Journal: Frontiers in Immunology

Article Title: PD0325901 alleviates thrombin-inhibited osteogenic differentiation through an IL-1β-activated feedback loop between MEK-Erk1/2 and NF-κB signal pathways: insights from bioinformatics and experimental verification

doi: 10.3389/fimmu.2026.1730337

Figure Lengend Snippet: Regulatory relationships between NF-κB and MEK-Erk1/2 pathways in thrombin-induced osteoblast differentiation. (A) Nuclear translocation of p65 in osteoblasts was assessed by immunofluorescence. (B) Phosphorylated levels of p65 in thrombin and PD03-treated osteoblasts at 15min, 30min, and 60min were determined by western blot. (C) Relative protein levels of p-p65 to p65 were quantified using ImageJ software. (D) Phosphorylated levels of Erk1/2 in osteoblasts following treatment with thrombin and QNZ for 15min, 30min, and 60min were assessed by western blot. (E) Relative protein levels of p-Erk1/2 to Erk1/2 were quantified using ImageJ software. (F) ALP staining (upper panel) and intracellular calcium signaling (lower panel) were evaluated by the test kits. (G) ALP activities in thrombin- and QNZ-treated osteoblasts for 7 days were measured by colorimetric assay. (H) Expression of osteogenic marker genes (Col1α1, Runx2, and OCN), proliferation-related genes (MCM2, PCNA) and hub genes (MMP-9, COX-2) in thrombin- and QNZ-treated osteoblasts was evaluated by western blot. (I) Relative protein levels of Col1α1/β-actin, Runx2/β-actin, OCN/β-actin, MCM2/β-actin, PCNA/β-actin, MMP-9/β-actin, and COX-2/β-actin were quantified using ImageJ software. (J) qRT-PCR was used to analyze the expression of osteogenic marker genes (e.g., Runx2, Osterix, and OCN) in thrombin- and QNZ-treated osteoblasts for 7 days. Data are presented as mean ± SD (n = 3). P-values were determined by one-way ANOVA (multi-group comparisons) (*p < 0.05; **p < 0.01; ***p < 0.001; ns, P >0.05). Scale bar: 100 μm.

Article Snippet: Based on the experimental design, osteoblasts were divided into the following groups: control group (osteoblasts were cultured in osteogenic induction medium), thrombin group (osteoblasts were cultured in osteogenic induction medium in the presence of 20 U/mL thrombin), PD03 group (osteoblasts were cultured in osteogenic induction medium in the presence of 20 U/mL thrombin and 0.1 μM PD03), C188 group (osteoblasts were cultured in osteogenic induction medium in the presence of 20 U/mL thrombin and 5μM C188-9), QNZ group (osteoblasts were cultured in osteogenic induction medium in the presence of 20 U/mL thrombin and 0.1μM QNZ) and Vorapaxar group [osteoblasts were cultured in osteogenic induction medium in the presence of 20 U/mL thrombin and 0.1 μM, 0.5 μM, or 1 μM Vorapaxar (HY-10119, MCE, USA)].

Techniques: Translocation Assay, Immunofluorescence, Western Blot, Software, Staining, Colorimetric Assay, Expressing, Marker, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: PD0325901 alleviates thrombin-inhibited osteogenic differentiation through an IL-1β-activated feedback loop between MEK-Erk1/2 and NF-κB signal pathways: insights from bioinformatics and experimental verification

doi: 10.3389/fimmu.2026.1730337

Figure Lengend Snippet: The modulation of PD03 on osteoblast differentiation mediated by the IL-1β-activated feedback loop between NF-κB and MEK-Erk1/2 pathways. (A) PAR-1, Col1α1, Runx2 and OCN protein levels in osteoblasts were assessed by western blot analysis. (B) The quantitative analysis of PAR-1, Col1α1, Runx2 and OCN expression. (C) Thrombin-stimulated IL-1β expression in osteoblasts was inhibited by PD03 and QNZ, as determined by immunofluorescence. (D) PD03 and QNZ inhibited thrombin-stimulated IL-1β secretion in osteoblasts, as measured by ELISA. (E) IL-1RA protein levels in osteoblasts were assessed by western blot analysis. (F) The quantitative analysis of IL-1RA expression. (G) PD03-mediated modulation of NF-κB signaling pathways activated by IL-1β in osteoblasts. (H) Relative protein levels of p-p65 to total p65 in osteoblasts were quantified using ImageJ software. (I) The regulatory effect of QNZ on the IL-1β-activated MEK-Erk1/2 signaling pathway in osteoblasts. (J) Relative protein levels of p-Erk1/2 to total Erk1/2 in osteoblasts were quantified using ImageJ software. (K) The regulatory effects of PD03 and QNZ on IL-1β-induced COX-2, MMP-9, Col1α1, Runx2 and OCN expression were evaluated by western blot analysis. (L) The ratios of MMP-9/β-actin, COX-2/β-actin, Col1α1/β-actin, Runx2/β-actin and OCN/β-actin were compared among different groups. (M) The expression of osteogenic markers (e.g., Runx2, Osterix, Col1a1, OPG, and OCN) was evaluated by RT-qPCR. Data are presented as mean ± SD (n = 3). P-values were determined by one-way ANOVA (multi-group comparisons) (*p < 0.05; **p < 0.01; ***p < 0.001; ns, P >0.05). Scale bar: 100 μm.

