AAV mediated carboxyl terminus of Hsp70 interacting protein overexpression mitigates the cognitive and pathological phenotypes of APP/PS1 mice

The E3 ubiquitin ligase,carboxyl terminus of heat shock protein 70(Hsp70)interacting protein(CHIP),also functions as a co-chaperone and plays a crucial role in the protein quality control system.In this study,we aimed to investigate the neuroprotective effect of overexpressed CHIP on Alzheimer’s disease.We used an adeno-associated virus vector that can cross the blood-brain barrier to mediate CHIP overexpression in APP/PS1 mouse brain.CHIP overexpression significantly ameliorated the performance of APP/PS1 mice in the Morris water maze and nest building tests,reduced amyloid-βplaques,and decreased the expression of both amyloid-βand phosphorylated tau.CHIP also alleviated the concentration of microglia and astrocytes around plaques.In APP/PS1 mice of a younger age,CHIP overexpression promoted an increase in ADAM10 expression and inhibitedβ-site APP cleaving enzyme 1,insulin degrading enzyme,and neprilysin expression.Levels of HSP70 and HSP40,which have functional relevance to CHIP,were also increased.Single nuclei transcriptome sequencing in the hippocampus of CHIP overexpressed mice showed that the lysosomal pathway and oligodendrocyte-related biological processes were up-regulated,which may also reflect a potential mechanism for the neuroprotective effect of CHIP.Our research shows that CHIP effectively reduces the behavior and pathological manifestations of APP/PS1 mice.Indeed,overexpression of CHIP could be a beneficial approach for the treatment of Alzheimer’s disease.

Effect of Ultraviolet Radiation on Hsp70 Protein Expression in HaCaT Cells

Ultraviolet radiation by its wavelength is divided into: UVA, UVB and UVC. Only UVA and UVB manage to penetrate the ozone layer, but due to anthropological activities, all of them are capable of interacting with humans to a greater or lesser extent, and can generate adverse effects such as cellular stress when interacting with intra-and extracellular biomolecules. The skin is the first organ in contact with UV radiation, and the stress it generates can be analyzed by the expression of a bioindicator of cellular damage such as Hsp70. Therefore, the objective of the project was: to determine the effect of UVA, UVB and UVC radiation on HaCaT epithelial cells, by analyzing the expression of Hsp70. Materials and methods: HaCaT cells were cultured in vitro, which were irradiated with UVA, UVB and UVC light at different doses, to subsequently determine the degree of Hsp70 expression by Immunodetection by PAGE-SDS and Western Blot. Results: Basal expression of Hsp70 was observed in no irradiated HaCaT cells. When HaCaT cells were irradiated with UVA, UVB, UVC, an increase in this Hsp70 protein was observed. With UVA, a higher degree of expression was observed at a time of 30 minutes of irradiation. With UVB the highest expression shifted to a time of 20 minutes. With UVC, overexpression was observed after 10 minutes. Conclusion: UV radiation generates cellular stress on HaCaT cells, evaluated by the stress bioindicator Hsp70. According to the wavelength of UV radiation, those that have a shorter wavelength have a greater potential for cellular damage, such as UVC.

Effect of chaperone–client interaction strength on Hsp70-mediated protein folding

Protein folding in crowding cellular environment often relies on the assistance of various chaperones. Hsp70 is one of the most ubiquitous chaperones in cells. Previous studies showed that the chaperone–client interactions at the open state tend to remodel the protein folding energy landscape and direct the protein folding as a foldase. In this work, we further investigate how the chaperone–client interaction strength modulates the foldase function of Hsp70 by using molecular simulations. The results showed that the time of substrate folding(including the whole folding step and substrate release step) has a non-monotonic dependence on the interaction strength. With the increasing of the chaperone–client interaction strength, the folding time decreases first, and then increases. More detailed analysis showed that when the chaperone–client interaction is too strong, even small number of chaperones–client contacts can maintain the substrate bound with the chaperone. The sampling of the transient chaperones–client complex with sparse inter-molecule contacts makes the client protein have chance to access the misfolded state even it is bound with chaperone. The current results suggest that the interaction strength is an important factor controlling the Hsp70 chaperoning function.

