Helicobacter pylori infection is related to the development of gastric diseases. Our previous studies showed that high thioredoxin-1 (Trx1) expression in H. pylori can promote gastric carcinogenesis. To explore the underlying molecular mechanisms, we performed an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis of stomach tissues from Mongolian gerbil infected with H. pylori expressing high and low Trx1. Differences in the profiles of the expressed proteins were analyzed by bioinformatics and verified using Western blot analysis. We found three candidate proteins, 14-3-3α/β, glutathione-S-transferase (GST), and heat shock protein 70 (HSP70), in high Trx1 tissues compared with low Trx1 tissues and concluded that cellular stress and redox activity-related proteins were involved in the pathogenesis of gastric cancer associated with H. pylori Trx1.
14-3-3 Proteins/physiology* ; Animals ; Computational Biology ; Gerbillinae ; Glutathione Transferase/physiology* ; HSP70 Heat-Shock Proteins/physiology* ; Helicobacter Infections/complications* ; Helicobacter pylori ; Oxidation-Reduction ; Stomach Neoplasms/etiology* ; Stress, Physiological ; Thioredoxins/physiology*

OBJECTIVETo investigate the protective effect of heat shock protein 70 (HSP70) against hypoxic pulmonary hypertension (HPH) in neonatal rats.

METHODSA total of 128 neonatal rats were randomly divided into blank control group, HPH model group, empty virus group, and HSP70 group, with 32 rats in each group. Before the establishment of an HPH model, the rats in the blank control group and HPH model group were given caudal vein injection of 5 μL sterile saline, those in the empty virus group were given caudal vein injection of 5 μL Ad-GFP (1 010 PFU/mL), and those in the HSP70 group were given caudal vein injection of 5 μL Ad-HSP70 (1 010 PFU/mL). HPH model was prepared in the HPH model, empty virus, and HSP70 groups after transfection. At 3, 7, 10, and 14 days after model establishment, a multi-channel physiological recorder was used to record mean pulmonary arterial pressure (mPAP), optical and electron microscopes were used to observe the structure and remodeling parameters of pulmonary vessels, and Western blot was used to measure the protein expression of HSP70, hypoxia-inducible factor-1α (HIF-1α), endothelin-1 (ET-1), and inducible nitric oxide synthase (iNOS) in lung tissues.

RESULTSAt 3, 7, 10, and 14 days after model establishment, the HPH model group and the empty virus group had a significantly higher mPAP than the blank control group (P<0.05). On days 7 and 10 of hypoxia, the blank control group and the HSP70 group had significantly lower MA% and MT% than the HPH model group and the empty virus group (P<0.01); on day 14 of hypoxia, the HPH model group, empty virus group, and HSP70 group had similar MA% and MT% (P>0.05), but had significantly higher MA% and MT% than the blank control group (P<0.01). On days 3, 7 and 10 of hypoxia, the HSP70 group had significantly higher protein expression of HSP70 than the HPH model group, empty virus group, and blank control group (P<0.01); the HSP70 group had significantly lower expression of HIF-1α, ET-1, and iNOS than the HPH model group and the empty virus group (P<0.05) and similar expression of HIF-1α, ET-1, and iNOS as the blank control group (P>0.05).

CONCLUSIONSIn neonatal rats with HPH, HSP70 transfection can increase the expression of HSP70 in lung tissues, downregulate the expression of HIF-1α, ET-1, and iNOS, alleviate pulmonary vascular remodeling, and reduce pulmonary artery pressure; therefore, it may become a new strategy for the treatment of HPH in neonates.

Animals ; Disease Models, Animal ; Endothelin-1 ; analysis ; HSP70 Heat-Shock Proteins ; genetics ; physiology ; Hypertension, Pulmonary ; prevention & control ; Hypoxia ; complications ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; Nitric Oxide Synthase Type II ; analysis ; Pulmonary Artery ; pathology ; Rats ; Rats, Wistar ; Transfection

Neurodegenerative disease is characterized by progressive loss of neurons in specific brain regions that results in neuronal dysfunction of the central nervous system. Although the pathological mechanism is not fully established, the activation of glial cells mediated neuroinflammation appears to be involved. Heat shock protein 70 (HSP70) is originally described as intracellular chaperone, which plays an important role in protein quality control in cells. However, recent study showed that up-regulation of HSP70 had anti-inflammatory effects in the brain. HSP70 protected neurons from damage and improved neurological function by decreasing inflammatory response as indicated by inactivation of glial cells and inhibition of pro-inflammatory cytokine release. So it is of great significance to find new compounds targeting at HSP70 as neuroprotective agents to delay the progress of neurodegenerative disease. This review will focus on the role of HSP70 in neuroinflammation and the recent advances in using HSP70 as a target for the treatment of neurodegenerative disease.
Brain ; physiopathology ; Cytokines ; HSP70 Heat-Shock Proteins ; physiology ; Humans ; Inflammation ; pathology ; Neurodegenerative Diseases ; physiopathology ; Neurons ; pathology ; Neuroprotection ; Up-Regulation

OBJECTIVETo explore the inhibition mechanism and safety of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives against proliferation of human lung cancer A549 cells, H322 cells, and human umbilical vein endothelial cell (HUVEC).

