Fig. 4
HZKL inhibits cellular pyroptosis by regulating LCN2 expression. (A,B) Validation of LCN2 overexpression in Caco-2 cells by Western blot (A) and qPCR (B) (mean ± SEM, n = 3 independent biological replicates). (C,D) Effects of HZKL on Caco-2 cell viability in the UC model and UC + LCN2 overexpression model assessed by CCK-8 assay. Groups included Blank, HZKL-L, HZKL-M, and HZKL-H (mean ± SEM, n = 5 independent experiments). (E) Flow cytometric analysis of cell death using 7-AAD/PI double staining in different treatment groups (Control, Model, HZKL, Model + LCN2-OE, Model + LCN2-OE + HZKL, Model + LCN2-OE + anti-LCN2). (F,G) Quantitative analysis of 7-AAD and PI-positive cell populations (mean ± SEM, n = 3 independent biological replicates). (H) Western blot analysis of pyroptosis-related proteins (NLRP3, ASC, Pro-Caspase-1, Caspase-1, GSDMD, and GSDMD-N) with GAPDH as a loading control. (I–N) Densitometric quantification of protein expression levels shown in (H) (mean ± SEM, n = 3 independent biological replicates). (O,P) Immunofluorescence double staining showing co-localization of LCN2 (green) with GSDMD or NLRP3 (red); nuclei were counterstained with DAPI (blue). Yellow signals indicate co-localization (scale bar: 100 μm). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
