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Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis
Published on: November 28, 2015
MK8617 inhibits M1 macrophage polarization and inflammation via the HIF-1α/GYS1/UDPG/P2Y14 pathway
Lingling Qian1, Xiao-Qin Chen2, Deyang Kong3
1Department of Nephrology, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.
Background:
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.
Methods:
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 P2Y14, 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, P2Y14 and inflammatory indexes of macrophages were detected by qRT-PCR and WB.
Results:
MK8617 reduced LPS-induced release of pro-inflammatory factors as well as UDPG secretion and P2Y14 expression. UDPG upregulated P2Y14 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.
Conclusions:
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/P2Y14 pathway, providing new therapeutic ideas for the study of inflammation.
Insights
MK8617, a novel hypoxia-inducible factor-prolyl hydroxylase inhibitor (HIF-PHI), reduces macrophage inflammation by inhibiting the HIF-1α/GYS1/UDPG/P2Y14 pathway. This discovery offers new therapeutic strategies for inflammatory diseases.
Area of Science:
- Immunology
- Molecular Biology
- Pharmacology
Background:
- Nonresolving inflammation is a significant health concern.
- Hypoxia-inducible factor (HIF) plays a crucial role in inflammatory processes.
- Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) show potential in blocking inflammation by stabilizing HIF.
Purpose of the Study:
- To investigate the anti-inflammatory effects of MK8617, a novel HIF-PHI, on macrophages.
- To elucidate the underlying molecular mechanisms of MK8617's action in regulating macrophage inflammation.
Main Methods:
- Assessed cell viability using CCK8 assay to determine optimal MK8617 concentrations.
- Induced macrophage inflammation with lipopolysaccharide (LPS) in MK8617-treated and untreated cells.
- Quantified inflammatory indicators, uridine diphosphate glucose (UDPG) secretion, and expression of P2Y14, HIF-1α, and GYS1 using qRT-PCR, WB, and ELISA.
- Investigated the effects of UDPG inhibition and knockdown of HIF-1α and GYS1 on macrophage inflammation.
Main Results:
- MK8617 significantly reduced LPS-induced pro-inflammatory factors, UDPG secretion, and P2Y14 expression.
- UDPG was found to upregulate P2Y14 and inflammatory markers, with UDPG inhibition suppressing inflammation.
- HIF-1α was identified as a direct regulator of GYS1, which synthesizes UDPG, thus influencing UDPG secretion.
- Knockdown of HIF-1α or GYS1 abolished the anti-inflammatory effects of MK8617.
Conclusions:
- MK8617 exhibits anti-inflammatory properties in macrophages.
- The mechanism involves the HIF-1α/GYS1/UDPG/P2Y14 pathway, highlighting a novel therapeutic target for inflammation.
