Am J Physiol Lung Cell Mol Physiol 292: L960-L971, 2007.
First published January 5, 2007; doi:10.1152/ajplung.00388.2006
1040-0605/07 $8.00
Hydrogen sulfide acts as an inflammatory mediator in cecal ligation and puncture-induced sepsis in mice by upregulating the production of cytokines and chemokines via NF-
B
Huili Zhang,1
Liang Zhi,1
Shabbir Moochhala,1,2
Philip K. Moore,1 and
Madhav Bhatia1
1Department of Pharmacology, National University of Singapore; and 2Center for Biomedical Science, Defence Medical and Environmental Research Institute, Defence Science Organization, Singapore
Submitted 3 October 2006
; accepted in final form 11 December 2006
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ABSTRACT
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Recent studies have implied that hydrogen sulfide (H2S) plays a crucial role in several inflammatory conditions. However, so far little is known about the mechanism by which H2S provokes the inflammatory response in sepsis. Thus the aim of this study was to investigate if H2S regulates sepsis-associated systemic inflammation and production of proinflammatory mediators via the activation of NF-
B. Male Swiss mice were subjected to cecal ligation and puncture (CLP)-induced sepsis and treated with DL-propargylglycine (PAG; 50 mg/kg ip), NaHS (10 mg/kg ip), or saline. PAG, an inhibitor of H2S formation, was administered either 1 h before or 1 h after CLP, whereas NaHS, an H2S donor, was given at the time of CLP. Some normal mice were given NaHS (10 mg/kg ip) to induce lung inflammation with or without pretreatment with the NF-B inhibitor BAY 11-7082. Eight hours after CLP, both prophylactic and therapeutic administration of PAG significantly reduced the mRNA and protein levels of IL-1
, IL-6, TNF-
, monocyte chemotactic protein-1, and macrophage inflammatory protein-2 in lung and liver coupled with decreased activation and translocation of NF-B in lung and liver. Inhibition of H2S formation also significantly reduced lung permeability and plasma alanine aminotransferase activity. In contrast, injection of NaHS significantly aggravated sepsis-associated systemic inflammation and increased NF-B activation. In addition, H2S-induced lung inflammation was blocked by BAY 11-7082. Therefore, H2S upregulates the production of proinflammatory mediators and exacerbates the systemic inflammation in sepsis through a mechanism involving NF-B activation.
DL-propargylglycine; nuclear factor-B
GASEOUS TRANSMITTERS, such as nitric oxide (NO) and carbon monoxide (CO), play important roles in both physiological and pathological conditions. In recent years, interest has been directed towards another naturally occurring gas, hydrogen sulfide (H2S). Two pyridoxal-5-phosphate-dependent enzymes, cystathionine -synthase (CBS; EC 4.2.1.22
[EC]
) and cystathionine 
-lyase (CSE; EC 4.4.1.1
[EC]
), are involved in the majority of the endogenous production of H2S in a wide range of mammalian cells and tissues (20, 24). H2S dilates blood vessels and gastrointestinal smooth muscles by opening muscle K+ATP channels and promotes hippocampal long-term potentiation by enhancing the sensitivity of N-methyl-D-aspartate receptors to glutamate (20, 24).
As a potent vasodilator and atypical neurotransmitter, H2S has been implicated to play a proinflammatory role in various animal models of hindpaw edema (3), acute pancreatitis (4), and lipopolysaccharide (LPS)-induced endotoxemia (19), as well as cecal ligation and puncture (CLP)-induced sepsis (30). In these studies, tissue CSE expression is upregulated, leading to increased H2S biosynthesis, whereas inhibition of H2S formation has been shown to display distinct anti-inflammatory activity (3, 4, 19, 30). In addition, exogenous H2S itself causes lung inflammation in normal mice as evidenced by the elevation in lung MPO (myeloperoxidase) activity and histological damage (19). However, the precise mechanism by which H2S exacerbates inflammatory response is not clear.
In experimental animal models of sepsis and clinical sepsis, a great number of bacterial compounds initiate excessive production and release of cytokines and chemokines (5, 7, 8, 9, 10, 21). The profiles of these chemokines and cytokines also correlate with the severity of sepsis (10). Overproduction and release of inflammatory mediators are mediated by the activation of inducible transcription factors, such as activating protein-1 and nuclear factor-B (NF-B) (6, 13, 22, 26). In light of these findings, the objective of the present study was to explore whether H2S upregulates the production of chemokines and cytokines via activation of NF-B in CLP-induced sepsis, and, if so, whether inhibition of endogenous H2S formation alleviates sepsis-associated systemic inflammation and multiple organ damage.
