Journal: Theranostics

Article Title: Melatonin Suppresses Neuropathic Pain via MT2-Dependent and -Independent Pathways in Dorsal Root Ganglia Neurons of Mice.

doi: 10.7150/thno.19500

Figure Lengend Snippet: Figure 3. MT2 activation by 100 μM 8MP suppressed calcium channel signaling via MAPK1 in DRG neurons (Part I). (A) The biological process of Gene Ontology analysis of MT activation by 100 μM 8MP in primary DRG neurons; (B) The pathway network analysis of 39 pain-related DEGs in the 100 μM 8MP group; (C) Real-time PCR and Western blotting results obtained from different groups with β-actin as an internal standard. Bar chart represents the fold increases in expression of different genes and proteins isolated from primary cultured DRG neurons following 24-h incubation with different treatments: Vel group (treated with vehicle), 100 μM 8MP group (treated with 100 μM 8MP) and 100 μM 8MP+L group (treated with 100 μM 8MP and 100 µM luzindole). (***P<0.001 vs. Vel; ###P<0.001 vs. 100 μM 8MP; n=8 for each group).

Article Snippet: Mel, luzindole (MT1 and MT2 antagonist), ramelteon (MT1 and MT2 agonist), 8-methoxy-2-propionamidotetralin (8MP, MT2 agonist) 4-phenyl-2-propionamidotetralin (4PP, MT2 antagonist), CGP52608 (RAR-related orphan receptor alpha [RORα] receptor agonist), and ML-176 (RORα receptor inverse agonist) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Isolation, Cell Culture, Incubation

Journal: Theranostics

Article Title: Melatonin Suppresses Neuropathic Pain via MT2-Dependent and -Independent Pathways in Dorsal Root Ganglia Neurons of Mice.

doi: 10.7150/thno.19500

Figure Lengend Snippet: Figure 5. Low-dose Mel suppressed calcium channel and MAPK1 expression in DRG neurons. Real-time PCR and Western blotting from different groups with β-actin as an internal standard: Primary cultured DRG neurons were subjected to different treatments as follows: (A) Vel group (treated with vehicle), 100 μM Mel group (treated with 100 μM Mel) and 100 μM Mel+L group (treated with 100 μM Mel and 100 µM luzindole), ***P<0.001 vs. Vel group, ###P<0.001 vs. 100 µM Mel; (B) Vel group (treated with vehicle) and 1 mM Mel group (treated with 1 mM Mel), ***P<0.001 vs. Vel group; n=8 in all groups

Article Snippet: Mel, luzindole (MT1 and MT2 antagonist), ramelteon (MT1 and MT2 agonist), 8-methoxy-2-propionamidotetralin (8MP, MT2 agonist) 4-phenyl-2-propionamidotetralin (4PP, MT2 antagonist), CGP52608 (RAR-related orphan receptor alpha [RORα] receptor agonist), and ML-176 (RORα receptor inverse agonist) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture

Journal: Theranostics

Article Title: Melatonin Suppresses Neuropathic Pain via MT2-Dependent and -Independent Pathways in Dorsal Root Ganglia Neurons of Mice.

doi: 10.7150/thno.19500

Figure Lengend Snippet: Figure 6. RORα activation by Mel inhibited MAPK1 and calcium channel down-regulation via MT2. Real-time PCR and Western blotting from different groups with β-actin as an internal standard: Primary cultured DRG neurons were subjected to different treatments as follows: (A) 1 mM Mel+Con siRNA group (treated with 1 mM Mel and control siRNA), 1 mM Mel+MAPK1 siRNA group (treated with 1 mM Mel and MAPK1 siRNA), ***P<0.001 vs. 1 mM Mel+Con siRNA; (B) and (C) 1 mM Mel group (treated with 1 mM Mel), 1 mM Mel+200 µM ML-176 group (treated with 1 mM Mel and 200 µM ML-176), 1 mM Mel+RORα siRNA group (treated with 1 mM Mel and RORα siRNA), **P<0.01 vs. 1 mM Mel ***P<0.001 vs. 1 mM Mel; (D) 100 µM Mel group (treated with 100 µM Mel), 100 µM Mel+C group (treated with 100 µM Mel and 200 µM CGP52608), 100 µM Mel+C+RORα siRNA group (treated with 100 µM Mel, 200 µM CGP52608 and RORα siRNA), 100 µM Mel+L group (treated with 100 µM Mel and 200 µM luzindole), ***P<0.001 vs. 100 µM Mel, ###P<0.001 vs. 100 µM Mel+C; (E) 100 µM 8MP group (treated with 100 µM 8MP), 100 µM 8MP+C group (treated with 100 µM 8MP and 200 µM CGP52608), 100 µM 8MP+C+RORα siRNA group (treated with 100 µM 8MP, 200 µM CGP52608 and RORα siRNA), ***P<0.001 vs. 100 µM Mel, ###P<0.001 vs. 100 µM Mel+C; n=8 for all groups.

