Journal: Frontiers in Pharmacology

Article Title: Selumetinib - a potential small molecule inhibitor for osteoarthritis treatment

doi: 10.3389/fphar.2022.938133

Figure Lengend Snippet: The molecular mechanism of selumetinib protects chondrocytes and inhibits osteoclast formation during OA. In ATDC5, selumetinib inhibited IL-1β-induced activation of NF-kB and MAPK signalling pathways and alleviated EMC degeneration. Selumetinib also attenuated the occurrence of inflammation by inhibiting necroptosis. For BMMs, selumetinib inhibited the activation of NF-kB and MAPK signaling pathways induced by RANKL, reducd osteoclast-associated gene expression and osteoclast formation.

Article Snippet: Nuclear factor-κB receptor activator ligand (RANKL) and mouse recombinant macrophage colony-stimulating factor (M-CSF) were purchased from LifeTein (Beijing, China).

Techniques: Activation Assay, Protein-Protein interactions, Gene Expression

Journal: Frontiers in Pharmacology

Article Title: Selumetinib - a potential small molecule inhibitor for osteoarthritis treatment

doi: 10.3389/fphar.2022.938133

Figure Lengend Snippet: Selumetinib inhibits RANKL-induced osteoclast-related gene expression and osteoblast differentiation in vitro . (A) We stimulated BMMs with MCSF (30 ng/mL), RANK (50 ng/mL) and different concentrations of selumetinib (10, 20, and 40 nM) for 7 days to investigate the concentration-dependent inhibition of osteoblast differentiation by selumetinib. (B,C) The area and number of TRAP-stained positive cells (nuclei ≥ 3) were recorded. (D–F) The time-dependent inhibition of osteoclast formation was investigated by treating 40 nM selumetinib at different time points (days 1-3, 3-5 or 5-7) to counteract the effect of RANKL on promoting osteoclast differentiation. (G) We stained BMMs for laser scanning confocal microscopy observation and quantified the number of formed actin rings and the area of osteoclasts actin ring (%) (H,I) . (J) We treated BMMs with RANKL and 10, 20 and 40 nM selumetinib for 5 days and performed RT-PCR to examine osteoclast-related mRNA expression levels, including NFATc1, c-fos, ACP5, DC-STAMP, V-ATPase D2 and CTSK. Data are presented as mean ± S.D. n = 3. Significant differences between groups are indicated as **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Nuclear factor-κB receptor activator ligand (RANKL) and mouse recombinant macrophage colony-stimulating factor (M-CSF) were purchased from LifeTein (Beijing, China).

Techniques: Gene Expression, In Vitro, Concentration Assay, Inhibition, Staining, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Frontiers in Pharmacology

Article Title: Selumetinib - a potential small molecule inhibitor for osteoarthritis treatment

doi: 10.3389/fphar.2022.938133

Figure Lengend Snippet: Selumetinib impairs RANKL-induced activation of the MAPK and NF-κB signalling pathway. (A) BMMs were pretreated for 1 h with or without selumetinib (40 nM) and then stimulated by RANKL (50 ng/mL) for 0, 5, 10, 20, 30 or 60 min. The proteins expression of NF-kB and MAPK signalling pathway was assessed by Western blot analysis, and quantified by image J (B–F) . (G) BMMs were treated with RANKL (50 ng/mL) and different concentrations of selumetinib (10, 20 or 40 nM) simultaneously for 3 days to observe the effect of different concentrations of selumetinib on the NF-kB pathway. (H) After treatment with RANKL (50 ng/ml) without or with selumetinib (40 nM), BMMs were stained for laser scanning confocal microscopy assay. (I,J) Treatment with 50 ng/mL RANKL and 40 nM Selumetinib simultaneously for 1, 3 or 5 days to observe the relative expression of NFATc1. Data are presented as mean ± S.D. n = 3. Significant differences between groups are indicated as **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Nuclear factor-κB receptor activator ligand (RANKL) and mouse recombinant macrophage colony-stimulating factor (M-CSF) were purchased from LifeTein (Beijing, China).

