Fig 2: Expression of NLRP3 and pyroptosis-related proteins increased after stimulation with different glucose concentrations in HK-2 cells. (A) Western blot showing the expression of NLRP3, caspase-1 p20, GSDMD-N, IL-1ß, and IL-18 increased in a glucose-concentration-dependent manner. (B) The protein fold change of NLRP3, caspase-1 p20, GSDMD-N, IL-1ß, and IL-18, using Gapdh as a reference gene. Data is presented as the average fold change compared to controls ± s.e.m. n = 3. *P < 0.05 vs. 5.6 mmol/L glucose group; #P < 0.05 vs. 15 mmol/L glucose group; ?P < 0.05 vs. 30 mmol/L glucose group. n = 3.

Fig 3: TLR4/ NF-?B and GSDMD-NT expressions were increased in HK-2 cells under HG condition. (A) WB and densitometric analysis of TLR4 in HK-2 cells exposed to different concentrations of D-glucose. (B) Protein expression of GSDMD-NT exposed to different concentrations of D-glucose. The protein level of TLR4 (A1) and GSDMD-NT(B1) in HK-2 cells exposured to D-glucose (5.5, 17.5, 30, and 45 mM) for 24 h. Each assay was representative of three independent experiments. Data were expressed as means ±SEM. (*P < 0.05 vs. control; **P < 0.05 vs. 17.5Glu group; ***P < 0.05 vs. 30Glu group).

Fig 4: EBE inhibited pyroptosis of cells model by BCL2. (A–F) BCL2 (A), CASPASE9 (B), CASPASE3 (C), CASPASE3-cleaved (D), GSDMD-N (E), and numbers of living cells (F) for RAW 264.7 of vehicle treatment (Control), RAW 264.7 of LPS (Model), model of 50 µg/mL EBE treatment (EBEH) and model of 12.5 µg/mL EBE treatment (EBEL). (G) Transmission electron microscopy images of cells for Control, Model, EBEH and EBEL. Red arrows feature mitochondrial morphology, blue arrows feature membrane pores and yellow arrows feature heterochromatin marginalizing. (A–E) were measured by western blotting. Unpaired t-test and ordinary one-way ANOVA were used on experimental data. Data are presented as mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 5: The expression differences of key molecules in NLRP3 inflammasome-mediated pyrolysis (NLRP3, ASC, caspase-1, IL-1ß, and GSDMD) in mRNA level (A) and in protein level (B) in the control, cisplatin, cisplatin+siNC, and cisplatin+siNLRP3 groups. (C) The difference in the IL-1ß concentrations in the supernatant of each group. Representative results of at least three repeated experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance.