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High fatty acids induce TNF-α as well as NO release mediated by P2X4 receptor and protective effects of puerarin in RAW264.7 cells


Circulating levels of free fatty acids (FFAs) are often found to be increased in patients with type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS). High plasma FFAs may give rise to maladaptive macrophage activation and promote inflammatory responses, which has been proposed as a potential mechanism for the development of DM and MS. P2X4 receptor (P2X4R), a ligand-gated cation channel activated by extracellular adenosine triphosphate (ATP), plays a primary role in regulation of inflammatory responses. Puerarin has been reported to possess a potential anti-inflammatory activity. However, the anti-inflammatory activity of puerarin and the underlying molecular mechanisms under the setting of high FFAs remain unknown. In this study, we found high FFAs increased the expression of P2X4R, cytosolic Ca2+ concentration as well as phosphorylation of extracellular signal-regulated kinase (ERK) and induced the expression of tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) mRNA and the release of TNF-α and nitric oxide (NO) in RAW264.7 macrophages. Such a high FFA-induced inflammation may be reversed by P2X4R selective antagonist 5 BDBD, which manifests the important role of P2X4R in TNF-α and NO release caused by high FFAs in RAW264.7 cells. Molecular docking data showed that puerarin could interfere with the activation of P2X4R by forming hydrogen bonding towards residue Arg267, an important residue essential for canonical activation of P2X4R. Treatment with puerarin dose-dependently reduced high FFA-elevated P2X4R expression and inhibited P2X4R-medicated inflammatory signalling, including high FFA-evoked [Ca2+]i, ERK phosphorylation, expression of TNF-α and iNOS mRNA and release of TNF-α and NO. Our findings emphasize the critical role of P2X4R in high FFA-induced TNF-α and NO release of RAW264.7 macrophages. Puerarin notably counteracts these high FFA-induced adverse effects through its inhibition of P2X4R expression and P2X4R-mediated inflammatory signalling.

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Publication details

The article was received on 09 Apr 2017, accepted on 02 Sep 2017 and first published on 04 Sep 2017

Article type: Paper
DOI: 10.1039/C7FO00544J
Citation: Food Funct., 2017, Accepted Manuscript
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    High fatty acids induce TNF-α as well as NO release mediated by P2X4 receptor and protective effects of puerarin in RAW264.7 cells

    Y. tu, C. Gong, L. ding, X. liu , T. li , F. hu, S. wang, C. xiong, S. liang and H. xu, Food Funct., 2017, Accepted Manuscript , DOI: 10.1039/C7FO00544J

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