Hsp70 Protein Expression Levels Research Articles

Chlorogenic acid ameliorates the heat stress-induced impairment of porcine Sertoli cells by suppressing oxidative stress and apoptosis

Sertoli cells are an important type of somatic cell in the testis that are in direct contact with spermatogonia in vivo and play an important role in the process of spermatogenesis. Chlorogenic acid (CGA) plays a pivotal role in the regulation of the mammalian cell heat stress response. For example, CGA treatment protects porcine oocytes from heat stress-induced apoptosis and prevents reduced embryo quality. However, the role of CGA treatment in protecting porcine testicular Sertoli cells against heat-induced damage has rarely been studied. This study aimed to identify the protective effects of CGA on oxidative stress and apoptosis in Sertoli cells under heat stress. The present results demonstrated that the addition of CGA significantly inhibited the accumulation of reactive oxygen species (ROS) and apoptosis in Sertoli cells induced by heat stress and decreased the expression of CASP3 protein and the BAX/BCL-2 protein ratio. CGA pretreatment also prevented the heat stress-induced reductions in the mitochondrial membrane potential, PCNA protein expression, and SOD and CAT activities. Moreover, CGA treatment reversed S phase cell cycle arrest and increased the HSP70 protein expression levels. Overall, these results suggest that oxidative damage participates in the inhibition of the proliferation of Sertoli cells and the increase in their apoptosis induced by heat stress, and the protective effects of CGA treatment on Sertoli cells under heat stress provide a theoretical basis for preventing heat stress injury in animals.

Extremely low-frequency electromagnetic field induces acetylation of heat shock proteins and enhances protein folding

The pervasive weak electromagnetic fields (EMF) inundate the industrialized society, but the biological effects of EMF as weak as 10 µT have been scarcely analyzed. Heat shock proteins (HSPs) are molecular chaperones that mediate a sequential stress response. HSP70 and HSP90 provide cells under undesirable situations with either assisting covalent folding of proteins or degrading improperly folded proteins in an ATP-dependent manner. Here we examined the effect of extremely low-frequency (ELF)-EMF on AML12 and HEK293 cells. Although the protein expression levels of HSP70 and HSP90 were reduced after an exposure to ELF-EMF for 3 h, acetylations of HSP70 and HSP90 were increased, which was followed by an enhanced binding affinities of HSP70 and HSP90 for HSP70/HSP90-organizing protein (HOP/STIP1). After 3 h exposure to ELF-EMF, the amount of mitochondria was reduced but the ATP level and the maximal mitochondrial oxygen consumption were increased, which was followed by the reduced protein aggregates and the increased cell viability. Thus, ELF-EMF exposure for 3 h activated acetylation of HSPs to enhance protein folding, which was returned to the basal level at 12 h. The proteostatic effects of ELF-EMF will be able to be applied to treat pathological states in humans.

Effects of carnosine on the embryonic development and TiO2 nanoparticles-induced oxidative stress on Zebrafish.

Oxidative stress is due to an unbalance between pro-oxidants, such as reactive oxygen (ROS) and nitrogen (RNS) species, and antioxidants/antioxidant system. Under physiological conditions these species are involved in different cellular processes such as cellular homeostasis and immune response, while an excessive production of ROS/RNS has been linked to the development of various diseases such as cancer, diabetes, and Alzheimer's disease. In this context, the naturally occurring dipeptide carnosine has shown the ability to scavenge ROS, counteract lipid peroxidation, and inhibit proteins oxidation. Titanium dioxide nanoparticles (TiO2-NPs) have been widely used to produce cosmetics, in wastewater treatment, in food industry, and in healthcare product. As consequence, these NPs are often released into aquatic environments. The Danio rerio (commonly called zebrafish) embryos exposure to TiO2-NPs did not affect the hatching rate, but induced oxidative stress. According to this scenario, in the present study, we first investigated the effects of carnosine exposure and of a sub-toxic administration of TiO2-NPs on the development and survival of zebrafish embryos/larvae measured through the acute embryo toxicity test (FET-Test). Zebrafish larvae represent a useful model to study oxidative stress-linked disorders and to test antioxidant molecules, while carnosine was selected based on its well-known multimodal mechanism of action that includes a strong antioxidant activity. Once the basal effects of carnosine were assessed, we then evaluated its effects on TiO2-NPs-induced oxidative stress in zebrafish larvae, measured in terms of total ROS production (measured with 2,7-dichlorodihydrofluorescein diacetate probe) and protein expression by immunohistochemistry of two cellular stress markers, 70 kDa-heat shock protein (Hsp70) and metallothioneins (MTs). We demonstrated that carnosine did not alter the phenotypes of both embryos and larvae of zebrafish at different hours post fertilization. Carnosine was instead able to significantly decrease the enhancement of ROS levels in zebrafish larvae exposed to TiO2-NPs and its antioxidant effect was paralleled by the rescue of the protein expression levels of Hsp70 and MTs. Our results suggest a therapeutic potential of carnosine as a new pharmacological tool in the context of pathologies characterized by oxidative stress such as neurodegenerative disorders.