Techniques: Activation Assay, Expressing, Western Blot, Staining, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Individual and Combined Effect of Bisphenol A and Bisphenol AF on Prostate Cell Proliferation through NF-κB Signaling Pathway

doi: 10.3390/ijms232012283

Figure Lengend Snippet: Localization observation of phosphorylated NF-κB p65 (p-NF-κB p65), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and epidermal growth factor receptor (EGFR) expression in VP. ( A ) Immunohistochemical images of p-NF-κB p65, COX-2, TNF-α and EGFR of the prostate tissues in the rats treated using BPA, BPAF and PDTC (400×, scale bar = 20 μm). Arrows indicate the positive expression. Effect of exposure to BPA, BPAF and PDTC on the rate of positive expression of p-NF-κB p65 ( B ); the semiquantitative expression levels of COX-2 ( C ), TNF-α ( D ) and EGFR ( E ) in ventral prostate (VP). Results were performed as means ± SD, analyzed using ANOVA followed by LSD post hoc test, n = 8. Comparison of the individual administration group and the control: * p < 0.05, ** p < 0.01; comparison of the combined administration group and the individual administration group: # p < 0.05, ## p < 0.01.

Article Snippet: In accordance with the protocol of the manufacturer of the human ELISA Kits (NF-κB p65, 022214CYF129780224, Jianglaibio, Shanghai, China; COX-2, 022214CYF194700224, Jianglaibio, Shanghai, China; TNF-α, EK0525, Boster, Wuhan, China; EGFR, EK0327, Boster, Wuhan, China), the cell samples were incubated with the corresponding antibodies and colored.

Techniques: Expressing, Immunohistochemical staining, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Individual and Combined Effect of Bisphenol A and Bisphenol AF on Prostate Cell Proliferation through NF-κB Signaling Pathway

doi: 10.3390/ijms232012283

Figure Lengend Snippet: BPA, BPAF and PDTC induced the quantitative changes of p-NF-κB p65, COX-2, TNF-α and EGFR in VP. Content of NF-κB p65 ( A ), COX-2 ( B ), TNF-α ( C ) and EGFR ( D ) was obtained using enzyme-linked immunosorbent assay (ELISA). Results are performed as means ± SD, analyzed using ANOVA followed by LSD post hoc test, n = 4. Comparison of the individual administration group and the control: * p < 0.05, ** p < 0.01; comparison of the combined administration group and the individual administration group: # p < 0.05, ## p < 0.01.

Article Snippet: In accordance with the protocol of the manufacturer of the human ELISA Kits (NF-κB p65, 022214CYF129780224, Jianglaibio, Shanghai, China; COX-2, 022214CYF194700224, Jianglaibio, Shanghai, China; TNF-α, EK0525, Boster, Wuhan, China; EGFR, EK0327, Boster, Wuhan, China), the cell samples were incubated with the corresponding antibodies and colored.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Individual and Combined Effect of Bisphenol A and Bisphenol AF on Prostate Cell Proliferation through NF-κB Signaling Pathway

doi: 10.3390/ijms232012283

Figure Lengend Snippet: PDTC influenced BPA- and BPAF-facilitated NF-κB p65, COX-2, TNF-α and EGFR expression in prostate cells. Effect of exposure to BPA, BPAF and the inhibitor of NF-κB (PDTC) on the expression of NF-κB p65 ( A ), COX-2 ( B ), TNF-α ( C ) and EGFR ( D ) in WPMY−1 cells and the levels of NF-κB p65 ( E ), COX-2 ( F ) and EGFR ( G ) in HPrF cells. Results are performed as means ± SD, analyzed using ANOVA followed by LSD post hoc test, n = 3. Comparison of the individual administration group and the control: * p < 0.05, ** p < 0.01; comparison of the combined administration group and the individual administration group: # p < 0.05, ## p < 0.01.

Article Snippet: In accordance with the protocol of the manufacturer of the human ELISA Kits (NF-κB p65, 022214CYF129780224, Jianglaibio, Shanghai, China; COX-2, 022214CYF194700224, Jianglaibio, Shanghai, China; TNF-α, EK0525, Boster, Wuhan, China; EGFR, EK0327, Boster, Wuhan, China), the cell samples were incubated with the corresponding antibodies and colored.

