Department of Nephrology, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.
Department of Nephrology, Taizhou School of Clinical Medicine, The Affiliated Taizhou People's Hospital of Nanjing Medical University, Taizhou, Jiangsu, China.
PeerJ. 2023 Jun 30;11:e15591. doi: 10.7717/peerj.15591. eCollection 2023.
Nonresolving inflammation is a major driver of disease and needs to be taken seriously. Hypoxia-inducible factor (HIF) is closely associated with inflammation. Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs), as stabilizers of HIF, have recently been reported to have the ability to block inflammation. We used MK8617, a novel HIF-PHI, to study its effect on macrophage inflammation and to explore its possible mechanisms.
Cell viability after MK8617 and lipopolysaccharide (LPS) addition was assessed by Cell Counting Kit-8 (CCK8) to find the appropriate drug concentration. MK8617 pretreated or unpretreated cells were then stimulated with LPS to induce macrophage polarization and inflammation. Inflammatory indicators in cells were assessed by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blot (WB) and immunofluorescence (IF). The level of uridine diphosphate glucose (UDPG) in the cell supernatant was measured by ELISA. Purinergic G protein-coupled receptor P2Y, as well as hypoxia-inducible factor-1α (HIF-1α) and glycogen synthase 1 (GYS1) were detected by qRT-PCR and WB. After UDPG inhibition with glycogen phosphorylase inhibitor (GPI) or knockdown of HIF-1α and GYS1 with lentivirus, P2Y and inflammatory indexes of macrophages were detected by qRT-PCR and WB.
MK8617 reduced LPS-induced release of pro-inflammatory factors as well as UDPG secretion and P2Y expression. UDPG upregulated P2Y and inflammatory indicators, while inhibition of UDPG suppressed LPS-induced inflammation. In addition, HIF-1α directly regulated GYS1, which encoded glycogen synthase, an enzyme that mediated the synthesis of glycogen by UDPG, thereby affecting UDPG secretion. Knockdown of HIF-1α and GYS1 disrupted the anti-inflammatory effect of MK8617.
Our study demonstrated the role of MK8617 in macrophage inflammation and revealed that its mechanism of action may be related to the HIF-1α/GYS1/UDPG/P2Y pathway, providing new therapeutic ideas for the study of inflammation.
未解决的炎症是疾病的主要驱动因素，需要认真对待。缺氧诱导因子（HIF）与炎症密切相关。缺氧诱导因子脯氨酰羟化酶抑制剂（HIF-PHI）作为 HIF 的稳定剂，最近被报道具有阻断炎症的能力。我们使用新型 HIF-PHI 药物 MK8617 研究其对巨噬细胞炎症的影响，并探索其可能的机制。
通过 Cell Counting Kit-8（CCK8）评估 MK8617 和脂多糖（LPS）添加后细胞的存活率，以找到合适的药物浓度。然后用 LPS 预处理或未预处理的 MK8617 刺激细胞，诱导巨噬细胞极化和炎症。通过实时定量逆转录聚合酶链反应（qRT-PCR）、Western blot（WB）和免疫荧光（IF）评估细胞内炎症指标。通过 ELISA 测量细胞上清液中的尿苷二磷酸葡萄糖（UDPG）水平。通过 qRT-PCR 和 WB 检测嘌呤能 G 蛋白偶联受体 P2Y 以及缺氧诱导因子-1α（HIF-1α）和糖原合酶 1（GYS1）。用糖原磷酸化酶抑制剂（GPI）抑制 UDPG 或用慢病毒敲低 HIF-1α 和 GYS1 后，通过 qRT-PCR 和 WB 检测巨噬细胞的 P2Y 和炎症指标。
MK8617 降低了 LPS 诱导的促炎因子释放以及 UDPG 分泌和 P2Y 表达。UDPG 上调了 P2Y 和炎症指标，而抑制 UDPG 则抑制了 LPS 诱导的炎症。此外，HIF-1α 直接调节 GYS1，后者编码糖原合酶，该酶通过 UDPG 介导糖原的合成，从而影响 UDPG 的分泌。敲低 HIF-1α 和 GYS1 破坏了 MK8617 的抗炎作用。
本研究证实了 MK8617 在巨噬细胞炎症中的作用，并揭示其作用机制可能与 HIF-1α/GYS1/UDPG/P2Y 通路有关，为炎症研究提供了新的治疗思路。
