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HO-1 signaling pathway
International Immunopharmacology 28 (2015) 643–654
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Molecular hydrogen protects mice against polymicrobial sepsis by ameliorating endothelial dysfunction via an Nrf2/HO-1 signaling pathway☆ Hongguang Chen a,1, Keliang Xie a,⁎,1, Huanzhi Han a,b,1, Yuan Li a, Lingling Liu a, Tao Yang a, Yonghao Yu a,⁎ a b
Department of Anesthesiology, General Hospital of Tianjin Medical University, Tianjin Institute of Anesthesiology, No.154 Anshan Street, Heping District, 300054 Tianjin, PR China Department of Anesthesiology, Dezhou City People's Hospital, No.1751, Xinhu Street, Decheng District, Dezhou 253014, Shandong Province, PR China
a r t i c l e
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Article history: Received 11 May 2015 Received in revised form 27 June 2015 Accepted 27 July 2015 Available online xxxx Keywords: Sepsis H2 Endothelial dysfunction HO-1 Nrf2
a b s t r a c t Endothelial injury is a primary cause of sepsis and sepsis-induced organ damage. Heme oxygenase-1 (HO-1) plays an essential role in endothelial cellular defenses against inflammation by activating nuclear factor E2related factor-2 (Nrf2). We found that molecular hydrogen (H2) exerts an anti-inflammatory effect. Here, we hypothesized that H2 attenuates endothelial injury and inflammation via an Nrf2-mediated HO-1 pathway during sepsis. First, we detected the effects of H2 on cell viability and cell apoptosis in human umbilical vein endothelial cells (HUVECs) stimulated by LPS. Then, we measured cell adhesion molecules and inflammatory factors in HUVECs stimulated by LPS and in a cecal ligation and puncture (CLP)-induced sepsis mouse model. Next, the role of Nrf2/HO-1 was investigated in activated HUVECs, as well as in wild-type and Nrf−/− mice with sepsis. We found that both 0.3 mmol/L and 0.6 mmol/L (i.e., saturated) H2-rich media improved cell viability and cell apoptosis in LPS-activated HUVECs and that 0.6 mmol/L (i.e., saturated) H2-rich medium exerted an optimal effect. H2 could suppress the release of cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1), and pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and high-mobility group box 1 protein (HMGB1). Furthermore, H2 could elevate anti-inflammatory cytokine IL-10 levels in LPS-stimulated HUVECs and in lung tissue from CLP mice. H2 enhanced HO-1 expression and activity in vitro and in vivo. HO-1 inhibition reversed the regulatory effects of H2 on cell adhesion molecules and inflammatory factors. H2 regulated endothelial injury and the inflammatory response via Nrf2-mediated HO-1 levels. These results suggest that H2 could suppress excessive inflammatory responses and endothelial injury via an Nrf2/HO-1 pathway. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sepsis and its sequelae represent a continuum of clinical and pathologic severity [1]. An important hallmark of sepsis is endothelial activation and dysfunction, which appear to play a pivotal role in the development of sepsis. Sepsis induces endothelial modulation Abbreviations: CLP, cecal ligation and puncture; DMEM, Dulbecco modified eagle medium; DMSO, cimethylsulfoxide; H2, hydrogen; HMGB1, high-mobility group box 1 protein; HO-1, heme oxygenase-1; HUVEC, human umbilical vein endothelial cell; ICAM, intercellular cell adhesion molecule; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MODS, multiple organ disfunction syndromes; MTT, 3-(4, 5)-dimethylthiahiazo (−z-y1)3, 5-di-phenytetrazoliumromide; NADPH, nicotinamide adenine dinucleotide phosphate; Nrf2, nuclear factor E2-related factor-2; PI, propidium iodide; PVDF, polyvinylidene fluoride; RIPA, radioimmunoprecipitation assay; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule-1; ZY, zymosan; Znpp-IX, zinc protoporphyrin IX. ☆ Competing interests: The authors declare that they have no competing interests. ⁎ Corresponding authors. E-mail addresses: [email protected] (K. Xie), [email protected] (Y. Yu). 1 Drs. Hongguang Chen, Keliang Xie and Huanzhi Han made equal contributions to this study.
http://dx.doi.org/10.1016/j.intimp.2015.07.034 1567-5769/© 2015 Elsevier B.V. All rights reserved.
via several different mechanisms that respond to various aspects of infection or to components of the bacterial wall (e.g., LPS) that accompany structural changes and vital functional changes. These aspects of infection include programmed cell death, adhesion molecule expression, inflammatory responses, and leukocyte trafficking [2–4]. Programmed cell death, or apoptosis, leads to an accentuated pro-inflammatory response in the endothelium and is subsequently involved in cross-talk between adhesion molecules and cytokines on the cell surface. In stimulated endothelial cells, adhesion molecules improve the action of leukocytes rolling along the endothelial layer and transmigrating across the endothelial barrier to locations of inflammation or injury [5]. Endothelial injury is a primary result of pathogenesis during sepsis and a critical target to inhibit or even prevent its development. Recently, hydrogen (H2) was shown to exert an effective therapeutic role in many disorders including sepsis, multiple organ dysfunction syndrome (MODS), LPS-induced acute lung injury, and stroke [6–10]. Its therapeutic effects were accomplished via anti-apoptotic, antiinflammatory and anti-oxidative stress mechanisms [11]. Our group
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reported that H2 could improve the survival rates, organ injuries and inflammatory responses in septic mice and regulate the expression of inflammatory factors in macrophages and during sepsis in a zymosan (ZY)-challenged generalized inflammation model [6,9,10]. Song et al. [12] confirmed that H2 reduced ICAM-1 and VCAM-1 expression in the arterial walls of mice administered a high-fat diet. However, the specific mechanisms of H2 have not been clarified. HO-1, a cytoprotective enzyme, is known to play a crucial role in protecting the body from excessive inflammatory processes [13]. Recently, we verified that H2-rich medium reduced TNF-α, HMGB1, and IL-1β expression via an HO-1 pathway in activated macrophages. In human umbilical vein endothelial cells (HUVECs), an inducer of HO decreased ox-LDL-induced VCAM-1 and ICAM-1 secretion and mRNA transcription, and an inhibitor of HO reversed these inhibitory effects [14]. Moreover, HO-1 was augmented by activating the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2). Kawamura et al. reported that H2 treatment increased HO-1 expression in lung grafts [15] and ameliorated hyperoxic lung injury by modulating the Nrf2 signaling pathway [16]. Here, we hypothesized that Nrf2/HO-1 signaling exerts a vital regulatory effect on the anti-inflammatory function of H2-rich media during endothelial injury resulting from sepsis.
Consistent with previously described methods [18], briefly, H2 was dissolved in DMEM supplemented with 10% FBS or saline for 4 h under 0.4 MPa pressure to reach a saturated state. The saturated H2-rich medium (0.6 mmol/L) or saline was maintained in an aluminum bag with no dead volume and stored at 4 °C under atmospheric pressure. The supersaturated H2 medium was diluted with normal medium to prepare 0.3 mmol/L H2-rich culture medium. To ensure a 0.6 mmol/L concentration, H2-rich medium was freshly prepared every week. A needle-type H 2 sensor (Unisense A/S, Aarhus, Denmark) was used to detect the H 2 concentration of the media following the process published by Hayashida et al. [18] According to previous studies and with several modifications [19] based on our preliminary experimental results, sepsis in mice was induced by CLP. H2-rich saline solution (5 mL/kg) or normal saline solution (5 mL/kg) was injected i.p. at 1 and 6 h after the sham and CLP operations.
2. Materials and methods
2.5. Nrf2 siRNA transfection
2.1. Animals
Nrf2 siRNA-transfected HUVECs were cultured in 6-well plates and were transfected with Nrf2 siRNA (Santa Cruz Biotechnology) diluted with siRNA LipofectAMINE™ 2000 transfection reagent (Invitrogen Company Carlsbad, CA, USA) according to the manufacturer's instructions. The cells were cultured at 37 °C with 5% CO2 for 24 h before treatment with LPS and H2.
All experimental procedures utilizing animals were approved by the Institutional Animal Care Committee of Tianjin Medical University and performed according to the “Policies on the Use of Animal and Humans in Neuroscience Research”. This protocol was in agreement with the Committee on the Ethics of Animal Experiments of Tianjin Medical University General Hospital, Tianjin, China (permit number: 2011-X6-18). Male wild-type (WT) and Nrf2-gene-deleted (Nrf2−/−) ICR mice weighing 20–25 g were obtained from the General Hospital of Nanjing Military Command (Nanjing, China). The mice were fed water and food ad libitum and maintained in an air-conditioned and individually ventilated environment on a 12 h light/dark cycle. 2.2. Cell culture and treatment HUVECs-12 (termed HUVECs) that were purchased from ATCC were cultured in cell culture medium (DMEM) supplemented with 10% heatinactivated fetal bovine serum (FBS; Life Technologies Corp., NY, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies Corp., NY, USA) at 37 °C in an atmosphere of 95% air and 5% CO2. HUVECs were maintained at a density of 1 × 106 cells/mL in DMEM and 10% FBS. HUVECs were treated with vehicle or varying concentrations of LPS (Escherichia coli 0111:B4; Sigma, St. Louis, MO, USA) combined with 1% FBS at 37 °C for further experiments. 2.3. Sepsis in mice induced by cecal ligation and puncture (CLP) following zinc protoporphyrin IX (ZnppIX) treatment CLP is a classic and clinically relevant sepsis model that can comprehensively mimic the disease processes related to sepsis. As described in a previous study [17], briefly, the mice were anesthetized with chloral hydrate, shaved, and disinfected, and a laparotomy was performed. Then, the cecum was ligated from the top and punctured twice on the antimesenteric side with a 20-gauge needle. A small amount of feces from the bowel was expelled from the puncture hole, and the cecum was replaced gently. Sham-operated mice underwent the same procedure but without ligation and puncture. After surgery, the mice were given warm normal saline to resuscitate them (0.1 mL/g body weight). The mice received an intraperitoneal (i.p.) ZnppIX (40 mg/kg) injection 1 h before cecum ligation and puncture. ZnppIX (Porphyrin Products,
Logan, UT, USA) was dissolved in 0.2 M sodium hydroxide, and HCl was added to adjust to a pH of 7.4. 2.4. H2-rich medium or saline treatment
2.6. MTT and LDH detection HUVECs (104 cells/well) were grown in a 96-well plate for at least 12 h and then incubated with LPS or H2. According to the manufacturer's instructions, cell viability was determined by MTT assay, and cytotoxicity was detected by lactate dehydrogenase (LDH) assay. Briefly, after the HUVECs were treated with LPS or H2, they were washed with 3-(4,5)dimethylthiahiazo (−z-y1)-3,5-di-phenyltetrazolium bromide (MTT) (5 mg/mL) in cell medium supplemented with 10% FBS. After the cells were incubated for 3 h at 37 °C, the medium was discarded, and formazan blue was dissolved in 100 μL of dimethyl sulfoxide (DMSO). The absorbance was measured at 490 nm using a microplate reader (Grodig, Austria). Relative cell viability was quantified compared with the non-treated group. Cell injury was measured via LDH release using a cytotoxicity detection kit according to the manufacturer's instructions (Roche, IN). Cell activity was expressed as a percentage of the LDH released in control cultures. 2.7. Apoptosis The percentage of apoptosis was determined using an Annexin V/PI Kit (BD Pharmingen, San Diego, CA, USA). Briefly, HUVECs were harvested after treatment, washed twice, rewashed in binding buffer, and then labeled with annexin V and propidium iodide (PI). The percentage of HUVECs undergoing apoptosis was determined using a Becton Dickinson FACStar flow cytometer. 2.8. Caspase-3 activity assay Experimental samples were collected to measure caspase-3 activity. After the samples were incubated with DEVD-AFC, caspase-3 activity was determined in a cyto-buffer as described previously [19]. Free AFC release was measured using a fluorescence microscope (Leica DM2500 system; Leica, Wetzlar, Germany) at excitation and emission wavelengths of 350 nm and 460 nm, respectively.
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2.9. ELISA After cells were treated, culture media and tissue homogenates were collected for the detection of cytokines and endocan. The cells were collected using a centrifuge, and supernatants were obtained for the detection of TNF-α, IL-1β, IL-10 (R&D Systems Inc., Minneapolis, MN, USA), HMGB1 (IBL, Hamburg, Germany) and endocan (R&D Systems, Minneapolis, MN) [9,10]. Briefly, the samples were added to microtiter plates pre-coated with anti-TNF-α, anti-IL-1β, anti-HMGB1 and anti-IL-10 monoclonal antibodies and incubated with horseradish peroxidase (HRP)-labeled TNF-α, IL-1β, HMGB1 and IL-10 IgG. The absorbance of each well was measured at 450 nm using a microplate reader (Grodig, Austria). Based on a previous study [20], the samples were incubated with anti-endocan antibodies and then incubated with peroxidaseconjugated anti-goat IgG antibodies. After stopping the reaction, absorbances were read at 490 nm. 2.10. HO-1 activity detection HO-1 activity was measured at different time points after LPS stimulation in vitro and at 24 h after sham or CLP in vivo as described previously in the Materials and methods section [21]. HUVECs were collected to prepare microsomes. Lung tissue was homogenized in volumes of potassium phosphate buffer containing sucrose and a protease inhibitor. After the
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samples were centrifuged, the microsomal pellets were collected. The reaction compound consisted of microsomes, biliverdin reductase, hemin and nicotinamide adenine dinucleotide phosphate (NADPH); biliverdin reductase was derived from the cytosolic fraction of rat liver. The reaction compound was incubated and then stopped with chloroform. The amount of extracted bilirubin in the chloroform layer was measured at absorbances between 464 and 530 nm. HO-1 activity was represented as picomoles of bilirubin formed per milligram of protein per hour. 2.11. Protein collection for western blot detection 2.11.1. Sample collection in vitro HO-1 and Nrf2 protein detection in HUVECs was performed at different time points in the presence or absence of ZnppIX (or Nrf2 siRNA) after LPS stimulation. After the experiment, the cells were collected for protein sample preparation and resuspended for use in a 200-μL radioimmunoprecipitation assay (RIPA) performed on ice for 30 min. The cells were centrifuged (15,000g, 10 min, 4 °C) and denatured (100 °C, 5 min), and protein was collected and maintained at −80 °C. 2.11.2. Sample collection in vivo Lungs were collected for homogenization in 1 mL of lysis buffer. The samples were centrifuged (15,000g, 20 min, 4 °C), and the supernatants were collected and stored at −80 °C for western blot analysis.
Fig. 1. The effect of H2 on cell viability, LDH release, caspase-3 activity and apoptosis in LPS-stimulated HUVECs. HUVECs were treated with 1 ng/mL, 10 ng/mL, 100 ng/mL, 1000 ng/mL or 10,000 ng/mL LPS, and then the cells were collected to detect cell viability by MTT (A) and LDH release (B). LPS caused a decrease in cell viability and excessive LDH release. Cells were challenged with 1 μg/mL LPS or PBS with or without 0.3 mmol/L (50% H2) or 0.6 mmol/L H2-rich medium. After treatment, cell viability (C), caspase-3 activity (D) and apoptosis (E) were tested. Fifty percent H2 and full-strength H2 improved cell viability, caspase-3 activity and apoptosis in endothelial cells; moreover, full-strength H2 was better than 50% H2. Cell viability, LDH release, caspase-3 activity and apoptosis were measured as described in the Materials and methods section. Each data point and error bar represents the mean ± SD. The data were analyzed by one-way ANOVA followed by Tukey's test. ***P b 0.001.
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2.11.3. Western blot analysis In total, 100 μg of denatured protein was separated on 10% acrylamide gels and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked and incubated overnight at 4 °C with primary antibodies against HO-1, Nrf2, and β-actin (Abcam, Cambridge, MA, USA, 1:2000 dilution). Subsequently, the membranes were washed and incubated with secondary antibodies (Abcam, Cambridge, MA, USA) at room temperature. The protein bands were visualized with an enhanced chemiluminescence (ECL) reagent and imaged using a Quantity One gel quantitation system (Bio-Rad, Tokyo, Japan). All sample detections and analyses were repeated three times. HO-1 and Nrf2 expressions were normalized to β-actin. 2.12. Real-time quantitative polymerase chain reaction (RT-PCR) Total RNA was extracted using a TRIzol RNA isolation reagent kit (Invitrogen, USA). Quantitative PCR analyses were performed using primers and probe sets commercially available from Applied Biosystems according to the manufacturer's instructions for reverse transcription reactions using a High Capacity Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA, USA). Amplification reactions were performed using a SYBR Green PCR Master Mix (Applied Biosystems). Each sample was analyzed in triplicate. The primers were as follows: Nrf2, forward 5′-TTCAGCCAGCCCAGCACATC-3′ and reverse 5′-CGTA GCCGAAGAAACCTCATTGTC-3′; β-actin, forward 5′-ATCAAGATCATTGC TCCTCCTG-3′ and reverse 5′-GCAACTAAGTCATAGTCCGCC-3′. Expression levels were normalized to β-actin expression levels.
2.13. Fluorescence microscopy analysis Cells were seeded on slides at an appropriate density and then treated with LPS (1 μg/mL) with or without H2. Then, the cells were washed, fixed in 4% paraformaldehyde and permeabilized with Triton X-100 (0.5%). After the fixed cells were washed, they were blocked and incubated with anti-Nrf2 antibodies. After the cells were washed again, they were incubated with IgG–FITC secondary antibodies, stained with DAPI and observed using an Olympus confocal microscope (Leica, DM4000 B, Germany). 2.14. Statistical analysis Statistical data analysis was performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA). In vitro experiments and cell viability, LDH release and caspase-3 activity assays were compared by one-way ANOVA followed by Tukey's post hoc test after H2 treatment. After ZnppIX or Nrf2 siRNA treatment, ICAM-1, VCAM-1, TNF-α, HMGB1, and HO-1 protein levels and activities were compared by one-way ANOVA followed by Tukey's post hoc test. For multiple group comparisons, two-way ANOVA followed by Tukey's post hoc tests were used to compare ICAM-1, VCAM-1 TNF-α, IL-1β, HMGB1, IL-10 and HO-1 protein levels and activities, as well as Nrf2 protein and mRNA levels at different time points and among different groups. The experimental data from septic mice for ICAM-1, VCAM-1, endocan, TNF-α, HMGB1, and HO-1 protein levels and activities were compared by one-way ANOVA followed by Tukey's post hoc
Fig. 2. H2 attenuated ICAM-1 and VCAM-1 expression at 6, 12 and 24 h in LPS-stimulated HUVECs. Cells were collected to measure ICAM-1 (A, C) and VCAM-1 (B, D) expression by western blot at 6, 12 and 24 h post-treatment when treated with LPS or H2 (n = 8 per group). H2 treatment decreased ICAM-1 and VCAM-1 expression in LPS-stimulated HUVECs. All values are expressed as the mean ± SD. ***P b 0.001 for two-way ANOVA followed by Tukey's test between more than two groups over time.
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3. Results
shown in Fig. 1, 0.3 mmol/L and 0.6 mmol/L H2-rich media increased cell viability and decreased caspase-3 activity and apoptosis in activated HUVECs in a concentration-dependent manner compared with the control group; furthermore, the 0.6 mmol/L H2-rich medium exerted a preferable protective effect with respect to endothelial cell injury.
3.1. The effects of different concentrations of LPS on HUVEC viability and cytotoxicity
3.3. H2-rich medium improved the release of cell adhesion molecules and inflammatory factors in LPS-induced endothelial cells
LPS, the primary component of the bacterial wall, was able activate endothelial cells to induce innate immune responses and inflammation, even leading to cell injury. Cell viability could be measured with an MTT assay; LDH, a direct determinant of cell death, is indicative of membrane integrity [6]. To detect the effects of LPS on cell viability and cytotoxicity in HUVECs, cells were treated with different concentrations of LPS (i.e., 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 μg/mL, or 10 μg/mL; E. coli 0111:B4; Sigma, St. Louis, MO, USA) for 24 h. As shown in Fig. 1, LPS weakened cell viability and increased LDH release in a concentrationdependent manner. Concentrations of 1–100 ng/mL LPS had little effect on cell viability and cytotoxicity compared with control cells, whereas 10 μg/mL LPS led to extensive cell viability breakdown and cell death such that the cells did not meet the demands of the experiments. Therefore, we chose 1 μg/mL LPS as the optimal concentration for stimulation in the following experimental scheme.
Based on the above results, 0.6 mmol/L H2-rich medium exerted a preferable protective effect compared with 0.3 mmol/L H2-rich medium. In the next experiment, 0.6 mmol/L H2-rich medium was used to incubate cells. LPS induced an excessive inflammatory response in endothelial cells. ICAM-1 and VCAM-1 protein expression was determined by western blot analysis; simultaneously, TNF-α, IL-1β, HMGB1 and IL-10 responses were determined by ELISA to assess the inflammatory state of the cells. Compared with the control group, LPS induced significantly increased ICAM-1 and VCAM-1 expression in a concentrationdependent manner at 6, 12 and 24 h following endothelial cell activation (two-way ANOVA; P b 0.001 for the effects of time and treatment, Fig. 2). Compared with the LPS group, incubating the HUVECs with H2 reversed the increased expression of ICAM-1 and VCAM-1 (two-way ANOVA; P b 0.001 for the effects of time and treatment, Fig. 2). TNF-α and IL-1β are produced early in the course of the inflammatory response, whereas HMGB1 exhibits a late kinetic profile during inflammation in response to endotoxemia [20]. Therefore, we investigated the release of cytokines from LPS-induced HUVECs from 3 to 24 h. The levels of the cytokines TNF-α, IL-1β, and HMGB1 significantly increased in cells from 3 to 24 h post-LPS stimulation (twoway ANOVA; P b 0.001 for the effects of time and treatment, Fig. 3). The time course and expression trends of anti-cytokine IL-10 were similar to those of HMGB1 in HUVECs (two-way ANOVA; P b 0.001 for the effects of time and treatment, Fig. 3). Furthermore, the peak concentrations of released TNF-α and IL-1β occurred at 6 h; for HMGB1 and IL-10,
test. Survival rates were analyzed using the Kaplan–Meier method followed by a log rank test. All data are expressed as the means ± SD; P b 0.05 was considered statistically significant.
3.2. H2-rich media improved cell viability and apoptosis in activated HUVECs in a concentration-dependent manner Based on the experiment described above, cells were stimulated with 1 μg/mL LPS. To assess the optimum concentration for H2-rich medium with respect to cell viability and apoptosis in activated HUVECs, we used 0.3 mmol/L H2-rich medium and 0.6 mmol/L H2-rich medium to culture HUVECs with or without LPS treatment for 24 h, and then cell viability, caspase-3 activity and apoptosis were measured. As
Fig. 3. The regulatory effects of H2 on cytokines at different time points in activated endothelial cells. After HUVECs were treated with LPS or H2, media was collected to quantify the proinflammatory factors TNF-α (A), IL-1β (B), and HMGB1 (C) and the anti-inflammatory factor IL-10 (D) at 3, 6, 12 and 24 h using an ELISA (n = 8 per group). H2 decreased the expression of pro-inflammatory factors and further increased the expression of anti-inflammatory factors in LPS-stimulated HUVECs. All data are expressed as the mean ± SD. ***P b 0.05 for two-way ANOVA followed by Tukey's test for four groups over time.
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the peak was at 24 h. H2 can markedly decrease TNF-α release (twoway ANOVA; P b 0.001 for the interaction between time and treatment, Fig. 3) and further increase IL-10 expression after LPS stimulation (twoway ANOVA; P b 0.001 for the interaction between time and treatment, Fig. 3). Based on these results, we concluded that H2 is able to regulate the inflammatory response by regulating the balance of pro- and antiinflammatory cytokines.
inhibitor of HO-1. As shown in Fig. 4, ZnppIX reversed the stimulatory effects of H2 on HO-1 expression and activity in activated HUVECs (expression: LPS + H2 + Znpp vs. LPS + H2, 0.21 ± 0.04 vs. 0.67 ± 0.08, P b 0.001; activity: LPS + H2 + Znpp vs. LPS + H2, 301.2 ± 42.7 vs. 856.4 ± 89.3, P b 0.001). 3.5. ZnppIX completely reversed the regulatory effects of H2 on cell adhesion molecules and inflammatory factors in LPS-stimulated HUVECs
3.4. Effect of H2 on HO-1 expression and activity in LPS-stimulated HUVECs HO-1 performs a vital function in the inflammatory response [6]. In our experimental protocol, HO-1 expression and activity were measured in HUVECs from 6 to 24 h post-LPS stimulation in HUVECs. As shown in Fig. 4, these levels were significantly increased at 6, 12 and 24 h in the LPS group compared with the control group (expression: two-way ANOVA, P b 0.001 for the interaction between time and treatment; activity: two-way ANOVA, P b 0.001 for the interaction between time and treatment). Compared with the group cultured without H2, culture with H2 could further enhance HO-1 expression and activity after LPS stimulation (expression: two-way ANOVA, P b 0.001 for the interaction between time and treatment; activity: two-way ANOVA, P b 0.001 for the interaction between time and treatment). To deepen our analysis of the role of HO-1, we selected ZnppIX (20 μg/mL) as an
Based on the previous results, H2 can inhibit the release of cell adhesion molecules and the expression of pro-inflammatory factors and promote the expression and release of anti-inflammatory factors. H2 was also shown to accelerate HO-1 expression and activity in activated HUVECs. In the next experiment, we sought to further verify the effect of HO-1 on cell adhesion molecules and inflammatory factors after H 2 -treated HUVECs were challenged with LPS. As shown in Fig. 5, we found that H 2-rich medium decreased the release of cell adhesion molecules and pro-inflammatory factors (one-way ANOVA, P b 0.001, Fig. 5). However, ZnppIX, a HO-1 inhibitor, reversed the protective effects of H2 on cell adhesion molecules and inflammatory factors. These results suggest that H 2 can regulate the inflammatory response via the HO-1 pathway in endothelial cells.
Fig. 4. HO-1 protein expression and HO-1 activity in endothelial cells. After cells were treated with LPS or H2, they were collected for protein extraction and HO-1 activity measurement. HO-1 protein expression was detected by western blot. HO-1 activity was determined as described in the Materials and methods section. (A) and (B) show HO-1 protein expression at 6, 12 and 24 h after LPS and H2 administration; HO-1 activity is shown in (C) (n = 8 per group). The HO-1 inhibitor ZnppIX (20 μmol/L) was added to the medium 24 h before the application of H2 and LPS. HO-1 protein levels (D) and activity (E) were detected at 24 h after LPS, H2 or ZnppIX administration (n = 8 per group). All data are expressed as the mean ± SD. HO-1 protein expression and activity were compared using a two-way ANOVA followed by Tukey's post hoc test for different treatments and timepoints. After ZnppIX treatment, two-way ANOVA followed by Tukey's post hoc test was used to compare HO-1 expression and activity. ***P b 0.001.
