姜黄素通过诱导热休克蛋白70保护多巴胺能细胞

目的 探讨姜黄素通过诱导热休克蛋白70(Hsp70)高表达,抑制α-突触核蛋白的异常表达和聚集,促进蛋白酶体系统降解异常α-突触核蛋白对多巴胺能细胞的保护作用.方法 选用大鼠嗜铬细胞瘤细胞株(PCI2细胞),鱼藤酮诱导其损伤建立帕金森病细胞模型,利用姜黄素进行干预:采用MTT法检测细胞活力,荧光酶标仪检测蛋白酶体水解酶活性,Western blot检测Hsp70和α-突触核蛋白的表达,免疫荧光法检测细胞内Hsp70的表达和α-突触核蛋白的聚集.结果 鱼藤酮组PC12细胞活力及蛋白酶体水解酶活性明显降低,Hsp70的表达轻度增加,α-突触核蛋白表达和聚集明显增加,与对照组比较差异有统计学意义(P<0.05).经不同浓度姜黄素预处理4h后与0.1 μmol/L鱼藤酮共同孵育PC12细胞24 h,与鱼藤酮组比较,0.5 μmol/L和1.0 μmol/L姜黄素使细胞活力以及蛋白酶体水解酶活性明显升高,Hsp70表达明显升高,α-突触核蛋白的表达和聚集明显减少,差异有统计学意义(P<0.05);5.0 μmol/L和10 μmol/L姜黄素对鱼藤酮的拮抗作用明显减弱,细胞活力与鱼藤酮组比较差异均无统计学意义(P>0.05),胰蛋白酶、多肽-谷氨酰-多肽水解酶活性与鱼藤酮组比较差异均无统计学意义(P>0.05);10 μmol/L姜黄素组糜蛋白酶样水解酶活性进一步降低,与鱼藤酮组比较差异有统计学意义(P<0.05).结论 低浓度姜黄素能够通过诱导PC12细胞表达Hsp70,诱导蛋白酶体水解酶活性表达,进而抑制α-突触核蛋白的表达和聚集,从而拮抗鱼藤酮诱导的PC12细胞的损伤.     

替普瑞酮诱导AD模型大鼠海马HSP70表达及其神经保护作用的研究

目的 研究替普瑞酮（Geranylgeranylacetone，GGA）诱导阿尔茨海默病（Alzheimer disease，AD）模型大鼠海马热休克蛋白70（heat shock protein70，HSP70）表达及其对AD大鼠的神经保护作用。方法 90只SD大鼠随机分为替普瑞酮组、模型组与生理盐水组，双侧海马注射Aβ1-42。建立阿尔茨海默病大鼠模型，替普瑞酮组给予替普瑞酮800mg·kg^-1·d^-1灌胃，余两组给予等量生理盐水灌胃；术后1d、7d、14d、21d并于Y迷宫行为学测试后处死大鼠；采用western-blot方法检测各组大鼠海马HSP70表达的变化，HE染色观察海马神经元形态学改变，TUNEL法检测海马神经元凋亡。结果术后14d、21d模型组大鼠学习记忆能力较生理盐水组明显减退（P〈0．05），替普瑞酮组较模型组明显改善（P〈0．05）；术后7d、14d、21d模型组大鼠海马HSP70表达较生理盐水组逐渐减少（P〈0．05），替普瑞酮组HSP70表达明显增加（P〈0．05）；术后21d模型组大鼠海马CA1区神经元结构紊乱，细胞减少，凋亡细胞较生理盐水组明显增加（P〈0．01），替普瑞酮组凋亡细胞较模型组显著减少（P〈0．05），细胞形态学改变减轻。结论替普瑞酮能够诱导AD模型大鼠海马HSP70表达，减少大鼠海马神经元凋亡而产生神经保护作用，改善大鼠的学习记忆能力。     

