Fig 1: Determination of the optimal dose of T+L+Bz-ATP (Bz) that stimulated NLRP3 and pro-IL-1β to a similar extent as T+L+A (5 mM), the and optimal dose of the P2X7R inhibitor (A4), the ROS inhibitor (NAC), and the caspase-1 inhibitor (YVAD) that did not affect the T+L+A-stimulated increase in NLRP3 or pro-caspase-1 (YVAD) or pro-IL-1β expression, when administered to HCMFs alone, after 8 h. (A) Representative Western blots of NLRP3 and pro-IL-1β expression from T-, T+L+A (5 mM)- or T+L+Bz (0.1 or 0.2 mM)-stimulated HCMFs after 8 h in culture. (C,E,G) Representative Western blots of NLRP3 and pro-IL-1β expression show the effects of T- vs. T+L+A-stimulation of HCMFs treated with increasing doses of A4 (0.05–0.5 µM) (C), NAC (1–10 µM) (E), or YVAD (0.05–0.5 µM) (G) for 8 h. (B,D,F,H) Also shown are the mean ± SEM of each end point measured from each treatment group studied, corrected for GAPDH (house-keeping protein) loading, and expressed relative to the value in the T-stimulated group, which was expressed as 1 in each case, from n = 3–5 separate experiments conducted in duplicate. The individual data sets for each group are represented by the white circles within each set of bar graphs. * p < 0.05, ** p < 0.01 compared with the T alone group.
Fig 2: The effects of RLX ± the P2X7R inhibitor (A4), TLR-4 inhibitor (TAK), ROS inhibitor (NAC) and caspase-1 inhibitor (YVAD) on the T+L+A-stimulated increase in myofibroblast NLRP3 inflammasome measures. (A,C,E,G) Representative Western blots of TLR-4, NLRP3 (or pro-caspase-1 for the studies involving YVAD), pro-IL-β and pro-IL-8 expression from HCMFs treated with TGF-β1 (T), T+L+A, T+L+A+A4, T+L+A+RLX or T+L+A+RLX +A4 (A); T, T+L+A, T+L+A+TAK, T+L+A+RLX or T+L+A+RLX +TAK (C); T, T+L+A, T+L+A+NAC, T+L+A+RLX or T+L+A+RLX+NAC (E); or T, T+L+A, T+L+A+YVAD, T+L+A+RLX or T+L+A+RLX+YVAD (G), after 8 h. (B,D,F,H) Also shown are the mean ± SEM of each end point measured from each treatment group studied, corrected for GAPDH (house-keeping protein) loading, and expressed relative to the value in the T-stimulated cell group, which was expressed as 1 in each case, from n = 6 separate experiments conducted in duplicate. The individual data sets for each group are represented by the white circles within each set of bar graphs. * p < 0.05, ** p < 0.01 compared with the T alone group; # p < 0.05 compared with the T+L+A group; ¶ p < 0.05 compared with the T+L+A+RLX group.
Fig 3: The effects of RLX ± the RXFP1 inhibitor (RXFP1-I), AT2R inhibitor (PD) or P2X7R inhibitor (A4) on the T+L+Bz-stimulated increase in TLR-4, NLRP3, pro-caspase-1, pro-IL-1β and pro-IL-18 levels from HCMFs. (A,C,E) Representative Western blots of TLR-4, NLRP3, pro-caspase-1, pro-IL-β and pro-IL-8 expression from HCMFs treated with TGF-β1 (T), T+L+Bz, T+L+Bz+RXFP1-I, T+L+Bz+RLX or T+L+Bz+RLX+RXFP1-I (A); T, T+L+Bz, T+L+Bz+PD, T+L+Bz+RLX or T+L+Bz+RLX+PD (C); or T, T+L+Bz, T+L+Bz+A4, T+L+Bz+RLX or T+L+Bz+RLX+A4 (E), after 8 h. (B,D,F) Also shown are the mean ± SEM of each end point measured from each treatment group studied, corrected for GAPDH (house-keeping protein) loading, and expressed relative to the value in the T-stimulated cell group, which was expressed as 1 in each case, from n=6 separate experiments conducted in duplicate. The individual data sets for each group are represented by the white circles within each set of bar graphs. (G) Representative immunofluorescence staining of RXFP1, AT2R and P2X7R, and colocalization of RXFP1 and P2X7R or AT2R and P2X7R in HCMFs. Scale bar = 20 μm. Nuclear staining was performed using DAPI, whilst merged images demonstrated that all three receptors were expressed by HCMFs. * p < 0.05, ** p < 0.01 compared to the T alone group; # p < 0.05 compared to the T+L+Bz group; ¶ p < 0.05 compared to the T+L+Bz+RLX group.
Fig 4: MFG-E8 inhibits the NETs-induced activation of NLRP3 inflammasome.a Representative immunofluorescence images of active caspase-1 and IL-1β in LPS-primed BMDMs from WT or Mfge8−/− mice (n = 4) treated with 2 μg/mL mCRAMP (LL-37) or 10 μg/mL NETs, and then administrated with or without rmFMG-E8. The active caspase-1+ and IL-1β+ BMDMs in five random microscopic fields in n = 4 mice per group was performed. b–c After administration with or without rmMFG-E8, quantified the mean fluorescence density (MFI) of active caspase-1 b and IL-1β c in LPS-primed BMDMs from WT or Mfge8−/− mice exposed to NETs or mCRAMP. d–e Quantification of IL-1β d and IL-18 e mRNA levels in LPS-primed BMDMs from WT or Mfge8−/− mice (n = 4) after stimulation with NETs or mCRAMP when addition with or without rmMFG-E8. f Exposure to NETs or mCRAMP, western blot analysis of the active caspase-1 and caspase-1 expression in BMDMs from WT or Mfge8−/− mice after treatment with or without rmMFG-E8. g The P2X7 receptor (P2X7 R+) macrophages in wound dermis from diabetic WT and Mfge8−/− mice were determined by immunofluorescence; more than five random microscopic fields in n = 6 mice per group was performed. For all experiments, data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5: A schematic illustration summarizing the main findings of this study in relation to those of previously published findings on the RLX signal transduction of myofibroblasts [29,30,31,34,35]. It is now proposed that RLX signals through its cognate receptor, RXFP1, the AT2R and P2X7R on HCMFs (when all three receptors are appropriately expressed) as well as a RXFP1-nNOS-(NO-sGC-cGMP)-mediated mechanism to disrupt the induction of the myofibroblast NLRP3 inflammasome at the level of the TLR-4 (and the TLR-4/TGF-β1 axis [42]) and ROS. This study confirmed that RLX can also directly inhibit the myofibroblast NLRP3 inflammasome (at the level of caspase-1) to mediate its anti-fibrotic actions in HCMFs and the LV of ISO-injured mice. This in turn would result in the RLX-induced inhibition of the pro-fibrotic TGF-β1/IL-1β and TGF-β1/IL-18 interactions on myofibroblast differentiation and myofibroblast-mediated ECM/collagen synthesis and deposition (fibrosis).
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