MK8617 inhibits M1 macrophage polarization and inflammation via the HIF-1α/GYS1/UDPG/P2Y14 pathway

Abstract

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.

Keywords: Glycogen synthase 1; Hypoxia-inducible factor-prolylhydroxylase inhibitor; Inflammation; Uridine diphosphate glucose/P2Y14 signaling pathway.

Figure 1. MK8617 attenuates M1 macrophage inflammation.

(A) Western blotting result of CD80. (B) Quantification of the data in (A). mRNA levels were determined using RT-PCR: (C) TNF-α, (D) IL-β and (E) IL-6. (F) Western blotting result of iNOS. (G) Quantification of the data in (F). (H) iNOS expression was determined by immunofluorescence. Scale bars, 100 μm. Data are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. “−” represents none and “+” represents yes.

Figure 2. MK8617 inhibits the production of UDPG and P2Y₁₄ in M1 macrophages.

(A) The supernatant was collected for determination of UDPG content by ELISA. (B) P2Y₁₄ mRNA level was determined using qRT-PCR. (C) Western blotting result of P2Y₁₄. (D) Quantification of the data in (C). (E) P2Y₁₄ expression was determined by IF. Scale bars, 100 μm. Data are mean ± SEM (n = 3). *p < 0.05, **p < 0.01.

Figure 3. UDPG binding to P2Y₁₄ receptors induces macrophage polarization and inflammation.

(A) mRNA expression of P2Y₁₄ by qRT-PCR. (B) Western blotting result of P2Y₁₄. (C) Quantification of the data in (B). (D) P2Y₁₄ expression was determined by IF. Scale bars, 100 μm. (E) Western blotting result of CD80. (F) Quantification of the data in (E). mRNA levels were determined by qRT-PCR: (G) TNF-α; (H) IL-1β and (I) IL-6. (J) Protein expression of iNOS by WB. (K) Quantification of the data in (J). (L) iNOS expression was determined by IF. Scale bars, 100 μm. Data are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4. MK8617 reduces UDPG and P2Y₁₄ production through upregulation of HIF-1α/GYS1.

mRNA levels were determined by qRT-PCR: (A) HIF-1α (B) GYS1. (C–E) WB results: HIF-1α and GYS1. (F–H) qRT-PCR and WB determination of mRNA and protein levels of GYS1 after HIF-1α was knocked down. (I) Supernatant was collected to determine UDPG content by ELISA. (J–L) mRNA and protein levels of P2Y₁₄ were determined by RT-PCR and WB. Data are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5. HIF-1α and GYS1 knockdown disrupt MK8617-induced suppression of inflammation.

HIF-1α was knocked out in macrophages and then LPS was used to induce inflammation. mRNA levels of inflammatory cytokines were determined using qRT-PCR: (A) TNF-α (B) IL-β (C) IL-6 (D, E) WB was used to determine iNOS expression. GYS1 was knocked out in macrophages and then LPS was used to induce inflammation. mRNA levels of inflammatory cytokines were determined using qRT-PCR: (F) TNF-α (G) IL-β (H) IL-6 (I, J) WB was used to determine iNOS expression. Data are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6. MK8617 inhibits M1 macrophage inflammation via the HIF-1α/GYS1/UDPG/P2Y₁₄ pathway.

HIF-PHI upregulated GYS1 by stabilizing HIF-1α, and GYS1 was able to mediate glycogen synthesis by UDPG. Secretion of extracellular UDPG bound to P2Y₁₄ was thus reduced and the initiation of inflammation was inhibited.