Article Snippet: Based on the experimental design, osteoblasts were divided into the following groups: control group (osteoblasts were cultured in osteogenic induction medium), thrombin group (osteoblasts were cultured in osteogenic induction medium in the presence of 20 U/mL thrombin), PD03 group (osteoblasts were cultured in osteogenic induction medium in the presence of 20 U/mL thrombin and 0.1 μM PD03), C188 group (osteoblasts were cultured in osteogenic induction medium in the presence of 20 U/mL thrombin and 5μM C188-9), QNZ group (osteoblasts were cultured in osteogenic induction medium in the presence of 20 U/mL thrombin and 0.1μM QNZ) and Vorapaxar group [osteoblasts were cultured in osteogenic induction medium in the presence of 20 U/mL thrombin and 0.1 μM, 0.5 μM, or 1 μM Vorapaxar (HY-10119, MCE, USA)].

Techniques: Western Blot, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Protein-Protein interactions, Software, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: (A-C) Immunofluorescence of PAR1 (green) in myometrium from a pregnant woman (A) , non-pregnant woman ( B ), and fetal membrane (C) . Nuclei were stained with DAPI (blue). Am, amnion, Cho, chorion, Deci, decidua. (D-G) Localization of hemorrhage, thrombin, and PAR1 in placental abruption at 25 weeks of gestation (D and E) , and 33 weeks gestation (F and G) resulting in disseminated intravascular coagulopathy and uterine bleeding requiring hysterectomy for hemostasis. (D and F) Hematoxylin and eosin staining of the myometrium adjacent to the placenta. Note that hemorrhage infiltrated the myometrium. Bars, 50 μm. (E and G) Immunofluorescence of PAR1 (green), thrombin (red), and DAPI (blue) at the same location of (A) . Bars, 50 μm.

Article Snippet: Primary antibodies used and concentration were as follows: thrombin (coagulation factor II, Novus Biologicals, NBP1-58268, Research Resource Identifier (RRID): AB_11023777, 1:100) and PAR1 (N2-11, Novus Biologicals, NBP1-71770, RRID: AB_11027203, 1:100).

Techniques: Immunofluorescence, Membrane, Staining

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: Thrombin increased contraction of primary human myometrial cells through PAR1. (A and B) (Left images) Representative images of collagen lattice assay of human myometrial cells at 30 min treated with PBS (Ctl) and thrombin (A) or PAR1 activating peptide, TFLLR (B) . (Right graphs) Quantification of myometrial contractions in collagen lattice assays (n = 3). **, p < 0.01 at each time point. (C) Collagen lattice assay of myometrial cells at 30 min with 2 U/mL of thrombin (Thr) pretreated with or without 100 nM PAR1 selective inhibitor (SCH79797, PAR1-i) for 1 h (n = 3). Representative image (upper panel) and quantification of gel areas (lower graph). The experiments were repeated three times. *, p < 0.05, and **, p < 0.01.

Article Snippet: Primary antibodies used and concentration were as follows: thrombin (coagulation factor II, Novus Biologicals, NBP1-58268, Research Resource Identifier (RRID): AB_11023777, 1:100) and PAR1 (N2-11, Novus Biologicals, NBP1-71770, RRID: AB_11027203, 1:100).

Techniques:

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: (A) Immunocytochemistry of PAR1 (red) and phosphorylated MLC2 (Ser19, green) in human myometrial cells. (B, C) Immunoblots of phosphorylated MLC2 (p-MLC2), total MLC2, and β-actin of myometrial cells. Myometrial cells were treated with thrombin (2 U/mL) as a function of time (B) , or pretreated with 100 nM PAR1 inhibitor (SCH79797) for 1 h, and then treated with 2 U/mL of thrombin for 30 min (C) . The experiments were repeated three times.

Article Snippet: Primary antibodies used and concentration were as follows: thrombin (coagulation factor II, Novus Biologicals, NBP1-58268, Research Resource Identifier (RRID): AB_11023777, 1:100) and PAR1 (N2-11, Novus Biologicals, NBP1-71770, RRID: AB_11027203, 1:100).

Techniques: Immunocytochemistry, Western Blot

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: (A) Collagen lattice assay of myometrial cells at 30 min with 2 U/mL of thrombin (Thr) pretreated with 1 μM progesterone (P4) for 1 h. Representative image (upper panel) and quantification of gel areas (lower graph). (B) Inhibition of thrombin-induced increases of PTGS2 , IL1B , and F2R mRNA by P4. Myometrial cells were pretreated with 1 μM of P4 for 1 h, and then treated with 2 U/mL of thrombin. (C) Gene expressions of progesterone receptor-A and–B ( PgR-A and PgR-B ) with 24 h treatment of 1 U/mL of thrombin, 10 nM of PGE2, and 10 nM of PGF2α (upper graphs) and PgR-A to PgR-B ratio (lower graphs). n = 3 in each group. *, p < 0.05, and **, p < 0.01. The experiments were repeated three times.

Article Snippet: Primary antibodies used and concentration were as follows: thrombin (coagulation factor II, Novus Biologicals, NBP1-58268, Research Resource Identifier (RRID): AB_11023777, 1:100) and PAR1 (N2-11, Novus Biologicals, NBP1-71770, RRID: AB_11027203, 1:100).

Techniques: Inhibition