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3.6. The effect of H2 on the expression of the transcription factor Nrf2 in LPS-stimulated HUVECs The transcription factor Nrf2 is an important regulator of cellular inflammation. However, whether Nrf2 plays a vital role in the processes that occur in H2-treated HUVECs remains unknown. Nrf2 expression and localization were detected by RT-PCR, western blot and immunofluorescence. Nrf2 mRNA and protein expression were significantly higher at 6 and 24 h post-treatment in the LPS group compared with the control group. H2 further heightened Nrf2 mRNA and protein expression after LPS stimulation. As shown in Fig. 6D, LPS induced increased localized expression at 24 h post-LPS stimulation, and H2 further promoted localized Nrf2 expression (P b 0.05, Fig. 6). 3.7. Effects of Nrf2 on HO-1 expression and activity in H2-treated HUVECs Nrf2, which controls HO-1 expression in response to noxious stimuli such as infection and tissue injury and in response to prooxidant stimuli, is regulated by cytosolic Keap1, which directly interacts with Nrf2 for proteasomal degradation via ubiquitination [21]. To assess whether H2 improved HO-1 expression via the Nrf2
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signaling pathway in activated HUVECs, Nrf2 siRNA was used to inhibit Nrf2 gene expression. As shown in Fig. 7, siRNA could clearly inhibit Nrf2 mRNA and protein expression in the LPS + Nrf2 siRNA group compared with the LPS + scrambled siRNA group. After inhibiting Nrf2 mRNA and protein expression, H2 exerted no significant effects on Nrf2 mRNA. The results shown in Fig. 8 demonstrate that Nrf2 reversed the regulatory effects of H 2 on HO-1 expression and activity in HUVECs stimulated by LPS and H2 treatment. Based on the above results, we suggest that H2 can promote Nrf2 and HO-1 expression in LPS-stimulated HUVECs and that Nrf2 regulates HO-1 expression and activity during this process. 3.8. H2-rich saline induced HO-1 expression and activity in septic mice Based on our findings regarding the role of HO-1 in LPS-stimulated HUVECs treated with H2, we also investigated HO-1 expression and activity in a clinically relevant septic model in vivo. In this model, sepsis was induced by CLP. The results revealed that LPS elevated HO-1 expression and activity in the lungs of septic mice (Fig. 9A and B) and that H2-rich saline treatment further improved HO-1 expression and activity in CLP-treated mice (Fig. 9A and B).
Fig. 5. The expression of ICAM-1, VCAM-1 and the cytokines TNF-α and HMGB1 in endothelial cells after LPS, H2 or ZnppIX treatment. The HO-1 inhibitor ZnppIX was added to the medium 24 h before the administration of H2 and LPS. ICAM (A, C), VCAM-1 (B, D), TNF-α (E) and HMGB1 (F) were detected after 24 h in LPS-stimulated HUVECs when HO-1 was inhibited by ZnppIX (n = 8 per group). All data are expressed as the mean ± SD. ***P b 0.001 for one-way ANOVA followed by Tukey's post hoc test for different groups.
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Fig. 6. Nrf2 protein and mRNA expression and localization in endothelial cells. After cells were treated with LPS or H2, they were collected for protein and mRNA extraction and localization assessment. Protein expression was detected by western blot. mRNA expression was detected by RT-PCR. Nrf2 localization was determined by an immunofluorescence assay. (A) and (B) show Nrf2 protein expression at 6, 12 and 24 h post-LPS and H2 administration; (C) shows mRNA expression (n = 8 per group). The HO-1 inhibitor ZnppIX (20 μmol/L) was added to the medium 24 h before the application of H2 and LPS. Nrf2 localization is shown in (D) (n = 8 per group). All data are expressed as the mean ± SD. ***P b 0.001 for twoway ANOVA followed by Tukey's post hoc test for different groups.
3.9. H2-rich saline improved survival rates in experimental septic mice via HO-1 To determine the role of HO-1 in experimentally septic mice, we observed the survival rate in the CLP animal model with or without treatment with the HO-1 inhibitor ZnppIX from the 1st to the 7th day of the experiment. CLP led to severe sepsis and resulted in a mortality
rate of up to 100% within 3 days in mice. As illustrated in Table 1, compared with septic mice, the administration of H2-rich saline resulted in protection against mortality in septic mice. However, the coadministration of H2-rich saline and ZnppIX attenuated the protective effects of H2-rich saline on the survival rate in the animal model of sepsis when compared with H2-rich saline administration in septic mice. The results suggest that HO-1 is critical for survival in septic mice.
Fig. 7. Nrf2 mRNA and expression in activated endothelial cells with or without Nrf2 siRNA treatment. Nrf2 siRNA was used to inhibit Nrf2 mRNA and protein expression in cells. Nrf2 mRNA and protein expression levels were determined by RT-PCR and western blot. All data are expressed as the mean ± SD. ***P b 0.001 for one-way ANOVA followed by Tukey's post hoc test for different groups.
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Fig. 8. HO-1 protein expression and activity in LPS- or siRNA-treated cells. Nrf2 siRNA was used to inhibit Nrf2 mRNA and protein expression in cells. After Nrf2 inhibition, HO-1 protein expression (A) and activity (B) were detected in endothelial cells (n = 8 per group). All data are expressed as the mean ± SD. We used one-way ANOVA followed by Tukey's post hoc test for different groups.
3.10. The effect of H2-rich saline on vascular inflammatory responses in the lungs of septic mice
regulatory effects of H2-rich saline on the excessive release of TNF-α and HMGB1 (Fig. 10E and F).
Endocan, also known as endothelial cell specific molecule-1, may be a biomarker of endothelial inflammatory responses, which play a critical role in the regulation of inflammatory disorders and cell adhesion. At 24 h after CLP, endocan levels significantly increased in CLP mice compared with the control group; H2-rich saline treatment clearly inhibited endocan overexpression in the lungs of septic mice. In contrast, ZnppIX reversed the inhibitory effects of H2-rich saline on endocan overexpression. Compared with control sham mice, ICAM-1 and VCAM-1 exhibited higher expression in septic mice as detected by western blot analysis (Fig. 10A, B and C). H2-rich saline markedly protected against increased ICAM-1 and VCAM-1 expression in septic mice. After exposure to ZnppIX, H2-rich saline lost its ability to regulate ICAM-1 and VCAM-1 in the lungs of CLP mice, indicating that HO-1 was required for regulating the increase in ICAM-1 and VCAM-1 expression in vivo. To elucidate the effect of HO-1 on inflammatory cytokines, we measured the release of TNF-α and HMGB1 at 24 h after CLP-induced sepsis in mice. As shown in the results presented in Fig. 5, ZnppIX hindered the
3.11. H2-rich saline improved HO-1 expression and activity in septic mice To further address the role of H2-rich saline in regulating the Nrf2/ HO-1 signaling pathway in vivo, we used Nrf2−/− knock-out mice and wild-type mice to induce sepsis. As shown in Fig. 11, H2-rich saline significantly induced HO-1 expression and activity in the Nrf2+/+ wild-type group after CLP treatment; however, no difference was observed between this group and the Nrf2−/− CLP-treated group with respect to HO-1 expression or activity. 4. Discussion In 2007, Oshawa et al. reported that H2 exerted protective effects on ischemia–reperfusion injury in the brain [7]. Numerous subsequent studies also demonstrated a protective effect for H2 in sepsis, MODS, LPS-induced acute lung injury, and stroke via reduced inflammatory responses, apoptosis and oxidative stress [6–10,18,22]. In a previous study, we found that H2 alleviated inflammation associated with sepsis,
Fig. 9. HO-1 expression and activity in CLP-induced septic mice. Sepsis was induced in mice by CLP. H2-rich saline (5 mL/kg) or the same volume of normal saline were injected i.p. at 1 and 6 h after sham and CLP operations. Lungs were collected to detect HO-1 expression (A) and activity (B) at 24 h after the CLP operation (n = 8 per group). All data are expressed as the mean ± SD. ***P b 0.05 for one-way ANOVA followed by Tukey's post hoc test for different groups. Con = sham; Con + H2 = sham + H2; CLP = septic mice; CLP + H2 = septic mice + H2.
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Table 1 H2-rich saline improved survival rates in experimental septic mice. Group
1d
2d
3d
5d
7d
Sham Sham + H2 CLPa CLP + H2a,b CLP + Znppa CLP + H2 + Znppa,c
100 100 80 90 70 80
100 100 30 70 30 30
100 100 0 60 0 20
100 100 0 50 0 10
100 100 0 50 0 10
H 2 -rich saline (5 mL/kg) was injected i.p. at 1 and 6 h after CLP operations. ZnppIX (40 mg/kg) was injected 1 h before CLP. Survival rates were measure at 1, 2, 3, 5 and 7 d after CLP (n = 20 per group). All data are expressed as the mean ± SD. a P b 0.05 vs. sham group. b P b 0.05 vs. CLP group. c P b 0.05 vs. CLP + H2 group.
MODS and RAW 264.7 macrophages induced by LPS in vitro [6]. However, the specific mechanism by which H2 lessens the burden of disease is unclear. In the present study, we focused on the effects of Nrf2/HO-1 on inflammation during H2 treatment in HUVECs and septic mice. We determined the following: 1) 0.3 mmol/L and 0.6 mmol/L (i.e., saturated) H2-rich medium improved cell viability and cell apoptosis in activated HUVECs, and 0.6 mmol/L (saturated) H2-rich medium was more protective; 2) H2 could inhibit the expression of the cell adhesion molecules VCAM-1 and ICAM-1 and the pro-inflammatory factors TNF-α, IL-1β and HMGB1 and elevate the levels of the anti-inflammatory factor IL10 in LPS-stimulated HUVECs; 3) H2 enhanced HO-1 expression and activity and Nrf2 expression and localization in HUVECs; 4) H2 protects against endothelial inflammatory disorders via a Nrf2-mediated HO-1 signaling pathway in vitro; 5) H2 improved VCAM-1, ICAM-1, endocan, TNF-α and IL-1β expression in septic mice; and 6) H2 improved endothelial inflammation via Nrf2/HO-1 activity. Finally, H2 could suppress an excessive inflammatory response in endothelial cells via Nrf2/HO-1 during sepsis.
Endothelial tissue is pervasive; when pathogens invade an organ or tissue, endothelial cells may undergo necrosis or apoptosis to allow tissue to be reabsorbed and repaired [1]. Under normal conditions, only a small percentage of endothelial cells are apoptotic (b0.1%). Once activated, endothelial cells subsequently trigger a variety of signaling cascades, including the activation of pro-apoptotic caspases [23,24]. LPS has been shown to induce endothelial apoptosis in vitro and in vivo [25]. Moreover, here, we observed that LPS led to an increased number of apoptotic cells and to a decrease in endothelial cell viability. Caspase3 is an essential protease that promotes cell death. Therefore, caspase-3 is termed the “death protease” [26]. Our results suggest that LPS activates endothelial cells to release additional caspase-3, which may trigger a downstream apoptotic cascade and lead to endothelial cell apoptosis. H2 exerts anti-apoptotic, anti-inflammatory and anti-oxidative stress effects on diseases such as sepsis, MODS, LPS-induced acute lung injury, and stroke [6–10,18,22]. Recently, Jiang et al. [18] found that advanced glycation end products induced apoptosis in endothelial cells and that decreased apoptosis was associated with an increase in the Bcl-2/Bax ratio in endothelial cells in vitro. Consistent with these results, our results suggest that H2-rich medium can improve cell viability and reduce the number of apoptotic cells and the expression of caspase-3 in a concentration-dependent manner in activated endothelial cells. Moreover, saturated H2-rich medium exerts a preferable protective effect. Endothelial cells are activated by a multistep cascade. First, loose adhesion molecules lead to leukocyte rolling; then, ICAM-1 and VCAM-1 mediate the firm adhesion of adhesion molecules on leukocytes and endothelial cells [27]. Exogenous IL-1β upregulates ICAM-1 and VCAM-1 during endothelial cell apoptosis; the latter is associated with increased expression of IL-1β-converting enzyme [28]. In clinical trials, plasma sICAM-1 and sVCAM-1 concentrations during sepsis were higher on the 1st day of diagnosis compared with healthy children and were
Fig. 10. ICAM-1, VCAM-1, endocan, TNF-α and HMGB1 expression in the lungs of septic mice with or without ZnppIX treatment. Sepsis was induced in mice by CLP. H2-rich saline (5 mL/kg) was injected i.p. at 1 and 6 h after CLP operations. Lungs were collected to detect ICAM-1, VCAM-1, endocan, TNF-α and HMGB1 expression at 24 h after the CLP operation. ICAM-1 (A, B) and VCAM-1 (A, C) protein expression was measured by western blot. Endocan (D), TNF-α (E) and HMGB1 (F) were detected by ELISA (n = 8 per group). All data are expressed as the mean ± SD. *P b 0.05 for one-way ANOVA followed by Tukey's post hoc test for different groups.