Chronic heat stress promotes liver inflammation in broilers via enhancing NF-κB and NLRP3 signaling pathway

BackgroundThis study investigated the effects of chronic heat stress on liver inflammatory injury and its potential mechanisms in broilers. Chickens were randomly assigned to the 1-week control group (Control 1), 1-week heat stress group (HS1), 2-week control group (Control 2), and a 2-week heat stress group (HS2) with 15 replicates per group. Broilers in the heat stress groups were exposed to heat stress (35 ± 2 °C) for 8 h/d for 7 or 14 consecutive days, and the rest of 26 hours/day were kept at 23 ± 2 °C like control group broilers. Growth performance and liver inflammatory injury were examined for the analysis of liver injury.ResultsThe results showed that heat stress for 2 weeks decreased the growth performance, reduced the liver weight (P < 0.05) and liver index (P < 0.05), induced obvious bleeding and necrosis points. Liver histological changes found that the heat stress induced the liver infiltration of neutrophils and lymphocytes in broilers. Serum levels of AST and SOD were enhanced in HS1 (P < 0.01, P < 0.05) and HS2 (P < 0.01, P < 0.05) group, compared with control 1 and 2 group broilers. The MDA content in HS1 group was higher than that of in control 1 group broilers (P < 0.05). Both the gene and protein expression levels of HSP70, TLR4 and NF-κB in the liver were significantly enhanced by heat stress. Furthermore, heat stress obviously enhanced the expression of IL-6, TNF-α, NF-κB P65, IκB and their phosphorylated proteins in the livers of broilers. In addition, heat stress promoted the activation of NLRP3 with increased NLRP3, caspase-1 and IL-1β levels.ConclusionsThese results suggested that heat stress can cause liver inflammation via activation of the TLR4-NF-κB and NLRP3 signaling pathways in broilers. With the extension of heat stress time, the effect of heat stress on the increase of NF-κB and NLRP3 signaling pathways tended to slow down.

Analgecine protects against cerebral ischemia-reperfusion through apoptosis inhibition and anti-neuroinflammation in rats

Stroke influence the quality of life of patients and leave big public health issues as acute cerebrovascular disease all over the world. Analgecine (AGC) relieves pain and accelerates repair of nerve injury. This current study aims to observe the pharmacological effects and related mechanisms of AGC in cerebral ischemic stroke among middle cerebral artery ischemia-reperfusion (MCAO) rats. After seven days of AGC administration, motor function was enhanced as evidenced by the prehensile traction test. Morphological ameliorations were observed by immunohistochemistry analysis. The protein expression levels of HSP70, Bcl-2, Bax, TRAF-6, MyD88, BDNF, NGF, pCREB, CREB, pTrkB, TrkB, pAKT and AKT were estimated by western blot. Meanwhile, AGC alleviated MCAO-induced inflammation chiefly by decreasing inflammatory cytokines in rat brain tissues. These results above suggested that MCAO-caused brain infarction was obviously alleviated by AGC. The immunohistochemistry data showed that AGC reduced neuronal injury and apoptosis, and inhibited microglia and astrocytes activation. The protein results suggested the expression of apoptosis-relevant proteins decreased among AGC treated groups and the neurotrophin related proteins were obviously enhanced by CREB/BDNF/TrkB/AKT and HSP70/Bcl-2/Bax pathways. Collectively, the results demonstrated that AGC primarily promoted neuro-nutrition, reduced the injury of nerve apoptosis and ameliorated neuroinflammation. In summary, AGC played a neuroprotective role, which had provided reliable evidence for AGC to be a potential drug in treating stroke.

