Neutrophilic granule protein (NGP) attenuates lipopolysaccharide-induced inflammatory responses and enhances phagocytosis of bacteria by macrophages.

Neutrophilic granule protein (NGP) belongs to the cystatin superfamily. Even though this superfamily is critically involved in cancer biology and adaptive immunity, the relationship of macrophage NGP to inflammation and phagocytosis remains poorly understood. In this study, we observed a significant increase of NGP in peritoneal macrophages (PMs) isolated from mice challenged with E. coli or lipopolysaccharide (LPS), as judged by NGP mRNA microarray. We also found changes in NGP to be mainly Toll-like receptor 4 (TLR4)-dependent. By western blot and electrophoretic mobility shift assay, we demonstrated NGP overexpression to reduce TNF-α and IL-1ß production by LPS-induced RAW264.7 cells (RAW) via suppression of the NF-κB (p65 and p50) signalling pathway, rather than the JNK1/AP-1 (fos and jun) signalling pathway. NGP overexpression by LPS-induced RAW also induced IL-10, an anti-inflammatory cytokine, which was partially involved in the anti-inflammatory effect produced by NGP overexpression. Moreover, upregulated NGP enhanced the phagocytosis of E. coli by RAW. Taken together, these results demonstrated NGP to be an important host defense component that regulates inflammatory responses and phagocytosis by activated macrophages. As such, NGP may be useful for the treatment of inflammatory based disease.

Effects of small interfering RNA targeting TLR4 on expressions of adipocytokines in obstructive sleep apnea hyponea syndrome with hypertension in a rat model.

We explored the effects of RNA interference-mediated silencing of TLR4 gene on expressions of adipocytokines in obstructive sleep apnea hyponea syndrome (OSAS) with hypertension in a rat model. Systolic blood pressure of caudal artery and physiological changes were observed when establishing rat models of OSAS with hypertension. Mature rat adipocytes were induced from separated and cultured primary rat adipocytes. To transfect rat mature adipocytes, TLR4 siRNA group and negative control (NC) siRNA group were established. Expressions of TLR4 mRNA of adipocytes were examined after silenced by siRNA by quantitative real-time polymerase chain reaction (qRT-PCR). By enzyme-linked immunosorbent assay (ELISA), expressions of inflammatory cytokines, and adipocytokines of adipocytes were detected. Blood pressure in rat caudal artery was higher in the intermittent hypoxia group than that of the blank control group by 29.87 mmHg, and cardiocytes in the former group showed physiological changes, which indicated successful establishment of rat models of OSAS with hypertension. Red particles could be seen in mature rat adipocytes when stained with Oil Red O. Transfection of TLR4 mRNA was significantly suppressed in the TLR4 siRNA group, which didn't happen in the untransfected control group. Rats in the TLR4 siRNA group had significantly reduced expressions of such inflammatory cytokines as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) and such adipocytokines as visfatin, adiponectin (ADN), and leptin than those in the untransfected control group. RNA interference-mediated silencing of TLR4 gene could regulate occurrence and development of OSAS with hypertension in rats by downregulating expressions of adipocytokines.

Downregulated SOCS1 expression activates the JAK1/STAT1 pathway and promotes polarization of macrophages into M1 type.

Macrophage polarization is flexible, and involves in different signaling pathways and various transcription factors. Suppressor of cytokine signaling (SOCS) is an important inhibitor of cytokine signaling pathways and also a key physiological regulator for natural and acquired immunity systems. Following transfection of SOCS1 short hairpin (sh)RNA into mouse macrophage cells, reverse transcription­quantitative polymerase chain reaction demonstrated that the mRNA levels of Janus kinase (JAK)1 and signal transducer and activator of transcription (STAT)1 increased significantly. In addition, western blotting indicated that JAK1, STAT1 and p­STAT1 expression was significantly enhanced. Fludarabine can inhibit phosphorylation of STAT1 and SOCS1 expression. When fludarabine was added and SOCS1 shRNA was transfected, the inhibition of fludarabine was weakened, and p­STAT1 expression was upregulated. Flow cytometry detection indicated that, following the downregulation of SOCS1 expression, M1­type cells significantly increased, but the proportion of M2­type cells did not change significantly. Fludarabine can reduce the effect of SOCS1 shRNA on promoting M1­type cell polarization, and macrophages can polarize into both M1 and M2 phenotypes. Further ELISA results presented that, when downregulating SOCS1 expression, interleukin (IL)­4 and IL­10 expression was both downregulated, and tumor necrosis factor (TNF)­α and interferon (IFN)­Î³ expression was significantly upregulated. When adding fludarabine or injecting with the traditional Chinese medicine Xuebijing, IL­4 and IL­10 expression was both significantly upregulated, and TNF­α and IFN­Î³ expression was significantly downregulated. When adding fludarabine and downregulating SOCS1, IL­4, IL­10, TNF­α and IFN­Î³ expression presented no significant changes. The above results indicated that, when SOCS1 expression is downregulated, it will activate the JAK1/STAT1 pathway, and thereby promote the polarization of macrophages into M1 type. The findings are of great importance for understanding occurrence, development and treatment of various immune­related diseases.