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MATERIALS AND METHODS
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Induction of sepsis.
All experiments were approved by the animal ethics committee of National University of Singapore and carried out in accordance with the established Guiding Principles for Animal Research. The previously described model of CLP-induced sepsis was used with minor modifications (2, 11). Male Swiss albino mice (25–30 g) were lightly anesthetized with the mixture of ketamine and medetomindine [0.75 ml ketamine (100 mg/ml) and 1 ml medetomindine (1 mg/ml) dissolved in 8.25 ml of distilled water] (7.5 ml/kg) under aseptic conditions. After shaving the abdominal fur and applying a topical disinfectant, a small midline incision was made through the skin and peritoneum of the abdomen to expose the cecum. The cecal appendage was ligated with Silkam 4/0 thread 3–5 mm below the ileocecal valve without occluding the bowel passage and then perforated in two locations with a 22-gauge needle distal to the point of ligation. Next, a small amount of stool was squeezed out through both holes. Finally, the bowel was repositioned, and the abdomen was stitched up with sterile Permilene 5/0 thread. Animals with sham operation underwent the same procedure without CLP.
DL-propargylglycine (PAG; 50 mg/kg ip, Sigma), an irreversible inhibitor of CSE (3, 19, 25), or saline were administered either 1 h before ("prophylactic") or 1 h after ("therapeutic") CLP. In the NaHS intervention experiment, mice underwent CLP operation and were simultaneously given NaHS (10 mg/kg ip, Sigma) or saline. Eight hours after the operation, animals were killed by an intraperitoneal injection of a lethal dose of pentabarbitone (90 mg/kg). Blood samples were drawn from the right ventricles using heparinized syringes and centrifuged (4,000 rpm for 10 min, 0–4°C). Thereafter, plasma was aspirated and stored at –80°C for ELISA and aminotransferase activity assay. Samples of lung and liver were stored at –80°C for subsequent measurement of RT-PCR, ELISA, and NF-B activity assay.
Induction of lung injury by H2S.
Male Swiss albino mice (25–30 g) were intraperitoneally injected with NaHS (10 mg/kg) or saline. BAY 11-7082 (Calbiochem), an inhibitor of NF-B, was dissolved in 1% DMSO and intraperitoneally administered to mice at doses of 5, 10, and 20 mg/kg 30 min before injection of NaHS. Control animals were only injected with vehicle (1% DMSO) 30 min before injection of saline or NaHS. One hour after injection of NaHS, animals were killed by an intraperitoneal injection of a lethal dose of pentabarbitone (90 mg/kg). Samples of lung were harvested and stored at –80°C for subsequent measurement of ELISA and NF-B activity assay.
RT-PCR analysis of tissue cytokine and chemokine mRNA.
Total RNA from lung and liver was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. One microgram of RNA was reverse transcribed using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) at 25°C for 5 min, 42°C for 30 min, followed by 85°C for 5 min. The cDNA was used as a template for PCR amplification by iQ Supermix (Bio-Rad). The primer sequences for detection of IL-1, IL-6, TNF-, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and 18S gene, optimal annealing temperature, optimal cycles, and product sizes are shown in Table 1. PCR amplification was carried out in MyCycler (Bio-Rad). The reaction mixture was first subjected to 95°C for 3 min, followed by an optimal cycle of amplifications consisting of 95°C for 50 s, optimal annealing temperature for 50 s, and 72°C for 1 min. Final extension was at 72°C for 7 min. PCR products were analyzed on 1.5% wt/vol agarose gels containing 0.5 µg/ml ethidium bromide.
Chemokine and cytokine analysis.
For the measurement of cytokines (IL-1, IL-6, and TNF-) and chemokines (MCP-1, MIP-2) in homogenized liver, lung, and plasma, ELISA kits from R&D Systems were used according to the manufacturer's instructions. The lower limits of detection of the levels of IL-1, IL-6, TNF-, MCP-1, and MIP-2 were 15.625, 15.625, 31.25, 3.91, and 15.625 pg/ml, respectively. The ELISA assay for the three cytokines and two chemokines gave reproducible results with interassay variability of less than 9.5% and intra-assay variability of less than 6.5%. Results were then corrected for the DNA content of the tissue samples (17) and were expressed as pg/µg DNA.
