To date, selective blockade of Toll-like receptor (TLR) signalling has been developed as a new approach for treatment for many inflammatory diseases. As β-D-mannuronic acid (M2000) has been known as an anti-inflammatory molecule in several experimental models, we investigated the antagonistic effects of M2000 on TLR2 and TLR4 downstream signalling transduction pathway in human embryonic kidney (HEK) 293 cell lines overexpressing TLR2/CD14 and the TLR4/MD2/CD14 complex, respectively. M2000 effectively inhibited mRNA expression of MyD88 and p65, major subunit of nuclear factor-κB, in HEK293 cells stimulated by lipoteichoic acid (LTA, a TLR2 agonist) and lipopolysaccharide (LPS, a TLR4 agonist) with no evidence of cytotoxicity. In addition, M2000 also suppressed LTA and LPS-induced production of TNF-α and IL-6 inflammatory cytokines in these cells. Furthermore, the results revealed that M2000 had no significant effect on Tollip mRNA expression as a negative regulator of TLR signalling in aforesaid cells. Overall, these data point to M2000 inhibitory effect on Toll-like receptor (TLR) 2, 4 signalling in HEK293 cells. This information might provide new insights into the possible roles of this small drug in order to introduce it as a TLR signalling pathway inhibitor. However, more studies are needed to confirm β-D-mannuronic acid antagonistic effects including the effects of M2000 on peritoneal isolated macrophages and also on blood cells in patients with inflammatory diseases such as ankylosing spondylitis.
The results of this study revealed that G2013 is able to 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.
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.
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