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The complex effects of the slow-releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells.

Li L, Fox B, Keeble J, Salto-Tellez M, Winyard PG, Wood ME, Moore PK, Whiteman M - J. Cell. Mol. Med. (2013)

Bottom Line: We have now assessed the effect of GYY4137 (a slow-releasing H2 S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro.GYY4137 (0.1-0.5 mM) decreased LPS-induced production of nitrite (NO2 (-) ), PGE2 , TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro.In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity and decreased TNF-α, IL-1β, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Science Research Division, King's College London, London, England.

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Effect of GYY4137 on LPS-induced expression of COX-2, iNOS and TNF-α-converting enzyme (TACE) expression and activity in human synoviocytes and human articular chondrocytes. HAC and HFLS were treated with GYY4137 at the concentrations stated for 1 hr prior to LPS (10 μg/ml) stimulation for 18 hr. After this time, cells were lysed and levels of COX-2 (A) and iNOS (B) determined by commercial ELISA. (C) Expression of pre- and mature TACE was determined in HFLS by Western blotting after 24 hr. (D–F) Effects of GYY4137 on isolated human recombinant COX-2 (D), iNOS (E) and (F) TACE activity. Recombinant enzyme activities are expressed as % control enzyme activity after 1 hr (iNOS and COX-2) and 4 hr (TACE). GYY4137 was added at the concentrations stated and enzyme activity determined. DuP697 (1 μM) was used as a positive control for COX-2 activity and L-NNA (supplied with the NOS activity kit; 100 μM) as positive control for iNOS activity. Western blots are representative of three separate determinations and data shown are mean ± SEM of at least three separate experiments. *P < 0.05 c.f. LPS-stimulated cells.
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fig03: Effect of GYY4137 on LPS-induced expression of COX-2, iNOS and TNF-α-converting enzyme (TACE) expression and activity in human synoviocytes and human articular chondrocytes. HAC and HFLS were treated with GYY4137 at the concentrations stated for 1 hr prior to LPS (10 μg/ml) stimulation for 18 hr. After this time, cells were lysed and levels of COX-2 (A) and iNOS (B) determined by commercial ELISA. (C) Expression of pre- and mature TACE was determined in HFLS by Western blotting after 24 hr. (D–F) Effects of GYY4137 on isolated human recombinant COX-2 (D), iNOS (E) and (F) TACE activity. Recombinant enzyme activities are expressed as % control enzyme activity after 1 hr (iNOS and COX-2) and 4 hr (TACE). GYY4137 was added at the concentrations stated and enzyme activity determined. DuP697 (1 μM) was used as a positive control for COX-2 activity and L-NNA (supplied with the NOS activity kit; 100 μM) as positive control for iNOS activity. Western blots are representative of three separate determinations and data shown are mean ± SEM of at least three separate experiments. *P < 0.05 c.f. LPS-stimulated cells.

Mentions: To determine whether GYY4137 affected the levels of COX-2 or iNOS enzymes, intracellular levels of these proteins were determined in LPS-treated cells by ELISA. Treatment of HFLS or HAC with GYY4137 for 1 hr prior to LPS significantly reduced LPS-induced COX-2 (Fig. 3A) and iNOS (Fig. 3B) protein levels in addition to reducing PGE2 and NO2− levels. Although treatment of HFLS with a cocktail of cytokines (TNF-α, IL-1β and IFN-γ) induced an increase in the levels of TACE protein (Fig. 3C), the level of this enzyme was unaffected by GYY4137. To examine further the possibility that the attenuation of LPS-stimulated increases in PGE2, •NO and TNF-α (Figs 1 and 2) were due to an inhibitory effect of GYY4137 on COX-2, iNOS and TACE activity, respectively, we incubated human recombinant COX-2, iNOS and TACE with GYY4137 and determined residual catalytic activity. GYY4137 and Na2S significantly inhibited COX-2, iNOS and TACE activity (Fig. 3D–F), respectively, suggesting that H2S could directly inhibit enzyme activity and consequently cellular synthesis/secretion of PGE2, NO2− and TNF-α which is independent of any effect of this drug on COX-2 or iNOS protein levels.


The complex effects of the slow-releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells.