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Fig. 11. HO-1 protein expression and activity in wild-type and Nrf2−/− CLP mice. CLP was induced in wild-type and Nrf2−/− CLP mice. Lungs were collected to detect HO-1 protein expression (A) and activity (B) at 24 h after the CLP operation and H2-rich saline treatment (n = 8 per group). All data are expressed as the mean ± SD. ***P b 0.001 for one-way ANOVA followed by Tukey's post hoc test for different groups.
predictive of mortality in this population. Additionally, changes in sICAM-1 and sVCAM-1 occur in not only children [29] but also adults [30]. According to previous studies, ICAM-1 and VCAM-1 are biomarkers of endothelial activation and are clearly modulated during the 24 h following the diagnosis of sepsis or an infectious disease. In the current study, endothelial cells secreted increasing levels of ICAM-1 and VCAM1 from 6 to 24 h after treatment with LPS, suggesting that these proteins may facilitate interactions with endothelial cells and leukocytes to remove exogenous pathogens. Endothelial barrier dysfunction destroys the protective balance of inflammatory responses, leading to sepsis [31]. Endothelial cell apoptosis also results in an accentuated proinflammatory response. TNF-α and IL-1β are among the early-stage inflammatory factors, whereas HMGB1 appears during the late stages of the inflammatory process. Therefore, we determined the release of the pro-inflammatory factors TNF-α, IL-1β, HMGB1 and the antiinflammatory factor IL-10 at 3, 6, 12 and 24 h post-treatment in activated endothelial cells. We found that H2 treatment could inhibit the release of TNF-α, IL-1β, HMGB1 and increase the levels of IL-10 in a time-dependent manner, suggesting that H2 exerted anti-inflammatory effects and permitted the host to regain a balanced and normal state of inflammation. Endocan, which plays a critical role in the regulation of inflammatory disorders and cell adhesion [32,33], was secreted by vascular endothelial cells of the lung during the inflammatory response. Our study revealed that sepsis caused excessive endocan release in the lungs of septic mice; concurrently, VCAM-1 and ICAM-1 expression also increased. Our results are consistent with the notion that endocan is a pro-inflammatory mediator that induces VCAM-1 and ICAM-1 expression [20]. The beneficial effects of HO-1 appear to be regulated primarily through the degradation of pro-inflammatory heme and the production of the anti-inflammatory molecules CO and bilirubin [34]. The pro-inflammatory mediator LPS has been shown to induce HO-1 gene expression in macrophages, mononuclear phagocytes and endothelial cells [6,34,35]. Previous studies have confirmed that H2 contributes to the induction of HO-1 mRNA and protein expression in injured lung tissue [11]. Our previous research demonstrated that H2 -rich medium boosts HO-1 protein expression and activity in a concentration-dependent manner and that H2 exerts anti-inflammatory properties via HO-1 expression in macrophages stimulated by LPS [6]. Our current observations that LPS or CLP stimulated increased HO-1 expression and activity and that H2 further augmented HO-1 expression in the endothelial cells or lungs of septic mice are consistent with previous reports. Metalloporphyrins, such as ZnppIX, are prototypical inhibitors of HO-1 and are frequently used in in vivo and in vitro experimental models. ZnppIX was used to inhibit HO-1 in our
experiments. We found that ZnppIX completely reversed the regulatory effects of H2 on the survival rate, cell adhesion molecule release and inflammatory factor expression in HUVECs stimulated by LPS and CLP-induced sepsis in mice. These results suggest the antiinflammatory action of H 2 involves HO-1 expression in vitro and in vivo. As is commonly known, HO-1 upregulation is mediated via a redox-dependent signaling pathway that involves activation of the transcription factor Nrf2. Nrf2 is present in the cytoplasm as an inactive complex bound to its partner, Keap1. After encountering harmful stimuli, Nrf2 dissociates from Keap1 to transfer into the nucleus and bind to regulatory sequences (termed antioxidant response elements) located in the promoter regions of genes encoding antioxidants and phase 2 detoxifying enzymes such as HO-1 [36]. The data presented here indicated Nrf2 mRNA and protein expression increased in HUVECs upon LPS stimulation. Nrf2 siRNA was transfected into HUVECs to inhibit Nrf2 expression and to alleviate HO-1 protein expression and activity. Moreover, H 2 had no effect on HO-1 expression and activity in Nrf2 knockout septic mice. The above results verify the pivotal role of H 2 in HO1 expression and activity mediated by the transcription factor Nrf2 during sepsis. These data suggest that molecular H 2 protects against endothelial inflammatory disorders in vitro and in vivo via an Nrf2-mediated HO-1 signaling pathway. Our study has several limitations. We detected the abovementioned indicators at 24 h after LPS stimulation or sepsis induction in mice. We believe that the inflammatory response occurs throughout the entire course of sepsis in endothelial tissues both in vitro and in vivo; however, we only focused on the acute phase of injury and the associated inflammatory response. In a future study, we will determine changes in the expression of inflammatory factors over a longer period. In conclusion, the current study demonstrates that H2 reduces the mass of apoptotic cells, the mortality rate due to sepsis, and the secretion of excessive cell adhesion molecules and inflammatory factors after LPS stimulation or CLP-induced sepsis. Furthermore, H2 exerts beneficial effects with respect to imbalanced inflammatory responses via Nrf2/HO-1 in activated HUVECs and during sepsis in mice. These observations provide new insights into the regulatory mechanisms of H2 in the inflammatory response. Acknowledgments This study was supported by a grant from the National Natural Science Foundation of China (Nos. 81372033 to Yonghao Yu; 81471842 to Keliang Xie), Beijing, China, the Natural Science
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Foundation of the Science Committee (Nos. 13JCQNJC11400 to Keliang Xie).
[19] [20]
References [21] [1] W.C. Aird, The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome, Blood 101 (2003) 3765–3777. [2] P. Henneke, D.T. Golenbock, Innate immune recognition of lipopolysaccharide by endothelial cells, Crit. Care Med. 30 (2002) S207–S213. [3] T. Volk, W.J. Kox, Endothelium function in sepsis, Inflamm. Res. 49 (2000) 185–198. [4] B. Vallet, E. Wiel, Endothelial cell dysfunction and coagulation, Crit. Care Med. 29 (2001) S36–S41. [5] A.V. Page, W.C. Liles, Biomarkers of endothelial activation/dysfunction in infectious diseases, Virulence 4 (2013) 507–516. [6] H.G. Chen, K.L. Xie, H.Z. Han, W.N. Wang, D.Q. Liu, G.L. Wang, et al., Heme oxygenase-1 mediates the anti-inflammatory effect of molecular hydrogen in LPSstimulated RAW 264.7 macrophages, Int. J. Surg. 11 (2013) 1060–1066. [7] I. Ohsawa, M. Ishikawa, K. Takahashi, M. Watanabe, K. Nishimaki, K. Yamagata, et al., Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals, Nat. Med. 13 (2007) 688–694. [8] K. Xie, W. Fu, W. Xing, A. Li, H. Chen, H. Han, et al., Combination therapy with molecular hydrogen and hyperoxia in a murine model of polymicrobial sepsis, Shock 38 (2012) 656–663. [9] K. Xie, Y. Yu, Y. Pei, L. Hou, S. Chen, L. Xiong, et al., Protective effects of hydrogen gas on murine polymicrobial sepsis via reducing oxidative stress and HMGB1 release, Shock 34 (2010) 90–97. [10] K. Xie, Y. Yu, Z. Zhang, W. Liu, Y. Pei, L. Xiong, et al., Hydrogen gas improves survival rate and organ damage in zymosan-induced generalized inflammation model, Shock 34 (2010) 495–501. [11] C.S. Huang, T. Kawamura, Y. Toyoda, A. Nakao, Recent advances in hydrogen research as a therapeutic medical gas, Free Radic. Res. 44 (2010) 971–982. [12] G. Song, H. Tian, S. Qin, X. Sun, S. Yao, C. Zong, et al., Hydrogen decreases atherosusceptibility in apolipoprotein B-containing lipoproteins and aorta of apolipoprotein E knockout mice, Atherosclerosis 221 (2012) 55–65. [13] P. Wiesel, A.P. Patel, N. DiFonzo, P.B. Marria, C.U. Sim, A. Pellacani, et al., Endotoxininduced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase-1-deficient mice, Circulation 102 (2000) 3015–3022. [14] H.P. Zhang, F.L. Zheng, J.H. Zhao, D.X. Guo, X.L. Chen, Genistein inhibits ox-LDLinduced VCAM-1, ICAM-1 and MCP-1 expression of HUVECs through heme oxygenase-1, Arch. Med. Res. 44 (2013) 13–20. [15] T. Kawamura, C.S. Huang, X. Peng, K. Masutani, N. Shigemura, T.R. Billiar, et al., The effect of donor treatment with hydrogen on lung allograft function in rats, Surgery 150 (2011) 240–249. [16] T. Kawamura, N. Wakabayashi, N. Shigemura, C.S. Huang, K. Masutani, Y. Tanaka, et al., Hydrogen gas reduces hyperoxic lung injury via the Nrf2 pathway in vivo, Am. J. Physiol. Lung Cell. Mol. Physiol. 304 (2013) L646–L656. [17] D. Rittirsch, M.S. Huber-Lang, M.A. Flierl, P.A. Ward, Immunodesign of experimental sepsis by cecal ligation and puncture, Nat. Protoc. 4 (2009) 31–36. [18] H. Jiang, P. Yu, D.H. Qian, Z.X. Qin, X.J. Sun, J. Yu, et al., Hydrogen-rich medium suppresses the generation of reactive oxygen species, elevates the Bcl-2/Bax ratio
[22]
[23]
[24] [25] [26]
[27] [28]
[29]
[30] [31]
[32]
[33]
[34]
[35]
[36]
and inhibits advanced glycation end product-induced apoptosis, Int. J. Mol. Med. 31 (2013) 1381–1387. G.M. Li, M.H. Ji, X.J. Sun, Q.T. Zeng, M. Tian, Y.X. Fan, et al., Effects of hydrogen-rich saline treatment on polymicrobial sepsis, J. Surg. Res. 181 (2013) 279–286. W. Lee, S.K. Ku, S.W. Kim, J.S. Bae, Endocan elicits severe vascular inflammatory responses in vitro and in vivo, J. Cell. Physiol. 229 (2014) 620–630. H. Wang, O. Bloom, M. Zhang, J.M. Vishnubhakat, M. Ombrellino, J. Che, et al., HMG1 as a late mediator of endotoxin lethality in mice, Science 285 (1999) 248–251. K. Hayashida, M. Sano, I. Ohsawa, K. Shinmura, K. Tamaki, K. Kimura, et al., Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia– reperfusion injury, Biochem. Biophys. Res. Commun. 373 (2008) 30–35. D.D. Bannerman, S.E. Goldblum, Mechanisms of bacterial lipopolysaccharideinduced endothelial apoptosis, Am. J. Physiol. Lung Cell. Mol. Physiol. 284 (2003) L899–L914. S.M. Dauphinee, A. Karsan, Lipopolysaccharide signaling in endothelial cells, Lab. Investig. 86 (2006) 9–22. R.S. Hotchkiss, K.W. Tinsley, P.E. Swanson, I.E. Karl, Endothelial cell apoptosis in sepsis, Crit. Care Med. 30 (2002) S225–S228. N. Liadis, K. Murakami, M. Eweida, A.R. Elford, L. Sheu, H.Y. Gaisano, et al., Caspase3-dependent beta-cell apoptosis in the initiation of autoimmune diabetes mellitus, Mol. Cell. Biol. 25 (2005) 3620–3629. K.D. Patel, S.L. Cuvelier, S. Wiehler, Selectins: critical mediators of leukocyte recruitment, Semin. Immunol. 14 (2002) 73–81. M.J. Hebert, S.R. Gullans, H.S. Mackenzie, H.R. Brady, Apoptosis of endothelial cells is associated with paracrine induction of adhesion molecules: evidence for an interleukin-1beta-dependent paracrine loop, Am. J. Pathol. 152 (1998) 523–532. F. Paize, R. Sarginson, N. Makwana, P.B. Baines, A.P. Thomson, I. Sinha, et al., Changes in the sublingual microcirculation and endothelial adhesion molecules during the course of severe meningococcal disease treated in the paediatric intensive care unit, Intensive Care Med. 38 (2012) 863–871. T.W. Stief, O. Ijagha, B. Weiste, I. Herzum, H. Renz, M. Max, Analysis of hemostasis alterations in sepsis, Blood Coagul. Fibrinolysis 18 (2007) 179–186. R.L. Qiao, H.S. Wang, W. Yan, L.E. Odekon, V.P. Del, T.J. Smith, et al., Extracellular matrix hyaluronan is a determinant of the endothelial barrier, Am. J. Physiol. 269 (1995) C103–C109. P. Lassalle, S. Molet, A. Janin, J.V. Heyden, J. Tavernier, W. Fiers, et al., ESM-1 is a novel human endothelial cell-specific molecule expressed in lung and regulated by cytokines, J. Biol. Chem. 271 (1996) 20458–20464. S. Sarrazin, E. Adam, M. Lyon, F. Depontieu, V. Motte, C. Landolfi, et al., Endocan or endothelial cell specific molecule-1 (ESM-1): a potential novel endothelial cell marker and a new target for cancer therapy, Biochim. Biophys. Acta 2006 (1765) 25–37. V. Vijayan, E. Baumgart-Vogt, S. Naidu, G. Qian, S. Immenschuh, Bruton's tyrosine kinase is required for TLR-dependent heme oxygenase-1 gene activation via Nrf2 in macrophages, J. Immunol. 187 (2011) 817–827. N. Wijayanti, S. Huber, A. Samoylenko, T. Kietzmann, S. Immenschuh, Role of NF-kappaB and p38 MAP kinase signaling pathways in the lipopolysaccharidedependent activation of heme oxygenase-1 gene expression, Antioxid. Redox Signal. 6 (2004) 802–810. M. Komatsu, H. Kurokawa, S. Waguri, K. Taguchi, A. Kobayashi, Y. Ichimura, et al., The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1, Nat. Cell Biol. 12 (2010) 213–223.