Preparation of nuclear extract and determination of NF-B activation.
Nuclear extracts from lung (50 mg) and liver (100 mg) were prepared by using Nuclear Extraction Kit as described by the manufacturer (Active Motif, Tokyo, Japan). Protein concentrations in nuclear extracts were determined using the Bradford assay (Bio-Rad). To monitor NF-B activation in lung and liver tissues, we used a TransAM NF-B p65 Transcription Factor Assay Kit (Active Motif). The kit consisted of a 96-well plate into which oligonucleotide containing the NF-B consensus site (5'-GGGACTTTCC-3') is bound. The active form of NF-B in the nuclear extract specifically binds to this consensus site and is recognized by a primary antibody specific for the activated form of p65 of NF-B. A horseradish peroxidase-conjugated secondary antibody provides the basis for the colorimetric quantification. The absorbance of the resulting solution was measured 2 min later (450 nm with a reference wavelength of 655 nm) using a 96-well microplate reader (Tecan Systems, San Jose, CA). The wild-type consensus oligonucleotide is provided as a competitor for NF-B binding to monitor the specificity of the assay. Results were expressed as fold increase over the control group.
Immunofluorescence.
Mice were anesthetized by the mixture of ketamine and medetomindine and fixed using cardiac perfusion with 4% paraformaldehyde (PFA) in PB (100 mM sodium phosphate buffer, pH 7.4). Lung and liver tissues were removed and fixed by immersion in 4% PFA in PB for 2 h at room temperature. The fixed tissues were cryoprotected in graded sucrose solution (10% and 20% sucrose in PB) for 2 h each, placed in 30% sucrose in PB overnight at 4°C, and were then stored at –80°C until sectioned.
Tissues were embedded in OCT medium (Tissue-Tek) and rapidly frozen in isopentane cooled with liquid nitrogen. Frozen tissues were sectioned at 8-µm thickness by Leica CM 1850 cryostat. The sections were attached on polylysine-coated slides. After the sections were washed three times with PBS for 5 min each, nonspecific staining was blocked by incubation with Ultra-V block (Labvision) for 5 min. The sections were incubated with rabbit anti-human NF-B p65 (Serotec) diluted 1:50 in PBS at 37°C for 1 h. These primary antibodies were detected using goat anti-rabbit IgG conjugated to rhodamine (Santa Cruz). Nuclei were counterstained with the DNA-specific fluorophore 4,6-diamidino-2-phenylindole (Vectashield, Vector Laboratories). Images were captured with an Olympus digital camera mounted on an Olympus microscope.
Measurement of pulmonary microvascular permeability.
Two hours before death, each animal received an intravenous bolus injection containing FITC-albumin (5 mg/kg–1, Sigma). FITC-albumin was dissolved in saline at a concentration of 150 mg/ml. Immediately after death, the lungs were lavaged three times with 1 ml of normal saline. The lavage fluid was combined, and FITC fluorescence was measured in the lavage fluid and plasma (excitation = 494 nm; emission = 520 nm). The ratio of fluorescence emission in lavage fluid to plasma was calculated and used as a measure of pulmonary microvascular permeability.
Alanine aminotransferase assay.
Plasma alanine aminotransferase (ALT) activity was measured using a kinetic spectrophotometric assay (Infinity ALT reagent, Thermo) according to the manufacturer's instructions.
Statistics.
The data were expressed as means ± SE. The significance of differences among groups was evaluated by ANOVA with post hoc Tukey's test when comparing three or more groups. The significance of differences between two groups was evaluated by Student's t-test. P < 0.05 was regarded as statistically significant.
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RESULTS
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Inhibition of H2S formation reduces mRNA expression and production of proinflammatory cytokines and chemokines during sepsis.
Pretreatment with PAG, a CSE inhibitor, 1 h before CLP and posttreatment with PAG 1 h after CLP, significantly decreased the mRNA expression levels of IL-1, IL-6, TNF-, MCP-1, and MIP-2 in both lung and liver (Fig. 1). We next assessed if reduced levels of mRNA expression in the tissues from septic mice with PAG treatment were associated with a corresponding decrease in circulating and tissue cytokine and/or chemokine levels. Levels of IL-1, IL-6, TNF-, MCP-1, and MIP-2 in plasma, lung, and liver were quantified by ELISA from the same mice assessed for mRNA expression. As shown in Figs. 2 and 3, 8 h after the onset of sepsis, plasma and tissue levels of IL-1, IL-6, TNF-, MCP-1, and MIP-2 in both PAG-pretreated and PAG-posttreated mice were significantly lower than those in saline-treated mice (Figs. 2 and 3). In addition, mild background increase in the tissue levels of cytokines and chemokines was observed. The elevation seemed to be correlated with the nonspecific inflammation caused by sham operation.
