Fig 2: GSDMD is involved in the heme-inducing SIRS in mice. (a) The expression of GSDMD, GSDMD-N, Casp1, and Casp1 p20 in HLMVECs after NLRP3 knockdown were assayed by Western blotting after heme treatment. (b) The levels of IL-1β and IL-18 in WT and GSDMD−/− mice serum were detected after heme treatment. (c) The wet-to-dry lung ratio in WT and GSDMD−/− mice was calculated after heme treatment. (d) WT and GSDMD−/− mice were intravenously injected with tetramethylrhodamine-dextran after heme treatment at 24 h and the lung vascular permeability was examined by multiphoton-microscope. (e) The inflammation score of WT and GSDMD−/− mice was measured at 0 h, 12 h, 24 h, and 48 h after heme treatment. (f) The neutrophil, monocyte, erythrocyte, and lymphocyte count in WT and GSDMD−/− mice were detected after heme treatment. There were 5 mice in each group. The data are expressed as mean ± SEM. ns: no significance, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: SIRS occurred and heme elevated after RFA of hepatic hemangioma in mice. (a) The orthotopic liver hemangioma model was established, and RFA was performed. (b) The liver hemangioma of mice was stained by CD31 immunohistochemical staining and H&E staining, black arrow indicates the positive CD31 staining. Scale bar = 200 µm. (c) The level of heme in mice was detected after RFA. (d) The levels of IL-1β and IL-18 were detected by ELISA. (e) The wet-to-dry lung ratio was calculated. (f) The neutrophil, monocyte, lymphocyte, and erythrocyte count were examined. There were 6 mice in each group. The data are expressed as mean ± SEM. ns: no significance, *P < 0.05, **P < 0.01.

Fig 4: HUA promoted HUVECs inflammation and pyroptosis through miR‐22/NLRP3. (A) Relative HOTAIR expression was measured by qPCR after incubation with AMO‐22. The expression of HOTAIR did not change after incubation in HUA+AMO‐22 group. p > 0.05 compared to the HUA group; n = 3 in each group. (B) Protein levels of caspase‐1, NLRP3, GSDMD‐N and GSDMD‐FL, as measured by Western blot analysis; quantification normalized to GAPDH after incubation with AMO‐22. After knocking down miR‐22 with AMO‐22, the expression of NLRP3, caspase‐1, GSDMD‐N and GSDMD‐FL increased in HUA+AMO‐22 group; *p < 0.05 compared to the HUA group; n = 3 in each group. (C,D) Levels of IL‐1β, IL‐18 and LDH in cell culture supernatants, measured by ELISA after incubation with AMO‐22. The levels of IL‐1β, IL‐18 and LDH increased in HUA+AMO‐22 group; *p < 0.05 compared to the HUA group; n = 3 in each group. (E) Protein levels of caspase‐1, NLRP3, GSDMD‐N and GSDMD‐FL, as measured by Western blot analysis; quantification normalized to GAPDH after incubation with a miR‐22 mimic. After incubation with the miR‐22 mimic in a HUA environment, the expression of NLRP3, caspase‐1, GSDMD‐N and GSDMD‐FL significantly decreased; *p < 0.05 compared to the HUA group; n = 3 in each group. (F,G) Levels of IL‐1β, IL‐18 and LDH in cell culture supernatants, measured by ELISA after incubation with a miR‐22 mimic. The levels of IL‐1β, IL‐18 and LDH significantly decreased in HUA+miR‐22 mimic group; *p < 0.05 compared to the HUA group; n = 3 in each group. (H) Protein levels of caspase‐1, NLRP3, GSDMD‐N and GSDMD‐FL, as measured by Western blot analysis; quantification normalized to GAPDH in the NLRP3‐OE group after incubation with a miR‐22 mimic. In the NLRP3‐OE group after incubation with the miR‐22 mimic, the expression of NLRP3, caspase‐1, GSDMD‐N and GSDMD‐FL significantly increased in NLRP3‐OE+miR‐22 mimic group compared to that in the Vector +miR‐22 mimic group. *p < 0.05 compared to the Vector +miR‐22 mimic group; n = 3 in each group. (I,J) Levels of IL‐1β, IL‐18 and LDH in cell culture supernatants, measured by ELISA using the NLRP3‐OE group after incubation with a miR‐22 mimic. The levels of IL‐1β, IL‐18 and LDH were significantly increased in NLRP3‐OE+miR‐22 mimic group compared to those in the Vector +miR‐22 mimic group; *p < 0.05 compared to the vector +miR‐22 mimic group; n = 3 in each group

Fig 5: Heme induces SIRS in mice. (a) The levels of IL-1β and IL-18 were detected after heme treatment. (b) The wet-to-dry lung ratio was calculated after heme treatment. (c) The neutrophil, monocyte, erythrocyte, and lymphocyte count were detected. (d) The inflammation score of mice was measured at 0 h, 12 h, 24 h, and 48 h after heme treatment. There were 6 mice in each group. The data are expressed as mean ± SEM. ns: no significance, *P < 0.05, **P < 0.01, ***P < 0.001.