Fig 1: HET0016 as a lipid peroxidation inhibition protects against sepsis.A Levels of plasma MDA from healthy control or patients. B Spearman correlation analysis for plasma MDA and D-lactate or APACHE II score in all patients (n = 28). C Kaplan–Meier analysis according to a cutoff value calculated from ROC analysis. D Representative histopathological section images, immunohistochemical or fluorescent images in the intestinal tissues of patients with sepsis. The scale bar represents 50 µm. The quantified fluorescence intensity of 4-HNE was shown in the lower left corner. E Survival analysis of the indicated mice in CLP-induced sepsis with or without treatment of 10 mg/kg HET0016, 20 mg/kg JSH-23, or 10 mg/kg Carnosol at 2 h before CLP and 12, 24, 48, and 72 h after CLP (n = 15 per group). F Immunoblot analysis of p-TBK1, TBK1, p-P65, and P65 in PBMCs from patients with sepsis or healthy control, supplemented with or without HET0016 (5 µM) for 24 h (n = 4 per group). G qPCR analysis of Il-6, Il-1b, and Tnf mRNA in PBMCs from patients with sepsis or healthy control, supplemented with or without HET0016 (5 µM) for 24 h (n = 6 per group). H Production of MDA in PBMCs from patients with sepsis supplemented with or without HET0016 (5 µM) for 24 h (n = 6 per group). Data are shown as mean ± SD, and analysis by one-way ANOVA test. Data in (G, H) are analyzed by paired Student’s t-test. PBMCs peripheral blood mononuclear cells, MODS multiple organ dysfunction syndromes, MDA malondialdehyde, FTH ferritin heavy chain, GPX4 glutathione peroxidase 4, 4-HNE 4-hydroxynonenal. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.
Fig 2: HNRNPA3 dimethylation and its correlation with inflammatory factors. (A) Greatly increased asymmetric arginine dimethylation of HNRNPA3 proteins in CP tissues. Protein asymmetric arginine dimethylation were quantitatively assessed by immunoprecipitation using antibodies targeting ADMA, followed by Western blot with the antibodies specifically recognizing HNRNPA3 (B). Relative HNRNPA3 dimethylation levels among four CP patients were compared with the control group containing four other cholangiocarcinoma patients. (C, Dand E) The positive correlation of HNRNPA3 asymmetric arginine dimethylation levels with the major inflammatory factors in serum of CP patients. Serum TGF-b, TNF-a and IL-6 levels in CP patients were analyzed via ELISA method. *P<0.05.
Fig 3: AMs develop osteolytic lesions and induce osteoclastogenesis. (A) Hematoxylin and eosin staining of histological sections of the tumors from two patients: One FT and a PT. Scale bar, 250 µm. (B) TRAP-positive cells were presented on the surface of the bone adjacent to tumor cells (blue arrowhead), but more commonly located at a distant site from the tumors (black arrow). Scale bar, 250 µm. (C) Expression of osteoclast-activating factors in AM tissues and cells. The polymerase chain reaction products were separated on a 2% agarose gel and stained with gold view and expression of each gene was normalized to GAPDH, which was used as a loading control. The experiment was performed in triplicate. PAM, primary AM cells; FT, follicular type; PT, plexiform type; NB, normal bone tissue; T, tumor; CB, cortical bone; BM, bone marrow; RANKL, receptor activator of NF-κB ligand; IL, interleukin; TNF, tumor necrosis factor; M-CSF, macrophage-colony stimulating factor; AM, ameloblastoma; TRAP, acid phosphatase 5, tartrate resistant.
Fig 4: AM-derived TNF-α upregulated IL-8 expression in bone marrow stromal cells and IL-8 directly triggered osteoclastogenesis. (A) HS-5 cells were treated with CM from hTERT-AM with or without TNF-α antibody for 24 h and reverse transcription-quantitative polymerase chain reaction was performed to detect IL-8 expression in HS-5 cells. *P<0.05 vs. cells treated with M-CSF, #P<0.05 vs. wells containing M-CSF+AM/HS-5 CM. (B) HS-5 Cells and hTERT-AM were cultured independently or directly cocultured with or without TNF-α antibody (50 ng/ml) for 24 h, IL-8 levels in the culture media were detected by ELISA. *P<0.05 vs. control, #P<0.05 vs. coculture of HS-5 and hTERT-AM. (C) Osteoclast formation stimulated by recombinant IL-8 or RANKL. Bone marrow-derived monocyte/macrophage precursor cells were cultured in osteoclastogenic medium with 25 ng/ml M-CSF, or in the same medium containing RANKL (50 ng/ml) or IL-8 (100 ng/ml). Scale bar, 250 µm. (D) TRAP positive MN cells (≥3 nuclei) were counted as mature osteoclasts. *P<0.05 vs. cells treated with M-CSF. The experiment was performed in triplicate. IL, interleukin; M-CSF, macrophage-colony stimulating factors; TNF, tumor necrosis factor; RANKL, receptor activator of NF-κB ligand; AM, ameloblastoma; CM, conditioned media; TRAP, acid phosphatase 5, tartrate resistant; MN, multinucleated; Ig, immunoglobulin.
Fig 5: Interactions between AM cells and BMSCs stimulated secretion of IL-8 and activin A. (A) Cytokine array of the conditioned media of HS-5 cells, hTERT-AM cells and the coculture of the two cell types. The hTERT-AM cells and HS-5 cells were directly co-cultured in 1:1 ratio or independently cultured at a density of 2.5×105 cells/ml for 24 h, the serum-free mediums were collected for array analysis. A1-A4: POS1; A5-A8: POS2; A9-A12: Activin A; B1-B4: FGF-1; B5-B8: Amphiregulin; B9-B12: bFGF; C1-C4: BMP-4; C5-C8: BMP-9; C9-C12: E-Selectin; D1-D4: CD54; D5-D8: IGF-1; D9-D12: IL-1α; E1-E4: IL-1β; E5-E8: IL-6; E9-E12: IL-8; F1-F4: IL-11; F5-F8: IL-17A; F9-F12: MCP-1; G1-G4: M-CSF; G5-G8: MIP-1α; G9-G12: MMP-2; H1-H4: MMP-9; H5-H8: MMP-13; H9-H12: Osteoactivin; I1-I4: P-Cadherin; I5-I8: RANK; I9-I12: Stromal-cell derived factor-1α; J1-J4: Sonic hedgehog-N; J5-J8: TGFβ1; J9-J12: TGFβ2; K1-K4: TNF-α; K5-K8: CD106; K9-K12: CDH5. A heat map (right) demonstrating the semiquantitative results of cytokine levels. (B) Protein levels of IL-8 in the serum-free culture media from AM cells (hTERT-AM cells, primary AM cells), HS-5 cells or direct or Transwell co-cultures measured by ELISA. *P<0.05 vs. wells containing HS-5 cells or the corresponding AM cells. (C) hTERT-AM cells and HS-5 cells were cultured separately with a Transwell plate for 24 h, RT-qPCR was performed to detect relative mRNA expression of IL-8 to GAPDH. *P<0.05 vs. HS-5 monoculture. (D) Protein levels of activin A in the serum-free culture media from AM cells (hTERT-AM cells, primary AM cells), HS-5 cells or direct or Transwell co-cultures measured by ELISA. *P<0.05 vs. wells containing HS-5 cells or the corresponding AM cells. (E) hTERT-AM cells and HS-5 cells were cocultured for 24 h. Following cell separation, RT-qPCR was performed to detect relative mRNA expression of INHβA to GAPDH. *P<0.05 vs. HS-5 monoculture. The experiment was performed in triplicate. pAM, primary AM cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; IL, interleukin; CD, cluster of differentiation; BMP, bone morpho-genic protein; AM, ameloblastoma; MMP, matrix metalloproteinase; TGF, transforming growth factor; TNF, tumor necrosis factor; bFGF, basic fibroblast growth factor; SDF, stromal-cell derived factor.
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