METHODSCells were treated with 40 Μmol/L of the ppo3a, ppo3b, ppo3i, and 0.1% DMSO (control) for 48 hours, respectively. Apoptosis was determined by Hoechst 33258 staining assay in H322 and A549 cells. Cell cycle distribution was determined by flow cytometry analysis in A549 cell. LC3-II, p53, and heat shock protein (HSP) 70 protein levels were detected by Western blotting in A549 cells treated with ppo3b for 48 hours. The morphology and viability of HUVEC were observed by inverted microscope and sulforhodamine B (SRB) assay.

RESULTSPpo3a, ppo3b, and ppo3i significantly induced apoptosis in H322 and A549 cells. A strong G1-phase arrest was concomitant with the growth inhibitory effect on A549 cells. Ppo3b effectively elevated the p53 protein level, but significantly reduced the HSP70 protein level. There were no significantly inhibitory effect on the morphology and viability of HUVEC when treated with ppo3a, ppo3b, and ppo3i.

CONCLUSIONSppo3a, ppo3b, and ppo3i could inhibit H322 proliferation through apoptosis and inhibit A549 through apoptosis and G1-phase arrest. The protein p53 and HSP70 might involve in the inhibition effects. These derivatives might be a clue to find effective and safe drug for lung cancers.

Apoptosis ; drug effects ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HSP70 Heat-Shock Proteins ; analysis ; physiology ; Humans ; Pyrazoles ; pharmacology ; Tumor Suppressor Protein p53 ; analysis ; physiology

OBJECTIVETo explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on the vitality and toxicity of E. coli ATCC 25922, and to analyze the potential mechanism.

METHODS(1) In vitro experiments. Standard strains of E. coli ATCC 25922 were divided into groups A (without addition), B, C, D, and E according to the random number table, and then the latter 4 groups were respectively cultured with 1.2 mmol/L CORM-2, 1.6 mmol/L CORM-2, 1.2 mmol/L inactive CORM-2 (iCORM-2), 1.6 mmol/L iCORM-2, with six samples in each group. After being cultured for 0, 3, 5, 8, 10, 12, 16, 20, 24, 27, 30, 48 hours, proliferative vitality of E. coli was examined (denoted as absorption value under 600 nm wavelength), and bacteria number was counted. Other standard strains of E. coli ATCC 25922 were divided into groups F (without addition) and G (cultured with 0.8 mmol/L CORM-2), the expressions of genes fliA, dnaK, marA, and waaQ related to E. coli were detected by quantitative real-time (qRT)-PCR. (2) In vivo experiments. Other standard strains of E. coli ATCC 25922 were grouped as A', B', C', D', and E' and treated with the same method as that in groups A, B, C, D, and E, and 0.5 mL bacterial liquid of each group were collected when the absorption value of bacterial liquid in group A' was equal to 0.4 (under 600 nm wavelength). Seventy-two C57BL/6 mice were divided into groups, namely blank control (without treatment), H, I, J, K, and L according to the random number table, with 12 mice in each group. The mice in the latter 5 groups were intraperitoneally injected with 0.5 mL bacterial suspension of groups A', B', C', D', and E' respectively. After injection, general condition of mice in groups H, I, J, K, and L was observed. The serum levels of TNF-α and IL-6 were determined at post injection hour (PIH) 6, 12. The liver and lung samples were harvested for determination of myeloperoxidase (MPO) activity at PIH 12. The same process was carried out in blank control group. Data were processed with repeated measure analysis of variance (ANOVA), factorial design ANOVA, one-way ANOVA, and t test.

