Journal: Advanced Biology
Article Title: HMGB1 Derived from the Pyroptotic Microenvironment Promotes Macrophage Extracellular Traps in Hirschsprung‐Associated Enterocolitis
doi: 10.1002/adbi.202400761
Figure Lengend Snippet: The TLR4‐P38 MAPK/P65 NF‐kB signaling pathways mediate the‐pyroptotic‐microenvironment‐induced MET formation. A) Western blot analysis of p‐ERK, p‐p38, p‐JNK and p‐p65 levels in macrophages cocultured with pyroptotic‐CM for 4 h. B,C) Macrophages were pretreated with inhibitors of the ERK, JNK, p38 MAPK, and p65 NF‐kB pathways prior to incubation with pyroptotic‐CM. The MET formation by macrophages was evaluated with SYTOX Green staining and detected by flow cytometry. The representative images are shown in B and the quantification of B is shown in C (one‐way ANOVA followed by Tukey's multiple‐comparison test, n = 3 per group). D) Macrophages were pretreated with inhibitors of TLR2, TLR4, TLR9 and RAGE prior to coculture with pyroptotic‐CM, and the p‐p38 and p‐p65 levels in macrophages were measured by western blotting. E,F) The MET formation of macrophages pretreated with inhibitors of TLR2, TLR4 and RAGE prior to incubation with pyroptotic‐CM was evaluated with SYTOX Green staining and detected by flow cytometry. The representative images are shown in E and the quantification of E is shown in F (one‐way ANOVA followed by Tukey's multiple‐comparison test, n = 3 per group). Data are expressed as mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Article Snippet: The inhibitors used in this study included an HMGB1 antagonist (HY‐N0184, MCE, USA), a p38 MAPK pathway inhibitor (HY‐12839, MCE, USA), a p65 NF‐kB pathway inhibitor (HY‐138537, MCE, USA), an ERK pathway inhibitor (HY‐112287, MCE, USA), a JNK inhibitor (HY‐12041, MCE, USA), a TLR2 antagonist (HY‐112146, MCE, USA), a TLR4 antagonist (HY‐11109, MCE, USA), a TLR9 antagonist ( HY131952 , MCE, USA), and a RAGE antagonist (HY‐P2268).
Techniques: Protein-Protein interactions, Western Blot, Incubation, Staining, Flow Cytometry, Comparison
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