Ischemic preconditioning induces chaperone hsp70 expression and inhibits protein aggregation in the CA1 neurons of rats

Objective To investigate the effect of ischemic preconditioning on chaperone hsp70 expression and protein aggregation in the CA1 neurons of rats, and to further explore its potential neuroprotective mechanism. Methods Two-vesseloccluded transient global ischemia rat model was used. The rats were divided into sublethal 3-min ischemia group, lethal 10- min ischemia group and ischemic preconditioning group. Neuronal death in the CA1 region was observed by hematoxylineosin staining, and number of live neurons was assessed by cell counting under a light microscope. Immunochemistry and laser scanning confocal microscopy were used to observe the distribution of chaperone hsp70 in the CA1 neurons. Differential centrifuge was used to isolate cytosol, nucleus and protein aggregates fractions. Western blot was used to analyze the quantitative alterations of protein aggregates and inducible chaperone hsp70 in cellular fractions and in protein aggregates under different ischemic conditions. Results Histological examination showed that ischemic preconditioning significantly reduced delayed neuronal death in the hippocampus CA1 region （P 〈 0.01 vs 10-min ischemia group）. Sublethal ischemic preconditioning induced chaperone hsp70 expression in the CA1 neurons after 24 h reperfusion following 10-min ischemia. Induced-hsp70 combined with the abnormal proteins produced during the secondary lethal 10-min ischemia and inhibited the formation of cytotoxic protein aggregates（P〈0.01 vs 10-min ischemia group）.Conelusion Ischemic preconditioning induced chaperone hsp70 expression and inhibited protein aggregates formation in the CA1 neurons when suffered secondary lethal ischemia, which may protect neurons from death.

Effects of moxibustion on heat-shock protein 70 expression in the spinal cord and colonic mucosa in a rat model of ulcerative colitis

Pathological changes in the colon are closely associated with the spinal cord, and innervation of spinal cord can regulate cellular functions. Our previous studies verified that moxibustion protects and restores the colonic mucosa, but the mechanisms of action remain unknown. The present study observed the effects of moxibustion and salicylazosulfapyridine on expression of heat-shock protein 70 （HSP70） and its mRNA in the spinal cord and colonic mucosa of ulcerative colitis rats. Results demonstrated that moxibustion and salicylazosulfapyridine increased HSP70 mRNA expression in the spinal cord and colonic mucosa of ulcerative colitis rats. The decreased transcriptional activity of HSP70 in the spinal cord and colonic mucosa might participate in damage to the colonic mucosa in ulcerative colitis rats. Moxibustion exerted protective effects on colonic mucosa by up-regulating HSP70 transcriptional activity in the spinal cord and colonic mucosa.

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c(Hsp70_(359–610)) fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.

Novel heat shock protein Hsp70L1 activates dendritic cells and acts as a Th1 polarizing adjuvant

Heat shock proteins (HSPs) are reported to act as effective adjuvants to elicit anti-tumor and anti-infection immunity. Here, we report that Hsp70-like protein 1 (Hsp70L1), a novel HSP derived from human dendritic cells (DCs), has potent adjuvant effects that polarize responses toward Th1. With a calculated molecular weight of 54.8 kDa, Hsp70L1 is smaller in size than Hsp70 but resembles it both structurally and functionally. Hsp70L1 shares common receptors on DCs with Hsp70 and can interact with DCs, promoting DC maturation and stimulating secretion of the proinflammatory cytokines interleukin 12p70 (IL-12p70), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and the chemokines IP-10, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and normal T cell expressed and secreted (RANTES). The induction of interferon-gamma-inducible protein 10 (IP-10) secretion by Hsp70L1 is not shared by Hsp70, and other functional differences include more potent stimulation of DC IL-12p70, CC-chemokine, and CCR7 and CXCR4 expression by Hsp70L1. Immunization of mice with the hybrid peptide Hsp70L1-ovalbumin(OVA)(257-264) induces an OVA(257-264)-specific Th1 response and cytotoxic T lymphocyte (CTL) that results in significant inhibition of E.G7-OVA tumor growth. The ability of Hsp70L1 to activate DCs indicates its potential as a novel adjuvant for use with peptide immunizations; the Hsp70L1 antigen peptide hybrid may serve as a more effective vaccine for the control of cancer and infectious diseases.

