Fig 2: 5-HMF inhibits NLRP3 inflammasome by down-regulating ER stress. BMDM were transfected with ATF4 and CHOP mRNA as indicated. After 48 h, cells were incubated with 5-HMF (50 µg/ml) or PBS for 30 min, followed by LPS (500 ng/ml) for 6 h and ATP (5 mM) for 1 h (A) IL-1ß levels in culture supernatants. (B) Immunoblot showing levels of indicated proteins in the culture supernatants (SN) and cell lysates (CL). (C,D) The relative band densities of IL-1ß (C), caspase-1 p10 (D) in supernatant. (E) Representative immunofluorescence images showing ASC speck formation (×400), Scale bar, 500 µm. (F) The percentage of cells containing ASC-specks in (E). (G,H) Immunoblot showing ASC oligomerization in pellets cross-linked by DSS.

Fig 3: Inhibition of ER stress by 5-HMF mediates amelioration of LPS-induced acute lung injury. C57BL/6J mice (5 mice/group) pretreated with PBS or 5-HMF (12 mg/kg, i. p.) and/or tunicamycin (TM, 0.2 mg/kg, i. p.) were challenged with LPS (1 mg/kg, i. p.). (A) Representative images of H&E-stained lung tissue sections from the indicated groups (×200), Scale bar, 100 µm. (B) Representative IHC images showing in-situ expression of CHOP and GPR78 in lung tissues of mice (×200), Scale bar, 10 µm. (C) Protein content in BALF. (D) MPO activity of lung tissues. (E–I) IL-6 (E), TNF-a (F), IL-1ß (G), IL-8/KC (H) and IL-10 (I) levels in sera. All results are expressed as the mean ± SD: * and ** indicate p < 0.05 and p < 0.01 respectively by Student’s t-test.