Fig. 6
Knockdown of GSDMD by lentiviral shRNA. Hydrogen mediated GSDMD triggers pyroptosis in endometrial cancer cells. Three pairs of GSDMD gene shRNA interference fragments: LV-GSDMD-RNA interference (i)-71,359–11 (RNAi-1), LV-GSDMD-RNAi-71,360-1 (RNAi-2), and LV-GSDMD-RNAi-71,361–1 (RNAi-3), and one negative control (NC) pair were designed and transfected into Ishikawa cells. A. GSDMD mRNA expression in Ishikawa, HEC1A, and AN3CA cells, and B. GSDMD mRNA expression in Ishikawa cells following transfection with RNAi-1 or RNAi-3 were decreased significantly compared with that in the NC group. Two shRNA vectors suppressed GSDMD mRNA expression by 73.7 and 59.6%, respectively. Western blotting analysis revealed that levels of GSDMDNterm protein in GSDMD depleted ishikawa cells were downregulated compared with NC group. C. RNAi-1-1 had an increased transfection efficiency (78.8%) and was selected for subsequent experiments. LDH release assay (d, e, f) and IL-1β release assay by ELISA (G. H. I.) under hydrogen stimulation from HEC1A, AN3CA, and Ishikawa cells primed with LPS (10 ng/mL for 4 h), and then stimulated for 30 min with nigericin (10 μM), NAC (5 mM), MCC950 (10 μM), or VX-765 (0.8 nM). d. Compared with CM group, hydrogen treatment after 4 h significantly induced the release of LDH from HEC1A and AN3CA cells. LDH release increased after hydrogen-treated HEC1A cells were primed with LPS and nigericin, but decreased after AN3CA cells were primed with NAC, MCC950, or VX-765. e. Compared with CM group, hydrogen treatment or primed with LPS and nigericin significantly induced the release of LDH from Ishikawa cells. Compared with H-CM group, LDH release decreased after cells were primed with NAC, MCC950, or VX-765. Even if GSDMD was deleted, H-CM-Ishikawa cells primed with LPS and nigericin produced a greater release of LDH. f. GSDMD depletion reduced the release of LDH with both H-CM and CM treatment or with LPS and nigericin in Ishikawa cells compared with the control group. G. Hydrogen treatment enhanced significantly the release of IL-1β from HEC1A and AN3CA cells compared with CM group. IL-1β release increased after hydrogen treatment in HEC1A and AN3CA cells primed with LPS and nigericin, while release diminished after AN3CA cells were primed with NAC, MCC950, or VX-765 compared with H-CM group. h. There were, however, no significant differences on the release of IL-1β among the different groups with or without hydrogen treatment when a GSDMD siRNA was transfected into Ishikawa cells. i. GSDMD depletion reduced the release of IL-1β in CM-, H-CM- and NAC-treated Ishikawa cells compared with the control group. I. *P < 0.05, # P > 0.05. Each experiment was performed in triplicate (See Additional file 8, 9, and 10)