Heat Shock Protein 70 Protein Research Articles

The Heat Shock Protein HSP70 Promotes Th17 Genes’ Expression via Specific Regulation of microRNA

T helper cells type 17 (Th17) are orchestrators of autoimmune conditions, including multiple sclerosis (MS), but mechanisms of Th17 pathogenicity remain unknown. MicroRNAs (miRNA) are known to control T cells. To understand the function of miRNA in Th17, we have established a T cell line, EL4-TCR+, that resembles the expression pattern of the Th17 cells. Subsequently, we have evaluated the crosstalk between miRNA and Th17 genes’ expression using a combination of gene expression profiling, gene expression manipulation, RNA and protein immunoprecipitation, as well as confocal microscopy. We have found that Th17-related miRNA were strongly expressed in EL4-TCR+ cells following the binding of the cluster of differentiation 3 (CD3) component of the T cell receptor (TCR). Furthermore, a specific inhibition of these miRNA resulted in downregulation of the critical Th17 genes’ expression. Surprisingly, this mechanism relied on the function of the stress signal regulator heat shock protein 70 (HSP70). Upon activation, HSP70 co-localized intracellularly with miRNA processing proteins. Precipitation of HSP70 resulted in enrichment of the Th17-associated miRNA. Finally, HSP70 inhibition led to downregulation of the Th17 genes’ expression and ameliorated development of autoimmune demyelination. Our study demonstrated that HSP70 facilitates specific miRNA function leading to Th17 genes’ expression, a mechanism linking stress and autoimmunity.

An Increase in Thermotolerance Is Associated with Elevated Levels of Heat Shock Protein 70 (HSP70) in a Mouse Exertional Heat Stroke Model

Recent data from our laboratory showedthat one exertional heat stroke (EHS) exposure improved thermal tolerance inmice who were exposed to the same conditions again within 24 h. This was evident by longer average runningtime, higher thermal load, higher maximum core temperature, and less severehypothermia during the second EHS exposure and recovery compared to the first. Theaim of this study was to determine if traditional biomarkers of EHS severitywere reflective of the improved thermoregulatory performance observed during the second EHS. We collected blood and liver samples from male C57BL/6J mice at30 min and 3 h into recovery from a second EHS event that occurred 24 h after the initial EHS exposure. Mice displayed significantly lower circulating cytokines and chemokines 30 min (MIP‐1β, MIP‐2; P<0.05) and 3 h (G‐CSF,IL‐10, IP‐10, KC, MCP‐1; P<0.05) following the second vs. first EHS exposure. Plasma corticosterone was also significantly lower in mice 3 h after the second vs. first EHS (36458 vs. 262914 pg/ml, respectively; P=0.0005). Liver heat shock protein (HSP)70 was significantly elevated in mice 24 h after the first EHS and was further increased after the second EHS at 30 min (5‐fold) and 3 h(2‐fold) of recovery. The elevated levels of liver HSP70 protein correlated with a reduction in HSP70 gene expression at these time points, suggestive of a negative feedback loop. Although HSP70 is thought to protect against heat‐induced organ damage, aspartate aminotransferase (AST; a clinical biomarker of liver damage) was significantly higher 30 min after the second vs. first EHS exposure (994 U/L vs. 374 U/L, respectively; P<0.05). Taken together, our results suggest that one EHS event induces partial thermal tolerance in mice that improves performance in the heat 24 h later and may protect against systemic inflammation during the subsequent heat exposure, as evidenced by changes in the expression of pro‐ and anti‐inflammatory factors, such as cytokines, chemokines and corticosterone. Elevated liver HSP70 suggests protection against organ damage, but this interpretation needs to be validated with histological analysis of the liver since elevated AST levels suggest increased stress to this organ following the second EHS. Perhaps the adaptive responses of improved run time and thermal tolerance in the heat 24 h after the initial EHS come at the cost of additional organ damage. Author views not official US Army or DOD policy.

Humoral immune response to heat shock protein 60 of Aggregatibacter actinomycetemcomitans and cross-reactivity with malondialdehyde acetaldehyde-modified LDL.