Exogenous H2S enhances mRNA expression and production of proinflammatory cytokines and chemokines during sepsis.
To explore the effect of H2S on CLP-associated systemic inflammation directly, an H2S donor drug, NaHS, was applied. Injection of NaHS at the time of CLP operation caused a further pronounced rise in the mRNA levels of IL-1, IL-6, TNF-, MCP-1, and MIP-2 in lung and liver compared with saline treatment (Fig. 4). Moreover, NaHS administration also resulted in a significant elevation in the protein levels of IL-1, IL-6, TNF-, MCP-1, and MIP-2 in plasma, lung, and liver 8 h after the induction of sepsis (Figs. 5 and 6).
Effect of H2S on pulmonary and hepatic NF-B activation during sepsis.
Since NF-B plays an essential role in the pathophysiological process of sepsis, H2S was tested for the ability to affect activation and translocation of NF-B. As expected, the activity of NF-B in nuclear extracts of lung and liver was significantly increased in the CLP group compared with sham controls (both P < 0.01, Fig. 7). The increase in the activity of NF-B in nuclear extracts of lung and liver 8 h after CLP was greatly inhibited by PAG prophylactic and therapeutic administration (Fig. 7A). On the other hand, exogenous H2S from injection of NaHS significantly augmented the pulmonary and hepatic NF-B activation in nuclear extract during sepsis compared with saline treatment (Fig. 7B).
Immunohistochemical analysis in lung and liver exhibited clear translocation of NF-B into the nucleus 8 h after the induction of sepsis, whereas minimal translocation of NF-B into the nucleus was seen in sham-operated mice (Figs. 8, A and B, and 9, A and B). PAG prophylactic as well as therapeutic administration markedly attenuated the activation of NF-B in lung and liver from septic mice, leading to less immunoreactivity in the nucleus compared with saline treatment (Figs. 8, C and D, and 9, C and D). In contrast, injection of NaHS at the time of CLP resulted in a further distinct elevation in the activation and recruitment of NF-B to its nuclear target gene in lung and liver (Figs. 8E and 9E). Furthermore, we used polyclonal antibody specific for NF-B p65 subunit to detect the activation and translocation of NF-B. Hence, the result obtained by NF-B activity assay and immunofluorescence also indicated that p65 subunit of NF-B played a crucial role in H2S-induced proinflammatory response during sepsis (Figs. 7–9).
Blockage of NF-B activation alleviates the lung inflammation induced by H2S.
Induction of lung inflammation by administration of NaHS (10 mg/kg ip) to normal mice significantly increased the pulmonary production of proinflammatory cytokines (IL-1, IL-6, and TNF-) and chemokines (MCP-1 and MIP-2) (Fig. 10, B–F). At the same time, a significant rise in the activation of NF-B in nuclear extract of lung was obtained (Fig. 10A). In contrast, inhibition of NF-B activation by BAY 11-7082, a selective inhibitor of NF-B that prevents a step in the phosphorylation of inhibitory protein IB bound to NF-B (15, 23), reduced the H2S-associated inflammatory response in lung in a dose-dependent manner. This was evidenced by the decrease in pulmonary levels of IL-1, IL-6, TNF-, MCP-1, and MIP-2 (Fig. 10, B–F). BAY 11-7082 at a dose of 10 mg/kg could substantially restrain H2S-induced lung inflammation with minimal adverse effect.
Inhibition of H2S formation improves multiple organ dysfunction during sepsis.
As shown in Fig. 11, induction of sepsis by CLP significantly increased the pulmonary microvascular permeability and plasma ALT activity. Injection of PAG 1 h before or 1 h after CLP significantly reduced the pulmonary microvascular permeability and plasma ALT activity and therefore alleviated sepsis-associated multiple organ dysfunction (MOD) (Fig. 11).