RESULTS(1) In vitro experiments. Compared with those of groups A and D, the proliferative vitality and bacteria number of E. coli in group B were all decreased (with F values respectively 1170.80, 217.52, P values all below 0.01). Compared with those of groups A and E, the proliferative vitality and bacteria number of E. coli in group C were also obviously decreased (with F values respectively 7948.34, 14 432.85, P values all below 0.01). Compared with those in group F, the expression of fliA was downregulated, while the expressions of dnaK, marA, and waaQ were upregulated in group G (with t values 30.28, -165.54, -168.88, -187.28, P values all below 0.01). (2) In vivo experiments. Symptoms including listlessness and tachypnea were observed in mice in groups H, K, and L, and they were ameliorated or not obvious in groups I and J. At PIH 6, the serum levels of TNF-α and IL-6 in groups H and K were respectively (647.3 ± 3.8) pg/mL, (3.44 ± 0.22) ng/mL and (639.3 ± 0.8) pg/mL, (2.47 ± 0.32) ng/mL, which were obviously higher than those in group I [(124.6 ± 10.7) pg/mL, (1.03 ± 0.16) ng/mL, with t values from 15.22 to 84.03, P values all below 0.01]. The serum levels of TNF-α and IL-6 in group J at PIH 6, 12 were also obviously decreased as compared with those in groups H and L (with t values from 19.27 to 245.34, P values all below 0.01). MPO activity of liver and lung tissues were significantly attenuated in group I at PIH 12 as compared with those in groups H and K, and it was also attenuated in group J when compared with those in groups H and L (with t values respectively from 17.36 to 18.92 and 2.35 to 3.61, P values all below 0.01).

CONCLUSIONSCORM-2 can obviously inhibit the vitality and toxicity of E. coli, which might be attributable to regulation of expressions of genes fliA, dnaK, marA, and waaQ of E. coli.

Animals ; Carbon Monoxide ; metabolism ; DNA-Binding Proteins ; metabolism ; Escherichia coli ; drug effects ; metabolism ; physiology ; Escherichia coli Proteins ; metabolism ; Glycosyltransferases ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Interleukin-6 ; blood ; Liver ; enzymology ; Lung ; enzymology ; Mice ; Mice, Inbred C57BL ; Organometallic Compounds ; pharmacology ; Peroxidase ; metabolism ; Sigma Factor ; metabolism ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo establish an effective and stable rabbit heat acclimatization model for the experiment of heat acclimatization mechanisms.

METHODSSixteen healthy male rabbits were divided into heat acclimatization group and control group randomly (n = 8). Heat acclimatization (HA) group was kept in simulation chamber with dry bulb temperature of (36 +/- 1) degrees C, wet bulb temperature of (29 +/- 0.5) degrees C, black-bulb temperature of (40 +/- 1.0) degrees C, 100 min/day for 21 days. Control group was kept in the room with temperature of 20 degrees C and relative humidity < 60% during 20 days, then removed into simulation chamber on day 21 to estimate and monitor the rectal temperature together with the heat acclimatization group. Venous blood of control and heat acclimatization group before and after heat exposure on the 1st day, 11th day and 21st day were collected to detect levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6) and heat shock protein 70 (HSP70) by ELISA analysis.

RESULTS(1) Rectal temperature: There was no significant change in control group during 21 days. In heat acclimatization group, it increased (2.07 +/- 0.43) degrees C after the 1st exposure, and increased (1.78 +/- 0.37) degrees C after the 11th exposure, the range of increasing decreased (0.29 +/- 0.09) degrees C. After the 21st exposure, it increased (1.52 +/- 0.29) degrees C, which was (0.55 +/- 0.14) degrees C lower than that of the 1st (P < 0.05),and (0.53 +/- 0.14) degrees C lower to that of the control group under 1st heat stress (P < 0.05); (2) The level of TNF-alpha after the 1st exposure increased significantly (P < 0.05), but didn't raise along with the exposure times. And fell back to the original level after the 11th and 21st exposure. Compared with control group, the level of IL-6 increased after the 1st, 11th and 21st exposure (P < 0.05), and maintained highly after the 11th and 21st exposure. Compared with the control group, the level of HSP70 increased dramatically with the heat exposure times. Significant increasing of (HSP70) could be detected after the 11th and 21st exposure (P < 0.05), but there was no difference to that of the 1st exposure.

CONCLUSIONProlonged or repeated exposure to heat stressful environmental conditions can reduce the physiological strain, improve heat tolerance, elicits heat acclimatization.

Acclimatization ; physiology ; Animals ; Body Temperature Regulation ; physiology ; Disease Models, Animal ; HSP70 Heat-Shock Proteins ; metabolism ; Heat Stress Disorders ; metabolism ; physiopathology ; Hot Temperature ; Male ; Rabbits

OBJECTIVETo investigate the expression and relationship between HSP70 and caspase-3 in knee osteoarthritis.

METHODSForty adult SD rats were divided into experimental group and control group. Thirty rats in experimental group, anterior cruciate ligament transaction (ACLT) was cut off and partial meniscectomy of 1/3 inside incision were performed to reproduce knee osteoarthritis (KOA) model according to Hulth methos, and the other 10 rats was treated with nothing as control group. The rats were sent to the cage and free to move. At 1, 2 and 4 weeks later, the arthritis cartilage of femoral and tibial end were observed through immunohistochemistry staining and light microscope. Meanwhile, Mankin scale system was adopted for histomorphology evaluation.

