The Inhibitory Role of M2000 (β-D-Mannuronic Acid) on Expression of Toll-like Receptor 2 and 4 in HT29 Cell Line

Abstract

Background/Objectives:Anti-inflammatory agents play a crucial role in controlling inflammatory diseases such as Inflammatory Bowel Disease (IBD) but their use is restricted due to their vast side effects. M2000 (β-D-mannuronic acid) is a new immunomodulatory drug. According to the capacity of M2000 in suppressing some molecules involved in Toll Like Receptors (TLRs) signaling and reducing oxidative stress we hypothesize that, this molecule may have a potential role in decreasing inflammatory responses in IBD. The aim of this study was to evaluate the cytotoxicity of M2000 and its effect on the gene expression of TLR2 and TLR4.Methods:HEK293 cell line was grown and divided into 96-well cell plate and MTT assay was performed. HT29 cells were cultured and treated with low and high doses of M2000. Total RNA was extracted and cDNA synthesized and quantitative real-time PCR was done to quantify the TLR2 and TLR4 mRNA expression.Results:We found that M2000 at the concentration of ≤ 1000µg/ml had no obvious cytotoxicity effect on the HEK293 cells. Also, low and high doses of M2000 could significantly down-regulate both TLR2 and TLR4 mRNA expression. Moreover, a significant reduction in gene expression of TLR2 and TLR4 in an inflammatory condition resulted in high doses of M2000 in the presence of LPS.Conclusion:Our study which was conducted in colonic epithelial cell model, shows that M2000 can be considered as a new anti-inflammatory agent in IBD. However, more comprehensive experimental and clinical studies are required to recognize the molecular mechanism of M2000 and also its safety and efficacy.

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“…Innate immune cells express Toll-like receptors (TLRs) responsible for the identification of pathogen-associated molecular patterns (PAMPs) and introduce an inflammatory immune response in response to them [78]. Sharifi and colleagues in 2019 showed a significant reduction in gene expression of TLR2 and TLR4 in an intestine cell-line, HT29, under an inflammatory condition and after curing the cells with mannuronic acid [79]. In the same year, Aletaha, et al showed that mannuronic acid can effectively prevent mRNA expression of TLR signaling molecules including MyD88 and NF-κB, in HEK293 cells triggered by a TLR2 agonist (LTA) and a TLR4 agonist (LPS); also, they showed that mannuronic acid decreased LTA and LPS-activated production of TNF-α and IL-6 inflammatory cytokines [80].…”

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“…M2000 is a new member of the group of immunosuppressive agents, with properties of nonsteroidal anti-inflammatory drugs [ 147 ]. The therapeutic benefits of M2000 include the treatment of oxidative stress and inflammation-related disorders such as diabetes, aging, neurodegeneration, inflammatory bowel disease, rheumatoid arthritis, tumors, and cardiac disorders [ 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 ]. M2000 showed protective effects against adjuvant-induced arthritis, experimental autoimmune encephalomyelitis, nephrotic syndrome, and acute glomerulonephritis during in vitro and in vivo examinations with maximum tolerance, and its efficacy was proved in various laboratory experimental models [ 157 , 158 , 159 ].…”