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DISCUSSION
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H2S has been well known for several decades as a toxic gas with the smell of rotten eggs. However, it is also generated endogenously during cysteine metabolism in many types of mammalian cells in the reaction catalyzed by CBS (EC4.2.1.22) and CSE (EC4.4.1.1) (20, 24). The expression of CSE and CBS have been identified in human cells and other mammalian cells, including those from liver, kidney, brain, skin fibroblasts, and blood lymphocytes (24).
We have previously reported that CLP-induced sepsis upregulates liver CSE mRNA expression and H2S biosynthesis. Therefore, it causes a significant increase in plasma H2S concentration (30). The metabolic fate of H2S in plasma is far from clear, and substantial amounts may be converted into sulfate or bound to hemoglobin. Blockage of H2S formation by administration of PAG, an inhibitor of CSE enzyme activity, alleviates sepsis-associated systemic inflammation, whereas exogenous H2S results in augmented MPO activity as well as histological changes in lung and liver (30). A similar proinflammatory effect of H2S has also been reported in other animal models of inflammatory diseases, such as hindpaw edema (3), acute pancreatitis (4), and LPS-induced endotoxemia (19). Based on these findings, we have proposed that H2S acts as a proinflammatory mediator in sepsis. However, the precise mechanism by which H2S exacerbates inflammatory response during sepsis remains unknown.
In sepsis, bacterial pathogens and their products trigger the inflammatory response by transcriptional activation of inflammatory genes, leading to the release and production of a large number of inflammatory mediators, including cytokines, chemokines, adhesion molecules, reactive oxygen species, and reactive nitrogen species. Induction of multiple proinflammatory genes is mediated by the activation of inducible transcriptional factors, such as NF-B. Therefore, it is rational to investigate whether endogenous H2S regulates sepsis-associated systemic inflammation and related proinflammatory mediators via the activation of NF-B.
Our findings indicate that the proinflammatory role of H2S in sepsis is mediated by the activation of NF-B and consequent upregulation of transcription of NF-B-dependent proinflammatory genes, including IL-1, IL-6, TNF-, MCP-1, and MIP-2 genes. These cytokines in turn cause activation of NF-B through their respective signaling pathways and exacerbate the disorder of host defense against invading bacteria. On the other hand, inhibition of endogenous H2S formation by administration of PAG significantly reduced the NF-B activation, as evidenced by the reduction in the activity of NF-B in nuclear extracts of lung and liver and immunoreactivity in nucleus. As a result, the mRNA expression levels as well as the production of IL-1, IL-6, TNF-, MCP-1, and MIP-2 in lung and liver were substantially decreased. In contrast, exogenous H2S resulted in a promising and further elevation in the pulmonary and hepatic activation of NF-B and thereby increased both the mRNA and protein expression levels of IL-1, IL-6, TNF-, MCP-1, and MIP-2. In addition, we used polyclonal antibody against p65 subunit to investigate the activation and translocation of NF-B. Hence, our results also demonstrate that NF-B dimers containing p65 seem to play a crucial role in H2S-associated upregulation of proinflammatory gene expression.
To directly investigate whether H2S upregulates inflammatory response via the activation of NF-B, an animal model of H2S-nduced lung inflammation was utilized. Application of NaHS in normal mice resulted in obvious lung inflammation, as evidenced by the elevated level of proinflammatory mediators in lung. H2S-associated lung inflammation also caused the activation of NF-B. Inhibition of the activation of NF-B by pretreatment with BAY 11-7082, a potent inhibitor of NF-B (15, 23), substantially decreased the pulmonary level of proinflammatory mediators and thus alleviated H2S-induced lung inflammation. Therefore, these findings once again ascertain that H2S may provoke inflammatory response via NF-B. Nevertheless, the underlying cellular signaling pathway by which H2S modulates the tissue activity of NF-B in sepsis warrants further investigation.
Recent reports show that direct exposure of human aorta smooth muscle cells (HASMC) to NaHS treatment (27) as well as overexpression of CSE cDNA in HEK-293 cells (28) and HASMC (29) activates ERK and p38 MAPK but not JNK. It is therefore possible that overproduction of endogenous H2S may participate in the activation of MAPK pathway, thereby promoting the activation of NF-B, leading to overproduction of proinflammatory mediators and exacerbating the resulting septic shock. Additionally, H2S causes a dose-dependent increase in intracellular calcium concentration in microglia cells, which may be mediated by cAMP/PKA (18). Since increase in intracellular Ca2+ and PKA facilitates NF-B nuclear translocation and its DNA binding activity (12, 16), it offers an original possibility that H2S may modulate the activity of NF-B and consequent inflammatory response via intracellular calcium homostasis or PKA.
