qnz  (MedChemExpress)

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: bioRxiv

Article Title: GluN2D-containing NMDA receptors regulate dentate gyrus function by facilitating granule cell activity and mediating synaptic plasticity

doi: 10.64898/2026.03.06.710109

Figure Lengend Snippet: (A) Bath application of the GluN2D antagonist DQP-1105 (30 μM for 15 min) caused a significant decrease in GC holding current (Control: −43.5 ± 1.3 pA, p < 0.01, n = 9, paired t-test), which was abolished in the presence of NMDAR antagonist D-APV (50 μM) (D-APV: 4.9 ± 4.7 pA, p = 0.3371, n = 7, paired t-test; Control vs D-APV: p < 0.001, unpaired t-test). Voltage-clamp recordings of GCs were performed at Vh = +40 mV in the presence of 10 μM NBQX and 100 μM picrotoxin. (B) The DQP-induced decrease in holding current was abolished Grin2d cKO mice (cKO: −0.4 ± 5.2 pA, p = 0.99479, n = 6, paired t-test) compared with controls (Control: −42.0 ± 11.5 pA, p < 0.05, n = 6 paired t-test; Control vs cKO: p < 0.01, unpaired t-test). (C) MPP NMDAR-EPSCs were recorded from GC (Vh = −45 mV) and evoked by stimulation electrode placed in the middle molecular layer (MML), in the presence of 10 μM NBQX and 100 μM picrotoxin. Bath application of the GluN2D antagonists DQP-1105 (30 μM) or QNZ46 (30 µM) for 30 mins had no effect on basal NMDAR synaptic transmission at MPP-GC synapses (99.9 ± 5.9 %, p = 0.9705, n = 9, paired t-test). The presence of NMDARs at the synapse was confirmed by bath applying D-APV (50 μM for 15 min) at the end of the recording. (D) Top , Representative NMDA outward currents (Vh = +40 mV) elicited by puffing a mix of 1 mM NMDA and 100 μM glycine on the MML of rat hippocampal slices. Bath application of the GluN2D antagonist DQP-1105 (30 μM) reduced the amplitude of these currents, which were abolished by subsequent application of D-APV (50 µM). Bottom left , Time-course plot (DQP-1105: 70.2 ± 3.7 % of baseline, p < 0.001, paired t-test; D-APV: 6.4 ± 2.3 % of baseline, p < 0.001, paired t-test; n = 6). Bottom right, summary plot showing the effects of DQP in rats, wildtype (WT: 63.9 ± 7.4 %, p < 0.01, n = 5, paired t-test), and Grin2d KO mice (95.1 ± 7.1 %, p = 0.5022, n = 7, paired t-test; Control vs KO: p < 0.01, unpaired t test). Data are presented as mean ± s.e.m.

Article Snippet: DQP-1105, QNZ46, D-APV, NMDA, and glycine were purchased from Tocris Biosciences.

Techniques: Control, Transmission Assay

Journal: bioRxiv

Article Title: GluN2D-containing NMDA receptors regulate dentate gyrus function by facilitating granule cell activity and mediating synaptic plasticity

doi: 10.64898/2026.03.06.710109

Figure Lengend Snippet: (A) Left, Representative average traces before (1) and after (2) pairing protocol application. Right, Time-course summary plot showing how pairing pre- and postsynaptic activity (pre-post: 6 pre pulses at 50 Hz followed by 5 post pulses at 100 Hz with a 10-ms interval, repeated 100 times every 0.5 s) induced robust NMDAR-LTP at MPP-GC synapses in rat hippocampal slices (Pre-post: 159.5 ± 7.3 %, p < 0.001, n = 6, paired t-test). In contrast, post-pre protocol (Post-pre: 5 post pulses at 100 Hz followed by 6 pre pulses at 50 Hz with a 10-ms interval, repeated 100 times every 0.5 s) did not induce significant long-term changes in NMDAR-mediated transmission (Post only: 96.9 ± 5.3 %, p = 0.58, n = 7, paired t-test). The pairing protocol was delivered at the time indicated by the vertical arrow. NMDAR-EPSCs were recorded (Vh = −45 mV) in the presence of 10 μM NBQX and 100 μM picrotoxin. (B) Neither presynaptic (Pre only: p = 0.51, n = 3, paired t-test) nor postsynaptic (Post only: p = 0.89, n = 8, paired t-test) bursts alone elicited any long-lasting change in NMDAR EPSC amplitude. (C) Bath application of the GluN2D antagonist DQP-1105 (30 µM) prevented NMDAR-LTP induction (Control: 149.1 ± 8.1 %, p < 0.001, n = 7, paired t-test; DQP-1105: 95.6 ± 8.8 %, p = 0.65, n = 4, paired t-test; DQP-1105 vs control: p < 0.01, unpaired t-test). (D) Pre-post pairing LTP induction protocol was delivered at MPP inputs. Bath application of either DQP-1105 (30 µM) or QNZ46 (30 µM) 30 min after NMDAR-LTP induction substantially (166.9 ± 8.2 %) significantly reduced NMDAR-mediated transmission (119.5 ± 8.8 %, n = 6; GluN2D antagonism vs post-LTP: p < 0.05, unpaired t-test). (E) Left, example traces of whole-cell current-clamp recordings in a GC (Vh –60 mV) in response to MPP burst-stimulation (5 pulses at 50 Hz, 20 V), before and 15–20 min after induction of NMDAR LTP. Right , average number of action potentials (APs) per train at different stimulation intensities, showing that induction of NMDAR LTP increases MPP-driven GC firing under control conditions (p < 0.001, n = 6, two-way ANOVA with Tukey’s comparison test). (F) This effect was abolished in the presence of DQP-1105 (30 µM) or QNZ46 (30 µM) (p = 0.42, n = 5, two-way ANOVA with Tukey’s comparison test). Data are presented as mean ± s.e.m.

Article Snippet: DQP-1105, QNZ46, D-APV, NMDA, and glycine were purchased from Tocris Biosciences.

Techniques: Activity Assay, Transmission Assay, Control, Comparison