Quinazoline Based HSP90 Inhibitors: Synthesis, Modeling Study and ADME Calculations Towards Breast Cancer Targeting

A new 2-thioquinazolinones series was designed and synthesized as HSP90 inhibitors based on the structure of hit compound VII obtained by virtual screening approach. Their in vitro anti-proliferative activity was evaluated against three human cancer cell lines rich in HSP90 namely; colorectal carcinoma (HCT-116), and cervical carcinoma (Hela), breast carcinoma (MCF-7). Compounds 5a, 5d, 5e and 9h showed a significant broad spectrum anti-proliferative activity against all tested cell lines. They were characterized by potent effect against breast cancer in particular with IC50 of 11.73, 8.56, 7.35 and 9.48 μM, respectively against Doxorubicin (IC50 4.17 μM). HSP90 ATPase activity inhibition assay were conducted where compound 5d exhibited the best IC50 with 1.58 μM compared to Tanespimycin (IC50 = 2.17 μM). Compounds 5a and 9h showed higher IC50 values of 3.21 and 3.41 μM, respectively. The effects of 5a, 5d and 9h on Her2 (a client proteins of HSP90) and HSP70 were evaluated in MCF-7 cells. All tested compounds were found to reduce Her2 protein expression levels and induce Hsp70 protein expression levels significantly, emphasizing that antibreast cancer effect is a consequence of HSP90 chaperone inhibition. Cell cycle analysis of MCF-7 cells treated with 5d showed cell cycle arrest at G2/M phase 38.89% and pro-apoptotic activity as indicated by annexin V-FITC staining by 22.42%. Molecular docking studies suggested mode of interaction to HSP90 via hydrogen bonding. ADME properties prediction of the active compounds suggested that they could be used as orally absorbed anticancer drug candidates.

Effect of Glutamine on Antioxidant Capacity and Lipid Peroxidation in the Breast Muscle of Heat-stressed Broilers via Antioxidant Genes and HSP70 Pathway.

Simple SummaryBroilers are heat stress sensitive animals. Heat stress can reduce the meat quality and increase the oxidative stress of broilers. The aim of this study was to investigate the effect of dietary supplementation with Glutamine (Gln) on meat quality, antioxidant capacity, lipid peroxidation, antioxidant genes, and the HSP70 gene and protein expression in broilers exposed to cyclic heat stress. Results showed that Gln improved the meat quality in heat-stressed broilers. In conclusion, our study suggested that dietary Gln supplementation alleviated antioxidant capacity and lipid peroxidation in the breast muscle of heat-stressed broilers by antioxidant genes and the HSP70 pathway.This study investigated whether Glutamine (Gln) could be used as an additive to improve antioxidant capacity in the breast muscle of heat-stressed broilers. Two hundred and forty 22-day-old Arbor Acres broilers in the G1, G2, G3, and G4 groups (n = 60 each) were housed in a cyclic hot environment and fed the basal diet with 0%, 0.5%, 1.0%, and 1.5% Gln, respectively. Compared with the G1 group, dietary 1.5% Gln increased (p < 0.05) pH and b* values, but decreased (p < 0.05) L* cooking loss, drip loss, and water loss rate in breast meat of heat-stressed broilers. Malondialdehyde levels in the breast muscle were lower (p < 0.05) in 1.0% and 1.5% Gln groups than that of the heat-stress group. Compared with the G1 group, dietary 1.5% Gln increased (p < 0.05) catalase (CAT), glutathione, glutathione peroxidase (GSH-Px,) and total antioxidant capacity in the breast muscle of heat-stressed broilers. Furthermore, the CAT, GSH-Px, HSP70 mRNA expression levels, and HSP70 protein expression levels were increased (p < 0.05) in the G3 and G4 groups compared with the G1 group. In sum, Gln alleviated antioxidant capacity and lipid peroxidation in the breast muscle of heat-stressed broilers through antioxidant genes and HSP70 pathways.

Thermal pretreatment promotes the protective effect of HSP70 against tendon adhesion in tendon healing by increasing HSP70 expression.