Atherosclerosis is a chronic inflammatory disease and major cause of mortality worldwide. One of the crucial steps for atherosclerotic plaque development is oxidation of low-density lipoprotein (LDL). Through the oxidation, highly immunogenic epitopes are created and the immune system is activated. Association between atherosclerosis and periodontal diseases is well documented, and one of the main oral pathogens common in periodontitis is Aggregatibacter actinomycetemcomitans (Aa). Heat shock protein 60 (HSP60) is an important virulence factor for Aa bacteria and a strong activator of the immune system. Cross-reactivity of HSP60 and oxidized LDL (OxLDL) antibodies could be a potential mechanism in the progression of atherosclerosis and one possible link between atherosclerosis and periodontitis. Human plasma samples from neonates and mothers were analyzed to determine if antibody titer to Aa-HSP60 protein is already present in newborns. Further objectives were to characterize antibody response in Aa-HSP60 immunized mice and to determine possible antibody cross-reaction with oxidized LDL. We demonstrated that newborns already have IgM antibody levels to Aa-HSP60. We also showed that in mice, Aa-HSP60 immunization provoked IgG and IgM antibody response not only to Aa-HSP60 but also to malondialdehyde acetaldehyde-modified LDL (MAA-LDL). Competition assay revealed that the antibodies were specific to Aa-HSP60 and cross-reacted with MAA-LDL. Our results suggest a possibility of molecular mimicry between Aa-HSP60 and MAA-LDL, making it intriguing to speculate on the role of HSP60 protein in atherosclerosis that manifests at young age.

Resveratrol Plays Protective Roles on Kidney of Uremic Rats via Activating HSP70 Expression.

Objective To investigate the protective effects of resveratrol on kidney of uremic rats and to explore whether the mechanism is associated with heat shock protein 70 (HSP70) expression. Methods Sixty male Sprague Dawley rats were randomly separated into 5 groups, including sham group, uremic model group, and different doses of resveratrol group (5 mg/kg, 10 mg/kg, and 20 mg/kg). The serum creatinine (Cr) and urea nitrogen (BUN) levels were detected by Automatic Biochemical Analyzer (ABA). The pathological changes of renal tissues and the renal interstitial fibrosis were analyzed by hematoxylin-eosin (HE) and Masson, respectively. The expression of HSP70 protein in renal tissues was detected by immunohistochemistry. The expression of HSP70 and NF-κB pathway-related proteins were detected by Western blot. To further validate the protective role of resveratrol through activating HSP70 in uremic rats, HSP70 activator (17-AAG) and HSP70 inhibitor group (MKT-077) were used. Results In the model group, the levels of Cr and BUN in serum were significantly increased, and the renal interstitial collagen deposition was also obviously increased (p < 0.05). Compared with the model group, the levels of Cr and BUN in different doses of resveratrol groups were remarkably declined, and the renal interstitial collagen deposition was declined (p < 0.05). Resveratrol also significantly improved the renal tissue lesions when compared with the model group. In renal tissues, different doses of resveratrol treatment remarkably raised HSP70 and p-IκBα expression and also remarkably declined the level of p-P65 protein (p < 0.05). Meanwhile, the effect of 17-AAG was similar to 20 mg/kg resveratrol on NF-κB pathway-related proteins expression. After the added MKT-077 in the resveratrol treatment group, the levels of HSP70 and p-IκBα in the renal tissue were remarkably declined; however, the levels of p-P65 protein was remarkably raised (p < 0.05). Conclusion Resveratrol played a protective role on the kidney of uremic rats through activating HSP70 expression.

Ovary Proteome Analysis Reveals RH36 Regulates Reproduction via Vitellin Uptake Mediated by HSP70 Protein in Hard Ticks.

Ticks are blood-sucking vector arthropods, which play an important role in transmitting pathogens between humans and animals. RH36 is an immunomodulatory protein expressed in the salivary glands, but not other organs, of partially fed Rhipicephalus haemaphysaloides ticks, and it reaches its peak on the day of tick engorgement. RH36 gene silencing inhibited tick blood feeding and induced a significant decrease in tick oviposition, indicating that another function of immunosuppressor RH36 was regulating tick reproduction. Why did RH36 protein expressed uniquely in the salivary gland regulate tick reproduction? RH36 regulated positively the expression of vitellogenin in ovary, which indicated RH36 protein played an important role in the integration of nutrition and reproduction. According to proteomic analysis, heat shock protein 70 (HSP70) was significantly down-regulated in the immature ovary of post-engorged ticks. In addition, gene silencing of HSP70 not only inhibited tick blood-sucking and the expression of vitellogenin, but also increased tick death rate. These results suggested RH36 affected tick vitellogenin uptake and then regulated ovary cell maturation by modulating the expression of HSP70 protein, and finally controlled tick oviposition.

HSP Transcript and Protein Accumulation in Brassinosteroid Barley Mutants Acclimated to Low and High Temperatures.

