ABSTRACT
INTRODUCTION: Inhibition of Toll-like receptors (TLRs) signaling plays a crucial role in suppressing the inflammation and available data presenting G2013 as an immunomodulatory agent, therefore, we designed this study to answer whether G2013 can affect the signaling pathway of TLR2 and TLR4. METHODS: Cytotoxicity study of G2013 was performed by MTT assay. HEK293 TLR2 and HEK293 TLR4 cell lines were cultured and treated with low dose (5µg/ml) and high dose (25µg/ml) of G2013 for 24 hours. Gene expressions of MyD88, Tollip, and NF-κB were defined by quantitative real-time PCR. RESULTS: The cytotoxicity assay showed that the concentrations lesser than 125µg/ml of G3012 had no apparent cytotoxicity, however, the concentrations of 5µg/ml and 25µg/ml could suppress the mRNA expression of MyD88, Tollip and NF-κB in HEK293 TLR2 and HEK293 TLR4 cell lines. CONCLUSION: in our study, we verified the linkage between the immunosuppressive property of G2013 and TLR2, TLR4 signaling cascade; but so far, the specific target of G2013 and its molecular mechanism has not been detected yet. We recommend further studies on other Patten Recognition Receptors (PRRs)and other mechanisms of inflammation like oxidative stress to be conducted in the future.
Subject(s)
Hexuronic Acids/pharmacology , Toll-Like Receptor 2/genetics , Toll-Like Receptor 4/genetics , Cell Line , Dose-Response Relationship, Drug , Gene Expression/drug effects , HEK293 Cells , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolismABSTRACT
BACKGROUND: The non-steroidal anti-inflammatory drugs (NSAIDs) play crucial role in the controlling of inflammatory diseases. Due to the vast side effects of NSAIDs, its use is limited. G2013 or α-L-Guluronic Acid is a new NSAID with immunomodulatory features. OBJECTIVES: Considering the leading role of TLRs in inflammatory responses, in this study, we aimed to evaluate G2013 cytotoxicity and its effect on the expression of TLR2 and TLR4 molecules. METHODS: HEK293-TLR2 and HEK293-TLR4 cells were cultured and seeded on 96-well cell plate, and MTT assay was performed for detecting the viability of the cells after treatment with different concentrations of G2013. HT29 cells were grown and treated with low and high doses of G2013. After total RNA extraction and cDNA synthesis, quantitative real-time PCR were performed to assess the TLR2 and TLR4 mRNA synthesis. RESULTS: We found that concentrations of ≤125 µg/ml of G2013 had no apparent cytotoxicity effect on the HEK293-TLR2 and -TLR4 cells. Our results indicated that after G2013 treatment (5 µg/ml) in HT29 cells, TLR2 and TLR4 mRNA expression decreased significantly compared with the untreated control group (p=0.02 and p=0.001 respectively). CONCLUSION: The results of this study revealed that G2013 can down regulate the TLR2 and TLR4 gene expression and exerts its inhibitory effect. Our findings are parallel to our previous finding which showed G2013 ability to down regulate the signaling pathway of TLRs. However, further studies are needed to identify the molecular mechanism of G2013.
Subject(s)
Hexuronic Acids/pharmacology , Immunologic Factors/pharmacology , Toll-Like Receptor 2/genetics , Toll-Like Receptor 4/genetics , Cell Survival/drug effects , Gene Expression Regulation/drug effects , HEK293 Cells , HT29 Cells , Humans , RNA, Messenger/metabolismABSTRACT
Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease characterized by the inflammation of sacroiliac joints and axial skeleton. A combination of genetic, environmental and immunological factors are involved in AS's pathogenesis. TLRs are type I transmembrane glycoproteins that play a crucial role in the innate immune responses against invading pathogens. Observational studies have demonstrated a possible association between TLR dysregulation and AS. The ß-d-mannuronic acid (M2000), as a novel NSAID with immunosuppressive property, has shown an inhibitory effect on Toll-like receptor (TLR) 2, 4 signaling in HEK293 cells. In the present study, we investigated the gene expression of Myd88, IKB-alpha, NF-kB and MAPK14 (genes of the TLR/NF-kB Signaling Pathway) in AS patients in comparison to healthy subjects and also the effect of ß-d-mannuronic acid on disease activity and mRNA expression of these molecules in affected patients. We showed for the first time that the gene expression level of Myd88, IKB-alpha, NF-kB and MAPK14 was higher in AS patients in comparison to healthy subjects. Moreover we confirmed that the ß-d-mannuronic acid not just reduced significantly the disease activity of AS individuals compared to placebo, but also it could significantly decrease the expression level of genes associated with TLR/NF-kB Signaling Pathway in treated patients with M2000. These results may provide a new therapeutic approach to attenuate inflammatory responses in AS patients, (Identified; IRCT 2013062213739N1).