Tendon adhesion is a substantial challenge for tendon repair. Thermal pretreatment (TP) may decrease inflammation by upregulating heat shock proteins (HSPs). The present study intends to identify the function that TP serves when combined with HSP70 overexpression in tendon healing and adhesion in rats. Sprague-Dawley male rats were used to establish a surgically ablative tendon postoperative suture model, and the positive expression of the HSP70 protein was measured using immunohistochemistry. Changes to the blood vessels and collagenous fiber, in addition to the maximum tensile strength and the tendon sliding distance, were detected under a microscope. Finally, HSP70, tumor growth factor β (TGF-β), and insulin-like growth factor 1 (IGF-1) mRNA and protein levels were all determined by employing reverse transcription-quantitative polymerase chain reaction and western blot analysis methods. The positive expression of the HSP70 protein increased following TP. Furthermore, TP reduced the infiltration of inflammatory cells and improved the collagenous arrangement, accompanied by an increased maximum tensile force and tendon gliding distance following surgery. In addition, TP increased the mRNA and protein expression levels of HSP70, TGF-β and IGF-1. Altogether, TP increases HSP70 expression, thereby reducing postoperative traumatic inflammation and establishing tendon adhesion and promoting tendon healing. Thus, TP may be a potential strategy for the treatment of tendon adhesion.

Heat shock protein (HSP) expression and mitogen-activated protein kinase (MAPK) phosphorylation during early embryonic developmental stages of the Gilthead sea bream (Sparus aurata)

Both heat shock proteins (HSPs), which have key roles in vital cell functions, as well as members of the mitogen-activated protein kinases (MAPKs), which adjust gene expression by transducing cellular signals to the nucleus, are necessary for normal embryonic development in vertebrates. Therefore, protein expression levels of HSP70 and HSP90 and the activation of members of the MAPK protein family, such as p38 MAPK, ERKs, and JNKs were studied in the early developmental stages of the Gilthead sea bream, Sparus aurata Linnaeus, 1758. The protein expression of HSP70 and the phosphorylation ratio of JNKs remained at equal levels at all examined developmental stages, while the other examined proteins exhibited a differential profile. HSP90 levels were mostly increased at the 16-cell stage and towards the morula stages, and the lowest values were observed at the two- to four-cell and one-half epiboly stages. While p38 MAPK phosphorylation ratio exhibited increased values mostly in the early developmental stages, the opposite was observed concerning ERK phosphorylation ratio, where increased values were observed in the later embryonic stages (high blastula to one-half epiboly stages). These differential profiles of the examined protein expression levels highlight the importance of these proteins during embryogenesis and pave the way for further research to unveil their distinct role in early development.

T-helper 2 cytokine-induced heat shock protein 70 secretion and its potential association with allergic rhinitis.

Various inflammatory mediators have been found to be involved in the pathogenesis of allergic disease (AR). The role of heat shock proteins in AR has not been studied. The aim of this study was to investigate the levels of heat shock protein 70 (Hsp70) in the nasal lavage fluids of AR patients and controls to elucidate the role of Hsp70 in the pathogenesis of AR. Using an enzyme-linked immunosorbent assay, the levels of Hsp70, Hsp90, interleukin (IL)-4, IL-13, and IL-8 in nasal lavage fluid from patients were measured and statistically analyzed. Primary human nasal epithelial cells were culturedin vitroand T-helper 2 (Th2) cytokines (IL-4, IL-13) were added to the culture medium. We evaluated the mRNA and protein expression levels of Hsp70 using real-time polymerase chain reaction and western blot assay. Hsp70 was easily detected in nasal lavage fluid and the levels of Hsp70 were higher in AR patients than in healthy controls. Other clinical characteristics of subjects were not significantly associated with Hsp70 levels. Furthermore, we found that treatment with IL-4 and IL-13 induced the secretion of Hsp70 in human nasal epithelial cells. We found that Hsp70 was abundant and positively detected in nasal lavage fluid samples from all subjects, and that Hsp70 levels were significantly higher in AR patients. We demonstrated, bothin vivoandin vitro,that Hsp70 could play an important role in the pathogenesis of AR, and we suggest that Hsp70 can be used as a disease marker for AR.