In temperature stress, the main role of heat-shock proteins (HSP) is to act as molecular chaperones for other cellular proteins. However, knowledge about the hormonal regulation of the production of the HSP is quite limited. Specifically, little is known about the role of the plant steroid hormones—brassinosteroids (BR)—in regulating the HSP expression. The aim of our study was to answer the question of how a BR deficit or disturbances in its signaling affect the accumulation of the HSP90, HSP70, HSP18, and HSP17 transcripts and protein in barley growing at 20 °C (control) and during the acclimation of plants at 5 °C and 27 °C. In barley, the temperature of plant growth modified the expression of HSPs. Furthermore, the BR-deficient mutants (mutations in the HvDWARF or HvCPD genes) and BR-signaling mutants (mutation in the HvBRI1 gene) were characterized by altered levels of the transcripts and proteins of the HSP group compared to the wild type. The BR-signaling mutant was characterized by a decreased level of the HSP transcripts and heat-shock proteins. In the BR-deficient mutants, there were temperature-dependent cases when the decreased accumulation of the HSP70 and HSP90 transcripts was connected to an increased accumulation of these HSP. The significance of changes in the accumulation of HSPs during acclimation at 27 °C and 5 °C is discussed in the context of the altered tolerance to more extreme temperatures of the studied mutants (i.e., heat stress and frost, respectively).

The Antagonistic Effects of Selenium Yeast (SeY) on Cadmium-Induced Inflammatory Factors and the Heat Shock Protein Expression Levels in Chicken Livers

Cadmium (Cd) is a ubiquitous toxic heavy metal in the natural environment that can cause multiple organ damage to the bodies of animals and humans. Selenium yeast (SeY) is a kind of organic selenium (Se) that has a very strong function against Cd-induced injury to tissues or organs. The aim of the current study was to investigate the roles of inflammatory factors and heat shock proteins (HSPs) in inflammatory injury triggered by Cd and to analyze the protective effects of SeY on Cd-induced damnification in the livers of chickens. Two hundred 120-day-old layers were randomly divided into four groups and raised on a conventional diet, or with Se (0.5mg/kg SeY), Cd (150mg/kg CdCl2), or Se + Cd (0.5mg/kg SeY and 150mg/kg CdCl2) in their basic diets for 120days. Then, the liver histopathology, production of nitric oxide (NO), activity of inducible NO synthase (iNOS), and mRNA and protein expression levels of inflammatory factors (iNOS, NF-κB, TNF-α, and PTGE) and heat shock proteins (HSPs 27, 40, 60, 70, and 90) were examined. The results showed that exposure to Cd obviously increased Cd accumulation, NO production, iNOS activity, inflammatory factor, and HSP mRNA and protein expression levels and further caused an inflammatory response. Supplementation with SeY had an antagonistic effect on Cd-induced inflammatory injury in chicken livers. Thus, the present study suggests that SeY can be taken as a potential therapeutic for Cd-induced liver inflammatory injury in chickens.

Heat shock protein 70 levels and post-harvest survival of eastern oysters following sublethal heat shock in the laboratory or conditioning in the field.

A major problem of storing and shipping eastern oysters (Crassostrea virginica) from the Northern Gulf of Mexico in summer and early fall is their elevated mortality. A study was therefore conducted to determine whether heat shocking the oysters or conditioning them to aerial exposure prior to harvest could reduce their mortality during cold storage. Increasing the levels of stress proteins in bivalves has been shown to reduce their mortality when exposed to additional stressors. In this study, the levels of heat shock protein 70 (HSP70) proteins and cumulative mortality during cold storage, out of water, of market-sized oysters were measured, in summer, following (1) sublethal heat shocks (41°C, 1h) in the laboratory or (2) 3weeks to 6weeks of daily exposures to air (0h, ~ 10h, or ~ 18h) in the field. In total, four heat shock and two aerial exposure studies were done. Consistently, heat shocks or 6weeks of daily aerial exposures increased HSP70 levels in oysters but did not reduce their mortality during cold storage. Three weeks of daily aerial exposure did not increase HSP70 levels and only marginally reduced mortality; a significant reduction in cumulative mortality occurred in one of the aerial exposure studies after 7days of cold storage (0h [26%], ~ 18h [8%]). In conclusion, upregulation of HSP70 proteins or aerial exposure during grow-out was not an effective tool in reducing the mortality of oysters harvested in summer and held in cold storage.

