑制细胞凋亡从而参与肾脏内源性保护机制。     

Association of heat shock protein 70 induction and the amelioration of experimental autoimmune uveoretinitis in mice

Experimental autoimmune uveoretinitis (EAU) serves as a model of human endogeneous uveitis. In the present study we examined whether induction of heat shock protein (HSP) 70 by oral geranylgeranylacetone (GGA) administration had a therapeutic effect on murine EAU. When C57BL/6 mice that had received oral administration of GGA (500mg/kg) were immunized with interphotoreceptor retinoid-binding protein (IRBP)-derived peptide plus adjuvants, the expression levels of HSP70 mRNA and protein were rapidly and transiently upregulated in eyes of the GGA-treated mice, compared with those from vehicle-pretreated and IRBP-immunized mice. The antigen-specific T cell proliferation was partially suppressed in these mice treated with GGA. The mean EAU scores of the GGA-treated mice on day 21 and 28 (2.4+/-0.2 and 2.1+/-0.2, respectively) were significantly lower than those in the controls (3.0+/-0.1 and 2.6+/-0.2, respectively p<0.01). The histopathological severity of the GGA-treated mice (average 0.33) was markedly milder than that in the controls (average 1.63, p<0.05) at day 21. The present findings demonstrate that the pharmacological induction of HSP70 may be applicable to the amelioration of ocular autoimmune diseases.     

Permanent focal and transient global cerebral ischemia increase glial and neuronal expression of heme oxygenase-1, but not heme oxygenase-2, protein in rat brain

Two heme oxygenase (HO) proteins have been identified to date; HO-1, a stress-induced protein, and HO-2, a constitutively expressed isoform. Recently, it was demonstrated that HO-1 mRNA expression is increased following transient global ischemia. The present study examined the effects of global and focal ischemia on HO-1 and HO-2 protein, using immunocytochemistry. Following 20 min of ischemia (rat 4 vessel occlusion model with hypotension) and 6 h of recirculation, increased HO-1 immunoreactivity was evident in hippocampal neurons. After 24 h of recirculation, HO-1 was observed in both hippocampal neurons and astroglial cells. By 72 h, expression was primarily glial and restricted to CA1 and CA3c. In addition to hippocampus, HO-1 was also evident in both neurons and glia in cerebral cortex and thalamus, and in striatal glial cells. Twenty-four hours following permanent focal ischemia, HO-1 immunoreactivity was observed in astroglial cells in the penumbra region surrounding the infarct. In contrast to HO-1, the pattern of HO-2 immunoreactivity was not altered following transient global or permanent focal ischemia. The increased expression of HO-1 following ischemia may confer protection against oxidative stress, but might also contribute to the subsequent neuronal degeneration.     

Immunohistochemical expression of heat shock protein 70 in vitiligo

Heat shock proteins (HSPs) are proteins that are expressed under variety of stresses including pathologic conditions. How stresses affect vitiligo is not fully understood and little is known about the role of HSPs generally and Hsp70 specifically in vitiligo. The current study investigated the expression of Hsp70 in vitiliginous (32) and normal skin (10) by immunohistochemistry together with correlating this expression with the clinicopathologic parameters in the studied vitiligo group. Hsp70 was expressed in the cytoplasm of epidermis in all normal skin compared with its localization to the cytoplasm in 35.5% and to the nuclei in 64.5% of epidermis in vitiligo lesions. Intense (P < .001) and diffuse (P < .001) expression of Hsp70 was in favor of vitiligo skin compared with normal skin. Nuclear form of Hsp70 tended to be expressed in progressive forms of the disease. The percentage of Hsp70 expression tended to be decreased with the duration of the disease. From the present study, up-regulation of HSP 70, in the form of its intense and diffuse expression, may be blamed in pathogenesis of vitiligo. Nuclear localization of HSP 70 may be more important than its presence or absence, beside it may be related to progression of the disease.     

Association between heat shock protein 70 gene polymorphisms and clinical outcomes in intensive care unit patients with sepsis

Objective:

The objective of the following study is to evaluate the associations between single nucleotide polymorphisms (SNPs) in the Heat Shock Protein 70 (HSP70) gene, gene expression of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) and medical intensive care unit (MICU) stay and organ failure in sepsis.

