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. 2022 Feb 9;71(12):2539–2550. doi: 10.1136/gutjnl-2021-325150

Figure 8.

Figure 8

Pyruvate kinase M2 (PKM2) activator inhibits follistatin-like protein 1 (FSTL1)-mediated inflammation and glycolysis in macrophages. Bone marrow-derived macrophages (BMDMs) with FSTL1 overexpression (FSTL1o/e) or normal expression (FSTL1n/e) and FSTL1o/e BMDMs treated with DASA-58 (50 μM, 1 hour) were treated by lipopolysaccharide (LPS) stimulation. BMDMs treated with 100 ng/mL LPS for 24 hours (A–G): (A–B) messenger RNA (mRNA) expression and protein expressions of tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-10 was determined; (C) dual-immunofluorescence staining of PKM2 and inducible nitric oxide synthase (iNOS); (D) protein expression of PKM2, p-PKM2, iNOS, p-STAT1 and p-STAT6; (E) mRNA expression of iNOS and Arg1 was measured; (F) immunofluorescence staining of p65 and DAPI; (G) protein expression of toll-like receptor (TLR)-4, p-nuclear factor kappa B (NF-κB) and p-Ikk. (H) Lactate acid production was measured from BMDMs in response to LPS (100 ng/mL, 6 hours). (I) Glycolytic rate and compacities of BMDMs were measured by real-time recording of extracellular acidification rates (ECAR) after successively injection of glucose (Glc), oligomycin (O) and 2-DG. (J) FSTL1FL/FL and FSTL1M-KO BMDMs treated with LPS (100 ng/mL) for 4 hours. These cells were washed twice and pretreated with 10 mM 2-DG for 1 hour, followed by treatment with or without LPS (100 ng/mL) for another 4 hours. mRNA levels or IL-1β and iNOS were determined. (K) Schematic illustration how FSTL1 complexed with PKM2 to promote phosphorylation and nuclear import of PKM2 and enhance cytoplasmic PKM2 stability in reprograming macrophage function during liver fibrosis. Data were presented as mean±SEM of at least three independent experiments; scale bars, 50 µm; *p<0.05, **p<0.01.