Heat Shock Proteins 27, 70 and 90 Protein Expression in Gastric Adenocarcinoma: Prognostic, Predictive and Cellular Differentiation Significances

Background: Gastric cancer is the second global leading cause of death from cancer and the most common gastrointestinal cancer in Iran. This condition is usually diagnosed at advance stages where treatment options are limited. Recently, heat shock proteins (HSPs) have been reported to be overexpressed in a wide range of malignancies and considered as promising candidate biomarkers and therapeutic targets for gastric adenocarcinoma. The aim of the present study was to compare HSPs protein expression between non-tumoral and tumoral sections from patients with gastric adenocarcinoma and determine HSPs protein expression correlation with histological stage, tumoral grade and prognosis. Methods: Immunohistochemistry was used to assess the expression levels of HSP-27, 70 and -90 proteins on both tumoral and non-tumoral (margin of tumor as control group) sections in 80 patients with gastric adenocarcinoma. Further analyses were histology, grade and stage of tumor (Tumor, node, and metastasis), HSPs expression level, clinicopathological significances, and survival rate. Results: The expression of HSPs was significantly increased in tumoral sections compared with non-tumoral sections (P<0.001). The HSP27 expression was correlated with tumors on the corpus of stomach (P=0.049). Patients younger than 63 years revealed higher expression levels of HSP70 (P=0.040). High expression levels of HSP90 were further assessed in well-differentiated and intestinal types of tumors (P=0.009 and P=0.019). Overexpressed levels of HSP27 and 90 were associated with the reduced survival rate of patients (P=0.017 and P=0.018). Conclusion: HSP27 and HSP 90 are potential prognostic biomarkers of patients’ survival rate. Patients harboring positive HSP27 and HSP 90 expression display worse disease-free survival compared to those with negative HSP27 and HSP 90 expression. Differential expression of HSPs may play crucial roles in the initiation and progression of gastric cancer and can be exploited as future therapeutic targets.

Identification and Expression of Heat Shock Protein 60 (HSP60) from the Ark Shell Scapharca broughtonii

Heat shock protein 60 (HSP60) is evolutionarily highly conserved, and it assists with protein folding in the mitochondria and serves as a molecular chaperone, increasing cell survival under various conditions. Studies of HSP60 in molluscs under stress conditions remain limited. Here, this study first characterize a full-length HSP60 cDNA from the ark shell Scapharca broughtonii (termed SbHSP60) containing a 1,725-bp open reading frame corresponding to a deduced protein of 574 amino acid residues with a predicted molecular mass of approximately 61.4 kDa. The deduced protein has 72.3%–80.3% amino acid identity with homologs from invertebrates and vertebrates. Phylogenetic analysis showed that SbHSP60 grouped with proteins from other molluscs, forming a sister cluster to the HSP60 proteins from vertebrates. The expression pattern of SbHSP60 as examined by quantitative real-time PCR suggested that these mRNA transcripts were distributed across all examined tissues. The highest levels of expression were found in the hepatopancreas and gill, with decreasing levels of expression in the mantle, foot, hemocytes, and adductor muscle. Expression of SbHSP60 after challenge with Vibrio anguillarum showed similar upregulation profiles in the hepatopancreas and gill, indicating that SbHSP60 is involved in the bacterial defense process as an acute-phase protein. Levels of SbHSP60 in the hepatopancreas and gill were higher in cadmium-exposed individuals than in controls. SbHSP60 was significantly upregulated by Cd stress in the gill, but expression was less affected in the hepatopancreas, implying different roles in cellular Cd stress protection. The effects of heat stress on SbHSP60 mRNA expression were duration- and tissue-specific. The induction of SbHSP60 in the heat-stressed group occurred predominantly in the hepatopancreas, suggesting that protein damage and the consequent requirement for the HSP60 protein are increased during the heat shock process.

Adenine Nucleotide Translocase 1 Expression is Coupled to the HSP27-Mediated TLR4 Signaling in Cardiomyocytes.

