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. 2023 Feb 27;14:1095457. doi: 10.3389/fimmu.2023.1095457

Figure 3.

Figure 3

Pyroptosis is activated following pneumonia-induced sepsis and Circ_0075723 inhibits pyroptosis of THP-1 in vitro. (A) Western blot analysis of TLR4, NLRP3, ASC1, caspase1, cleaved caspase-1, Pro-IL1β, IL-1β, GSDMD and GAPDH in CD14+ monocytes from pneumonia-induced sepsis, pneumonia without sepsis and healthy people. (B) ELISA of IL-18 and IL-1β in the plasma from pneumonia-induced sepsis, pneumonia without sepsis and healthy people. Each group has 4 samples. Data are presented as means ± SD; significant difference was identified with one-way ANOVA. *p < 0.05 vs. Pneumonia; **p < 0.01 vs. Pneumonia; ***p < 0.001 vs. Control. (C) Western blot analysis of TLR4, NLRP3, ASC1, caspase1, cleaved caspase-1, Pro-IL1β, IL-1β, GSDMD and GAPDH in THP-1 cells primed with different doses of LPS (0.1, 0.5 and 1 μg/ml) for 4 h and stimulated with nigericin (10 μM) for 2h. Data are presented as means ± SD; significant difference was identified with two-way ANOVA. ***p < 0.001 vs. PBS; ns: no significant. (D) qRT-PCR analysis of the expression of circ_0075723 primed with different doses of LPS (0.1, 0.5 and 1 μg/ml) for 4 h and stimulated with nigericin (10 μM) for 2h in THP-1 cells. Data are presented as means ± SD; significant difference was identified with Student t-tests. *p < 0.05 vs. PBS; **p < 0.01 vs. PBS. THP-1 cells were transfected with vector or shRNA scrambled control (shRNA NC) or were transfected with circ_0075723-overexpressing lentivirus plasmids (OE-circ_0075723), sh-circ_0075723-expressing lentivirus plasmids (sh-circ_0075723), vector or shRNA scrambled control (shRNA NC) and then were primed with LPS (1 μg/ml) for 4 h and stimulated with nigericin (10 μM) for 2h. (E) qRT-PCR analysis of the expression of circ_0075723 in THP-1 cells. Data are presented as means ± SD; significant difference was identified with Student t-tests. ***p < 0.001 vs. vector or shRNA NC; ###p < 0.001 vs. LPS/nigericin + Vector; &&p < 0.01 vs. LPS/nigericin + shRNA NC. (F) Western blot analysis of TLR4, NLRP3, ASC1, caspase1, cleaved caspase-1, Pro-IL1β, IL-1β, GSDMD and GAPDH in THP-1 cells. (G) ELISA of IL-18 and IL-1β in THP-1 supernatant. Data are presented as means ± SD; significant difference was identified with Student t-tests. **p < 0.01 vs. vector; ***p < 0.001 vs. vector or shRNA NC; #p < 0.05 vs. LPS/nigericin + Vector; ##p < 0.01 vs. LPS/nigericin + Vector; &p < 0.05 vs. LPS/nigericin + shRNA NC; &&p < 0.01 vs. LPS/nigericin + shRNA NC.