Techniques: Expressing, Comparison, Control

Journal: bioRxiv

Article Title: NF-κB Decoy ODN-Loaded Poly lactic-co-glycolic Acid Nanospheres Inhibit Alveolar Ridge Resorption

doi: 10.1101/2022.08.30.505814

Figure Lengend Snippet: A/E , IL-1β; B/F , TNF-α; C/G , TGF-β1; D/H , RANKL. On D7 and D28, PLGA-NfD demonstrated a lower immunopositive stained ratio for IL-1β, TNF-α, and RANKL and a high ratio for TGF-β1. The NfD group showed significantly lower IL-1β and RANKL expression only on D7; the expression of other factors in this group was similar to that in the PBS, ScD, and PLGA-ScD groups on D7 and D28. Abbreviations: D7, post-extraction day 7; D28, post-extraction day 28; PBS, PBS group; ScD, naked scrambled decoy group; NfD, naked NF-κB decoy group; PLGA-ScD, Scrambled decoy ODN-loaded PLGA nanosphere group; PLGA-NfD, NF-κB decoy ODN-loaded PLGA nanosphere group. Values are presented as mean ± standard deviation, (n = 5). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001. Original magnification 40x and 200x were shown in each representative histological image. Scale Bar = 100 μm.

Article Snippet: Appropriate specific TaqMan Gene Expression Assay primers (Applied Biosystems; Thermo Fisher Scientific, Foster City, CA, USA) were chosen for real-time PCR amplification of rat GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA (GAPDH; Rn01775763_g1), rat IL-1β (interleukin 1 beta) mRNA (Rn00580432_m1), rat TNF-α (tumor necrosis factor-alpha) mRNA (Rn01525859_g1), rat RANKL (Receptor Activation of Nuclear Factor-κB ligand) mRNA (Tnfsf11; Rn00589289_m1), rat osteoprotegerin (OPG) mRNA (Tnfrsf11b; Rn00563499_m1), and rat TGF-β1 (transforming growth factor-beta 1) mRNA (Rn00572010_m1).

Techniques: Staining, Expressing, Extraction, Standard Deviation

Journal: bioRxiv

Article Title: NF-κB Decoy ODN-Loaded Poly lactic-co-glycolic Acid Nanospheres Inhibit Alveolar Ridge Resorption

doi: 10.1101/2022.08.30.505814

Figure Lengend Snippet: Relative gene expression of (A) IL-1β, (B) TNF-α (C) TGF-β1 (D) Tnfsf11 (RANKL) (E) OPG (Tnfrsf11b) / GAPDH (fold change) and (F) Tnfsf11/Tnfrsf11b (RANKL/OPG) ratio is presented in the graph. On D7, the PLGA-NfD group showed lower relative gene expression of IL-1β and TNF-α than the PBS, ScD, and PLGA-ScD groups; lower RANKL and OPG expression than the PBS, ScD, and PLGA-ScD groups; lower RANKL/OPG ratio than the PLGA-ScD group. On D28, the PLGA-NfD group showed lower IL-1β expression than the PBS group; lower TNF-α expression than the ScD group; lower RANKL expression than the PBS group; and lower OPG expression than the ScD group. No significant difference in the RANKL/OPG ratio was found among the groups on D28. The PLGA-NfD group demonstrated greater relative gene expression of TGFβ1 on both D7 and D28 than the ScD group. The TGFβ1 expression on D28 was also significantly greater in the PLGA-NfD group than in the PBS group. Values are presented as mean ± standard deviation, (n = 5). *: P < 0.05, **: P < 0.01. Abbreviations: D7, post-extraction day 7; D28, post-extraction day 28; PBS, PBS group; ScD, naked scrambled decoy group; NfD, naked NF-κB decoy group; PLGA-ScD, Scrambled decoy ODN-loaded PLGA nanosphere group; PLGA-NfD, NF-κB decoy ODN-loaded PLGA nanosphere group.

Article Snippet: Appropriate specific TaqMan Gene Expression Assay primers (Applied Biosystems; Thermo Fisher Scientific, Foster City, CA, USA) were chosen for real-time PCR amplification of rat GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA (GAPDH; Rn01775763_g1), rat IL-1β (interleukin 1 beta) mRNA (Rn00580432_m1), rat TNF-α (tumor necrosis factor-alpha) mRNA (Rn01525859_g1), rat RANKL (Receptor Activation of Nuclear Factor-κB ligand) mRNA (Tnfsf11; Rn00589289_m1), rat osteoprotegerin (OPG) mRNA (Tnfrsf11b; Rn00563499_m1), and rat TGF-β1 (transforming growth factor-beta 1) mRNA (Rn00572010_m1).

