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Reactive oxygen species downregulate the expression of pro-inflammatory genes by human chondrocytes
M Mathy-Hartert1, G Martin, P Devel
1University of Liege, Institute of Pathology, CHU Sart-Tilman, B-4000 Liège, Belgium.
Objectives:
To determine the regulatory effects of reactive oxygen species (ROS) on the expression by human osteoarthritic chondrocytes of interleukin (IL)-1beta, -6 and -8, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene in response to interleukin (IL)-1beta or lipopolysaccharide (LPS).
Methods:
Human chondrocytes in monolayer culture were incubated for 3 h with ROS generating molecules such as S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 100 microM), 3-morpholinosydnonimine (SIN-1, 100 microM), with chemically synthesised peroxynitrite (ONOO-, 10 microM) or hydrogen peroxide (H2O2, 100 microM). After treatment by ROS, chondrocytes were washed and then cultured for the next 24 h with or without lipopolysaccharide LPS (10 microg/ml) or IL-1beta (1.10(-11) M). IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression was analysed by real time and quantitative RT PCR. IL-6, IL-8 and prostaglandin (PG) E2 productions were assayed by specific immunoassays. Nitrite was measured in the culture supernatants by the Griess procedure.
Results:
LPS and IL-1beta stimulated IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression. SNAP significantly downregulated LPS induced overall gene expressions, whereas SIN-1 had no effect. ONOO- inhibited iNOS and COX-2 gene expression but not that of the cytokine genes. When chondrocytes were incubated with IL-1beta, SIN-1 and ONOO dramatically decreased all gene expressions while SNAP was inefficient. H2O2 treatment inhibited both LPS and IL-1beta induced gene expressions.
Conclusions:
These data provide an evidence that ROS may have anti-inflammatory properties by depressing inflammatory gene expression. Further, we demonstrate that ROS effects are dependent on the nature of radical species and the signalling pathway that is activated. These findings should be taken into consideration for the management of antioxidant therapy in treatment of inflammatory joint diseases.
Insights
Reactive oxygen species (ROS) may possess anti-inflammatory properties by reducing inflammatory gene expression in osteoarthritis. The effects of ROS depend on the specific radical and activated signaling pathway, informing antioxidant therapy for joint diseases.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Osteoarthritis (OA) is characterized by inflammation and cartilage degradation.
- Interleukin-1beta (IL-1beta) and lipopolysaccharide (LPS) are key inflammatory mediators in OA.
- Reactive oxygen species (ROS) play complex roles in cellular processes.
Purpose of the Study:
- To investigate the regulatory effects of ROS on inflammatory gene expression in human OA chondrocytes.
- To determine how different ROS molecules modulate the expression of IL-1beta, IL-6, IL-8, iNOS, and COX-2.
- To understand the impact of ROS on inflammatory pathways activated by IL-1beta or LPS.
Main Methods:
- Human OA chondrocytes were treated with various ROS-generating agents (SNAP, SIN-1, peroxynitrite, hydrogen peroxide).
- Cells were subsequently stimulated with IL-1beta or LPS.
- Gene expression (IL-1beta, IL-6, IL-8, iNOS, COX-2) was analyzed using RT-qPCR.
- Protein production (IL-6, IL-8, PGE2) and nitrite levels were quantified.
Main Results:
- LPS and IL-1beta significantly upregulated inflammatory gene expression.
- S-nitroso-N-acetyl-D,L-penicillamine (SNAP) downregulated LPS-induced gene expression.
- Peroxynitrite (ONOO-) and hydrogen peroxide (H2O2) inhibited specific inflammatory gene expressions.
- The effects of ROS were dependent on the type of ROS and the activating stimulus (IL-1beta or LPS).
Conclusions:
- ROS exhibit potential anti-inflammatory properties by suppressing inflammatory gene expression in OA chondrocytes.
- The anti-inflammatory effects of ROS are species- and pathway-dependent.
- These findings suggest a role for ROS modulation in the therapeutic management of inflammatory joint diseases.