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Molecular hydrogen protects mice against polymicrobial sepsis by ameliorating endothelial dysfunction via an Nrf2/HO-1 signaling pathway☆ Hongguang Chen a,1, Keliang Xie a,⁎,1, Huanzhi Han a,b,1, Yuan Li a, Lingling Liu a, Tao Yang a, Yonghao Yu a,⁎ a b
Department of Anesthesiology, General Hospital of Tianjin Medical University, Tianjin Institute of Anesthesiology, No.154 Anshan Street, Heping District, 300054 Tianjin, PR China Department of Anesthesiology, Dezhou City People's Hospital, No.1751, Xinhu Street, Decheng District, Dezhou 253014, Shandong Province, PR China
a r t i c l e
i n f o
Article history: Received 11 May 2015 Received in revised form 27 June 2015 Accepted 27 July 2015 Available online xxxx Keywords: Sepsis H2 Endothelial dysfunction HO-1 Nrf2
a b s t r a c t Endothelial injury is a primary cause of sepsis and sepsis-induced organ damage. Heme oxygenase-1 (HO-1) plays an essential role in endothelial cellular defenses against inflammation by activating nuclear factor E2related factor-2 (Nrf2). We found that molecular hydrogen (H2) exerts an anti-inflammatory effect. Here, we hypothesized that H2 attenuates endothelial injury and inflammation via an Nrf2-mediated HO-1 pathway during sepsis. First, we detected the effects of H2 on cell viability and cell apoptosis in human umbilical vein endothelial cells (HUVECs) stimulated by LPS. Then, we measured cell adhesion molecules and inflammatory factors in HUVECs stimulated by LPS and in a cecal ligation and puncture (CLP)-induced sepsis mouse model. Next, the role of Nrf2/HO-1 was investigated in activated HUVECs, as well as in wild-type and Nrf−/− mice with sepsis. We found that both 0.3 mmol/L and 0.6 mmol/L (i.e., saturated) H2-rich media improved cell viability and cell apoptosis in LPS-activated HUVECs and that 0.6 mmol/L (i.e., saturated) H2-rich medium exerted an optimal effect. H2 could suppress the release of cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1), and pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and high-mobility group box 1 protein (HMGB1). Furthermore, H2 could elevate anti-inflammatory cytokine IL-10 levels in LPS-stimulated HUVECs and in lung tissue from CLP mice. H2 enhanced HO-1 expression and activity in vitro and in vivo. HO-1 inhibition reversed the regulatory effects of H2 on cell adhesion molecules and inflammatory factors. H2 regulated endothelial injury and the inflammatory response via Nrf2-mediated HO-1 levels. These results suggest that H2 could suppress excessive inflammatory responses and endothelial injury via an Nrf2/HO-1 pathway. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sepsis and its sequelae represent a continuum of clinical and pathologic severity [1]. An important hallmark of sepsis is endothelial activation and dysfunction, which appear to play a pivotal role in the development of sepsis. Sepsis induces endothelial modulation Abbreviations: CLP, cecal ligation and puncture; DMEM, Dulbecco modified eagle medium; DMSO, cimethylsulfoxide; H2, hydrogen; HMGB1, high-mobility group box 1 protein; HO-1, heme oxygenase-1; HUVEC, human umbilical vein endothelial cell; ICAM, intercellular cell adhesion molecule; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MODS, multiple organ disfunction syndromes; MTT, 3-(4, 5)-dimethylthiahiazo (−z-y1)3, 5-di-phenytetrazoliumromide; NADPH, nicotinamide adenine dinucleotide phosphate; Nrf2, nuclear factor E2-related factor-2; PI, propidium iodide; PVDF, polyvinylidene fluoride; RIPA, radioimmunoprecipitation assay; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule-1; ZY, zymosan; Znpp-IX, zinc protoporphyrin IX. ☆ Competing interests: The authors declare that they have no competing interests. ⁎ Corresponding authors. E-mail addresses: [email protected] (K. Xie), [email protected] (Y. Yu). 1 Drs. Hongguang Chen, Keliang Xie and Huanzhi Han made equal contributions to this study.
http://dx.doi.org/10.1016/j.intimp.2015.07.034 1567-5769/© 2015 Elsevier B.V. All rights reserved.
via several different mechanisms that respond to various aspects of infection or to components of the bacterial wall (e.g., LPS) that accompany structural changes and vital functional changes. These aspects of infection include programmed cell death, adhesion molecule expression, inflammatory responses, and leukocyte trafficking [2–4]. Programmed cell death, or apoptosis, leads to an accentuated pro-inflammatory response in the endothelium and is subsequently involved in cross-talk between adhesion molecules and cytokines on the cell surface. In stimulated endothelial cells, adhesion molecules improve the action of leukocytes rolling along the endothelial layer and transmigrating across the endothelial barrier to locations of inflammation or injury [5]. Endothelial injury is a primary result of pathogenesis during sepsis and a critical target to inhibit or even prevent its development. Recently, hydrogen (H2) was shown to exert an effective therapeutic role in many disorders including sepsis, multiple organ dysfunction syndrome (MODS), LPS-induced acute lung injury, and stroke [6–10]. Its therapeutic effects were accomplished via anti-apoptotic, antiinflammatory and anti-oxidative stress mechanisms [11]. Our group
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reported that H2 could improve the survival rates, organ injuries and inflammatory responses in septic mice and regulate the expression of inflammatory factors in macrophages and during sepsis in a zymosan (ZY)-challenged generalized inflammation model [6,9,10]. Song et al. [12] confirmed that H2 reduced ICAM-1 and VCAM-1 expression in the arterial walls of mice administered a high-fat diet. However, the specific mechanisms of H2 have not been clarified. HO-1, a cytoprotective enzyme, is known to play a crucial role in protecting the body from excessive inflammatory processes [13]. Recently, we verified that H2-rich medium reduced TNF-α, HMGB1, and IL-1β expression via an HO-1 pathway in activated macrophages. In human umbilical vein endothelial cells (HUVECs), an inducer of HO decreased ox-LDL-induced VCAM-1 and ICAM-1 secretion and mRNA transcription, and an inhibitor of HO reversed these inhibitory effects [14]. Moreover, HO-1 was augmented by activating the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2). Kawamura et al. reported that H2 treatment increased HO-1 expression in lung grafts [15] and ameliorated hyperoxic lung injury by modulating the Nrf2 signaling pathway [16]. Here, we hypothesized that Nrf2/HO-1 signaling exerts a vital regulatory effect on the anti-inflammatory function of H2-rich media during endothelial injury resulting from sepsis.
Consistent with previously described methods [18], briefly, H2 was dissolved in DMEM supplemented with 10% FBS or saline for 4 h under 0.4 MPa pressure to reach a saturated state. The saturated H2-rich medium (0.6 mmol/L) or saline was maintained in an aluminum bag with no dead volume and stored at 4 °C under atmospheric pressure. The supersaturated H2 medium was diluted with normal medium to prepare 0.3 mmol/L H2-rich culture medium. To ensure a 0.6 mmol/L concentration, H2-rich medium was freshly prepared every week. A needle-type H 2 sensor (Unisense A/S, Aarhus, Denmark) was used to detect the H 2 concentration of the media following the process published by Hayashida et al. [18] According to previous studies and with several modifications [19] based on our preliminary experimental results, sepsis in mice was induced by CLP. H2-rich saline solution (5 mL/kg) or normal saline solution (5 mL/kg) was injected i.p. at 1 and 6 h after the sham and CLP operations.
2. Materials and methods
2.5. Nrf2 siRNA transfection
2.1. Animals
Nrf2 siRNA-transfected HUVECs were cultured in 6-well plates and were transfected with Nrf2 siRNA (Santa Cruz Biotechnology) diluted with siRNA LipofectAMINE™ 2000 transfection reagent (Invitrogen Company Carlsbad, CA, USA) according to the manufacturer's instructions. The cells were cultured at 37 °C with 5% CO2 for 24 h before treatment with LPS and H2.
All experimental procedures utilizing animals were approved by the Institutional Animal Care Committee of Tianjin Medical University and performed according to the “Policies on the Use of Animal and Humans in Neuroscience Research”. This protocol was in agreement with the Committee on the Ethics of Animal Experiments of Tianjin Medical University General Hospital, Tianjin, China (permit number: 2011-X6-18). Male wild-type (WT) and Nrf2-gene-deleted (Nrf2−/−) ICR mice weighing 20–25 g were obtained from the General Hospital of Nanjing Military Command (Nanjing, China). The mice were fed water and food ad libitum and maintained in an air-conditioned and individually ventilated environment on a 12 h light/dark cycle. 2.2. Cell culture and treatment HUVECs-12 (termed HUVECs) that were purchased from ATCC were cultured in cell culture medium (DMEM) supplemented with 10% heatinactivated fetal bovine serum (FBS; Life Technologies Corp., NY, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies Corp., NY, USA) at 37 °C in an atmosphere of 95% air and 5% CO2. HUVECs were maintained at a density of 1 × 106 cells/mL in DMEM and 10% FBS. HUVECs were treated with vehicle or varying concentrations of LPS (Escherichia coli 0111:B4; Sigma, St. Louis, MO, USA) combined with 1% FBS at 37 °C for further experiments. 2.3. Sepsis in mice induced by cecal ligation and puncture (CLP) following zinc protoporphyrin IX (ZnppIX) treatment CLP is a classic and clinically relevant sepsis model that can comprehensively mimic the disease processes related to sepsis. As described in a previous study [17], briefly, the mice were anesthetized with chloral hydrate, shaved, and disinfected, and a laparotomy was performed. Then, the cecum was ligated from the top and punctured twice on the antimesenteric side with a 20-gauge needle. A small amount of feces from the bowel was expelled from the puncture hole, and the cecum was replaced gently. Sham-operated mice underwent the same procedure but without ligation and puncture. After surgery, the mice were given warm normal saline to resuscitate them (0.1 mL/g body weight). The mice received an intraperitoneal (i.p.) ZnppIX (40 mg/kg) injection 1 h before cecum ligation and puncture. ZnppIX (Porphyrin Products,
Logan, UT, USA) was dissolved in 0.2 M sodium hydroxide, and HCl was added to adjust to a pH of 7.4. 2.4. H2-rich medium or saline treatment
2.6. MTT and LDH detection HUVECs (104 cells/well) were grown in a 96-well plate for at least 12 h and then incubated with LPS or H2. According to the manufacturer's instructions, cell viability was determined by MTT assay, and cytotoxicity was detected by lactate dehydrogenase (LDH) assay. Briefly, after the HUVECs were treated with LPS or H2, they were washed with 3-(4,5)dimethylthiahiazo (−z-y1)-3,5-di-phenyltetrazolium bromide (MTT) (5 mg/mL) in cell medium supplemented with 10% FBS. After the cells were incubated for 3 h at 37 °C, the medium was discarded, and formazan blue was dissolved in 100 μL of dimethyl sulfoxide (DMSO). The absorbance was measured at 490 nm using a microplate reader (Grodig, Austria). Relative cell viability was quantified compared with the non-treated group. Cell injury was measured via LDH release using a cytotoxicity detection kit according to the manufacturer's instructions (Roche, IN). Cell activity was expressed as a percentage of the LDH released in control cultures. 2.7. Apoptosis The percentage of apoptosis was determined using an Annexin V/PI Kit (BD Pharmingen, San Diego, CA, USA). Briefly, HUVECs were harvested after treatment, washed twice, rewashed in binding buffer, and then labeled with annexin V and propidium iodide (PI). The percentage of HUVECs undergoing apoptosis was determined using a Becton Dickinson FACStar flow cytometer. 2.8. Caspase-3 activity assay Experimental samples were collected to measure caspase-3 activity. After the samples were incubated with DEVD-AFC, caspase-3 activity was determined in a cyto-buffer as described previously [19]. Free AFC release was measured using a fluorescence microscope (Leica DM2500 system; Leica, Wetzlar, Germany) at excitation and emission wavelengths of 350 nm and 460 nm, respectively.
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2.9. ELISA After cells were treated, culture media and tissue homogenates were collected for the detection of cytokines and endocan. The cells were collected using a centrifuge, and supernatants were obtained for the detection of TNF-α, IL-1β, IL-10 (R&D Systems Inc., Minneapolis, MN, USA), HMGB1 (IBL, Hamburg, Germany) and endocan (R&D Systems, Minneapolis, MN) [9,10]. Briefly, the samples were added to microtiter plates pre-coated with anti-TNF-α, anti-IL-1β, anti-HMGB1 and anti-IL-10 monoclonal antibodies and incubated with horseradish peroxidase (HRP)-labeled TNF-α, IL-1β, HMGB1 and IL-10 IgG. The absorbance of each well was measured at 450 nm using a microplate reader (Grodig, Austria). Based on a previous study [20], the samples were incubated with anti-endocan antibodies and then incubated with peroxidaseconjugated anti-goat IgG antibodies. After stopping the reaction, absorbances were read at 490 nm. 2.10. HO-1 activity detection HO-1 activity was measured at different time points after LPS stimulation in vitro and at 24 h after sham or CLP in vivo as described previously in the Materials and methods section [21]. HUVECs were collected to prepare microsomes. Lung tissue was homogenized in volumes of potassium phosphate buffer containing sucrose and a protease inhibitor. After the
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samples were centrifuged, the microsomal pellets were collected. The reaction compound consisted of microsomes, biliverdin reductase, hemin and nicotinamide adenine dinucleotide phosphate (NADPH); biliverdin reductase was derived from the cytosolic fraction of rat liver. The reaction compound was incubated and then stopped with chloroform. The amount of extracted bilirubin in the chloroform layer was measured at absorbances between 464 and 530 nm. HO-1 activity was represented as picomoles of bilirubin formed per milligram of protein per hour. 2.11. Protein collection for western blot detection 2.11.1. Sample collection in vitro HO-1 and Nrf2 protein detection in HUVECs was performed at different time points in the presence or absence of ZnppIX (or Nrf2 siRNA) after LPS stimulation. After the experiment, the cells were collected for protein sample preparation and resuspended for use in a 200-μL radioimmunoprecipitation assay (RIPA) performed on ice for 30 min. The cells were centrifuged (15,000g, 10 min, 4 °C) and denatured (100 °C, 5 min), and protein was collected and maintained at −80 °C. 2.11.2. Sample collection in vivo Lungs were collected for homogenization in 1 mL of lysis buffer. The samples were centrifuged (15,000g, 20 min, 4 °C), and the supernatants were collected and stored at −80 °C for western blot analysis.