Soluble Egg Antigen Activates M2 Macrophages via the STAT6 and PI3K Pathways, and Schistosoma Japonicum Alternatively Activates Macrophage Polarization to Improve the Survival Rate of Septic Mice.

Sepsis is one of the most challenging health problems worldwide. Our previous study showed that chronic schistosoma japonica (SJ) infection might increase serum anti-inflammatory factors to play a protective role, thus improving the survival rate of septic mice. Further research revealed that SJ infection promoted J774A.1 macrophage differentiation into M2 macrophages; suppressed LPS-induced activation of M1 macrophages; up-regulated CD163, IL-10, and TGF-ß1 expression; inhibited TNF-α and iNOS expression; and blocked the effect of LPS-promoted TNF-α and iNOS expression. Furthermore, adoptive transfer of ex vivo programed M2 macrophages significantly increased the survival rate of septic mice. In vitro studies suggested that soluble egg antigen (SEA) from SJ played the same role as worm infection but had no impact on M1 macrophages. SEA reduced LPS-induced TNF-α and iNOS expression, decreased the inhibitory effect of LPS on IL-10 and TGF-ß1 expression, increased STAT6 phosphorylation, and up-regulated PI3K and Akt expression but inhibited SOCS1 expression. When PI3K inhibitors were added, SEA-induced expression of CD163, IL-10, and arg1 might be reduced. Therefore, worm infection has a protective effect in septic mice in which SEA may play a key role via the STAT6 and PI3K pathways. This finding may provide a favorable solution for the treatment of sepsis, especially early cases. J. Cell. Biochem. 118: 4230-4239, 2017. © 2017 Wiley Periodicals, Inc.

Recombinant Trichinella spiralis 53-kDa protein activates M2 macrophages and attenuates the LPS-induced damage of endotoxemia.

Sepsis is a serious clinical condition of excessive systemic immune response to microbial infection. The pro-inflammatory stage of sepsis is generally launched by innate cells such as macrophages. They release inflammatory cytokines, activate other immune cells and cause severe tissue/organ damage. In this study, we have revealed that recombinant Trichinella spiralis (TS) excretory-secretory protein (rTsP53) exhibited anti-inflammatory properties and rescued mice from LPS-induced endotoxemia, which is a common model for sepsis study, potentially through the induction of M2 macrophages. rTsP53 treatment significantly decreased inflammatory cytokines (IL-6, IFN-γ and TNF-α) and increased IL-4, IL-10, IL-13 and TGF-ß secretion, both in circulation and in tissues. rTsP53 also induced the activation and infiltration of F4/80(+)CD163(+) macrophages to inflammatory tissues, increased M2 macrophage-related Arg1 and Fizz1 expression, and decreased M1 macrophage-related iNOS expression. PCR array showed that rTsP53 activated several genes that involve the survival of macrophages and also anti-inflammatory genes such as SOCS3. Together, our results show that rTsP53 activates M2 macrophages, which has strong anti-inflammatory potential to prevent LPS-induced lethal sepsis.

Micrometam C Protects against Oxidative Stress in Inflammation Models in Zebrafish and RAW264.7 Macrophages.

Micrometam C is a core of novel marine compound isolated from the mangrove associates Micromelum falcatum. In this study, we investigated the protective effects of micrometam C in inflammation models in the transgenic zebrafish line Tg (corola: eGFP) and RAW264.7 macrophages. We found that micrometam C significantly suppressed the migration of immune cells in tail-cutting-induced inflammation in transgenic zebrafish and reduced lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) in both zebrafish and macrophages. In addition, micrometam C also restored LPS-induced reduction of endogenous antioxidants, such as catalase (CAT), glutathione (GSH) and superoxide dismutase (SOD). The protective effects of micrometam C were in parallel to its inhibition of NADPH oxidase and nuclear factor-kappa-binding (NF-κB) activity. Thus, the present results demonstrate that micrometam C protects against LPS-induced inflammation possibly through its antioxidant property.

