Fig 2: Disruption of hepatocyte necroptosis, and not apoptosis, was required for the action of YQJPF in vivo. (A–D) MDA, ATP, GSH, and SOD levels in the rat livers were measured. (E) Western blot and quantitative analysis of cleaved caspase 8, MLKL, RIPK1, and RIPK3 expression in the rat livers. (F) Immunochemical staining and quantitative analysis of caspase 8, MLKL, RIPK1, and RIPK3 in liver tissues. Scale bar, 100 μm. (G–J) Expression levels of IL-1β, IL-6, TNF-α, and IL-18 in the rat livers were detected by ELISA. Data are expressed as mean ± SD (n = 8) *vs. control group, #vs. ACLF group; *p < 0.05, **p < 0.01; # p < 0.05, ## p < 0.01.

Fig 3: Silencing of AMPK-SIRT1 signaling eliminates the protective effect of colchicine on endothelial cells. HUVECs were transfected with siRNA targeting AMPa1 (si-AMPKa1) and SIRT1 (si-SIRT1) or a control siRNA (Si NC) or were not transfected (-). Cells were then added to cholesterol crystal (0.5 mg/ml) in the presence of colchicine (10 nM) for 10 or 24 hrs. Real-time PCR analysis of the mRNA expression of AMPKa and SIRT1 after transfection with si-AMPKa1, si-SIRT1, and si-NC for 24 hrs without (a) or with (b) the presence of cholesterol crystals and colchicine. (c) Intracellular ROS level was detected using a DCFH-DA probe, and (d) mitochondrial membrane potential was tested by JC-1. (e) Pyroptotic cell death was measured with Hoechst 33342 (blue)/PI (red) double-fluorescent staining. (f, g) The indicators of oxidative stress GSSG and MDA were tested by assay kits. (h) Real-time PCR analysis of the mRNA levels of GSDMD and proinflammatory cytokines (IL-18, IL-1ß, IL-6, IL-8, and MCP-1). Scale bars = 100 µm. Data was expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. the si-NC group or CC+COL+si-NC group.

Fig 4: YQJPF inhibited hepatocyte necroptosis through the inhibition of mitochondrial ROS generation and depolarization in vitro. Human LO2 cells were treated with LPS + D-Gal for 4 h and further pre-treated with 50 µM Nec-1 for 1 h, followed by YQJPF or atractylone treatment for 24 h. (A) Mitochondrial superoxide was detected and quantified through immunofluorescence by using MitoSox Red staining. Scale bar, 50 µm. (B) ROS production was assessed and quantified by DCFH-DA staining, Scale bar, 50 µm. (C–E) MDA, SOD, and GSH levels were measured by corresponding test kits. (F) ROS levels were assessed using ROS test kit. (G) Mitochondrial membrane potential was detected by JC-1 staining. Scale bar, 50 µm. (H) Western blot and quantitative analysis of the expression of IL-1ß, IL-6, TNF-a, and IL-18. Data are expressed as mean ± SD (n = 3). *vs. control group, #vs. LPS + D-Gal group; *p < 0.05, **p < 0.01; # p < 0.05, ## p < 0.01.

Fig 5: Colchicine alleviates NLRP3 inflammasome activation relevant in pyroptosis. HUVECs were treated for 24 h with different doses of colchicine (0–10 nM) in the presence of cholesterol crystal (0.5 mg/ml) or were left untreated (control). (a) Immunofluorescence was used for qualitative detection of NLRP3 (400x). (b) Western blotting was dedicated to examine the protein expression levels of NLRP3, ASC-1, procaspase-1, caspase-1, IL-18, and IL-1ß. (c) Quantitative analysis of pyroptosis-associated protein expression. (d–i) Real-time PCR analysis of the mRNA levels of GSDMD and proinflammatory cytokines (IL-18, IL-1ß, IL-6, IL-8, and MCP-1). Data was expressed as the mean ± SD of three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. the control group. #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 vs. the cholesterol crystal group.