Fig 2: K193 lactylation augmented Eno1 enzymatic activity. (A) The levels of lactate in the culture medium of PBS‐ and LPS‐treated MPMECs over time (n = 3). (B) PBS‐ and LPS‐treated MPMECs lysates were prepared to measure Eno1 enzymatic activity by monitoring phosphoenolpyruvate concentrations over time (n = 3). (C) Eno1 enzymatic activity was measured in lysates from shEno1+oe‐WT and shEno1+oe‐K193R MPMEC (n = 6). *p < 0.05. In C, *compared with the untreated shEno1+oe‐WT group, *p < 0.05; #compared with the lactate‐treated shEno1+oe‐K193R group, # p < 0.05.

Fig 3: ENO1 was increased in KCs after HS. KCs were isolated from Sham and HS mice, and ENO1 mRNA (A) was assessed by qPCR. Isolated KCs were stained with the surface marker F4/80 and CD11b, along with intracellular ENO1, and ENO1 protein expression (B–E) was analyzed by flow cytometry. Results are displayed as mean ± SEM (n = 5–6/group). KCs were isolated from healthy mice and subjected to H/R, and ENO1 mRNA (F) was measured by qPCR, while protein expression (G) was assessed by Western blot. Results are displayed as mean ± SEM (n = 3–5/group). All data were subjected to statistical analysis by an unpaired t-test. * p < 0.05 vs. Sham.

Fig 4: Increased protein lactylation in the PECs of ARDS mice, with Eno1 as a substrate. (A) Histogram of the ratio distribution of quantifiable Klac sites in the PECs of control and LPS‐induced ARDS mice. (B) GO items associated with Klac sites (ARDS/Control > +∞) located proteins. (C) Venn diagram of predicted CXCL12 3′ UTR binding proteins and proteins in picked GO items and the average protein copies of 13 screened proteins in PECs. (D) Ribbon diagram of the crystal structure of mouse Eno1 and lactylation at the K193 residue. (E) Mass spectrometry of Eno1 lactylated at K193. (F) Purified mouse Eno1 (100 µg/mL) was incubated with different concentrations of lactyl‐CoA for 1 h at 37°C. Then, the mixtures were added to protein loading buffer for denaturation, and K‐lactylation was assessed by western blotting. (G) The K193 site in Eno1 is conserved in different species. Conserved K193 sites are marked in red in the sequences among different species. (H) Western blotting analysis of the K193 lactylated level in PECs isolated from the lungs of control and LPS‐induced ARDS mice. (I) In vitro, the K193 lactylation levels in PBS‐ and LPS‐activated MPMECs were measured by western blotting.

Fig 5: ENOblock alleviates liver inflammation and ENO1 activity in HS. Liver tissues from Sham and HS mice treated with either Vehicle or ENOblock were harvested, and mRNA was extracted for analysis. The mRNA expression of cytokines IL-1β (A), TNF-α (B), and IL-6 (C), as well as chemokines MIP-2 (D) and KC (E), was measured by qPCR. Additionally, KCs were isolated from different groups, and ENO1 glycolytic activity was determined by an enolase activity assay kit (F). Results are presented as mean ± SEM (n = 8–9/group for mRNA assessment, n = 6–7/group for activity assay) and were subjected to statistical analysis through ANOVA and the SNK test. * p < 0.05 vs. Sham; # p < 0.05 vs. HS-Veh.