Dexamethasone down-regulates the expression of microRNA-155 in the livers of septic mice.

To investigate the expression of microRNA-155 (miRNA-155) in the livers of mice with lipopolysaccharide (LPS)-induced sepsis and to determine the role of dexamethasone (DXM) in the regulation of miRNA-155 expression, we pretreated mice with or without DXM prior to LPS exposure. Our study demonstrated that the expression of miRNA-155 and inflammatory factors increased in the liver tissues of mice with LPS-induced sepsis and that DXM down-regulated their expression in a dose-dependent manner. Moreover, DXM alone inhibited the expression of miRNA-155 to below the baseline level, but did not impact the expression of inflammatory factors, suggesting that the down-regulation of miRNA-155 by DXM may partially, but not completely, depend on the suppression of pro-inflammatory cytokines by DXM. Our data indicate that the overexpression of miRNA-155 in the livers of mice with LPS-induced sepsis may play an important role in the pathological processes of sepsis and that the down-regulation of miRNA-155 by DXM may be a novel mechanism regulating inflammation and immunity.

[Expression and effects of microRNA-155 in the livers of septic mice].

OBJECTIVE: To evaluate the effects of microRNA-155 (miR-155) on liver injury in mice with sepsis. METHODS: One hundred and twenty BALB/c mice were randomly divided into two groups of equal number according to random number table. Sepsis was induced by intraperitoneal injection of lipopolysaccharide (LPS,20 mg/kg). The mice were sacrificed at the time-points of 0, 2, 6, 12, 24, 48 hours. Blood and liver tissue were collected, and the levels of tumor necrosis factor- α (TNF- α ), interleukin (IL-6, IL-10) in serum and liver homogenate and alanine transaminase (ALT) in serum were determined by enzyme linked immunosorbent assay(ELISA). The injury of liver tissue was evaluated by histopathology. The expression of miR-155 in liver tissue was assessed by fluorescent quantitation reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The levels of TNF- α , IL-6 and IL-10 in serum and liver homogenate of septic mice increased with passage of time, and then the levels of TNF-α and IL-6 lowered after reaching the peak value, but remained higher than that of control group.TNF-α (ng/L) reached the peak value at 2 hours post-LPS-injection (serum: 1538.46 ± 102.12 vs. 64.52 ± 18.44,liver homogenate: 255.26 ± 41.23 vs. 60.21 + 13.55, both P<0.05). The level of IL-6 (µg/L) reached the peak value at 6 hours post-LPS-injection (serum: 875.33 ± 102.37 vs. 153.72 ± 20.67, liver homogenate: 9.22 + 0.82 vs. 3.35 ±0.36, both P<0.05), and that of IL-10 (ng/L) reached the peak value at 48 hours post-LPS-injection (serum: 520.13 ± 88.52 vs. 23.43 3.01, liver homogenate: 260.12 + 50.38 vs. 16.37 ± 3.71, both P<0.05). There were significant differences in above indexes between septic and control group (all P<0.05). The serum level of ALT (U/L) rose with passage of time, reaching the peak value at 48 hours post-LPS-injection (603.26 + 70.21 vs. 45.84 + 5.64, P<0.05). The values showed significant differences between septic and control group (P<0.05). A large number of leucocytic infiltration was found in liver. Hepatic tissue showed architectural distortion. Hepatocyte vacuolation and nodular necrosis were obvious at 12 hours post-LPS-injection. Relative expression of miR-155 was found to be increased at 2 hours post-LPS-injection, reaching its peak value at 12 hours post-LPS-injection [(72.96 ± 9.34)-fold of control group, P<0.05]. CONCLUSION: The increase in miR-155 expression might play an important role in the mechanism of liver injury during sepsis.

[The protective effect of chronic schistosoma japonica infestation against sepsis in mice].