Techniques: Gene Expression, Expressing, Standard Deviation, Extraction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Skimmianine Attenuates Osteoclast Activity by Suppressing ERp57‐ Driven Calcium Oscillations/Calcineurin/Nfatc1 Signalling in Postmenopausal Osteoporosis

doi: 10.1111/jcmm.70777

Figure Lengend Snippet: Ski maintains bioactivity in BMMs and impedes RANKL‐induced osteoclastogenesis. (A) CCK‐8 assay for cytotoxicity test. Bone marrow‐derived monocytes (BMMs) were treated with various concentrations of Ski (0–45 μM) stimuli in proliferating medium containing M‐CSF (10 ng/mL) only or in (B) osteoclast differentiation medium including M‐CSF and RANKL (30 ng/mL) for 24 h ( n = 3). (C) TRAP staining of osteoclasts derived from BMMs under different catalyses. BMMs were induced with M‐CSF (10 ng/mL) and RANKL (30 ng/mL) for a 5‐day period under Ski (0, 35 and 40 μM) treatment. Scale bar = 200 μm. (D) Quantitative analysis of TRAP‐positive cells (containing more than three nuclei) ( n = 3). Data were presented as mean ± SD. * p < 0.05; *** p < 0.001.

Article Snippet: Other biochemistry reagents for cell stimulation and differentiation included Skimmianine (Ski, 83‐95‐4, MCE), dimethyl sulphoxide (DMSO, D2650, Sigma‐Aldrich), macrophage colony‐stimulating factor (M‐CSF, 576406; BioLegend) and receptor activator of nuclear factor‐κB (RANK) ligand (RANKL, 462‐TEC‐010, R&D Systems).

Techniques: CCK-8 Assay, Derivative Assay, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Skimmianine Attenuates Osteoclast Activity by Suppressing ERp57‐ Driven Calcium Oscillations/Calcineurin/Nfatc1 Signalling in Postmenopausal Osteoporosis

doi: 10.1111/jcmm.70777

Figure Lengend Snippet: Ski imposes restrictions on resorptive function of osteoclasts. (A) F‐Actin ring staining. BMMs were induced with M‐CSF (10 ng/mL) and RANKL (30 ng/mL) for a 5‐day differentiation with or without Ski (35 and 40 μM) treatment. The F‐Actin rings (red) and nuclei (blue) of RANKL and Ski‐treated groups were stained using phalloidin‐ifour 594 and DAPI and observed under a fluorescence microscope. Scale bar = 300 μm. (B) The bar graph showed the proportion of osteoclasts with F‐Actin rings ( n = 3). Data were presented as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. (C) Acridine orange (AO) staining. BMMs were induced with M‐CSF (10 ng/mL) and RANKL (30 ng/mL) for a 4‐day differentiation with or without Ski (35 and 40 μM) treatment. The function of acid secretion in osteoclasts of acidic vesicles stained with AO solution was exhibited through the fluorescence microscope images. Scale bar = 300 μm. (D) Each fluorescence intensity was expressed by the ratio of red to green light and measured by ImageJ software (1.53c, National Institutes of Health, United States) ( n = 3). Data were presented as mean ± SD. ** p < 0.01. *** p < 0.001.

Article Snippet: Other biochemistry reagents for cell stimulation and differentiation included Skimmianine (Ski, 83‐95‐4, MCE), dimethyl sulphoxide (DMSO, D2650, Sigma‐Aldrich), macrophage colony‐stimulating factor (M‐CSF, 576406; BioLegend) and receptor activator of nuclear factor‐κB (RANK) ligand (RANKL, 462‐TEC‐010, R&D Systems).

Techniques: Staining, Fluorescence, Microscopy, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Skimmianine Attenuates Osteoclast Activity by Suppressing ERp57‐ Driven Calcium Oscillations/Calcineurin/Nfatc1 Signalling in Postmenopausal Osteoporosis

doi: 10.1111/jcmm.70777

Figure Lengend Snippet: Ski impedes the expression of osteoclast‐specific marker genes. (A–E) RT‐qPCR illustrated the mRNA levels of Traf6 (A), Nfatc1 (B), Ctsk (C) MMP9 (D) and Atp6v0d2 (E) in RANKL and Ski (40 μM) groups. Quantitative results were normalised to GAPDH and presented as means ± SD ( n = 3), ** p < 0.01 and *** p < 0.001. (F–H) WB showed the protein level of Nfatc1 and Ctsk in RANKL and Ski (40 μM) groups. Quantitative results were normalised to β‐Actin and presented as means ± SD ( n = 3). ** p < 0.01.