Cross-talk between H2S and NO raises another possible way that H2S may interact with NF-B in response to inflammation. One latest study has showed that H2S enhances NO production and inducible NO synthase expression by potentiating IL-1-induced NF-B activation through a mechanism involving ERK1/2 signaling cascade in rat vascular smooth muscle cells (14). NO provided by nitroflurbiprofen reduces the biosynthesis of H2S in LPS-induced endotoxemia, which may be due to the inhibition of transduction via the NF-B pathway (1). Thus NO and H2S may function together to modulate the inflammatory response and the activity of NF-B in sepsis.
Of even greater significance, we provide the evidence that PAG pretreatment and posttreatment not only blunts systemic inflammation in sepsis but also improves the sepsis-associated multiple organ damage including lung injury and liver dysfunction. As a result, sepsis-associated mortality is also significantly reduced. After 12 days of follow up, nearly 30–40% of septic mice with PAG prophylactic or therapeutic administration survived, whereas 
100% of septic animals without PAG intervention died within 7 days after CLP operation (30). It suggests that inhibition of H2S formation at early stage of sepsis may partially reverse the pathophysiological progression of sepsis. It highlights a potential novel approach to the development of new drugs in this clinical condition. Whether inhibition of H2S will ultimately prove useful and what is the appropriate time to block H2S overproduction in the clinic warrants further investigation.
In addition, investigation of the role of H2S in pathological conditions has been hampered by the paucity of specific pharmacological and genetic tools. Genetic deletion of CSE in mice or rats was not available until now. Most irreversible inhibitors of CSE, including PAG, have been suggested to nonspecifically interfere with other enzymes (24, 25). As with any pharmacological agent, we cannot exclude the possibility that PAG could exert nonspecific effect in sepsis. For these reasons, we carried out H2S donor experiment to increase the veracity of our results. This situation also calls for the necessity to develop specific inhibitors of H2S formation.
In summary, our results show that H2S upregulates the release and production of proinflammatory mediators and exacerbates the systemic inflammation in sepsis through a mechanism involving NF-B activation. Inhibition of H2S formation alleviates sepsis-associated systemic inflammation and MOD. Of course, sepsis is multifactorial and will involve many different mediators other than H2S. Nevertheless, it is important to note that H2S by itself may play a proinflammatory role via the activation of NF-B. The present study may contribute to the understanding of precise mechanism underlying the proinflammatory role of H2S in inflammatory diseases and the development of new drugs to these diseases.
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GRANTS
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This work was supported by Biomedical Research Council Grant R-184-000-094-305 and Office of Life Sciences Cardiovascular Biology Program Grant R-184-000-074-712, National University of Singapore.
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ACKNOWLEDGMENTS
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We thank Mei Leng Shoon for help with the animal experiments and Akhil Kumar Hegde Rama for reading the manuscript.
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FOOTNOTES
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Address for reprint requests and other correspondence: M. Bhatia, Cardiovascular Biology Research Programme, Dept. of Pharmacology, Center for Life Sciences, National Univ. of Singapore, 28 Medical Drive, #03-02, Singapore 117456 (e-mail: mbhatia{at}nus.edu.sg)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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T. Li, B. Zhao, C. Wang, H. Wang, Z. Liu, W. Li, H. Jin, C. Tang, and J. Du
Regulatory Effects of Hydrogen Sulfide on IL-6, IL-8 and IL-10 Levels in the Plasma and Pulmonary Tissue of Rats with Acute Lung Injury
Experimental Biology and Medicine,
September 1, 2008;
233(9):
1081 - 1087.
[Abstract]
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H. Zhang, L. Zhi, S. M. Moochhala, P. K. Moore, and M. Bhatia
Endogenous hydrogen sulfide regulates leukocyte trafficking in cecal ligation and puncture-induced sepsis
J. Leukoc. Biol.,
October 1, 2007;
82(4):
894 - 905.
[Abstract]
[Full Text]
[PDF]
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Copyright © 2007 by the American Physiological Society.
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ABSTRACT
MATERIALS AND METHODS
P < 0.01 when PAG-treated animals were compared with saline-treated animals. 
P < 0.05 when PAG-treated animals were compared with saline-treated animals.