OBJECTIVE: To preliminarily study the protective effect of chronic schistosoma japonica (SJ) infestation against sepsis in mice and its mechanism. METHODS: BALB/c male mice were used, and the experiment was divided into three parts. Experiment 1: chronic SJ infestation model was reproduced by SJ cercaria inoculation through abdominal skin for 8 weeks. Twenty mice were randomly grouped into normal group (n=10) and SJ group (n=10). The levels of interleukins (IL-4, IL-10),tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in serum were detected by enzyme linked immunosorbent assay (ELISA). Real-time polymerase chain reaction (PCR) was employed to detect the levels of IL-10 mRNA and TNF-αmRNA in abdominal macrophages. This experiment was meant to evaluate immune state in mice with chronic SJ infestation. Experiment 2: lipopolysaccharide (LPS) was intraperitoneally injected to reproduce sepsis model. Thirty mice were randomly grouped into LPS group (n=15) and SJ-LPS group (n=15). The levels of cytokines were determined by ELISA at 0, 24, 48 and 72 hours after LPS injection. This experiment was meant to detect the effect of chronic SJ infestation in mice during the septic process. Experiment 3: two types of sepsis model were reproduced by cecal ligation and puncture (CLP) and LPS injection, respectively. The survival rate of mice with chronic SJ infestation in 72 hours in either type of sepsis was evaluated. RESULTS: Experiment 1: compared with normal group [IL-4 (56.32±8.66) ng/L, IL-10 (48.17±7.23) ng/L], chronic SJ infestation showed an increase in serum IL-4 [(151.35±12.24) ng/L] and IL-10 [(133.22±11.09) ng/L, both P<0.05]. Chronic SJ infestation also resulted in an increase in IL-10 mRNA expression (SJ group 4.46±1.82, normal group 1.52±0.60) and inhibited TNF-α mRNA expression (SJ group 1.61±0.93, normal group 2.32±1.03) in abdominal macrophages (both P<0.05), indicating that macrophages could be differentiated into alternative activated macrophages. Experiments 2 and 3 showed that the levels of serum IL-4 and IL-10 were increased at 0 hour after LPS injection, and then gradually decreased in SJ-LPS group, but the levels were still higher than those in LPS group at 72 hours [IL-4 (ng/L): 92.2±7.6 vs. 41.5±4.5; IL-10 (ng/L): 92.1±7.8 vs. 35.6±4.0, both P<0.05]; the levels of TNF-α and IFN-γ were increased at 24 hours, and then decreased in SJ-LPS group, and the levels were lower than those in LPS group at 72 hours [TNF-α (ng/L): 82.9±5.6 vs. 91.5±5.2; IFN-γ (ng/L): 44.1±4.8 vs. 52.6±4.0, both P<0.05]. Therefore, chronic SJ infestation could improve the survival rate of mice with sepsis induced by CLP or LPS (CLP: 80% vs. 20%, LPS: 70% vs. 30%, both P<0.05). CONCLUSION: Chronic SJ infestation could elevate anti-inflammatory factors in septic mice, thus ameliorating the survival rate, so it has protective effect on mice with sepsis.

[Study of the effect of p53 and Bcl-2 protein expression in myocardial apoptosis in septic rats].

OBJECTIVE: To explore the significance of p53 and Bcl-2 gene in myocardial apoptosis in septic rats. METHODS: Adult male Sprague-Dawley (SD) rats were randomly divided into normal control group (n=6), sham operation group (n=6) and cecal ligation and puncture (CLP) group (n=24). The sepsis model was reproduced by CLP. Rats in CLP group were divided into four subgroups according to the time points of 3, 9, 12, and 24 hours after operation, with 6 rats in each subgroup, and their hearts were examined according to the experimental protocol. Myocardial apoptosis was detected with electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique. Bcl-2 and p53 protein expression was measured by immunohistochemistry method. RESULTS: Myocardial apoptosis index (AI) in septic rats were increased, higher than that in normal control group [(1.500+/- 0.141)%] and sham operation group [(1.567+/-0.258)%, all P<0.05]. The myocardial AI peaked at 12 hours [(55.633+/-2.073)%], and lowered at 24 hours [(33.683+/-2.070)å]. The expression level of p53 protein in CLP group was lowest at 3 hours [(13.817+/-0.964)%], peaked at 12 hours [(80.567+/-5.055)%], and higher than that in normal control group [(0.617+/-0.232)%] and sham operation group [(0.600+/-0.297)%, all P<0.05 ]. The trend was parallel with that of the results of TUNEL. However, the expression level of Bcl-2 protein peaked at 3 hours [(31.650+/-1.799)%], and was lowest at 12 hours [(0.650+/-0.308)%], and lower than that in normal control group [(47.017+/-0.691)%] and sham operation group [(46.817+/-0.567)%, all P<0.05]. The trend was opposite with that of the results of TUNEL. CONCLUSION: Myocardial apoptosis may be a mechanism of the pathogenesis of myocardial injury in sepsis. The change in the modulating genes p53 and Bcl-2 of apoptosis may serve as indicators of myocardial injury. p53 and Bcl-2 gene may be used as an interventional means in sepsis to change its outcome.