Fig. 1. The effect of H2 on cell viability, LDH release, caspase-3 activity and apoptosis in LPS-stimulated HUVECs. HUVECs were treated with 1 ng/mL, 10 ng/mL, 100 ng/mL, 1000 ng/mL or 10,000 ng/mL LPS, and then the cells were collected to detect cell viability by MTT (A) and LDH release (B). LPS caused a decrease in cell viability and excessive LDH release. Cells were challenged with 1 μg/mL LPS or PBS with or without 0.3 mmol/L (50% H2) or 0.6 mmol/L H2-rich medium. After treatment, cell viability (C), caspase-3 activity (D) and apoptosis (E) were tested. Fifty percent H2 and full-strength H2 improved cell viability, caspase-3 activity and apoptosis in endothelial cells; moreover, full-strength H2 was better than 50% H2. Cell viability, LDH release, caspase-3 activity and apoptosis were measured as described in the Materials and methods section. Each data point and error bar represents the mean ± SD. The data were analyzed by one-way ANOVA followed by Tukey's test. ***P b 0.001.
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2.11.3. Western blot analysis In total, 100 μg of denatured protein was separated on 10% acrylamide gels and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked and incubated overnight at 4 °C with primary antibodies against HO-1, Nrf2, and β-actin (Abcam, Cambridge, MA, USA, 1:2000 dilution). Subsequently, the membranes were washed and incubated with secondary antibodies (Abcam, Cambridge, MA, USA) at room temperature. The protein bands were visualized with an enhanced chemiluminescence (ECL) reagent and imaged using a Quantity One gel quantitation system (Bio-Rad, Tokyo, Japan). All sample detections and analyses were repeated three times. HO-1 and Nrf2 expressions were normalized to β-actin. 2.12. Real-time quantitative polymerase chain reaction (RT-PCR) Total RNA was extracted using a TRIzol RNA isolation reagent kit (Invitrogen, USA). Quantitative PCR analyses were performed using primers and probe sets commercially available from Applied Biosystems according to the manufacturer's instructions for reverse transcription reactions using a High Capacity Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA, USA). Amplification reactions were performed using a SYBR Green PCR Master Mix (Applied Biosystems). Each sample was analyzed in triplicate. The primers were as follows: Nrf2, forward 5′-TTCAGCCAGCCCAGCACATC-3′ and reverse 5′-CGTA GCCGAAGAAACCTCATTGTC-3′; β-actin, forward 5′-ATCAAGATCATTGC TCCTCCTG-3′ and reverse 5′-GCAACTAAGTCATAGTCCGCC-3′. Expression levels were normalized to β-actin expression levels.
2.13. Fluorescence microscopy analysis Cells were seeded on slides at an appropriate density and then treated with LPS (1 μg/mL) with or without H2. Then, the cells were washed, fixed in 4% paraformaldehyde and permeabilized with Triton X-100 (0.5%). After the fixed cells were washed, they were blocked and incubated with anti-Nrf2 antibodies. After the cells were washed again, they were incubated with IgG–FITC secondary antibodies, stained with DAPI and observed using an Olympus confocal microscope (Leica, DM4000 B, Germany). 2.14. Statistical analysis Statistical data analysis was performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA). In vitro experiments and cell viability, LDH release and caspase-3 activity assays were compared by one-way ANOVA followed by Tukey's post hoc test after H2 treatment. After ZnppIX or Nrf2 siRNA treatment, ICAM-1, VCAM-1, TNF-α, HMGB1, and HO-1 protein levels and activities were compared by one-way ANOVA followed by Tukey's post hoc test. For multiple group comparisons, two-way ANOVA followed by Tukey's post hoc tests were used to compare ICAM-1, VCAM-1 TNF-α, IL-1β, HMGB1, IL-10 and HO-1 protein levels and activities, as well as Nrf2 protein and mRNA levels at different time points and among different groups. The experimental data from septic mice for ICAM-1, VCAM-1, endocan, TNF-α, HMGB1, and HO-1 protein levels and activities were compared by one-way ANOVA followed by Tukey's post hoc
Fig. 2. H2 attenuated ICAM-1 and VCAM-1 expression at 6, 12 and 24 h in LPS-stimulated HUVECs. Cells were collected to measure ICAM-1 (A, C) and VCAM-1 (B, D) expression by western blot at 6, 12 and 24 h post-treatment when treated with LPS or H2 (n = 8 per group). H2 treatment decreased ICAM-1 and VCAM-1 expression in LPS-stimulated HUVECs. All values are expressed as the mean ± SD. ***P b 0.001 for two-way ANOVA followed by Tukey's test between more than two groups over time.
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3. Results
shown in Fig. 1, 0.3 mmol/L and 0.6 mmol/L H2-rich media increased cell viability and decreased caspase-3 activity and apoptosis in activated HUVECs in a concentration-dependent manner compared with the control group; furthermore, the 0.6 mmol/L H2-rich medium exerted a preferable protective effect with respect to endothelial cell injury.
3.1. The effects of different concentrations of LPS on HUVEC viability and cytotoxicity
3.3. H2-rich medium improved the release of cell adhesion molecules and inflammatory factors in LPS-induced endothelial cells
LPS, the primary component of the bacterial wall, was able activate endothelial cells to induce innate immune responses and inflammation, even leading to cell injury. Cell viability could be measured with an MTT assay; LDH, a direct determinant of cell death, is indicative of membrane integrity [6]. To detect the effects of LPS on cell viability and cytotoxicity in HUVECs, cells were treated with different concentrations of LPS (i.e., 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 μg/mL, or 10 μg/mL; E. coli 0111:B4; Sigma, St. Louis, MO, USA) for 24 h. As shown in Fig. 1, LPS weakened cell viability and increased LDH release in a concentrationdependent manner. Concentrations of 1–100 ng/mL LPS had little effect on cell viability and cytotoxicity compared with control cells, whereas 10 μg/mL LPS led to extensive cell viability breakdown and cell death such that the cells did not meet the demands of the experiments. Therefore, we chose 1 μg/mL LPS as the optimal concentration for stimulation in the following experimental scheme.
Based on the above results, 0.6 mmol/L H2-rich medium exerted a preferable protective effect compared with 0.3 mmol/L H2-rich medium. In the next experiment, 0.6 mmol/L H2-rich medium was used to incubate cells. LPS induced an excessive inflammatory response in endothelial cells. ICAM-1 and VCAM-1 protein expression was determined by western blot analysis; simultaneously, TNF-α, IL-1β, HMGB1 and IL-10 responses were determined by ELISA to assess the inflammatory state of the cells. Compared with the control group, LPS induced significantly increased ICAM-1 and VCAM-1 expression in a concentrationdependent manner at 6, 12 and 24 h following endothelial cell activation (two-way ANOVA; P b 0.001 for the effects of time and treatment, Fig. 2). Compared with the LPS group, incubating the HUVECs with H2 reversed the increased expression of ICAM-1 and VCAM-1 (two-way ANOVA; P b 0.001 for the effects of time and treatment, Fig. 2). TNF-α and IL-1β are produced early in the course of the inflammatory response, whereas HMGB1 exhibits a late kinetic profile during inflammation in response to endotoxemia [20]. Therefore, we investigated the release of cytokines from LPS-induced HUVECs from 3 to 24 h. The levels of the cytokines TNF-α, IL-1β, and HMGB1 significantly increased in cells from 3 to 24 h post-LPS stimulation (twoway ANOVA; P b 0.001 for the effects of time and treatment, Fig. 3). The time course and expression trends of anti-cytokine IL-10 were similar to those of HMGB1 in HUVECs (two-way ANOVA; P b 0.001 for the effects of time and treatment, Fig. 3). Furthermore, the peak concentrations of released TNF-α and IL-1β occurred at 6 h; for HMGB1 and IL-10,
test. Survival rates were analyzed using the Kaplan–Meier method followed by a log rank test. All data are expressed as the means ± SD; P b 0.05 was considered statistically significant.
3.2. H2-rich media improved cell viability and apoptosis in activated HUVECs in a concentration-dependent manner Based on the experiment described above, cells were stimulated with 1 μg/mL LPS. To assess the optimum concentration for H2-rich medium with respect to cell viability and apoptosis in activated HUVECs, we used 0.3 mmol/L H2-rich medium and 0.6 mmol/L H2-rich medium to culture HUVECs with or without LPS treatment for 24 h, and then cell viability, caspase-3 activity and apoptosis were measured. As
Fig. 3. The regulatory effects of H2 on cytokines at different time points in activated endothelial cells. After HUVECs were treated with LPS or H2, media was collected to quantify the proinflammatory factors TNF-α (A), IL-1β (B), and HMGB1 (C) and the anti-inflammatory factor IL-10 (D) at 3, 6, 12 and 24 h using an ELISA (n = 8 per group). H2 decreased the expression of pro-inflammatory factors and further increased the expression of anti-inflammatory factors in LPS-stimulated HUVECs. All data are expressed as the mean ± SD. ***P b 0.05 for two-way ANOVA followed by Tukey's test for four groups over time.
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the peak was at 24 h. H2 can markedly decrease TNF-α release (twoway ANOVA; P b 0.001 for the interaction between time and treatment, Fig. 3) and further increase IL-10 expression after LPS stimulation (twoway ANOVA; P b 0.001 for the interaction between time and treatment, Fig. 3). Based on these results, we concluded that H2 is able to regulate the inflammatory response by regulating the balance of pro- and antiinflammatory cytokines.
inhibitor of HO-1. As shown in Fig. 4, ZnppIX reversed the stimulatory effects of H2 on HO-1 expression and activity in activated HUVECs (expression: LPS + H2 + Znpp vs. LPS + H2, 0.21 ± 0.04 vs. 0.67 ± 0.08, P b 0.001; activity: LPS + H2 + Znpp vs. LPS + H2, 301.2 ± 42.7 vs. 856.4 ± 89.3, P b 0.001). 3.5. ZnppIX completely reversed the regulatory effects of H2 on cell adhesion molecules and inflammatory factors in LPS-stimulated HUVECs
3.4. Effect of H2 on HO-1 expression and activity in LPS-stimulated HUVECs HO-1 performs a vital function in the inflammatory response [6]. In our experimental protocol, HO-1 expression and activity were measured in HUVECs from 6 to 24 h post-LPS stimulation in HUVECs. As shown in Fig. 4, these levels were significantly increased at 6, 12 and 24 h in the LPS group compared with the control group (expression: two-way ANOVA, P b 0.001 for the interaction between time and treatment; activity: two-way ANOVA, P b 0.001 for the interaction between time and treatment). Compared with the group cultured without H2, culture with H2 could further enhance HO-1 expression and activity after LPS stimulation (expression: two-way ANOVA, P b 0.001 for the interaction between time and treatment; activity: two-way ANOVA, P b 0.001 for the interaction between time and treatment). To deepen our analysis of the role of HO-1, we selected ZnppIX (20 μg/mL) as an
Based on the previous results, H2 can inhibit the release of cell adhesion molecules and the expression of pro-inflammatory factors and promote the expression and release of anti-inflammatory factors. H2 was also shown to accelerate HO-1 expression and activity in activated HUVECs. In the next experiment, we sought to further verify the effect of HO-1 on cell adhesion molecules and inflammatory factors after H 2 -treated HUVECs were challenged with LPS. As shown in Fig. 5, we found that H 2-rich medium decreased the release of cell adhesion molecules and pro-inflammatory factors (one-way ANOVA, P b 0.001, Fig. 5). However, ZnppIX, a HO-1 inhibitor, reversed the protective effects of H2 on cell adhesion molecules and inflammatory factors. These results suggest that H 2 can regulate the inflammatory response via the HO-1 pathway in endothelial cells.
Fig. 4. HO-1 protein expression and HO-1 activity in endothelial cells. After cells were treated with LPS or H2, they were collected for protein extraction and HO-1 activity measurement. HO-1 protein expression was detected by western blot. HO-1 activity was determined as described in the Materials and methods section. (A) and (B) show HO-1 protein expression at 6, 12 and 24 h after LPS and H2 administration; HO-1 activity is shown in (C) (n = 8 per group). The HO-1 inhibitor ZnppIX (20 μmol/L) was added to the medium 24 h before the application of H2 and LPS. HO-1 protein levels (D) and activity (E) were detected at 24 h after LPS, H2 or ZnppIX administration (n = 8 per group). All data are expressed as the mean ± SD. HO-1 protein expression and activity were compared using a two-way ANOVA followed by Tukey's post hoc test for different treatments and timepoints. After ZnppIX treatment, two-way ANOVA followed by Tukey's post hoc test was used to compare HO-1 expression and activity. ***P b 0.001.
