Fig 2: Effects of THP on ER stress and inflammasome activation in golden hamsters with HLP induced by a high-fat diet. (a) The mRNA levels of caspase 12, GRP78, and CHOP in liver tissues were evaluated by qRT-PCR. (b) The protein levels of caspase 12, GRP78, and CHOP in liver tissues were measured by western blotting, with ß-actin as a loading control. (c) Serum IL-18 was determined by ELISA, and the results are expressed as the mean ± SD (n = 8). (d) The protein levels of NLRP3, ASC, pro-IL-1ß, and IL-1ß in liver tissues were measured by western blotting, with ß-actin as a loading control. The results of at least three independent experiments are shown as the mean ± SD. M versus NC, ***P < 0.001; LD, MD, HD, and PC versus (M) #P < 0.05, ##P < 0.01, and ###P < 0.001.

Fig 3: Melatonin treatment inhibits NLRP3 inflammasome activation. (A) Representative Western blot bands of NLRP3, ASC, and cleaved caspase-1. (B–D) The relative band densities of NLRP3, ASC, and cleaved caspase-1; n = 6 per group. The cropped bands had been run under the same experimental conditions. *P < 0.05 versus sham group, # P < 0.05 versus SAH + vehicle group, and & P < 0.05 versus SAH + Mel group.

Fig 4: The effects of RLX on Lipofectamine-induced CTL siRNA transfection in NLRP3 inflammasome activated BJ3 HDMFs after 72 h. (A) Representative Western blots of NLRP3, ASC, pro-caspase-1, pro-IL-1ß, pro-IL-18, a-SMA, and collagen-I protein expression in HDMFs treated with CTL siRNA ± T+L+A ± R treatment after 72 h. (B) Also shown are the mean ± SEM of each end-point measured from each treatment group studied, corrected for GAPDH loading and expressed relative to the value in the CTL siRNA+T+L+A-treated HDMF group, which was expressed as 1 in each case; from n=4–6 separate experiments conducted in duplicate. #p<0.05 ##p<0.01 vs. CTL siRNA+T+L+A-treated group. (C) Additionally shown are the mean ± SEM levels of active caspase-1 (pg/ml) (top panel) and active IL-1ß (pg/ml) (bottom panel) in CTL siRNA transfected HDFs treated with T+L+A or T+L+A+R after 72 h.

Fig 5: Free fatty acid-mediated macrophage M1 polarization in vitro. Mouse abdominal macrophage were isolated by high-sugar DMEM and then inoculated into a sterile Petri dish (2.5 × 105/ml). Palmitic acid (PA, 500 µM) was used for fatty acid stimulation. A Inflammation factors were detected by ELISA in macrophage culture medium, which was stimulated by PA, and B quantitative cellular expression was measured. Data are shown as the mean ± SEM, n = 3. *P < 0.05, ANOVA. Protein levels of ASC, IL-1ß, IL-18 and NLRP3 were performed by western blot in macrophage stimulated by PA (C). Flow cytometry to assess polarization in macrophage stimulated by PBS (D, E) or PA (F, G). Anti-F4/80 (D, F), anti-CD11c, and anti-CD206 (E, G). CD11c indicates M1 macrophage, CD206 indicates M2 macrophage. Marker genes of macrophage polarization were detected by qRT-PCR (H) and western blotting (I). Data are shown as the mean ± SEM, n = 3. *P < 0.05, ANOVA