The cardiac-specific overexpression of the adenine nucleotide translocase 1 (ANT1) has cardioprotective effects in various experimental heart disease models. Here, we analyzed the link between ANT1 expression and heat shock protein 27 (HSP27)-mediated toll-like receptor 4 (TLR4) signaling, which represents a novel communication pathway between mitochondria and the extracellular environment. The interaction between ANT1 and HSP27 was identified by co-immunoprecipitation from neonatal rat cardiomyocytes. ANT1 transgenic (ANT1-TG) cardiomyocytes demonstrated elevated HSP27 expression levels. Increased levels of HSP27 were released from the ANT1-TG cardiomyocytes under both normoxic and hypoxic conditions. Extracellular HSP27 stimulated TLR4 signaling via protein kinase B (AKT). The HSP27-mediated activation of the TLR4 pathway was more pronounced in ANT1-TG cardiomyocytes than in wild-type (WT) cardiomyocytes. HSP27-specific antibodies inhibited TLR4 activation and the expression of HSP27. Inhibition of the HSP27-mediated TLR4 signaling pathway with the TLR4 inhibitor oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) reduced the mitochondrial membrane potential (∆ψm) and increased caspase 3/7 activity, which are both markers for cell stress. Conversely, treating cardiomyocytes with recombinant HSP27 protein stimulated TLR4 signaling, induced HSP27 and ANT1 expression, and stabilized the mitochondrial membrane potential. The activation of HSP27 signaling was verified in ischemic ANT1-TG heart tissue, where it correlated with ANT1 expression and the tightness of the inner mitochondrial membrane. Our study shows a new mechanism by which ANT1 is part of the cardioprotective HSP27-mediated TLR4 signaling.

Differential DAMP release was observed in the sputum of COPD, asthma and asthma-COPD overlap (ACO) patients

Asthma-COPD overlap (ACO) has been under intensive focus; however, the levels of damage-associated molecular patterns (DAMPs) that can activate the innate and adaptive immune responses of ACO are unknown. The present study aimed to examine the levels of some DAMPs in asthma, COPD, and ACO and to identify the associations between clinical characteristics and DAMPs in ACO. Sputum from subjects with asthma (n = 87) or COPD (n = 73) and ACO (n = 68) or from smokers (n = 62) and never-smokers (n = 62) was analyzed for high mobility group protein B1 (HMGB1), heat shock protein 70 (HSP70), LL-37, S100A8, and galectin-3 (Gal-3). The concentration of HMGB1, HSP70, LL-37, and S100A8 proteins in sputum from ACO patients was significantly elevated, whereas that of Gal-3 was reduced, compared to that of smokers and never-smokers. The levels of HMGB1 and Gal-3 proteins in ACO patients were elevated compared to those in asthma patients. The sputum from ACO patients showed an increase in the levels of LL-37 and S100A8 proteins compared to that of asthma patients, whereas the levels decreased compared to those of COPD patients. The concentrations of HMGB1, HSP70, LL-37, and S100A8 proteins in the sputum of 352 participants were negatively correlated, whereas the levels of Gal-3 were positively correlated, with FEV1, FEV1%pred, and FEV1/FVC. Sputum HMGB1 had a high AUC of the ROC curve while distinguishing ACO patients from asthma patients. Meanwhile, sputum LL-37 had a high AUC of the ROC curve in differentiating asthma and COPD. The release of sputum DAMPs in ACO may be involved in chronic airway inflammation in ACO; the sputum HMGB1 level might serve as a valuable biomarker for distinguishing ACO from asthma, and the sputum LL-37 level might be a biomarker for differentiating asthma and COPD.

Comparative modeling and mutual docking of structurally uncharacterized heat shock protein 70 and heat shock factor-1 proteins in water buffalo

Aim:In this study, a wide range of in silico investigation of Bubalus bubalis (BB) heat shock protein 70 (HSP70) and heat shock factor-1 (HSF1) has been performed, ranging from sequence evaluation among species to homology modeling along with their docking studies to decipher the interacting residues of both molecules.Materials and Methods:Protein sequences of BB HSP70 and HSF1 were retrieved from NCBI database in FASTA format. Primary and secondary structure prediction were computed using Expasy ProtParam server and Phyre2 server, respectively. TMHMM server was used to identify the transmembrane regions in HSP70. Multiple sequence alignment and comparative analysis of the protein was carried out using MAFFT and visualization was created using ESPript 3.0. Phylogenetic analysis was accomplished by COBALT. Interactions of HSP70 with other proteins were studied using STRING database. Modeller 9.18, RaptorX, Swiss-Modeller, Phyre2, and I-TASSER were utilized to design the three-dimensional structure of these proteins followed by refinement; energy minimization was accomplished using ModRefiner and SPDBV program. Stereochemical quality along with the accuracy of the predicted models and their visualization was observed by PROCHECK program of PDBsum and UCSF Chimera, respectively. ClusPro 2.0 server was accessed for the docking of the receptor protein with the ligand.Results:The lower value of Grand Average of Hydropathy indicates the more hydrophilic nature of HSP70 protein. Value of the instability index (II) classified the protein as stable. No transmembrane region was reported for HSP70 by TMHMM server. Phylogenetic analysis based on multiple sequence alignments (MSAs) by COBALT indicated more evolutionarily closeness of Bos indicus (BI) with Bos taurus as compared to BI and BB. STRING database clearly indicates the HSF1 as one of the interacting molecules among 10 interacting partners with HSP 70. The best hit of 3D model of HSP70 protein and HSF1 was retrieved from I-TASSER and Phyre2, respectively. Interacting residues and type of bonding between both the molecules which were docked by ClusPro 2.0 were decoded by PIC server. Hydrophobic interactions, protein-protein main-chain-side-chain hydrogen bonds, and protein-protein side-chain-side-chain hydrogen bonds were delineated in this study.Conclusion:This is the first-ever study on in silico interaction of HSP70 and HSF1 proteins in BB. Several bioinformatics web tools were utilized to study secondary structure along with comparative modeling, physicochemical properties, and protein-protein interaction. The various interacting amino acid residues of both proteins have been indicated in this study.