大鼠热休克蛋白72基因真核表达载体构建及在COS7细胞中的表达

背景：体外克隆、表达热休克蛋白,尤其正常时少量或不表达,而应激时大量表达的热休克蛋白72对研究其缺血再灌注损伤中的作用尤为重要。 目的：构建热休克蛋白72基因真核表达载体并于COS7细胞内表达,为HSP72蛋白免疫调节功能的研究奠定基础。 方法：采用RT-PCR技术从BABL/C大鼠肝细胞中扩增出热休克蛋白72 cDNA,经限制性核酸内切酶消化后,插入真核表达载体pcDNA3.1(+)的相应酶切位点,并将重组质粒转染COS7细胞,进行基因表达鉴定。 结果与结论：重组质粒插入基因序列检测证实为热休克蛋白72 cDNA,并能在COS7细胞稳定表达。成功构建热休克蛋白72真核表达载体,并于COS7细胞中成功转录与表达。     

甲基强地松龙对大鼠脊髓损伤后热休克蛋白70表达的影响

目的 探讨大剂量甲基强地松龙㈣对大鼠脊髓损伤后脊髓内热休克蛋白70(HSP70)表达的影响.方法 125只SD大鼠按随机数字表法分为五组:单纯手术组30只、MP对照组30只、脊髓损伤组30只、MP治疗组30只、空白对照组5只,在实验开始后6个时间点:2h、6 h、12h、24h、48 h、72h(空白对照组任选一个时间点)取出脊髓标本进行脊髓病理形态学观察.并通过免疫组化染色法检测脊髓组织中HSP70的表达.结果 使用MP治疗后,大体标本及HE染色观察显示脊髓的继发性损伤减轻.免疫组化染色显示:空白对照组和MP对照组的脊髓组织中基本没有HSP70的表达:单纯手术组各时间点见少量HSP70表达;脊髓损伤组损伤后2h脊髓组织内出现HSP70的表达,损伤后24 h脊髓组织中HSP70表达达到高峰,并维持至损伤后72 h,HSP70表达主要出现在脊髓灰质的神经细胞和胶质细胞中;MP治疗组脊髓组织中HSP70的表达在所有的时间点较脊髓损伤组明显增强,差异有统计学意义(P<0.05),表达高峰提前至12h.同时脊髓白质神经纤维网内也可见HSP70的表达.结论 大剂量MP可以显著增强损伤的脊髓组织中HSP70的表达,提前其表达高峰时间,扩大其表达范围.通过诱导损伤后脊髓组织大量表达HSP70可能是MP保护脊髓的作用机理之一.     

Intracellular and extracellular expression of the major inducible 70kDa heat shock protein in experimental ischemia-reperfusion injury of the spinal cord

Inflammatory responses exacerbate ischemia-reperfusion (IR) injury of spinal cord, although understanding of mediators is incomplete. The major inducible 70kDa heat shock protein (hsp70) is induced by ischemia and extracellular hsp70 (e-hsp70) can modulate inflammatory responses, but there is no published information regarding e-hsp70 levels in the cerebrospinal fluid (CSF) or serum as part of any neurological disease state save trauma. The present work addresses this deficiency by examining e-hsp70 in serum and CSF of dogs in an experimental model of spinal cord IR injury. IR injury of spinal cord caused hind limb paraplegia within 2–3 h that was correlated to lumbosacral poliomalacia with T cell infiltrates at 3 d post-ischemia. In this context, we showed a 5.2-fold elevation of e-hsp70 in CSF that was induced by ischemia and was sustained for the following 3 d observation interval. Plasma e-hsp70 levels were unaffected by IR injury, indicating e-hsp70 release from within the central nervous system. A putative source of this e-hsp70 was ependymal cells in the ischemic penumbra, based upon elevated i-hsp70 levels detected within these cells. Results warrant further investigation of e-hsp70's potential to modulate spinal cord IR injury.     