Titanium Dioxide Nanoparticles-Mediated In Vitro Cytotoxicity Does Not Induce Hsp70 and Grp78 Expression in Human Bronchial Epithelial A549 Cells

Titanium dioxide nanoparticles (TiO(2)NPs) are increasingly being used in various industrial applications including the production of paper, plastics, cosmetics and paints. With the increasing number of nano-related products, the concern of governments and the general public about the health and environmental risks, especially with regard to occupational and other environmental exposure, are gradually increasing. However, there is insufficient knowledge about the actual affects upon human health and the environment, as well as a lack of suitable biomarkers for assessing TiO(2)NP-induced cytotoxicity. Since the respiratory tract is likely to be the main exposure route of industrial workers to TiO(2)NPs, we investigated the cytotoxicity of the anatase and rutile crystalline forms of TiO(2)NPs in A549 cells, a human alveolar type II-like epithelial cell line. In addition, we evaluated the transcript and protein expression levels of two heat shock protein (HSP) members, Grp78 and Hsp70, to ascertain their suitability as biomarkers of TiO(2)NP-induced toxicity in the respiratory system. Ultrastructural observations confirmed the presence of TiO(2)NPs inside cells. In vitro exposure of A549 cells to the anatase or rutile forms of TiO(2)NPs led to cell death and induced intracellular ROS generation in a dose-dependent manner, as determined by the MTS and dichlorofluorescein (DCF) assays, respectively. In contrast, the transcript and protein expression levels of Hsp70 and Grp78 did not change within the same TiO(2)NPs dose range (25-500 μg/ml). Thus, whilst TiO(2)NPs can cause cytotoxicity in A549 cells, and thus potentially in respiratory cells, Hsp70 and Grp78 are not suitable biomarkers for evaluating the acute toxicological effects of TiO(2)NPs in the respiratory system.

Synthesis and biological evaluation of 2,4-diaminoquinazoline derivatives as novel heat shock protein 90 inhibitors

Novel 2,4-diaminoquinazoline derivatives originating from a virtual screening approach were designed, synthesized and their biological activities as heat shock protein 90 (Hsp90) inhibitors were evaluated. The prepared compounds exhibited significant anti-proliferative activities against DU-145, HT-29, HCT-116, A375P and MCF-7 cancer cell lines. The selected compounds were tested against Her2, a client protein of Hsp90, and showed significant reduction in Her2 protein expression. Compound 6b was found the most potent, reduced Her2 protein expression levels and induced Hsp70 protein expression levels significantly.

The role of heat shock protein 27 in bronchiolitis obliterans syndrome after lung transplantation

Heat shock proteins (Hsps) are a family of evolutionary conserved proteins classified according to their size as small and large Hsps. They have a cytoprotective role and have been shown to be immunogenic molecules. In addition, self-reactivity to Hsps has been implicated in various autoimmune diseases and in the development of alloimmunity. This study examined the relationship of large and small Hsps and anti-Hsp antibodies in lung transplant (LTx) recipients who have bronchiolitis obliterans syndrome (BOS). Anti-Hsp27 and Hsp70 antibodies, and Hsp27, Hsp60, and Hsp70 protein expression levels were evaluated in serum and bronchoalveolar lavage samples collected from 8 LTx recipients with established BOS and 8 recipients without BOS (controls). Serum from 8 healthy individuals was examined for Hsp levels as a comparison. Elevated serum Hsp27 levels were observed in recipients with BOS compared with controls or healthy individuals, whereas Hsp70 and Hsp60 expression showed no difference. Anti-Hsp27 antibody levels were significantly higher in the BAL of recipients with BOS than in those without BOS. In contrast, anti-Hsp70 antibodies levels in serum or BAL showed no difference between groups. These results support the novel concept that Hsp27, but not the classic Hsp60 and Hsp70, may be associated with the development BOS. The expression of anti-Hsp27 antibodies found only in the BAL fluid suggests a local response occurring at the level of the alveoli and terminal airways.