Effect of moxibustion on expressions of HSP70 mRNA and protein in gastric cancer-bearing rats

Objective: To observe the effect of moxibustion on the mRNA and protein expressions of heat-shock protein 70 (HSP70) in gastric cancer-bearing rats. Methods: A total of 40 healthy Sprague-Dawley (SD) rats were adaptively fed for one week. The gastric cancer model was prepared by Walker-256 cancer tissue transplantation. After 7 d, 10 rats were randomly selected to verify the successful modeling, and the remaining 30 rats were divided into a model group, a moxibustion group and an infrared group by the random number table method, with 10 rats in each group. After enrollment, the moxibustion group received suspended moxibustion at Zhongwan (CV 12), Guanyuan (CV 4) and bilateral Zusanli (ST 36), (the first group of acupoints) on the 1st day, and suspended moxibustion at bilateral Pishu (BL 20) and Weishu (BL 21), (the second group of acupoints) on the 2nd day, 20 min each time, once a day. Moxibustion was alternately performed every other day at the two groups of acupoints for 21 d. From the day of enrollment, rats in the infrared group were irradiated with the infrared radiation at the stomach area on the 1st day, and at the T12-T13 interspinous region on the 2nd day, 20 min each time, once a day, and the two locations were alternately irradiated every other day for 21 d. During the treatment, rats in the model group were intervened by grasping and fixation without treatment. At the end of the treatment, blood was collected from the inner eye orbit, and the HSP70 expression in peripheral blood was determined by enzyme linked immunosorbent assay (ELISA). Rats were sacrificed, the tumor volume and growth inhibition rate were measured. The position and changes of HSP70 in gastric cancer were observed by streptavidin-perosidase (SP); HSP70 protein expression was determined by ELISA; HSP70 mRNA expression in cancer tissues was determined by reverse transcription-polymerase chain reaction (RT-PCR) assay. Results: In comparison of the model group, the volume growth of the gastric cancer in the moxibustion group was significantly restricted (P<0.01); the volume growth inhibition rate in the moxibustion group was 37.93%; the HSP70 expression in peripheral blood and the cancer tissues was significantly increased (both P<0.01); the expression of HSP70 mRNA and HSP70 content in gastric tumor were both obviously increased in the moxibustion group (P<0.01); and a large amount of HSP70 was released to the outside of cancer cells in the moxibustion group. In comparison of the model group, the volume growth of the gastric cancer in the infrared group was slightly restricted (P<0.05) with a volume growth inhibition rate of 15.89%; the HSP70 expression in the infrared group was increased significantly in peripheral blood (P<0.01) and in the gastric cancer tissues (P<0.05); more HSP70 was released outside of the cancer cells in the infrared group. In comparison of the infrared group, the volume growth of gastric cancer was more restricted in the moxibustion group (P<0.05), and the HSP70 expression in the gastric cancer tissues was also higher (P<0.05); more HSP70 was released outside of the cancer cells in the moxibustion group. Conclusion: Moxibustion and infrared treatment inhibit the gastric cancer growth in the gastric cancer-bearing rats, up-regulate the HSP70 expression in gastric cancer tissues, and promote the production and extracellular release of HSP70, and the effect of moxibustion is more obvious. Conclusion: Moxibustion and infrared treatment inhibit the gastric cancer growth in the gastric cancer-bearing rats, up-regulate the HSP70 expression in gastric cancer tissues, and promote the production and extracellular release of HSP70, and the effect of moxibustion is more obvious.