Induction of heat shock hsp70 mRNA and HSP70 kDa protein in neurons in the ‘penumbra’ following focal cerebral ischemia in the rat

Induction of hsp70 heat shock protein (HSP70) and hsp70 mRNA was examined using adjacent sections in the same rat brain following permanent middle cerebral artery (MCA) occlusions. hsp70 mRNA was induced within 4 h of MCA occlusion and persisted for at least 24 h. Cellular resolution autoradiographs suggested that hsp70 mRNA was induced primarily in neurons in the periphery of ischemia both outside and inside of the infarction, with small amounts of hsp70 mRNA being induced in the core of the infarction. HSP70 protein was localized in neurons outside the infarction and in endothelial cells within the infarction at 24 h but not at 4 h following permanent MCA occlusions. It is proposed that the penumbra, one of the areas that can be rescued by pharmacological agents, can be defined anatomically as the volume of tissue outside the area of infarction in which HSP70 protein is expressed primarily in neurons.     

Presence of molecular chaperones, heat shock cognate (Hsc) 70 and heat shock proteins (Hsp) 40, in the postsynaptic structures of rat brain

The synaptic localization of molecular chaperones, heat shock cognate protein 70 (Hsc70) and Hsp40, was investigated immunohistochemically in the normal rat brain. Postsynaptic density (PSD) fractions contained a constitutive form of HSP70, heat shock cognate protein 70 (Hsc70 or p73) but not inducible form of HSP70 (p72). The immunoreactivities of Hsc70 (p73) were distributed throughout the rat brain, in neuronal somata, dendrites and axons. Their immunoreactivity in neurons was localized in the cytoplasmic matrix, dendrites, and spines at the electron microscopic level. Presynaptic terminals, but less frequently than postsynaptic staining, were also reactive. Postsynaptic areas immediately beneath the synaptic contact or PSDs were immunoreactive for Hsc70. The Hsp40 was highly concentrated in PSD fractions. The staining of Hsp40 immunoreactivity was punctate and distributed widely in the brain. Hsp40 immunoreactivity was localized in dendritic spines, especially in the subsynaptic web, with weak staining of PSDs at the electron microscopic level. Double immunofluorescent staining and confocal microscopy revealed that Hsc70 and Hsp40 were co-localized on somata and neuronal processes of cultured cerebral neurons, on which synaptophysin immunoreactive spots were scattered. These results suggest that Hsp40 and Hsc70 are co-localized at postsynaptic sites and postsynaptic chaperone activity may be mediated by these two heat shock proteins.     

Temporal and spatial distribution of heat shock mRNA and protein (hsp70) in the rabbit cerebellum in response to hyperthermia

We have previously investigated the expression of hsp70 genes in the hyperthermic rabbit brain at the mRNA level by Northern blot and in situ hybridization procedures. Our studies have now been extended to the protein level utilizing Western blot and immunocytochemistry. Using an antibody which is specific to inducible hsp70, a prominent induction of hsp70 protein in glial cells of hyperthermic animals was noted. In particular, Bergmann glial cells in the cerebellum are strongly immunoreactive while adjacent Purkinje neurons are immunonegative. Extension of our in situ hybridization studies to a time course analysis revealed that the initial glial induction events were followed by a delayed accumulation of inducible hsp70 mRNA in Purkinje neurons at 10 hr post-heat shock. In control animals, high levels of constitutively expressed hsc70 mRNA and protein were observed in Purkinje neurons. Similar hsc70 and hsp70 mRNA observations were also made in neurons of the deep cerebellar nuclei and in motor neurons of the spinal cord. Our results suggest that these neuronal cell types accmulate hsp70 mRNA in response to hyperthermic treatment; however, the response is delayed when compared to the rapid response seen in glial cells. The high constitutive levels of hsc70 in certain neuronal cell types may play a role in the initial dampening of the hsp70 induction response in these cells. © 1993 Wiley-Liss, Inc.     

Neuroprotective effect of heat shock protein 70 Inhibition of Tau protein expression

BACKGROUND： Previous studies have shown that heat shock protein 70 （HSP70） has neuroprotective effects by decreasing phosphorylation of Tau protein, thereby reducing the expression of Tau protein and proper aggregation. OBJECTIVE： To observe and verify expressional changes of HSP70 and Tau in retinal ganglion cells following stretch injury to the right optic nerve in rats, and to determine the effect of heat stress pretreatment on HSP70 and Tau protein expressions. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Neurology Research Institute of the First Affiliated Hospital of Chongqing Medical University from March to June 2006. MATERIALS： Instant SABC immunohistochemistry kit, as well as mouse anti-HSP70 and rabbit anti-Tau polyclonal antibodies, were purchased from Wuhan Boster Bioengineering Limited, China. METHODS： A total of 57 male, Wistar rats were randomly assigned to 4 groups： control （n = 3）; 150-180 g stretch force was induced in the right optic nerves in stretch-only group （n = 18） to establish optic nerve stretch injury model; heat stress was applied to 18 animals in heat-stress treatment group; 18 rats in the heat-stress pretreatment plus stretch group were subjected to identical stretch injury as stretch-only group after 24-hour heat-stress pretreatment. According to sacrifice time, the groups were assigned to 6 subgroups at different time points of 4, 8, and 16 hours, and 1,3, and 5 days, with 3 rats in each subgroup. No treatment was performed in the control group except anesthesia. MAIN OUTCOME MEASURES： Morphological changes of optic nerves and retinal ganglion cells following stretch injury were observed by light microscopy following hematoxylin-eosin staining. HSP70 and Tau protein expression levels were observed in retinal ganglial cells from each group using im munohistochemistry. RESULTS： （1） Compared with the control group, morphological axonal and retinal ganglial cell changes, as well as a decreased number of retinal ganglial cells, were identified in the stretch-only group （P 〈 0.01）. Pathological damage in optical nerve and retinal ganglial cells were not remarkable in the heat-stress pretreatment plus stretch group, with no statistical difference in the number of retinal ganglial cells compared with the control group （P 〉 0.05）. （2） Compared with the control group significantly increased HSP70 expression in retinal ganglial cells occurred in the stretch-only, heat-stress treatment, and heat-stress pretreatment plus stretch groups （P 〈 0.05 or P 〈 0.01 ）. The peak of HSP70 expression was earlier in the heat-stress pretreatment plus stretch group compared with the stretch-only and heat-stress treatment groups, and was expressed over a longer period of time compared with the heat-stress treatment group. Compared with the control group, Tau expression in the retinal ganglial cells rapidly increased 4-16 hours following stretch injury in the stretch-only group （P 〈 0.05 or P 〈 0.01）, and obviously decreased in the heat-stress pretreatment plus stretch group （P 〈 0.05 or P 〈 0.01 ）. CONCLUSION： Tau expression increased following stretch injury, with an earlier expression peak than HSP70, which indicated that stretch injury-induced HSP70 expression was not strong or quick enough to sufficiently protect the nerve. A much more enhanced HSP70 expression, with an earlier peak and longer expression period, was observed in rats subjected to stretch injury following heat stress, which demonstrated that HSP70 exhibited neuroprotective functions by reducing abnormal aggregation of Tau.     

大鼠局灶性脑缺血再灌流后HSP70表达及其mRNA变化

目的 探讨热休克蛋白７０（ＨＳＰ７０）在局灶脑缺血再灌流后的变化和作用。方法 采用线栓法制备大脑中动脉缺国知再灌流模型。应用免疫组化方法观察ＨＳＰ７０的组织学分布,利用ＲＴ－ＰＣＲ方法检测缺血皮层与基底节区脑组织ＨＳＰ７０ｍＲＮＡ相对含量。结果 基底节区ＨＳＰ７０阳性细胞较少,持续时间短,眼层区ＨＳＰ７０阳性细胞较多,持续时间长。ＲＴ－ＰＣＲ结果表明ＨＳＰ７０ｍＲＮＡ相对含量与免疫组化结果相一致。     

Regionally different effects of scopolamine on NMDA antagonist-induced heat shock protein HSP70

Using immunohistochemical technique, we investigated the regionally different roles of muscarinic receptors in the induction of HSP-70 by NMDA receptor antagonists. The administration of memantine and phencyclidine induced HSP-70 in the retrosplenial cortex of rat brain. Pretreatment with the muscarinic receptor antagonist scopolamine (0.1–1 mg/kg) blocked induction of HSP-70 in layer III of the retrosplenial cortex. However, induction of HSP-70 in layer V was augmented by scopolamine. These results suggest a regional difference in the mechanism of neurotoxicity induced by NMDA receptor antagonists.     