松材线虫Hsp70基因的克隆与原核表达

热激蛋白70（Hsp70）是已知热休克蛋白家族中最重要的一种,它在细胞内的大量表达可以明显改善细胞的生存能力,提高对环境胁迫或伤害的耐受性。采用RACE-PCR技术,从松材线虫（Bursaphelenchus xylophilus）中克隆了Hsp70基因的全长cDNA序列（共2 061 bp）（GenBank登录号为：DQ785812）。其编码一个分子量为70 kD的642个氨基酸的蛋白序列,含有3段Hsp70家族的签名序列。同源性分析表明,氨基酸序列与其它真核生物的Hsp70序列具有很高的相似性,并与秀丽隐杆线虫（Caenorhabditis elegans）热激蛋白70家族中的hsp-1基因编码的氨基酸序列更为相似。因此,将克隆的松材线虫Hsp70基因命名为Bx-hsp-1。构建了一个原核表达载体Bx70pEASY-E1,当IPTG终浓度为0.4-0.8 mmol/L时,能诱导表达融合蛋白。Bx-hsp-1基因的克隆和表达,将为松材线虫的生态适应性机理研究奠定基础。

大鼠脑震荡伤后脑组织HSP70、bFGF及TGF-β1表达变化

目的研究脑震荡后脑组织损伤修复相关因子表达变化,为推断脑损伤时间提供法医学依据。方法建立SD大鼠脑震荡伤模型,提取脑组织行常规HE染色及热休克蛋白70(heat shock protein 70,HSP70)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和转化生长因子β1(transforming growth factor beta1,TGF-β1)免疫组织化学染色,在光学显微镜下观察并进行图像分析及相应统计学处理。结果伤后30min,在大脑皮质、海马区见少量呈弥散性分布的HSP70免疫组化染色阳性细胞,12h达高峰,伤后1d阳性细胞数量开始减少;伤后3h,在大脑皮质、海马区bFGF免疫组化染色阳性细胞开始增多,至12h时达高峰,然后开始下降;伤后6h～1d,在大脑皮质、海马区TGF-β1免疫组化染色阳性细胞逐渐增多,至3d时达高峰,然后开始下降。结论HSP70、bFGF、TGF-β1在脑震荡后脑组织中表达呈时序性改变。多指标综合应用为法医学损伤时间的推断提供了全新的视角。

Aβ_(25～35)诱导阿尔茨海默病模型大鼠海马神经元HSP70的表达

目的利用β淀粉样蛋白(Aβ25～35)毒性作用诱导大鼠阿尔茨海默病(AD)模型,研究热休克蛋白70(HSP70)在海马神经元损伤中的表达。方法将大鼠随机分为2组:AD模型组、溶媒体组,在双侧海马分别注射2μl(10μg)Aβ25～35、2μl生理盐水;于海马注射第1,7,14,21天采用免疫组织化学、积分光密度分析等手段对各组大鼠脑HSP70的表达进行观察。结果AD模型组手术第7天大鼠记忆明显减退(P<0.05),且HSP70在海马神经元内表达第1天时最高,第1～21天呈递减趋势,其中第14天锐减。结论Aβ25～35通过氧化应激和损伤海马神经元等过程使HSP70的表达明显降低,HSP70表达是AD大鼠脑内神经元存活的重要标志。