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3.6. The effect of H2 on the expression of the transcription factor Nrf2 in LPS-stimulated HUVECs The transcription factor Nrf2 is an important regulator of cellular inflammation. However, whether Nrf2 plays a vital role in the processes that occur in H2-treated HUVECs remains unknown. Nrf2 expression and localization were detected by RT-PCR, western blot and immunofluorescence. Nrf2 mRNA and protein expression were significantly higher at 6 and 24 h post-treatment in the LPS group compared with the control group. H2 further heightened Nrf2 mRNA and protein expression after LPS stimulation. As shown in Fig. 6D, LPS induced increased localized expression at 24 h post-LPS stimulation, and H2 further promoted localized Nrf2 expression (P b 0.05, Fig. 6). 3.7. Effects of Nrf2 on HO-1 expression and activity in H2-treated HUVECs Nrf2, which controls HO-1 expression in response to noxious stimuli such as infection and tissue injury and in response to prooxidant stimuli, is regulated by cytosolic Keap1, which directly interacts with Nrf2 for proteasomal degradation via ubiquitination [21]. To assess whether H2 improved HO-1 expression via the Nrf2
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signaling pathway in activated HUVECs, Nrf2 siRNA was used to inhibit Nrf2 gene expression. As shown in Fig. 7, siRNA could clearly inhibit Nrf2 mRNA and protein expression in the LPS + Nrf2 siRNA group compared with the LPS + scrambled siRNA group. After inhibiting Nrf2 mRNA and protein expression, H2 exerted no significant effects on Nrf2 mRNA. The results shown in Fig. 8 demonstrate that Nrf2 reversed the regulatory effects of H 2 on HO-1 expression and activity in HUVECs stimulated by LPS and H2 treatment. Based on the above results, we suggest that H2 can promote Nrf2 and HO-1 expression in LPS-stimulated HUVECs and that Nrf2 regulates HO-1 expression and activity during this process. 3.8. H2-rich saline induced HO-1 expression and activity in septic mice Based on our findings regarding the role of HO-1 in LPS-stimulated HUVECs treated with H2, we also investigated HO-1 expression and activity in a clinically relevant septic model in vivo. In this model, sepsis was induced by CLP. The results revealed that LPS elevated HO-1 expression and activity in the lungs of septic mice (Fig. 9A and B) and that H2-rich saline treatment further improved HO-1 expression and activity in CLP-treated mice (Fig. 9A and B).
Fig. 5. The expression of ICAM-1, VCAM-1 and the cytokines TNF-α and HMGB1 in endothelial cells after LPS, H2 or ZnppIX treatment. The HO-1 inhibitor ZnppIX was added to the medium 24 h before the administration of H2 and LPS. ICAM (A, C), VCAM-1 (B, D), TNF-α (E) and HMGB1 (F) were detected after 24 h in LPS-stimulated HUVECs when HO-1 was inhibited by ZnppIX (n = 8 per group). All data are expressed as the mean ± SD. ***P b 0.001 for one-way ANOVA followed by Tukey's post hoc test for different groups.
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Fig. 6. Nrf2 protein and mRNA expression and localization in endothelial cells. After cells were treated with LPS or H2, they were collected for protein and mRNA extraction and localization assessment. Protein expression was detected by western blot. mRNA expression was detected by RT-PCR. Nrf2 localization was determined by an immunofluorescence assay. (A) and (B) show Nrf2 protein expression at 6, 12 and 24 h post-LPS and H2 administration; (C) shows mRNA expression (n = 8 per group). The HO-1 inhibitor ZnppIX (20 μmol/L) was added to the medium 24 h before the application of H2 and LPS. Nrf2 localization is shown in (D) (n = 8 per group). All data are expressed as the mean ± SD. ***P b 0.001 for twoway ANOVA followed by Tukey's post hoc test for different groups.
3.9. H2-rich saline improved survival rates in experimental septic mice via HO-1 To determine the role of HO-1 in experimentally septic mice, we observed the survival rate in the CLP animal model with or without treatment with the HO-1 inhibitor ZnppIX from the 1st to the 7th day of the experiment. CLP led to severe sepsis and resulted in a mortality
rate of up to 100% within 3 days in mice. As illustrated in Table 1, compared with septic mice, the administration of H2-rich saline resulted in protection against mortality in septic mice. However, the coadministration of H2-rich saline and ZnppIX attenuated the protective effects of H2-rich saline on the survival rate in the animal model of sepsis when compared with H2-rich saline administration in septic mice. The results suggest that HO-1 is critical for survival in septic mice.
Fig. 7. Nrf2 mRNA and expression in activated endothelial cells with or without Nrf2 siRNA treatment. Nrf2 siRNA was used to inhibit Nrf2 mRNA and protein expression in cells. Nrf2 mRNA and protein expression levels were determined by RT-PCR and western blot. All data are expressed as the mean ± SD. ***P b 0.001 for one-way ANOVA followed by Tukey's post hoc test for different groups.
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Fig. 8. HO-1 protein expression and activity in LPS- or siRNA-treated cells. Nrf2 siRNA was used to inhibit Nrf2 mRNA and protein expression in cells. After Nrf2 inhibition, HO-1 protein expression (A) and activity (B) were detected in endothelial cells (n = 8 per group). All data are expressed as the mean ± SD. We used one-way ANOVA followed by Tukey's post hoc test for different groups.
3.10. The effect of H2-rich saline on vascular inflammatory responses in the lungs of septic mice
regulatory effects of H2-rich saline on the excessive release of TNF-α and HMGB1 (Fig. 10E and F).
Endocan, also known as endothelial cell specific molecule-1, may be a biomarker of endothelial inflammatory responses, which play a critical role in the regulation of inflammatory disorders and cell adhesion. At 24 h after CLP, endocan levels significantly increased in CLP mice compared with the control group; H2-rich saline treatment clearly inhibited endocan overexpression in the lungs of septic mice. In contrast, ZnppIX reversed the inhibitory effects of H2-rich saline on endocan overexpression. Compared with control sham mice, ICAM-1 and VCAM-1 exhibited higher expression in septic mice as detected by western blot analysis (Fig. 10A, B and C). H2-rich saline markedly protected against increased ICAM-1 and VCAM-1 expression in septic mice. After exposure to ZnppIX, H2-rich saline lost its ability to regulate ICAM-1 and VCAM-1 in the lungs of CLP mice, indicating that HO-1 was required for regulating the increase in ICAM-1 and VCAM-1 expression in vivo. To elucidate the effect of HO-1 on inflammatory cytokines, we measured the release of TNF-α and HMGB1 at 24 h after CLP-induced sepsis in mice. As shown in the results presented in Fig. 5, ZnppIX hindered the
3.11. H2-rich saline improved HO-1 expression and activity in septic mice To further address the role of H2-rich saline in regulating the Nrf2/ HO-1 signaling pathway in vivo, we used Nrf2−/− knock-out mice and wild-type mice to induce sepsis. As shown in Fig. 11, H2-rich saline significantly induced HO-1 expression and activity in the Nrf2+/+ wild-type group after CLP treatment; however, no difference was observed between this group and the Nrf2−/− CLP-treated group with respect to HO-1 expression or activity. 4. Discussion In 2007, Oshawa et al. reported that H2 exerted protective effects on ischemia–reperfusion injury in the brain [7]. Numerous subsequent studies also demonstrated a protective effect for H2 in sepsis, MODS, LPS-induced acute lung injury, and stroke via reduced inflammatory responses, apoptosis and oxidative stress [6–10,18,22]. In a previous study, we found that H2 alleviated inflammation associated with sepsis,
Fig. 9. HO-1 expression and activity in CLP-induced septic mice. Sepsis was induced in mice by CLP. H2-rich saline (5 mL/kg) or the same volume of normal saline were injected i.p. at 1 and 6 h after sham and CLP operations. Lungs were collected to detect HO-1 expression (A) and activity (B) at 24 h after the CLP operation (n = 8 per group). All data are expressed as the mean ± SD. ***P b 0.05 for one-way ANOVA followed by Tukey's post hoc test for different groups. Con = sham; Con + H2 = sham + H2; CLP = septic mice; CLP + H2 = septic mice + H2.
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Table 1 H2-rich saline improved survival rates in experimental septic mice. Group
1d
2d
3d
5d
7d
Sham Sham + H2 CLPa CLP + H2a,b CLP + Znppa CLP + H2 + Znppa,c
100 100 80 90 70 80
100 100 30 70 30 30
100 100 0 60 0 20
100 100 0 50 0 10
100 100 0 50 0 10
H 2 -rich saline (5 mL/kg) was injected i.p. at 1 and 6 h after CLP operations. ZnppIX (40 mg/kg) was injected 1 h before CLP. Survival rates were measure at 1, 2, 3, 5 and 7 d after CLP (n = 20 per group). All data are expressed as the mean ± SD. a P b 0.05 vs. sham group. b P b 0.05 vs. CLP group. c P b 0.05 vs. CLP + H2 group.
MODS and RAW 264.7 macrophages induced by LPS in vitro [6]. However, the specific mechanism by which H2 lessens the burden of disease is unclear. In the present study, we focused on the effects of Nrf2/HO-1 on inflammation during H2 treatment in HUVECs and septic mice. We determined the following: 1) 0.3 mmol/L and 0.6 mmol/L (i.e., saturated) H2-rich medium improved cell viability and cell apoptosis in activated HUVECs, and 0.6 mmol/L (saturated) H2-rich medium was more protective; 2) H2 could inhibit the expression of the cell adhesion molecules VCAM-1 and ICAM-1 and the pro-inflammatory factors TNF-α, IL-1β and HMGB1 and elevate the levels of the anti-inflammatory factor IL10 in LPS-stimulated HUVECs; 3) H2 enhanced HO-1 expression and activity and Nrf2 expression and localization in HUVECs; 4) H2 protects against endothelial inflammatory disorders via a Nrf2-mediated HO-1 signaling pathway in vitro; 5) H2 improved VCAM-1, ICAM-1, endocan, TNF-α and IL-1β expression in septic mice; and 6) H2 improved endothelial inflammation via Nrf2/HO-1 activity. Finally, H2 could suppress an excessive inflammatory response in endothelial cells via Nrf2/HO-1 during sepsis.
Endothelial tissue is pervasive; when pathogens invade an organ or tissue, endothelial cells may undergo necrosis or apoptosis to allow tissue to be reabsorbed and repaired [1]. Under normal conditions, only a small percentage of endothelial cells are apoptotic (b0.1%). Once activated, endothelial cells subsequently trigger a variety of signaling cascades, including the activation of pro-apoptotic caspases [23,24]. LPS has been shown to induce endothelial apoptosis in vitro and in vivo [25]. Moreover, here, we observed that LPS led to an increased number of apoptotic cells and to a decrease in endothelial cell viability. Caspase3 is an essential protease that promotes cell death. Therefore, caspase-3 is termed the “death protease” [26]. Our results suggest that LPS activates endothelial cells to release additional caspase-3, which may trigger a downstream apoptotic cascade and lead to endothelial cell apoptosis. H2 exerts anti-apoptotic, anti-inflammatory and anti-oxidative stress effects on diseases such as sepsis, MODS, LPS-induced acute lung injury, and stroke [6–10,18,22]. Recently, Jiang et al. [18] found that advanced glycation end products induced apoptosis in endothelial cells and that decreased apoptosis was associated with an increase in the Bcl-2/Bax ratio in endothelial cells in vitro. Consistent with these results, our results suggest that H2-rich medium can improve cell viability and reduce the number of apoptotic cells and the expression of caspase-3 in a concentration-dependent manner in activated endothelial cells. Moreover, saturated H2-rich medium exerts a preferable protective effect. Endothelial cells are activated by a multistep cascade. First, loose adhesion molecules lead to leukocyte rolling; then, ICAM-1 and VCAM-1 mediate the firm adhesion of adhesion molecules on leukocytes and endothelial cells [27]. Exogenous IL-1β upregulates ICAM-1 and VCAM-1 during endothelial cell apoptosis; the latter is associated with increased expression of IL-1β-converting enzyme [28]. In clinical trials, plasma sICAM-1 and sVCAM-1 concentrations during sepsis were higher on the 1st day of diagnosis compared with healthy children and were
Fig. 10. ICAM-1, VCAM-1, endocan, TNF-α and HMGB1 expression in the lungs of septic mice with or without ZnppIX treatment. Sepsis was induced in mice by CLP. H2-rich saline (5 mL/kg) was injected i.p. at 1 and 6 h after CLP operations. Lungs were collected to detect ICAM-1, VCAM-1, endocan, TNF-α and HMGB1 expression at 24 h after the CLP operation. ICAM-1 (A, B) and VCAM-1 (A, C) protein expression was measured by western blot. Endocan (D), TNF-α (E) and HMGB1 (F) were detected by ELISA (n = 8 per group). All data are expressed as the mean ± SD. *P b 0.05 for one-way ANOVA followed by Tukey's post hoc test for different groups.