Article Snippet: Other biochemistry reagents for cell stimulation and differentiation included Skimmianine (Ski, 83‐95‐4, MCE), dimethyl sulphoxide (DMSO, D2650, Sigma‐Aldrich), macrophage colony‐stimulating factor (M‐CSF, 576406; BioLegend) and receptor activator of nuclear factor‐κB (RANK) ligand (RANKL, 462‐TEC‐010, R&D Systems).

Techniques: Expressing, Marker, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Skimmianine Attenuates Osteoclast Activity by Suppressing ERp57‐ Driven Calcium Oscillations/Calcineurin/Nfatc1 Signalling in Postmenopausal Osteoporosis

doi: 10.1111/jcmm.70777

Figure Lengend Snippet: Ski negatively affects calcium oscillations/calcineurin/Nfatc1 signalling during osteoclastogenesis via the suppression of ERp57 expression. (A–D) Molecular docking ichnography and 3D molecular model between Ski and ERp57 (C), calcineurin (D). (E) Protein‐ligand interactions between Ski (blue) and calcineurin (violet, only interacting protein residues are shown). Interactions are drawn as coloured solid or dashed lines (mazarine, grey and orange). BMMs were induced with M‐CSF (10 ng/mL) and RANKL (30 ng/mL) with or without Ski (40 μM) for 48 h. 10 cells were selected from each treatment group (F, G) and indicated intensity of calcium oscillations. Images were captured every 5 s for 20 min through a fluorescence microscope (EVOS M7000). Quantitative analysis of fluorescence intensity was measured by ImageJ software. (H, J) WB showed the protein level of ERp57 in RANKL and Ski groups. (I, K) WB showed the protein level of PP2B‐Aα in RANKL and Ski groups. Quantitative results were normalised to β‐Actin and presented as means ± SD ( n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: Other biochemistry reagents for cell stimulation and differentiation included Skimmianine (Ski, 83‐95‐4, MCE), dimethyl sulphoxide (DMSO, D2650, Sigma‐Aldrich), macrophage colony‐stimulating factor (M‐CSF, 576406; BioLegend) and receptor activator of nuclear factor‐κB (RANK) ligand (RANKL, 462‐TEC‐010, R&D Systems).

Techniques: Expressing, Fluorescence, Microscopy, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Skimmianine Attenuates Osteoclast Activity by Suppressing ERp57‐ Driven Calcium Oscillations/Calcineurin/Nfatc1 Signalling in Postmenopausal Osteoporosis

doi: 10.1111/jcmm.70777

Figure Lengend Snippet: A working schematic diagram demonstrates Ski mitigates osteoclastogenesis through suppressing ERp57‐driven calcium oscillations–calcineurin–NFATc1 signalling. RANKL–RANK interaction initiates intracellular calcium oscillations, a signalling cascade that subsequently activates calcineurin‐dependent NFATc1 transcriptional activity. Ski inhibited ERp57‐driven intracellular calcium oscillations and the expression of calcineurin, which attenuated NFATc1 activation, subsequently leading to the reduction of osteoclast‐specific gene expression, including Ctsk, MMP9 and Atp6v0d2. RANK: TNF receptor superfamily member 11a; RANKL: Receptor activator of nuclear factor‐κB ligand; Traf6: TNF receptor‐associated factor 6; Nfatc1: Nuclear factor of activated T‐cells 1; Ctsk: Cathepsin K; MMP9: Matrix metalloproteinase 9; Atp6v0‐d2: ATPase H+ transporting V0 subunit d2.

Article Snippet: Other biochemistry reagents for cell stimulation and differentiation included Skimmianine (Ski, 83‐95‐4, MCE), dimethyl sulphoxide (DMSO, D2650, Sigma‐Aldrich), macrophage colony‐stimulating factor (M‐CSF, 576406; BioLegend) and receptor activator of nuclear factor‐κB (RANK) ligand (RANKL, 462‐TEC‐010, R&D Systems).

Techniques: Activity Assay, Expressing, Activation Assay, Gene Expression