RESULTSChanges of KOA such as hyperplastic synovium,erosion on the surface of cartilage and so on were found in experiment group, the expression of HSP70 was augmentation all the time, but the expression of caspase-3 was reduction 1 week later; no similar changes were found in control group. Mankin scale system showed that there were significant differences in the first week as compared with the second week and 4th week (both P < 0.01).

CONCLUSIONHeat shock protein inhibit the apoptosis of cartilage cells and protect the cartilage cells in knee osteoarthritis, the conservative treatment for clinical provide objective scientific basis.

Animals ; Apoptosis ; Cartilage ; pathology ; Caspase 3 ; physiology ; Chondrocytes ; pathology ; Female ; HSP70 Heat-Shock Proteins ; physiology ; Male ; Osteoarthritis, Knee ; pathology ; Rats ; Rats, Sprague-Dawley

Animals ; HSP70 Heat-Shock Proteins ; metabolism ; Male ; Physical Conditioning, Animal ; Physical Endurance ; physiology ; Quadriceps Muscle ; metabolism ; physiology ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism

The purpose of the present study is to investigate whether glucose-regulated protein 75 (GRP75) overexpression inhibits apoptosis induced by glucose deprivation through Raf/Mek/Erk1/2 signaling pathway. After pretreatment with Mek-specific inhibitor U0126, GRP75 overexpressing PC12 cells were incubated in glucose-free DMEM medium for indicated time (6, 12 and 24 h). And DMSO-treated GRP75 overexpressing PC12 cells were applied as control. Western blot was used to determine the expression and phosphorylation level of Erk1/2. MTT assay was used to measure cell viability. Hoechst 33258 staining and flow cytometry using propidium iodide (PI) staining was used to analysis apoptosis. Immunofluorescence with antibody against cytochrome c (Cyt c) was used to detect Cyt c release from mitochondrion. The results showed U0126 prevented the activation of Erk1/2 maintained by GRP75, but the total Erk1/2 expression was not affected. U0126-treated group showed lower cell viability and higher apoptotic rate compared with control group. Immunofluorescence indicated the delay in release of Cyt c was blocked by U0126. These results suggest U0126 prevents protective effect of GRP75 on PC12 cells by inhibiting Erk1/2 phosphorylation, which certifies that GRP75 can inhibit the mitochondria-dependent apoptotic pathway through Raf/Mek/Erk1/2 signaling cascade.
Animals ; Apoptosis ; physiology ; Butadienes ; pharmacology ; Cells, Cultured ; Culture Media ; Glucose ; pharmacology ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; MAP Kinase Signaling System ; physiology ; Membrane Proteins ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Nitriles ; pharmacology ; PC12 Cells ; Phosphorylation ; Rats ; raf Kinases ; metabolism

BACKGROUNDGlucocorticoid (GC) insensitivity/GC resistance is an important etiological and prognostic factor in multiple diseases and pathophysiological processes such as scald, shock and asthma. The function of GC was mediated by glucocorticoid receptor (GR). Scald not only decreased the expression of GR but also reduced the affinity of GR, which played an important role in GC resistance in scalded rats. Whereas the molecular mechanism responsible for the decrease of GR affinity resulted from scald remains unclear. Recent studies showed that the changes of heat shock proteins (hsp) especially hsp90 and hsp70 of GR heterocomplex were associated with GR low affinity in vitro.

METHODSThe affinity of GR in hepatic cytosols and in the cytosols of SMMC-7721 cells were determined by radioligand binding assay and scatchard plot. GR heterocomplex in cytosols were captured by coimmunoprecipation and the levels of hsp90 and hsp70 of GR complex were detected by quantitative Western blotting.

RESULTSSimilar with that of hepatic cytosol of scalded rats, a remarkable decrease of GR affinity was also found in the cytosol of heat stressed SMMC-7721 cells. The level of hsp70 of GR complex in hepatic cytosol of scalded rats (30% total body surface area immersion scald) and in cytosol of heat stressed human hepatocarcinoma cell line SMMC-7721 were both increased by 1.5 fold, whereas no change of hsp90 in GR heterocomplex was found. According to the correlation analysis, there may be a positive relationship between increased hsp70 of GR complex and decreased GR affinity in the cytosols.

CONCLUSIONSThe primary results indicated that the level of hsp70 of GR heterocomplex was increased in the hepatic cytosol of scalded rats and the cytosol of heat stressed SMMC-7721 cells. The increase of hsp70 of GR complex might be associated with the decrease of GR affinity.

Animals ; Blotting, Western ; Burns ; physiopathology ; Cell Line ; HSP70 Heat-Shock Proteins ; metabolism ; Heat-Shock Response ; physiology ; Immunoprecipitation ; Protein Binding ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptors, Glucocorticoid ; metabolism