Cytokine induction of heat shock protein expression in human oligodendrocytes: An interleukin-1-mediated mechanism

In this study, we examined the role of cytokines, known to be in elevated levels in multiple sclerosis (MS) plaques, in regulating oligodendrocyte (ODC) expression of heat shock protein (hsp) in human brain-derived glial cell cultures. Using dual-stain immunohistochemistry, we initially compared the ability of a mixture of cytokines (IL-1α, IL-1β, IL-2, IL-6, IL-8, TNF-α, TNF-β, IFN-β and IFN-γ) with that of physical stimuli such as heat shock and peroxide, to increase cellular expression of the mainly inducible hsp72 species in mixed glial cell cultures (containing ODC, astrocytes and microglia). Similar to heat shock and peroxide, the cytokine mixture induced hsp72 expression only in ODC (70 ± 5% vs. a baseline of 3 ± 1% positive cells). When used individually, however, only IL-1α (79 ± 3%), IFN-γ (70 ± 2%) and TNF-α (65 ± 5%) induced ODC hsp72 expression in mixed glial cell cultures. In purified ODC preparations, only IL-1α induced hsp72 expression (84 ± 4%). An IL-1 receptor antagonist (IL-1ra), abrogated hsp72 induction by IL-1α (16 ± 3%) as well as that due to IFN-γ (14 ± 1%) and TNF-α (13 ± 2%) in mixed glial cell cultures. Furthermore, ODC express IL-1 receptors, detected by confocal laser scanning microscopy. Our data indicate that cytokines mediate hsp induction in ODC possibly via a final common pathway involving IL-1 binding to its receptor on ODC. Such interaction could enhance any putative ODC-immune interactions which are dependent on hsp molecule recognition.     

Pattern of heat shock factor and heat shock protein expression in lymphocytes of bipolar patients: Increased HSP70-glucocorticoid receptor heterocomplex

Bipolar disorder (BD), a stress-related disease, is characterized by altered glucocorticoid receptor (GR) signalling. Stress response includes activation of heat shock factor (HSF) and subsequent heat shock protein (HSP) synthesis which regulate GR folding and function. The objective of this study was to investigate the possible role of HSFs, HSPs and their interaction with GR in BD. We applied immunoprecipitation, SDS-PAGE/Western blot analysis and electrophoretic mobility shift assay (EMSA) in lymphocytes (whole cell or nuclear extracts) from BD patients and healthy subjects and determined the HSPs (HSP90 and HSP70), the heterocomplexes HSP90-GR and HSP70-GR, the HSFs (HSF1 and HSF4) as well as the HSF-DNA binding. The HSP70-GR heterocomplex was elevated (p < 0.05) in BD patients vs healthy subjects, and nuclear HSP70 was reduced (p ≤ 0.01) in bipolar manic patients. Protein levels of HSF1, HSF4, HSP90, HSP90-GR heterocomplex, and HSF-DNA binding remained unaltered in BD patients vs healthy subjects. The corresponding effect sizes (ES) indicated a large ES for HSP70-GR, HSP70, HSF-DNA binding and HSF4, and a medium ES for HSP90, HSF1 and HSP90-GR between healthy subjects and bipolar patients. Significant correlations among HSFs, HSPs, GR and HSP70-GR heterocomplex were observed in healthy subjects, which were abrogated in bipolar patients. The higher interaction between GR and HSP70 and the disturbances in the relations among heat shock response parameters and GR as observed in our BD patients may provide novel insights into the contribution of these factors in BD aetiopathogenesis.     