[Role of nuclear factor-KappaB in endothelial injury in acute myocardial infarction].

OBJECTIVE: To examine the change in nuclear factor-KappaB (NF-KappaB) activity, tumor necrosis factor-alpha (TNF-alpha) and soluble thrombomodulin (sTM) levels at different time following reperfusion in acute myocardial infarction (AMI), and to identify the role of ischemia/reperfusion after ischemia in injury to endothelial cells and its relevant mechanism. METHODS: AMI group included 8 randomly selected patients with AMI, and a normal control group (n=8) composing individuals who underwent health check. NF-KappaB activity in monocytes was determined by electrophoretic mobility shift assays (EMSA). The level of TNF-alpha was measured by radio-immunity and sTM was measured by enzyme linked immunosorbent assay (ELISA). RESULTS: The NF-KappaB activity, TNF-alpha and sTM levels raised dramatically at 0.5 hour after reperfusion, reaching peak at 1 hour and declined gradually at 3, 12 and 24 hours. The levels of all the determined parameters at every time point were significantly higher than that of normal control group, and their levels at 1 hour were significantly higher than that at 24 hours (all P<0.05). There was a positive correlation between the NF-KappaB activity and the levels of TNF-alpha and sTM (all P<0.05). CONCLUSION: These results indicate that NF-KappaB is activated and the levels of TNF-alpha and sTM rise significantly after reperfusion in AMI. The activation of NF-KappaB maybe one of the most important pathogenic mechanism of endothelial injury.

[E1A gene transfection of human undifferentiated thyroid cancer cell line HTC/3 by nanoparticles].

OBJECTIVE: To prepare nanoparticles containing E1A gene and observe the efficiency and feasibility of transfecting E1A gene into human undifferentiated thyroid cancer cell line HTC/3. To examine the sensitivity of transgene cells to X-ray and X-ray-induced apoptosis in those cells. METHODS: Nanoparticle-DNA complex was prepared with PLGA coating adenoviral early expression gene E1A, and the package efficiency, release progress in vitro, and size of the complex were determined. The nanoparticle-DNA was transfected into the HTC/3 cells. Lipofectamine was used to transfect E1A gene as a control. RT-PCR was used to examine E1A gene mRNA expression in the transfected cells. The survival ratio of HTC/3-E1A and control cells, and the growth inhibition ratio induced by different doses of X-ray in HTC/3-E1A cells were examined by MTT assay. The apoptosis in HTC/3-E1A cells induced by 2 Gy X-ray iradiation was examined by flow cytometry and DNA electrophoresis. RESULTS: The package efficiency, release progress in vitro, and size of the nanoparticle-DNA complex were 0.78%, 18 days, and 150-280 nm, respectively when transfected the plasmid at the same level, the nanoparticle group got more positive transgene cell clones than that in lipofectamine group, with a statistically significant difference (P < 0.05). RT-PCR showed that transgenic cells from both nanoparticle-DNA and lipofectamine groups had E1A gene mRNA expression. The HTC/3-E1A cells grew slowly, and their doubling time was prolongated (1.44 times in comparison with that in parental cells). According to IC50, the sensitivity of HTC/3-E1A cells to X-ray was improved 2.9 and 2.8 times, respectively, in comparison with that in HTC/3-Vect and HTC/3 cells. The ratio of subG0/G1 phase of HTC/3-E1A cells was significantly higher than that in HTC/3-Vect and HTC/3 cells (P < 0.01). The ratio of S phase of HTC/3-E1A cells was significantly lower than that in HTC/3-Vect and HTC/3 cells (P < 0.01). A typical DNA ladder pattern of apoptosis in HTC/3-E1A cells was observed by electrophoresis, but not found in HTC/3-Vect and HTC/3 cells. CONCLUSION: A nanoparticle-DNA complex has been successfully prepared, and it may carry a foreign gene into cells. The sensitivity of HTC/3-E1A cells to X-ray is significantly improved. Moreover, apoptosis is induced by x-ray in the E1A gene-transfected cells.