Larval excretory-secretory products from the parasite Schistosoma mansoni modulate HSP70 protein expression in defence cells of its snail host, Biomphalaria glabrata

Synthesis of heat shock proteins (HSPs) following cellular stress is a response shared by many organisms. Amongst the HSP family, the approximately 70 kDa HSPs are the most evolutionarily conserved with intracellular chaperone and extracellular immunoregulatory functions. This study focused on the effects of larval excretory-secretory products (ESPs) from the parasite Schistosoma mansoni on HSP70 protein expression levels in haemocytes (defence cells) from its snail intermediate host Biomphalaria glabrata. S. mansoni larval stage ESPs are known to interfere with haemocyte physiology and behaviour. Haemocytes from two different B. glabrata strains, one which is susceptible to S. mansoni infection and one which is resistant, both showed reduced HSP70 protein levels following 1 h challenge with S. mansoni ESPs when compared to unchallenged controls; however, the reduction observed in the resistant strain was less marked. The decline in intracellular HSP70 protein persisted for at least 5 h in resistant snail haemocytes only. Furthermore, in schistosome-susceptible snails infected by S. mansoni for 35 days, haemocytes possessed approximately 70% less HSP70. The proteasome inhibitor, MG132, partially restored HSP70 protein levels in ESP-challenged haemocytes, demonstrating that the decrease in HSP70 was in part due to intracellular degradation. The extracellular signal-regulated kinase (ERK) signalling pathway appears to regulate HSP70 protein expression in these cells, as the mitogen-activated protein-ERK kinase 1/2 (MEK1/2) inhibitor, U0126, significantly reduced HSP70 protein levels. Disruption of intracellular HSP70 protein expression in B. glabrata haemocytes by S. mansoni ESPs may be a strategy employed by the parasite to manipulate the immune response of the intermediate snail host.

Hsp70- and p53-reponses after heat treatment and/or X-irradiation mediate the susceptibility of hematopoietic cells to undergo apoptosis

Purpose: The effect of heat treatment in combination with X-irradiation was examined with regard to expression of p53, a tumor suppressor gene product, and Hsp70, a heat-shock protein, in association with the occurrence of programmed cell death (apoptosis).Materials and methods: Three hematopoietic cell lines (HSB2, HL60 and Kasumi-1), which differ in p53 status, were exposed to 42.5°C during one hour and/or X-radiation (total dose 8 Gy). After exposure, both mRNA and protein expression levels of Hsp70 and p53 were investigated by real-time PCR (polymerase chain reaction) and Western blotting. Apoptosis was simultaneously analyzed by observation of cell morphology as well as flowcytometric determination of Annexin V binding to phosphatidylserine and propidium iodide exclusion.Results: Both HL60 and HSB2 cell lines with a low p53 status and a quick response to heat treatment with Hsp70 over-expression are less susceptible to heat-induced apoptosis compared to Kasumi-1 cells with wild-type p53 protein and no Hsp70 response. The combination of first applying X-irradiation followed by heat treatment resulted in the most effective induction of apoptosis due to impairment of the Hsp70 response in all three cell lines.Conclusion: These results indicate that the Hsp70 response and p53 status mediate the susceptibility of hematopoietic cells to undergo heat-induced apoptosis. Therefore, these parameters can be used as markers to predict the effectiveness of hyperthermia in cancer treatment.

Unilateral optic nerve transection up-regulate Hsp70 protein expression in lateral geniculate nucleus of rats

Studies have demonstrated that optic nerve transection results in apoptotic cell death of retinal ganglion cells (RGCs) and neurons within lateral geniculate nucleus (LGN). Heat shock protein (Hsp) 70 was reported to be involved in protecting cells from injury under various pathological conditions in vivo and in vitro. To determine the involvement of Hsp70 in protecting neurons within LGN against damage or loss induced by optic nerve injuries, we observed the changes in protein expression and distribution of Hsp70 in LGN at days 1, 3, 7, 14 and 28 after unilateral optic nerve transection in the left eye of Sprague–Dawley rats by using Western blot analysis and immunohistochemical staining. We found that the levels of Hsp70 protein expression increased significantly ( p < 0.05, n = 6 for each group) in both right and left LGN of rats following left optic nerve transection 1–7 days. The maximum of Hsp70 expression reached at day 3. However, Hsp70 protein expression levels in both right and left LGN returned to control levels at 14 and 28 days after left optic nerve lesion. In addition, the increased Hsp70 expression, which mainly localized in the intergeniculate leaflet of LGN, was also observed by immunostaining in right LGN at the end of day 3 after the lesion. These results suggest that increased expression of Hsp70 may be involved in protecting neurons within LGN against damage or loss induced by left optic nerve transection at early stage.