Fig 8. Alcohol binges plus MASH diet activates NLRP3 inflammasome and inhibition of NLRP3 attenuates neutrophil infiltration and NETs formation in mice.

Liver RNA was used to determine Nlrp3 (A), Casp1 (B) and Il1b (C) mRNA levels by qPCR. 18s was used to normalize Cq values (n=6–8/group). Liver lysates were used to detect IL-1β by ELISA (D) and by western blot (E-F). Liver lysates were used to detect Cleaved Caspase-1 by western blot (G-H) (n=6–8/group). (I) Feeding schematics for combined liver injury with a therapeutic intervention of MCC950 or NLRP3-KO mice fed on MASH diet plus alcohol as described in Methods. (J-K) ALT & AST levels were measured from serum. (L) Formalin-fixed liver sections were stained with Hematoxylin and eosin representative slides are shown, scale bar =50 μm. (M) IL-1β level in the mice serum was detected by ELISA. (N) Flow cytometry analysis of neutrophils (CD45⁺CD11b⁺Ly6G⁺) in liver immune cells. Levels of CXCL1 (O), NE (P), and LCN2 (Q), in serum as measured by ELISA. Whole-cell liver lysates were used to detect CXCL1 (R), NE (S), and LCN2 (T), and Cit-H3 (U) by ELISA. (V) Formalin-fixed liver sections were stained with Sirius red stain, and representative slides are shown. (n=6–8 mice/group and average of 3–5 images per mice). Scale bar=50 μm. (W-X) Liver lysates were analyzed by western blotting for α-SMA, using GAPDH as a loading control. The densitometric analysis of α-SMA is shown as bar graph. * p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005 (n=6mice/NLRP3-KO group and n=8 mice/all other group).