Intrarectally administered polaprezinc attenuates the development of dextran sodium sulfate-induced ulcerative colitis in mice.

Polaprezinc (PZ), a chelate of zinc and L-carnosine, has been widely used in the treatment of gastric ulcers since 1994. In recent years, researchers have found PZ to have a beneficial effect on various experimentally induced models of colitis in mice. In the present study, 6% dextran sodium sulfate (DSS) was used to induce a model of ulcerative colitis (UC) in Institute of Cancer Research mice. The therapeutic effect and mechanism of PZ action in a model of UC was studied in order to provide an experimental basis for the clinical application of PZ in UC treatment. The effect of PZ on UC was evaluated in five groups of mice: A vehicle control only group, a DSS model control group (DSS, 6%), a validated treatment control group (DSS 6% + Mesalamine), a low-dose PZ treatment group (DSS 6% + PZ 60 mg/kg) and a high-dose PZ group (DSS 6% + PZ 120 mg/kg). After the animals were sacrificed, blood was collected and the serum levels of NF-κB and tumor necrosis factor-α (TNF-α) were measured. Changes in histology were observed by light microscopy. The protein levels of AKT, phosphorylated AKT and heat shock protein 70 (HSP70) were determined by western blot analysis. The results suggested that PZ reduced the DSS-induced increase in the inflammatory proteins TNF-α and NF-κB in the UC model. The high-dose of PZ also increased the HSP70 protein level, inhibited AKT phosphorylation in a DSS-induced UC animal model, and decreased white blood cell and neutrophil % counts compared to levels in an untreated DSS control group. Histopathology indicated that the mice of the DSS model group had irregular colonic villi, a large number of inflammatory cells and mucosal damage, whereas mice of the group treated with PZ had small intestinal villus morphology and their villi showed signs of recovery from the damage of UC. The results of the present study indicated that PZ significantly alleviates DSS-induced UC in mice, relieves diarrhea, and inhibits the phosphorylation of inflammatory factors and the inflammatory AKT signaling pathway.

Protodioscin protects PC12 cells against oxygen and glucose deprivation-induced injury through miR-124/AKT/Nrf2 pathway.

The purpose of the current study was to demonstrate the neuroprotective effect of protodioscin (Prot) in an in vitro model of ischemia/reperfusion (I/R) and investigate the underlying molecular mechanism. After PC12 cells were exposed to oxygen and glucose deprivation (OGD) reperfusion, PI staining by flow cytometry was used to quantify the rate of apoptosis. The levels of hypoxia-inducible factor 1-alpha (HIF-1α), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were determined using commercially available kits. Intracellular reactive oxygen species (ROS) level was detected using the 20,70-dichlorodihy-drofluorescein diacetate (DCFH-DA) fluorescence assay. The expression levels of heat-shock proteins (HSP), PI3K, AKT, Nrf2, and miR-124 were tested by western blot or quantitative PCR. Prot significantly attenuated oxygen-glucose deprivation/reperfusion (OGD/R)-induced apoptotic death. Prot also reduced the oxidative stress as revealed by increasing the activities of SOD and GSH-Px, decreasing the levels of ROS and MDA. Moreover, mechanism investigations suggested that Prot prevented the decrease of HSP70, HSP32 (hemeoxygenase-1, HO-1), and PI3K protein expression, phosphorylation of AKT, and the accumulation of nuclear Nrf2. The level of miR-124 was decreased in PC12 cells, which was also effectively reversed by Prot treatment. Prot protected PC12 cells against OGD/R-induced injury through inhibiting oxidative stress and apoptosis, which could be associated with increasing HSP proteins expression via activating PI3K/AKT/Nrf2 pathway and miR-124 modulation.

4-HNE Induces Apoptosis of Human Retinal Pigment Epithelial Cells by Modifying HSP70.