[Study of apoptosis of peripheral blood mononuclear cells in patients with multiple organ dysfunction syndrome].

OBJECTIVE: To study apoptosis of peripheral blood mononuclear cells (PBMCs) in patients with multiple organ dysfunction syndrome (MODS) and its associated gene Bcl-2 and p53 expression. METHODS: Twenty-five patients with MODS and 18 healthy volunteers were enrolled for the study. Flow cytometry assay, electron microscopy, acridine orange-ethidium bromide staining and fluorescence microscopy, DNA agarose gel electrophoresis were used to identify and quantify apoptosis of PBMCs. Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify Bcl-2 mRNA and p53 mRNA expression. RESULTS: Typical morphological features of apoptotic PBMCs were observed in the patients. The apoptosis rate in MODS group was (25.4+/-9.2)%, and it was higher than that of control group (15.9+/-6.8)% (P<0.01). The number of apoptotic cells was (1.040+/-0.096)/high power field in MODS group, and it was higher than that in control group (0.235+/-0.028)/high power field (P<0.05). Bcl-2 mRNA expression of PBMCs in patients was significantly lower than that of healthy volunteers (0.11+/-0.09 vs. 0.19+/-0.06, P<0.05), while p53 mRNA expression was higher in patients than that of healthy volunteers (0.45+/-0.09 vs. 0.25+/-0.12, P<0.05). CONCLUSION: PBMCs apoptosis in patients with MODS is increased abnormally. The expression of Bcl-2 mRNA in patients is decreased while p53 mRNA expression is increased. The results suggest that abnormal apoptosis of monocytes as well as abnormal expression of apoptosis associated genes occur in patients with MODS.

[Protective effect of p38 mitogen activated protein kinase inhibitor on organs in sepsis in rats].

OBJECTIVE: To investigate the pathogenesis of multiorgan injury and the protective of p38 mitogen activated protein kinase(p38MAPK) inhibitor on organs in sepsis. METHODS: Cecal ligation and puncture was adopted to reproduce sepsis model. The levels of serum biochemical parameters [including alanine aminotransferase (ALT), blood urea nitrogen(BUN), creatinine(Cr), MB isoenzyme of creatine phosphokinase (CPK-MB), tumor necrosis factor-alpha(TNF-alpha) and interleukin-1beta(IL-1beta) were determined at different time points. RESULTS: The levels of ALT, BUN, Cr, CPK-MB, TNF-alpha and IL-beta rose progressively after the cecal ligation operation. The levels of TNF-alpha and IL-1beta showed a significant correlation with levels of ALT, BUN, Cr, CPK-MB. After the administration of p38MAPK inhibitor, SB203580, the level of TNF-alpha and IL-1beta were found to decrease evidently, and the injury to multiple organs was alleviated. CONCLUSION: Excessive secretion of TNF-alpha and IL-beta may be the main cause of multiorgan injury in sepsis. Modulation of the p38MAPK pathway may protect multiorgan injury in sepsis.

[Redox-dependent changes in structure and function of hHSF1].

To evaluate the effects of cysteine-SH-directed regents on the redox status, structure and function of human heat shock transcription factor 1 (hHSF1), treatment in vitro of hHSF1 with 0.3 0.5 mmol/L oxidizing reagent diamide (DM) and treatment in vivo of HeLa cells with 1 mmol/L buthionine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, promoted the formation of a compact, intramolecularly disulfide-crosslinked, stable monomeric form of ox-hHSF1, and blocked the trimerization and activation of HSF1. The effects of diamine were dose-dependent and readily could be completely reversed by adding 0.4 0.5 mmol/L reducing reagent dithiothreitol (DTT) to the samples prior to gel electrophoresis. Computer modeling of the alpha-helical coiled-coil domains of the HSF1 monomer and trimer showed that the alignment of the N- and C-terminal hydrophobic repeats of HSF1 monomer could bring C(3)(Cys(153))close to C(4) and C(5)(Cys(373) and Cys(378), respectively), in positions permissible for disulfide bond formation under appropriate experimental conditions. The results suggest that redox-dependent thiol-disulfide exchange can provide a mechanism for regulation the conformation and activity of hHSF1.