Li L, Fox B, Keeble J, Salto-Tellez M, Winyard PG, Wood ME, Moore PK, Whiteman M - J. Cell. Mol. Med. (2013)

Effect of GYY4137 on LPS-induced expression of COX-2, iNOS and TNF-α-converting enzyme (TACE) expression and activity in human synoviocytes and human articular chondrocytes. HAC and HFLS were treated with GYY4137 at the concentrations stated for 1 hr prior to LPS (10 μg/ml) stimulation for 18 hr. After this time, cells were lysed and levels of COX-2 (A) and iNOS (B) determined by commercial ELISA. (C) Expression of pre- and mature TACE was determined in HFLS by Western blotting after 24 hr. (D–F) Effects of GYY4137 on isolated human recombinant COX-2 (D), iNOS (E) and (F) TACE activity. Recombinant enzyme activities are expressed as % control enzyme activity after 1 hr (iNOS and COX-2) and 4 hr (TACE). GYY4137 was added at the concentrations stated and enzyme activity determined. DuP697 (1 μM) was used as a positive control for COX-2 activity and L-NNA (supplied with the NOS activity kit; 100 μM) as positive control for iNOS activity. Western blots are representative of three separate determinations and data shown are mean ± SEM of at least three separate experiments. *P < 0.05 c.f. LPS-stimulated cells.
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Related In: Results  -  Collection

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fig03: Effect of GYY4137 on LPS-induced expression of COX-2, iNOS and TNF-α-converting enzyme (TACE) expression and activity in human synoviocytes and human articular chondrocytes. HAC and HFLS were treated with GYY4137 at the concentrations stated for 1 hr prior to LPS (10 μg/ml) stimulation for 18 hr. After this time, cells were lysed and levels of COX-2 (A) and iNOS (B) determined by commercial ELISA. (C) Expression of pre- and mature TACE was determined in HFLS by Western blotting after 24 hr. (D–F) Effects of GYY4137 on isolated human recombinant COX-2 (D), iNOS (E) and (F) TACE activity. Recombinant enzyme activities are expressed as % control enzyme activity after 1 hr (iNOS and COX-2) and 4 hr (TACE). GYY4137 was added at the concentrations stated and enzyme activity determined. DuP697 (1 μM) was used as a positive control for COX-2 activity and L-NNA (supplied with the NOS activity kit; 100 μM) as positive control for iNOS activity. Western blots are representative of three separate determinations and data shown are mean ± SEM of at least three separate experiments. *P < 0.05 c.f. LPS-stimulated cells.
Mentions: To determine whether GYY4137 affected the levels of COX-2 or iNOS enzymes, intracellular levels of these proteins were determined in LPS-treated cells by ELISA. Treatment of HFLS or HAC with GYY4137 for 1 hr prior to LPS significantly reduced LPS-induced COX-2 (Fig. 3A) and iNOS (Fig. 3B) protein levels in addition to reducing PGE2 and NO2− levels. Although treatment of HFLS with a cocktail of cytokines (TNF-α, IL-1β and IFN-γ) induced an increase in the levels of TACE protein (Fig. 3C), the level of this enzyme was unaffected by GYY4137. To examine further the possibility that the attenuation of LPS-stimulated increases in PGE2, •NO and TNF-α (Figs 1 and 2) were due to an inhibitory effect of GYY4137 on COX-2, iNOS and TACE activity, respectively, we incubated human recombinant COX-2, iNOS and TACE with GYY4137 and determined residual catalytic activity. GYY4137 and Na2S significantly inhibited COX-2, iNOS and TACE activity (Fig. 3D–F), respectively, suggesting that H2S could directly inhibit enzyme activity and consequently cellular synthesis/secretion of PGE2, NO2− and TNF-α which is independent of any effect of this drug on COX-2 or iNOS protein levels.

Bottom Line: We have now assessed the effect of GYY4137 (a slow-releasing H2 S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro.GYY4137 (0.1-0.5 mM) decreased LPS-induced production of nitrite (NO2 (-) ), PGE2 , TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro.In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity and decreased TNF-α, IL-1β, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Science Research Division, King's College London, London, England.

Show MeSH
Related in: MedlinePlus