Fig 1: HMGB1 is released through sufficiently large membrane pores.BMDM were primed with LPS (0.5 μg ml−1; 4.5 h) and then treated with nigericin (Ng; 20 μM for 30 min), potassium depletion (K+ dep; 2 h) or treated with pneumolysin (PL) alone. Untreated controls and pneumolysin-treated cells were not primed with LPS. Cells were treated with or without 5 mM glycine (Gly), necrosulfonamide (NSA) (5 μM), or PL (0.5 μg ml−1). a Immunoblot analysis of released HMGB1 and cleaved IL-1β (p17) into the cell culture supernatant. PL causes release of HMGB1, but not IL-1 β processing. b LDH present in the extracellular media was quantified as a measure of cytotoxicity, with LPS + Ng and PL causing similar amounts of cell lysis. Data shown from n = 3 independent experiments. c Immunofluorescence of control and PL-treated BMDM. HMGB1 (green) and nuclei (DAPI; blue) indicate loss of nuclear HMGB1 in response to PL treatment. Data with error bars are represented as mean ± SEM. Each panel is a representative experiment of at least three independent experiments. *p < 0.0001 and n.s. not significant as determined by ANOVA with Tukey’s multiple comparison correction, two-sided.
Fig 2: Detection of pro-inflammatory factors in glial cells. (A) Expression of IL-1b mRNA in NC, DT, DO and DI group of glial cells. (B) Expression of IL-6 mRNA in NC, DT, DO and DI group of glial cells. (C) Expression of TNF-A in NC, DT, DO and DI group of glial cells. (D) Expression of PTGS2 in NC, DT, DO and DI group of glial cells. Data was presented as mean ± SD, each experiment was repeated for three times. *P<0.05 vs NC group of cells. #P<0.05 vs signal dezocine treatment group. NC: normal group, DT: dezocine treatment group, DO: dezocine treatment combined with RAPGEF3 overexpression group, DI: dezocine treatment combined with RAPGEF3 inhibition group. SH: sham group, TD: dezocine treatment group, TO: dezocine treatment combined with RAPGEF3 overexpression group, TI: dezocine treatment combined with RAPGEF3 inhibition group.
Fig 3: Detection of pro-inflammatory factors in brain tissues of rats. (A) Expression of IL-1b mRNA in SH, TD, TO and TI group of rats. (B) Expression of IL-6 mRNA in SH, TD, TO and TI group of rats. (C) Expression of TNF-A mRNA in SH, TD, TO and TI group of rats. (D) Expression of PTGS2 mRNA in SH, TD, TO and TI group of rats. Data was presented as mean ± SD, each experiment was repeated for three times. *P<0.05 vs SH group of rats. #P<0.05 vs signal dezocine treatment group. NC: normal group, DT: dezocine treatment group, DO: dezocine treatment combined with RAPGEF3 overexpression group, DI: dezocine treatment combined with RAPGEF3 inhibition group. SH: sham group, TD: dezocine treatment group, TO: dezocine treatment combined with RAPGEF3 overexpression group, TI: dezocine treatment combined with RAPGEF3 inhibition group.
Fig 4: Hypoxia and glutamine metabolism correlate to hair follicle development in WIHN.(A) Heatmap for canonical keratinocyte marker genes in each cluster of the wound center from scRNA-seq of SD0 samples. (B) The t-SNE plot shows clustering of SD0 wound center keratinocytes subpopulations. (C) H&E, Col17a1, Cox-2, Ube2c, and Krt17 staining of SD0 wounds to establish location of basal1, basal2, proliferous, and HG keratinocytes. Scale bars, 50 μm. (D) Expression of HG marker genes to identify the HG cluster in t-SNE plots. (E) The scores for hypoxia, glutamine, and glutamate metabolism and IL-1 signaling in keratinocytes subpopulations. (F and G) Pseudotime analysis plotting the development pattern of basal, spinous, proliferative, and HG keratinocytes. (H) The heatmap of differentiation markers with pseudotime. (I) Pseudotime plot depicting the abundance of HG markers Krt17, Krt79, and Sox9 as divided by keratinocyte subpopulation. (J) Pseudotime analysis divided all keratinocytes (top) and HG (bottom) into six stages according to the developmental sequence; the different stages are indicated by different colors labeled A to F. (K) The gene expression scores for hypoxia, glutamine, and glutamate metabolism, and IL-1 signaling pathways as divided by HG developmental stage. Box plot graphs indicated the value of minimum, first, quartile, median, third quartile, and maximum. Statistics as in Fig. 1.
Fig 5: PDG inhibited the release of inflammatory cytokines from brain tissue of MCAO mice. (a–c) ELISA kit was used to detect the effects of PDG on the release of inflammatory cytokines TNF‐α, IL‐1 β, and IL‐6 in brain tissues of the mice in the Sham group, MCAO group, MCAO + 5 mg/kg PDG group, and MCAO + 10 mg/kg PDG group. (d–h) Western blot was used to detect the effects of PDG on the expressions of p‐IKKβ, IKKβ, p‐IkBα, IkBα, NF‐κB p65 and p‐p65 in brain tissues of the mice in the Sham group, MCAO group, MCAO + 5 mg/kg PDG group, and MCAO + 10 mg/kg PDG group. **p < .01, and ***p < .001 versus the MCAO group
Supplier Page from Abcam for Mouse IL-1 beta ELISA Kit