Anti-Inflammatory Activity of Adenosine 5′-Trisphosphate in Lipopolysaccharide-Stimulated Human Umbilical Vein Endothelial Cells Through Negative Regulation of Toll-Like Receptor MyD88 Signaling

摘要

Activation of TLR4-MyD88-NF-κB signaling by lipopolysaccharide (LPS) evokes a proinflammatory immune response, and plays a pivotal role in initiation and progression of atherosclerosis (AS). ATP (adenosine 5′-trisphosphate), a powerful extracellular signal transduction molecule, functions to regulate immune inflammatory responses depending on the type of P2 receptors and cell lines. In this study, we first performed RT-PCR to detect the mRNA expression of monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-1β induced by different concentrations of LPS in human umbilical vein endothelial cells (HUVECs). Protein level of TLR4 signaling including TLR4, myeloid differentiation factor (MyD88), and CD14 induced by LPS (1?μg/mL) at different times (0, 10, 30, 60, 120?min) was analyzed by Western blot. Then, RT-PCR was performed to detect the effect of different concentrations of ATP on mRNA expression of IL-1β and MCP-1 induced by LPS (1?μg/mL) and the TLR4 signaling pathway. Western blot was performed to detect the effect of low concentrations of ATP on phosphorylation of p65 induced by 1?μg/mL LPS. Finally, we used P2Y receptor blocker Suramin to verify whether the role of ATP on LPS-induced inflammatory cytokine expression was through P2Y receptors. The results showed that LPS upregulated the expression of MCP-1, IL-8, and IL-1β in a dose-dependent manner accompanied by the activation of TLR4-MyD88 signaling in HUVECs. Only low concentration ATP (1, 10?μM) inhibited LPS-induced mRNA expression of IL-1β and MCP-1. ATP at low concentrations also downregulated the mRNA expression of TLR4, CD14, and MyD88 and inhibited LPS-induced phosphorylation of p65. Furthermore, Suramin, a nonspecific P2Y receptor antagonist, did not attenuate the inhibition of ATP on LPS-induced IL-1β and MCP-1 expression. Taking this together, low concentration ATP inhibited LPS-induced inflammation in HUVECs by negatively regulating TLR4-MyD88 signaling, and P2Y receptors were not involved in this process, which might provide new ideas for prevention and treatment of inflammatory diseases such as AS.