涎腺肿瘤中HSP27、HSP70与PCNA、CD9蛋白表达的相关性

目的研究热休克蛋白(HSP)27和70在涎腺肿瘤中的表达特征及与增殖细胞核抗原(PCNA)和移动相关蛋白-1(MRP-1,CD9)的相关性,探讨其对涎腺肿瘤发生发展的可能作用。方法采用免疫组化SP法和免疫组化双标双染法检测41例良性和17例恶性肿瘤中HSPs的表达。结果 (1)涎腺肿瘤中HSP27和HSP70高表达,但表达定位和强度存在差异。(2)涎腺恶性肿瘤中HSP27和CD9的阳性率和阳性强度均降低(P<0.05和P<0.01)而HSP70和PCNA的阳性率和阳性强度均增高(P均<0.05)。(3)HSP70的阳性率与阳性强度均与PCNA正相关(相关系数分别为Cramer’sV=3797和r=0.5123,P均<0.01);HSP27的表达率与CD9的阳性率正相关(Cramer’sV=0.3477),但阳性强度比较无相关(r=0.1762,P>0.05)。结论 (1)HSP70可能促进PCNA高表达,有用于涎腺肿瘤细胞的增殖。(2)HSP27可能不完全抑制肿瘤细胞的增殖。(3)HSP27和CD9的表达减少均可能是肿瘤细胞分化降低的标志,协同于增殖的恶性肿瘤细胞的迁移。

电击死兔骨骼肌及心肌HSP 70 mRNA和c-fos mRNA表达

目的研究电击死兔骨骼肌与心肌HSP 70 mRNA和c-fos mRNA表达变化,探究生前电击与死后电击的鉴别方法。方法15只新西兰兔,随机分电击死组、死后电击组和对照组,每组5只,用荧光RT-PCR技术检测骨骼肌与心肌热休克蛋白70(HSP 70)mRNA与c-fos mRNA表达水平,对所得结果进行统计学分析。结果生前电击兔骨骼肌及心肌HSP 70 mRNA与c-fos mRNA表达高于死后即刻电击者(P<0.05)。结论检测骨骼肌及心肌HSP 70 mRNA与c-fos mRNA表达变化有助于于生前电击与死后电击的鉴别。

Graves眼病眼眶组织HLA-DR和HSP-70表达的研究

目的 了解Graves眼病患者眼部组织的组织相容性抗原DR(HLA-DR)和热休克蛋白(HSP-70)的表达极其与病程和治疗的关系。方法 收集Graves眼病患者的眼外肌19例,通过免疫组化的方法观察HLA-DR和HSP-70在不同病程,治疗与未治疗患者眼外肌的表达差异。结果 HLA-DR在眼外肌的纤维母细胞、血管内皮细胞和浸润的淋巴细胞表达,以未治病人的眶组织为明显;HSP-70在肌细胞和纤维母细胞表达,病程短的病人其表达增强。结论 眼外肌细胞和纤维母细胞可能都是Graves眼病自身免疫反应的重要靶细胞;HLA-DR和HSP-70在自身免疫反应中起重要作用;病程和治疗影响HLA-DR和HSP-70的表达。

针刺对急性高血压脑出血大鼠HSP_(70)表达的良性调整作用

目的 :探索针刺对急性高血压脑出血大鼠治疗作用机制。方法 :复制急性高血压脑出血大鼠模型 ,动态观察针刺对脑组织热休克蛋白 70表达的影响。结果与结论 :针刺能够增加热休克蛋白 70在脑组织中的表达是针刺治疗急性脑出血的一个重要机制。

HSP70和HSP90α在人膀胱癌中的表达及意义

目的 探讨热休克蛋白70(heat- shockprotein70,HSP70)和热休克蛋白90α(heat shockprotein90α,HSP90α)在 膀胱癌中的表达及临床意义。方法 应用免疫组化技术检测HSP70、HSP90α和增殖细胞核抗原(proliferatingcellnuclearan tigen,PCNA)在50例膀胱癌组织和14例正常膀胱粘膜组织的表达。结果 膀胱癌中HSP70、HSP90α阳性表达率分别为 56%(28 50)和66%(33 50)。HSP70和HSP90α在膀胱癌病理分级、临床分期、生存率等方面具有显著性差异(P<0.05)。 HSP70、HSP90α和PCNA在膀胱癌中表达呈正相关。结论 HSP70、HSP90α与膀胱癌细胞分化,肿瘤的浸润深度有关,在膀 胱癌发生和发展中起重要作用。HSP70、HSP90α可以作为判断膀胱癌预后的新指标。