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Fig. 11. HO-1 protein expression and activity in wild-type and Nrf2−/− CLP mice. CLP was induced in wild-type and Nrf2−/− CLP mice. Lungs were collected to detect HO-1 protein expression (A) and activity (B) at 24 h after the CLP operation and H2-rich saline treatment (n = 8 per group). All data are expressed as the mean ± SD. ***P b 0.001 for one-way ANOVA followed by Tukey's post hoc test for different groups.
predictive of mortality in this population. Additionally, changes in sICAM-1 and sVCAM-1 occur in not only children [29] but also adults [30]. According to previous studies, ICAM-1 and VCAM-1 are biomarkers of endothelial activation and are clearly modulated during the 24 h following the diagnosis of sepsis or an infectious disease. In the current study, endothelial cells secreted increasing levels of ICAM-1 and VCAM1 from 6 to 24 h after treatment with LPS, suggesting that these proteins may facilitate interactions with endothelial cells and leukocytes to remove exogenous pathogens. Endothelial barrier dysfunction destroys the protective balance of inflammatory responses, leading to sepsis [31]. Endothelial cell apoptosis also results in an accentuated proinflammatory response. TNF-α and IL-1β are among the early-stage inflammatory factors, whereas HMGB1 appears during the late stages of the inflammatory process. Therefore, we determined the release of the pro-inflammatory factors TNF-α, IL-1β, HMGB1 and the antiinflammatory factor IL-10 at 3, 6, 12 and 24 h post-treatment in activated endothelial cells. We found that H2 treatment could inhibit the release of TNF-α, IL-1β, HMGB1 and increase the levels of IL-10 in a time-dependent manner, suggesting that H2 exerted anti-inflammatory effects and permitted the host to regain a balanced and normal state of inflammation. Endocan, which plays a critical role in the regulation of inflammatory disorders and cell adhesion [32,33], was secreted by vascular endothelial cells of the lung during the inflammatory response. Our study revealed that sepsis caused excessive endocan release in the lungs of septic mice; concurrently, VCAM-1 and ICAM-1 expression also increased. Our results are consistent with the notion that endocan is a pro-inflammatory mediator that induces VCAM-1 and ICAM-1 expression [20]. The beneficial effects of HO-1 appear to be regulated primarily through the degradation of pro-inflammatory heme and the production of the anti-inflammatory molecules CO and bilirubin [34]. The pro-inflammatory mediator LPS has been shown to induce HO-1 gene expression in macrophages, mononuclear phagocytes and endothelial cells [6,34,35]. Previous studies have confirmed that H2 contributes to the induction of HO-1 mRNA and protein expression in injured lung tissue [11]. Our previous research demonstrated that H2 -rich medium boosts HO-1 protein expression and activity in a concentration-dependent manner and that H2 exerts anti-inflammatory properties via HO-1 expression in macrophages stimulated by LPS [6]. Our current observations that LPS or CLP stimulated increased HO-1 expression and activity and that H2 further augmented HO-1 expression in the endothelial cells or lungs of septic mice are consistent with previous reports. Metalloporphyrins, such as ZnppIX, are prototypical inhibitors of HO-1 and are frequently used in in vivo and in vitro experimental models. ZnppIX was used to inhibit HO-1 in our
experiments. We found that ZnppIX completely reversed the regulatory effects of H2 on the survival rate, cell adhesion molecule release and inflammatory factor expression in HUVECs stimulated by LPS and CLP-induced sepsis in mice. These results suggest the antiinflammatory action of H 2 involves HO-1 expression in vitro and in vivo. As is commonly known, HO-1 upregulation is mediated via a redox-dependent signaling pathway that involves activation of the transcription factor Nrf2. Nrf2 is present in the cytoplasm as an inactive complex bound to its partner, Keap1. After encountering harmful stimuli, Nrf2 dissociates from Keap1 to transfer into the nucleus and bind to regulatory sequences (termed antioxidant response elements) located in the promoter regions of genes encoding antioxidants and phase 2 detoxifying enzymes such as HO-1 [36]. The data presented here indicated Nrf2 mRNA and protein expression increased in HUVECs upon LPS stimulation. Nrf2 siRNA was transfected into HUVECs to inhibit Nrf2 expression and to alleviate HO-1 protein expression and activity. Moreover, H 2 had no effect on HO-1 expression and activity in Nrf2 knockout septic mice. The above results verify the pivotal role of H 2 in HO1 expression and activity mediated by the transcription factor Nrf2 during sepsis. These data suggest that molecular H 2 protects against endothelial inflammatory disorders in vitro and in vivo via an Nrf2-mediated HO-1 signaling pathway. Our study has several limitations. We detected the abovementioned indicators at 24 h after LPS stimulation or sepsis induction in mice. We believe that the inflammatory response occurs throughout the entire course of sepsis in endothelial tissues both in vitro and in vivo; however, we only focused on the acute phase of injury and the associated inflammatory response. In a future study, we will determine changes in the expression of inflammatory factors over a longer period. In conclusion, the current study demonstrates that H2 reduces the mass of apoptotic cells, the mortality rate due to sepsis, and the secretion of excessive cell adhesion molecules and inflammatory factors after LPS stimulation or CLP-induced sepsis. Furthermore, H2 exerts beneficial effects with respect to imbalanced inflammatory responses via Nrf2/HO-1 in activated HUVECs and during sepsis in mice. These observations provide new insights into the regulatory mechanisms of H2 in the inflammatory response. Acknowledgments This study was supported by a grant from the National Natural Science Foundation of China (Nos. 81372033 to Yonghao Yu; 81471842 to Keliang Xie), Beijing, China, the Natural Science
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Foundation of the Science Committee (Nos. 13JCQNJC11400 to Keliang Xie).
[19] [20]
References [21] [1] W.C. Aird, The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome, Blood 101 (2003) 3765–3777. [2] P. Henneke, D.T. Golenbock, Innate immune recognition of lipopolysaccharide by endothelial cells, Crit. Care Med. 30 (2002) S207–S213. [3] T. Volk, W.J. Kox, Endothelium function in sepsis, Inflamm. Res. 49 (2000) 185–198. [4] B. Vallet, E. Wiel, Endothelial cell dysfunction and coagulation, Crit. Care Med. 29 (2001) S36–S41. [5] A.V. Page, W.C. Liles, Biomarkers of endothelial activation/dysfunction in infectious diseases, Virulence 4 (2013) 507–516. [6] H.G. Chen, K.L. Xie, H.Z. Han, W.N. Wang, D.Q. Liu, G.L. Wang, et al., Heme oxygenase-1 mediates the anti-inflammatory effect of molecular hydrogen in LPSstimulated RAW 264.7 macrophages, Int. J. Surg. 11 (2013) 1060–1066. [7] I. Ohsawa, M. Ishikawa, K. Takahashi, M. Watanabe, K. Nishimaki, K. Yamagata, et al., Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals, Nat. Med. 13 (2007) 688–694. [8] K. Xie, W. Fu, W. Xing, A. Li, H. Chen, H. Han, et al., Combination therapy with molecular hydrogen and hyperoxia in a murine model of polymicrobial sepsis, Shock 38 (2012) 656–663. [9] K. Xie, Y. Yu, Y. Pei, L. Hou, S. Chen, L. Xiong, et al., Protective effects of hydrogen gas on murine polymicrobial sepsis via reducing oxidative stress and HMGB1 release, Shock 34 (2010) 90–97. [10] K. Xie, Y. Yu, Z. Zhang, W. Liu, Y. Pei, L. Xiong, et al., Hydrogen gas improves survival rate and organ damage in zymosan-induced generalized inflammation model, Shock 34 (2010) 495–501. [11] C.S. Huang, T. Kawamura, Y. Toyoda, A. Nakao, Recent advances in hydrogen research as a therapeutic medical gas, Free Radic. Res. 44 (2010) 971–982. [12] G. Song, H. Tian, S. Qin, X. Sun, S. Yao, C. Zong, et al., Hydrogen decreases atherosusceptibility in apolipoprotein B-containing lipoproteins and aorta of apolipoprotein E knockout mice, Atherosclerosis 221 (2012) 55–65. [13] P. Wiesel, A.P. Patel, N. DiFonzo, P.B. Marria, C.U. Sim, A. Pellacani, et al., Endotoxininduced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase-1-deficient mice, Circulation 102 (2000) 3015–3022. [14] H.P. Zhang, F.L. Zheng, J.H. Zhao, D.X. Guo, X.L. Chen, Genistein inhibits ox-LDLinduced VCAM-1, ICAM-1 and MCP-1 expression of HUVECs through heme oxygenase-1, Arch. Med. Res. 44 (2013) 13–20. [15] T. Kawamura, C.S. Huang, X. Peng, K. Masutani, N. Shigemura, T.R. Billiar, et al., The effect of donor treatment with hydrogen on lung allograft function in rats, Surgery 150 (2011) 240–249. [16] T. Kawamura, N. Wakabayashi, N. Shigemura, C.S. Huang, K. Masutani, Y. Tanaka, et al., Hydrogen gas reduces hyperoxic lung injury via the Nrf2 pathway in vivo, Am. J. Physiol. Lung Cell. Mol. Physiol. 304 (2013) L646–L656. [17] D. Rittirsch, M.S. Huber-Lang, M.A. Flierl, P.A. Ward, Immunodesign of experimental sepsis by cecal ligation and puncture, Nat. Protoc. 4 (2009) 31–36. [18] H. Jiang, P. Yu, D.H. Qian, Z.X. Qin, X.J. Sun, J. Yu, et al., Hydrogen-rich medium suppresses the generation of reactive oxygen species, elevates the Bcl-2/Bax ratio
[22]
[23]
[24] [25] [26]
[27] [28]
[29]
[30] [31]
[32]
[33]
[34]
[35]
[36]
and inhibits advanced glycation end product-induced apoptosis, Int. J. Mol. Med. 31 (2013) 1381–1387. G.M. Li, M.H. Ji, X.J. Sun, Q.T. Zeng, M. Tian, Y.X. Fan, et al., Effects of hydrogen-rich saline treatment on polymicrobial sepsis, J. Surg. Res. 181 (2013) 279–286. W. Lee, S.K. Ku, S.W. Kim, J.S. Bae, Endocan elicits severe vascular inflammatory responses in vitro and in vivo, J. Cell. Physiol. 229 (2014) 620–630. H. Wang, O. Bloom, M. Zhang, J.M. Vishnubhakat, M. Ombrellino, J. Che, et al., HMG1 as a late mediator of endotoxin lethality in mice, Science 285 (1999) 248–251. K. Hayashida, M. Sano, I. Ohsawa, K. Shinmura, K. Tamaki, K. Kimura, et al., Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia– reperfusion injury, Biochem. Biophys. Res. Commun. 373 (2008) 30–35. D.D. Bannerman, S.E. Goldblum, Mechanisms of bacterial lipopolysaccharideinduced endothelial apoptosis, Am. J. Physiol. Lung Cell. Mol. Physiol. 284 (2003) L899–L914. S.M. Dauphinee, A. Karsan, Lipopolysaccharide signaling in endothelial cells, Lab. Investig. 86 (2006) 9–22. R.S. Hotchkiss, K.W. Tinsley, P.E. Swanson, I.E. Karl, Endothelial cell apoptosis in sepsis, Crit. Care Med. 30 (2002) S225–S228. N. Liadis, K. Murakami, M. Eweida, A.R. Elford, L. Sheu, H.Y. Gaisano, et al., Caspase3-dependent beta-cell apoptosis in the initiation of autoimmune diabetes mellitus, Mol. Cell. Biol. 25 (2005) 3620–3629. K.D. Patel, S.L. Cuvelier, S. Wiehler, Selectins: critical mediators of leukocyte recruitment, Semin. Immunol. 14 (2002) 73–81. M.J. Hebert, S.R. Gullans, H.S. Mackenzie, H.R. Brady, Apoptosis of endothelial cells is associated with paracrine induction of adhesion molecules: evidence for an interleukin-1beta-dependent paracrine loop, Am. J. Pathol. 152 (1998) 523–532. F. Paize, R. Sarginson, N. Makwana, P.B. Baines, A.P. Thomson, I. Sinha, et al., Changes in the sublingual microcirculation and endothelial adhesion molecules during the course of severe meningococcal disease treated in the paediatric intensive care unit, Intensive Care Med. 38 (2012) 863–871. T.W. Stief, O. Ijagha, B. Weiste, I. Herzum, H. Renz, M. Max, Analysis of hemostasis alterations in sepsis, Blood Coagul. Fibrinolysis 18 (2007) 179–186. R.L. Qiao, H.S. Wang, W. Yan, L.E. Odekon, V.P. Del, T.J. Smith, et al., Extracellular matrix hyaluronan is a determinant of the endothelial barrier, Am. J. Physiol. 269 (1995) C103–C109. P. Lassalle, S. Molet, A. Janin, J.V. Heyden, J. Tavernier, W. Fiers, et al., ESM-1 is a novel human endothelial cell-specific molecule expressed in lung and regulated by cytokines, J. Biol. Chem. 271 (1996) 20458–20464. S. Sarrazin, E. Adam, M. Lyon, F. Depontieu, V. Motte, C. Landolfi, et al., Endocan or endothelial cell specific molecule-1 (ESM-1): a potential novel endothelial cell marker and a new target for cancer therapy, Biochim. Biophys. Acta 2006 (1765) 25–37. V. Vijayan, E. Baumgart-Vogt, S. Naidu, G. Qian, S. Immenschuh, Bruton's tyrosine kinase is required for TLR-dependent heme oxygenase-1 gene activation via Nrf2 in macrophages, J. Immunol. 187 (2011) 817–827. N. Wijayanti, S. Huber, A. Samoylenko, T. Kietzmann, S. Immenschuh, Role of NF-kappaB and p38 MAP kinase signaling pathways in the lipopolysaccharidedependent activation of heme oxygenase-1 gene expression, Antioxid. Redox Signal. 6 (2004) 802–810. M. Komatsu, H. Kurokawa, S. Waguri, K. Taguchi, A. Kobayashi, Y. Ichimura, et al., The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1, Nat. Cell Biol. 12 (2010) 213–223.