Materials and Methods:

MICU patients with sepsis were genotyped for rs1061581, rs2227956, rs1008438 and rs1043618 polymorphisms in HSP70 gene using polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis or allele-specific PCR. Messenger ribonucleic acid (mRNA) expression of IL-6 and TNF-α were quantitated in peripheral blood lymphocytes. Outcomes were recorded.

Results:

108 patients (48 male) aged 40.7 ± 16.0 (mean ± standard deviation) years included H1N1 infection (36), scrub typhus (29) and urosepsis (12). Seventy-one (65.7%) had dysfunction of three or more organ systems, 66 patients (61.1%) were treated by mechanical ventilation, 21 (19.4%) needed dialysis. ICU stay was 9.3 ± 7.3 days. Mortality was 38.9%. One or more SNPs were noted in 101/108 (93.5%) and organ failure was noted in only 1/7 patients without a single SNP. The A allelotypes of rs1061581 and rs1008438 were associated with hematological dysfunction (P = 0.03 and 0.07) and longer ICU stay (P = 0.05 and 0.04), whereas IL-6 and TNF-α mRNA levels were associated with central nervous system dysfunction.

Conclusions:

HSP70 genotypes may determine some adverse outcomes in patients with sepsis.     

脑死亡致肺损伤家兔热休克蛋白70的表达与作用

背景：脑死亡供体已成为当前器官移植的主要来源。研究有效的方法与手段,保护供体器官,提高器官质量至关重要。 目的：观察热休克蛋白70在脑死亡状态致肺损伤中的作用。 方法：60只家兔随机均分为3组,即正常对照组：不行手术;假手术组：行股动脉插管、气管插管及颅骨钻孔置管术,不行颅内加压脑死亡术;脑死亡组：行股动脉插管、气管插管、颅骨钻孔置管及颅内加压脑死亡术,呼吸机维持脑死亡状态。各组均在术后2,4,6,8 h记录动脉血压及心率的变化。苏木精-伊红染色光镜观察肺脏结构改变,RT-PCR和免疫组化检测各组肺脏热休克蛋白70的mRNA及蛋白表达。 结果与结论：脑死亡组较假手术组血压与心率尚未发现显著变化(P > 0.05)。假手术组各时间点肺脏组织损伤不明显。2-6 h内,随着时间延长,脑死亡组家兔肺脏损伤逐渐加重(P < 0.05),但8 h出现一定程度好转。热休克蛋白70 mRNA与蛋白表达呈时间依赖性增高(P < 0.05),始于2 h,于8 h最高。结果证实,热休克蛋白70可能介入脑死亡状态诱导的肺脏损伤,发挥自身防御性保护作用。     

Oxidative stress and induction of heme oxygenase-1 in the kidney in sickle cell disease

Chronic nephropathy is a recognized complication of sickle cell disease. Using a transgenic sickle mouse, we examined whether oxidative stress occurs in the sickle kidney, the origins and functional significance of such oxidant stress, and the expression of the oxidant-inducible, potentially protective gene, heme oxygenase-1 (HO-1); we also examined the expression of HO-1 in the kidney and in circulating endothelial cells in sickle patients. We demonstrate that this transgenic sickle mouse exhibits renal enlargement, medullary congestion, and a reduced plasma creatinine concentration. Oxidative stress is present in the kidney as indicated by increased amounts of lipid peroxidation; heme content is markedly increased in the kidney. Exacerbation of oxidative stress by inhibiting glutathione synthesis with buthionine-sulfoximine dramatically increased red blood cell sickling in the sickle kidney: in buthionine-sulfoximine-treated sickle mice, red blood cell sickling extended from the medulla into the cortical capillaries and glomeruli. HO activity is increased in the sickle mouse kidney, and is due to induction of HO-1. In the human sickle kidney, HO-1 is induced in renal tubules, interstitial cells, and in the vasculature. Expression of HO-1 is increased in circulating endothelial cells in patients with sickle cell disease. These results provide the novel demonstration that oxidative stress occurs in the sickle kidney, and that acute exacerbation of oxidative stress in the sickle mouse precipitates acute vaso-occlusive disease. Additionally, the oxidant-inducible, heme-degrading enzyme, HO-1, is induced regionally in the murine and human sickle kidney, and systemically, in circulating endothelial cells in sickle patients.