大鼠局灶性脑缺血再灌注损伤白藜芦醇与HSP70蛋白表达的关系

目的探讨大鼠局灶性脑缺血再灌注损伤白藜芦醇与热休克蛋白(HSP)70的关系和作用。方法随机设立假手术组、对照组和白藜芦醇预处理组3个组,后两组采用线栓法建立大鼠大脑中动脉闭塞模型(MCAO)缺血2 h再灌注,且设立6 h和24 h两个再灌注时间点。白藜芦醇预处理组按40 mg/kg经腹腔注射。分别在术后不同时间点进行神经功能学评分,采用免疫组化和免疫印迹法测定HSP70蛋白表达。结果对照组和白藜芦醇预处理组与假手术组比较神经功能评分显著增高,白藜芦醇预处理组又明显低于对照组(P<0.05);免疫组化和免疫印迹结果一致,对照组和白藜芦醇预处理组与假手术组比较HSP70蛋白表达显著增高,白藜芦醇预处理组HSP70蛋白表达又明显高于对照组(P<0.05)。结论大鼠局灶性脑缺血再灌注损伤白藜芦醇的神经保护作用可以通过热休克蛋白HSP70蛋白的表达增多来产生。

肿瘤细胞HSP-70多肽诱导CTL及其抗瘤作用机制的研究

目的 探讨鼠S1 80 细胞HSP 70多肽诱导杀伤性T淋巴细胞 (CTL)及其特异性抗瘤作用机制。方法 从 43℃ 9h热休克诱导HSP高表达的S1 80 细胞提纯 80 μg mlHSP 70多肽 ,体外刺激小鼠脾淋巴细胞扩增、活化 ,然后用MTT法测定其杀瘤活性 ,并用电镜观察其培养上清诱导S1 80 细胞的形态学变化。结果 S1 80 细胞HSP 70多肽体外免疫扩增的小鼠脾淋巴细胞对S1 80 和H2 2 细胞的杀瘤率分别为83 .61%和 9.98% ,其培养上清能诱导S1 80 细胞出现程序化死亡的形态学变化。结论 S1 80 细胞HSP 70多肽能剌激小鼠脾淋巴细胞产生特异性杀伤S1 80 细胞的免疫活性细胞 ,该细胞还能分泌免疫活性物质诱导肿瘤细胞凋亡。

HeLa细胞HSP-70多肽体外诱导免疫活性细胞及其抗瘤机理

[目的]探讨用HeLa细胞HSP 70多肽诱导免疫活性细胞 (immunecompetentcells ,ICC)的特异性抗肿瘤机制。[方法]用热休克处理的HeLa细胞提取的HSP 70多肽剌激正常人外周血单个核细胞 ,在剌激扩增前后测定T淋巴细胞表型 ,并测定扩增的免疫活性细胞对HeLa细胞的杀瘤活性。[结果]在扩增的细胞中CD3+、CD8+淋巴细胞百分率明显增高 (P<0.01)。该免疫活性细胞对Hela细胞和来源于病人的宫颈癌细胞都具有较高的杀伤活性 ,而对经HSP 70单抗封闭后的HeLa细胞的杀伤率则明显下降。[结论]HeLa细胞HSP 70多肽能有效刺激PBMCs诱导活化免疫活性细胞 。

扶正护脑胶囊对脑出血大鼠心肌组织HSP70蛋白表达的影响

目的:观察脑出血大鼠心肌组织热休克蛋白70(HSP70)蛋白表达的变化及扶正护脑胶囊的干预作用。方法:动物随机分为假手术组、模型组、扶正护脑组和安宫牛黄组,分别于术后6 h、24 h、72 h 3个时相点取材,采用免疫组织化学SABC法测定心肌组织HSP70蛋白表达,并进行图像分析,观测其IOD值;并与血清CK-MB、cTnI含量作相关性分析。结果:HSP70阳性颗粒主要在心内膜下的心肌细胞和血管内皮细胞的胞浆中表达。模型组各时间点心肌组织HSP70蛋白表达均显著升高,其IOD值亦明显升高,扶正护脑组和安宫牛黄组均能明显增加HSP70蛋白的表达,升高其IOD值。IOD值与cTnI、CK-MB含量呈正相关。结论:HSP70对脑出血诱发的心肌损伤具有保护作用;扶正护脑胶囊通过提高HSP70的水平,减轻脑出血后心肌的损伤。