Department of Geriatric Cardiology, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, China.
School of Stomatological, WuHan University, Wuhan, Hubei, China.
DNA Cell Biol. 2019 Dec;38(12):1557-1563. doi: 10.1089/dna.2019.4773. Epub 2019 Oct 3.
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.
脂多糖（LPS）激活 TLR4-MyD88-NF-κB 信号通路会引发促炎免疫反应，并在动脉粥样硬化（AS）的发生和发展中起关键作用。三磷酸腺苷（ATP）是一种强大的细胞外信号转导分子，根据 P2 受体和细胞系的类型发挥作用，调节免疫炎症反应。在这项研究中，我们首先通过 RT-PCR 检测不同浓度 LPS 诱导的人脐静脉内皮细胞（HUVEC）中单核细胞趋化蛋白-1（MCP-1）、IL-8 和 IL-1β 的 mRNA 表达。通过 Western blot 分析 LPS（1μg/ml）在不同时间（0、10、30、60、120min）诱导的 TLR4 信号通路中 TLR4、髓样分化因子（MyD88）和 CD14 的蛋白水平。然后，通过 RT-PCR 检测不同浓度的 ATP 对 LPS（1μg/ml）诱导的 IL-1β 和 MCP-1mRNA 表达及 TLR4 信号通路的影响。通过 Western blot 检测低浓度 ATP 对 1μg/ml LPS 诱导的 p65 磷酸化的影响。最后，我们使用 P2Y 受体阻滞剂苏拉明验证 ATP 对 LPS 诱导的炎症细胞因子表达的作用是否通过 P2Y 受体。结果表明，LPS 呈剂量依赖性地上调 MCP-1、IL-8 和 IL-1β 的表达，并激活 HUVEC 中的 TLR4-MyD88 信号通路。只有低浓度的 ATP（1、10μM）抑制 LPS 诱导的 IL-1β 和 MCP-1mRNA 表达。低浓度的 ATP 还下调 TLR4、CD14 和 MyD88 的 mRNA 表达，并抑制 LPS 诱导的 p65 磷酸化。此外，P2Y 受体非特异性拮抗剂苏拉明不能减弱 ATP 对 LPS 诱导的 IL-1β 和 MCP-1 表达的抑制作用。综上所述，低浓度的 ATP 通过负调控 TLR4-MyD88 信号通路抑制 LPS 诱导的 HUVEC 炎症，而 P2Y 受体不参与这一过程，这可能为 AS 等炎症性疾病的防治提供新的思路。