The role of heat shock protein 70 (HSP70) in apoptosis of human retinal pigment epithelial cells (ARPE-19) induced by 4-hydroxy-2-nonenal (4-HNE) was explored. Different concentrations of 4-HNE were used to stimulate ARPE-19 cells, and apoptosis was measured by flow cytometry. The expression of apoptotic-related proteins, HSP70, X-linked inhibitor-of-apoptosis (XIAP), Bcl-2, and Bax were quantified by Western blotting. HSP70 and XIAP overexpression plasmids, or their corresponding siRNAs were transfected into ARPE-19 cells using Lipofectamine™ 2000. Co-immunoprecipitation and Western blotting were used to detect the effect of 4-HNE on the expression of HSP70 and the binding level between 4-HNE and HSP70. The results showed that 4-HNE induced late apoptosis in ARPE-19 cells, accompanied by elevated levels of 4-HNE-modified HSP70, but it did not affect HSP70 protein expression. 4-HNE-modified HSP70 down-regulated the expression of the apoptosis inhibitory protein XIAP. Overexpression of HSP70 or XIAP inhibited 4-HNE-induced apoptosis of ARPE-19 cells. It was suggested that 4-HNE could promote XIAP degradation by modification of HSP70 to induce late apoptosis of human retinal pigment epithelial cells.

Delineation of crosstalk between HSP27 and MMP-2/MMP-9: A synergistic therapeutic avenue for glioblastoma management

BackgroundEpithelial to mesenchymal transition (EMT) and extracellular matrix (ECM) remodeling, are the two elemental processes promoting glioblastoma (GBM). In the present work we propose a mechanistic modelling of GBM and in process establish a hypothesis elucidating critical crosstalk between heat shock proteins (HSPs) and matrix metalloproteinases (MMPs) with synergistic upregulation of EMT-like process and ECM remodeling. MethodsThe interaction and the precise binding site between the HSP and MMP proteins was assayed computationally, in-vitro and in GBM clinical samples. ResultsA positive crosstalk of HSP27 with MMP-2 and MMP-9 was established in both GBM patient tissues and cell-lines. This association was found to be of prime significance for ECM remodeling and promotion of EMT-like characteristics. In-silico predictions revealed 3 plausible interaction sites of HSP27 interacting with MMP-2 and MMP-9. Site-directed mutagenesis followed by in-vitro immunoprecipitation assay (IP) with 3 mutated recombinant HSP27, confirmed an interface stretch containing residues 29–40 of HSP27 to be a common interaction site for both MMP-2 and MMP-9. This was further validated with in-vitro IP of truncated (sans AA 29–40) recombinant HSP27 with MMP-2 and MMP-9. ConclusionThe association of HSP27 with MMP-2 and MMP-9 proteins along with the identified interacting stretch has the potential to contribute towards drug development to inhibit GBM infiltration and migration. General significanceCurrent findings provide a novel therapeutic target for GBM opening a new horizon in the field of GBM management.

Effects of exercise preconditioning and HSP72 on diaphragm muscle function during mechanical ventilation.

BackgroundMechanical ventilation (MV) is a life‐saving measure for patients in respiratory failure. However, prolonged MV results in significant diaphragm atrophy and contractile dysfunction, a condition referred to as ventilator‐induced diaphragm dysfunction (VIDD). While there are currently no clinically approved countermeasures to prevent VIDD, increased expression of heat shock protein 72 (HSP72) has been demonstrated to attenuate inactivity‐induced muscle wasting. HSP72 elicits cytoprotection via inhibition of NF‐κB and FoxO transcriptional activity, which contribute to VIDD. In addition, exercise‐induced prevention of VIDD is characterized by an increase in the concentration of HSP72 in the diaphragm. Therefore, we tested the hypothesis that increased HSP72 expression is required for the exercise‐induced prevention of VIDD. We also determined whether increasing the abundance of HSP72 in the diaphragm, independent of exercise, is sufficient to prevent VIDD.MethodsCause and effect was determined by inhibiting the endurance exercise‐induced increase in HSP72 in the diaphragm of exercise trained animals exposed to prolonged MV via administration of an antisense oligonucleotide targeting HSP72. Additional experiments were performed to determine if increasing HSP72 in the diaphragm via genetic (rAAV‐HSP72) or pharmacological (BGP‐15) overexpression is sufficient to prevent VIDD.ResultsOur results demonstrate that the exercise‐induced increase in HSP72 protein abundance is required for the protective effects of exercise against VIDD. Moreover, both rAAV‐HSP72 and BGP‐15‐induced overexpression of HSP72 were sufficient to prevent VIDD. In addition, modification of HSP72 in the diaphragm is inversely related to the expression of NF‐κB and FoxO target genes.ConclusionsHSP72 overexpression in the diaphragm is an effective intervention to prevent MV‐induced oxidative stress and the transcriptional activity of NF‐κB and FoxO. Therefore, overexpression of HSP72 in the diaphragm is a potential therapeutic target to protect against VIDD.

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 200μmol/L DHA for 48hr 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 100μmol/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 48hr. 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.