Journal: British Journal of Pharmacology

Article Title: Melatonin up‐regulates bone marrow mesenchymal stem cells osteogenic action but suppresses their mediated osteoclastogenesis via MT 2 ‐inactivated NF‐κB pathway

doi: 10.1111/bph.14972

Figure Lengend Snippet: Melatonin promoted osteogenesis of bone marrow mesenchymal stem cells (BMMSCs) and inhibited RANKL expression which is involved in osteoclastogenesis. (a–c) alkaline phosphatase (ALP) levels were detected after osteogenic induction with melatonin for 7 days. (d) Mineralization nodes were observed by alizarin red staining after 21 days of osteogenic induction. (e, f) Protein (e) and gene (f) levels of Runx2, Osterix, and collagen I (Col‐I) were detected on Day 3 and Day 7 after melatonin treatment. (g) osteocalcin (OCN) expression was detected on Day 14 after melatonin treatment. (h, i) RANKL protein level (h) and the ratio of opg/rankl gene (i) were detected in bone marrow mesenchymal stem cells after melatonin treatment. Data are expressed as the mean ± SD and n = 5 in each group; *P < .05 significant differences between each indicated group. NS, not significant

Article Snippet: The level of osteocalcin produced by mouse Bone marrow mesenchymal stem cells was measured by the Mouse Osteocalcin ELISA Kit (Elabscience Biotechnology, Wuhan, China).

Techniques: Expressing, Staining

Journal: British Journal of Pharmacology

Article Title: Melatonin up‐regulates bone marrow mesenchymal stem cells osteogenic action but suppresses their mediated osteoclastogenesis via MT 2 ‐inactivated NF‐κB pathway

doi: 10.1111/bph.14972

Figure Lengend Snippet: MT2 receptor played the main role in melatonin‐regulated osteogenesis. After MT1 or MT2 silencing with shRNA, bone marrow mesenchymal stem cells (BMMSCs) were treated with melatonin for different days as indicated before parameter determination. (a–c) alkaline phosphatase (ALP) staining (a), ALP activity (b), and alp mRNA (c) were determined in Bone marrow mesenchymal stem cells after melatonin treatment for 7 days. (d) Mineralization nodes were measured by alizarin red staining on the Day 21. (e, f) The expressions of Runx2 and Osterix were detected on Day 3 at the protein (e) and gene level (f). (g) osteocalcin (OCN) was detected on Day 14 at the protein and gene level. (h) RANKL production was measured by western blot. (i) The opg/rankl ratio was measured by qRT‐PCR, respectively. Data are expressed as the mean ± SD and n = 5 in each group; *P < .05, significant differences between each indicated group. NS, not significant. NC, negative control

Article Snippet: The level of osteocalcin produced by mouse Bone marrow mesenchymal stem cells was measured by the Mouse Osteocalcin ELISA Kit (Elabscience Biotechnology, Wuhan, China).

Techniques: shRNA, Staining, Activity Assay, Western Blot, Quantitative RT-PCR, Negative Control

Journal: British Journal of Pharmacology

Article Title: Melatonin up‐regulates bone marrow mesenchymal stem cells osteogenic action but suppresses their mediated osteoclastogenesis via MT 2 ‐inactivated NF‐κB pathway

doi: 10.1111/bph.14972

Figure Lengend Snippet: Melatonin promoted osteogenesis by inhibiting the MT2‐mediated NF‐κB pathway. (a–b) Expression levels of NF‐keppa B pathway proteins including p‐p65, p65, p‐IκBα, IκBα, p‐IKKα/β, IKKα, and IKKβ were detected in bone marrow mesenchymal stem cells (BMMSCs) treated without or with melatonin (a) or in MT1/MT2 pre‐silenced Bone marrow mesenchymal stem cells (b). (c–e) bone marrow mesenchymal stem cells were pretreated with or without the NF‐κB inhibitor JSH‐23 for 1 hr and then treated with or without melatonin before parameter detection. Runx2, Osterix (c) and RANKL (d) were examined by western blot, and the opg/rankl ratio (e) was examined by qRT‐PCR. Data are expressed as the mean ± SD and n = 5 in each group; *P < .05, significant differences between each indicated group

Article Snippet: The level of osteocalcin produced by mouse Bone marrow mesenchymal stem cells was measured by the Mouse Osteocalcin ELISA Kit (Elabscience Biotechnology, Wuhan, China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: British Journal of Pharmacology

Article Title: Melatonin up‐regulates bone marrow mesenchymal stem cells osteogenic action but suppresses their mediated osteoclastogenesis via MT 2 ‐inactivated NF‐κB pathway

doi: 10.1111/bph.14972

Figure Lengend Snippet: Osteoclastogenesis negatively regulated by melatonin was BMMSC dependent and was mediated by the MT2/NF‐κB pathway. (a) Osteoclastogenesis of primary bone marrow monocytes treated with different concentrations of melatonin was visualized by TRAP staining, and the number of osteoclasts and their area were determined. (b–c) In direct‐contact and indirect‐contact co‐culture systems, osteoclastogenesis of primary BMMs was visualized by TRAP staining, the number of osteoclasts and their area were determined (b), and the expression of cathepsin K and c‐Fos (osteoclastogenic markers) was measured by western blot (c). (d) In the indirect‐contact co‐culture system, bone marrow mesenchymal stem cells (BONE MARROW MONOCYTESCs)were pretreated with lentiviral shRNA transfer or JSH‐23, and the osteoclastogenesis of Bone marrow monocytes was determined by TRAP staining (d, e) and western blot (f). In b–f, the concentration of melatonin was 10 nM. Data are expressed as the mean ± SD, and n = 5 in each group; *P < .05, significant differences between each indicated group. NS, not significant. NC, negative control

Article Snippet: The level of osteocalcin produced by mouse Bone marrow mesenchymal stem cells was measured by the Mouse Osteocalcin ELISA Kit (Elabscience Biotechnology, Wuhan, China).

Techniques: Staining, Co-Culture Assay, Expressing, Western Blot, shRNA, Concentration Assay, Negative Control

Journal: British Journal of Pharmacology

Article Title: Melatonin up‐regulates bone marrow mesenchymal stem cells osteogenic action but suppresses their mediated osteoclastogenesis via MT 2 ‐inactivated NF‐κB pathway

doi: 10.1111/bph.14972

Figure Lengend Snippet: A schematic model depicting MT2‐mediated NF‐κΒ signalling regulates the functions of bone marrow mesenchymal stem cells (BMMSCs) and the relationship between bone marrow mesenchymal stem cells and osteoclasts in the bone micro‐environment. Melatonin augments the osteogenic differentiation of Bone marrow mesenchymal stem cells and declines the production of RANKL in Bone marrow mesenchymal stem cells by inhibiting MT2‐mediated NF‐κΒ signalling. Then bone marrow mesenchymal stem cells affect the maturation and functions of osteoclasts via RANKL paracrine secretion

Article Snippet: The level of osteocalcin produced by mouse Bone marrow mesenchymal stem cells was measured by the Mouse Osteocalcin ELISA Kit (Elabscience Biotechnology, Wuhan, China).

Techniques: