Journal: Cellular & Molecular Biology Letters
Article Title: GPR43 deficiency aggravates sepsis by promoting gut microbiota–dependent barrier disruption and HIF-1α–ENO1 axis–mediated M1 polarization of macrophages
doi: 10.1186/s11658-025-00833-4
Figure Lengend Snippet: GPR43 deficiency promotes glycolysis-driven macrophage M1 polarization activation. A , B Heat map of proinflammatory cytokine gene ( A ) and glycolysis-related gene ( B ) expression in WT and Gpr43 −/− BMDMs ± LPS + IFN-γ. C qRT–PCR validation of glycolytic enzyme gene expression in WT and Gpr43 −/− BMDMs following M1 polarization ( n = 3). D Representative immunoblots of glycolysis related proteins levels in M1-polarized WT and Gpr43 −/− BMDMs. The protein levels are shown in Supplementary Fig. S5D. E Lactate production in culture supernatants of M1-polarized WT and Gpr43 −/− BMDMs ( n = 3). F , G Glucose uptake (2-NBDG) measured by flow cytometry in M1-polarized WT and Gpr43 −/− BMDMs ( n = 3). Representative histograms ( G ) and quantification of 2-NBDG MFI ( F ). H–K Seahorse XF glycolysis stress test in M1-polarized WT and Gpr43 −/− BMDMs ( n = 3). ECAR traces ( H ) with sequential injections: glucose (10 mM), rotenone/antimycin A (0.5 µM), and 2-deoxyglucose (50 mM). Quantification of glycolysis ( I ), glycolytic capacity ( J ), and glycolytic reserve ( K ) from ECAR data. L – O Seahorse XF mitochondrial stress test in M1-polarized WT and Gpr43 −/− BMDMs ( n = 3). OCR traces ( L ) with sequential injections: oligomycin (1.5 µM), FCCP (1 µM), and rotenone/antimycin A (0.5 µM each). Quantification of basal respiration ( M ), maximal respiration ( N ), and spare respiratory capacity ( O ) from OCR data. Data in C , E , F , I – K and M–O were obtained from three independent experiments are presented as means ± SD. Statistical analysis by two-tailed unpaired Student’s t -test or one-way ANOVA with Tukey’s post hoc test, as appropriate. The data in A , B were obtained from RNA-seq analysis. ns, not significant; * p < 0.05, **** p < 0.0001
Article Snippet: For the inhibitor or agonist treatments, BMDMs were pretreated with glycolysis inhibitor 2-deoxyglucose (2-DG) (2 mM, 6 h, MCE, cat. no. HY-13966), ENO1 inhibitor AP-III-a4 (20 μM, 6 h, MCE, cat. no. HY-15858), HIF-1α inhibitor acriflavine (5 μM, 6 h, MCE, cat. no. HY-100575), or the GPR43 agonist TUG-1375 (20 μM, 6 h, MCE, cat. no. HY-112813) before M1 polarization.
Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Biomarker Discovery, Gene Expression, Western Blot, Flow Cytometry, Two Tailed Test, RNA Sequencing
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