Hypoxia-induced ABR change and heat shock protein expression in the pontine auditory pathway of young rabbits

The auditory brainstem response (ABR) was compared with the immunohistochemical expression of heat shock protein (HSP-72) and microtubule-associated protein 2 (MAP-2) of the brainstem auditory pathway in young rabbits subjected to hypoxic stress. Severe hypoxia for 2 h produced significant prolongation and decreased amplitude of the later component of ABR. HSP-72 expression was distinctly increased in the cochlear nucleus, but there was less induction in the inferior colliculus under severe hypoxia. MAP-2 immunostaining of neuropiles in the inferior collicular nucleus was decreased slightly after severe-long hypoxia, but cytoplasmic staining did not change. The present ABR change, which was produced by brainstem hypoxia-ischemia and acidosis, may be due to the neural cytoarchitectural derangement and less induction of stress proteins in the upper brainstem.     

The pattern of 72-kDa heat shock protein-like immunoreactivity in the rat brain following flurothyl-induced status epilepticus

The inducible 72-kDa heat shock protein (HSP72) is a highly conserved stress protein that is expressed in CNS cells and may play a role in protection from neural injury. We used a monoclonal antibody to HSP72 and immunocytochemistry to localize HSP72 in the rat brain 24 h following either 30 or 60 min of flurothyl-induced status epilepticus. Sprague-Dawley rats were anesthetized with halothane, paralyzed, and ventilated, and remained normotensive and well oxygenated for the duration of the seizures. Seizure activity was quantified via analysis of the scalp EEG pattern. HSP72-like immunoreactivity (HSP72-LI) was induced in specific brain regions in a graded fashion that correlated, in part, with the duration and degree of seizure activity. Milder seizures produced HSP72-LI limited to layers 2 and 3 of frontoparietal cortex, dentate hilus cells, and CA3 pyramidal neurons. More extensive seizures led to HSP72-LI in layers 2, 3 and 5 of frontoparietal and visual cortex, dentate hilus cells, CA1 and CA3 pyramidal neurons, and certain thalamic and amygdaloid nuclei. These are similar to many, but not all, of the brain regions known to be injured with this model. No HSP72-LI was observed in sham-treated controls or flurothyl-treated animals whose seizures were controlled with pentobarbital. HSP72-LI thus localizes to certain regions of seizure-induced injury, and may provide a sensitive method of detecting neuronal 'stress' or injury relatively soon after status epilepticus. Whether or not HSP72 synthesis plays a protective role in the pathogenesis of seizures, or is only a marker for cell injury, remains to be determined.     

Neuronal survival is associated with 72-kDa heat shock protein expression after transient middle cerebral artery occlusion in the rat

Induction of the 72-kDa heat shock protein expression is thought to protect neurons against the subsequent effects of ischemia. However, it is not clear whether the induction of 72-kDa heat shock protein expression by an ischemic event improves neuronal survival. To address this question, we outlined the temporal profile of neuronal induction and expression of the 72-kDa heat shock protein in a model of transient focal ischemia in the rat. Fifty two adult Wistar rats were subjected to middle cerebral artery occlusion of 2 h duration. At 0.5, 3, 6, 9, 12, 24, 48, 96 and 168 h after reopening the artery, coronal brain sections were analyzed using both immunohistochemical methods and hematoxylin and eosin staining to determine the topographic and cellular distribution of the 72-kDa heat shock protein, as well as the extent of neuronal damage. Immunoreactivity to the 72-kDa heat shock protein was not detected in neurons that were destined to become necrotic, and were located in the ischemic core of the brain lesions. However, 72-kDa heat shock protein expression was evident in morphologically intact neurons located in the peripheral zone. The earliest neuronal expression of 72-kDa heat shock protein was detected in animals in which the 2 h occlusion of the middle cerebral artery was followed by 6 h recirculation; the intensity of the 72-kDa heat shock protein immunoreactivity peaked at 48 h, and progressively disappeared 7 days after the ischemic reperfusion event. These studies suggest that (1) 72-kDa heat shock protein is not expressed in morphologically intact neurons destined to become necrotic after 2 h of focal ischemia; (2) the 72-kDa heat shock protein is expressed only in morphologically intact neurons located at the periphery of the ischemic territory where they may be subjected to only sublethal stress; these neurons preserved their integrity 7 days after the ischemic episode. These data support the hypothesis that the expression of 72-kDa heat shock protein in ischemic brain may confer “protection” to the neurons.