Article Snippet: Mel, luzindole (MT1 and MT2 antagonist), ramelteon (MT1 and MT2 agonist), 8-methoxy-2-propionamidotetralin (8MP, MT2 agonist) 4-phenyl-2-propionamidotetralin (4PP, MT2 antagonist), CGP52608 (RAR-related orphan receptor alpha [RORα] receptor agonist), and ML-176 (RORα receptor inverse agonist) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Control

Journal: Theranostics

Article Title: Melatonin Suppresses Neuropathic Pain via MT2-Dependent and -Independent Pathways in Dorsal Root Ganglia Neurons of Mice.

doi: 10.7150/thno.19500

Figure Lengend Snippet: Figure 7. Regulation of NOS1 signaling in the DRG neurons was independent from the MT2 receptor and RORα nuclear receptor. (A) Gene Ontology analysis of MT activation by 1 mM Mel in primary DRG neurons; (B) Pathway network analysis of 19 pain-related DEGs in the 1 mM Mel group; (C) The Venn diagram analysis of the 46 pain-related DEGs in the 1 mM Mel group and 100 μM 8MP group (the black number represents the overall DEGs and the red number represents the pain-related DEGs); (D) Real-time PCR and Western blotting of NOS1 under different treatments in DRG neurons. The primary cultured DRG neurons were treated with different reagents as follows: Vel group (treated with vehicle), 1 mM Mel group (treated with 1 mM Mel), 1 mM Mel+L group (treated with 1 mM Mel and 200 µM luzindole), 100 μM Mel+ML-176 group (treated with 100 μM Mel and 200 µM ML-176), 100 μM 8MP group (treated with 100 μM 8MP), and 200 μM CGP52608 group (treated with 200 µM CGP52608); ***P<0.001 vs. Vel; n=8 for each group.

Article Snippet: Mel, luzindole (MT1 and MT2 antagonist), ramelteon (MT1 and MT2 agonist), 8-methoxy-2-propionamidotetralin (8MP, MT2 agonist) 4-phenyl-2-propionamidotetralin (4PP, MT2 antagonist), CGP52608 (RAR-related orphan receptor alpha [RORα] receptor agonist), and ML-176 (RORα receptor inverse agonist) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture

Journal: Theranostics

Article Title: Melatonin Suppresses Neuropathic Pain via MT2-Dependent and -Independent Pathways in Dorsal Root Ganglia Neurons of Mice.

doi: 10.7150/thno.19500

Figure Lengend Snippet: Figure 8. Impact of MT and 8MP treatment on expression of c-fos, CGRP, and neuro-inflammatory cytokines in the DRG of the cuff-implanted mice. (A) Western blotting for c-fos and CGRP obtained from different groups with β-actin as an internal standard: Sham group (sham-operated mice), Cuff+Vel group (i.p. injection of vehicle), Cuff+Mel (i.p. injection of 100mg/kg Mel), Cuff+Mel+L group (i.p. injection of 100 mg/kg Mel and 20 mg/kg luzindole), Cuff+8MP group (i.p. injection of 100mg/kg 8MP) and Cuff+8MP+L group (i.p. injection of 00 mg/kg 8MP and 20 mg/kg luzindole), ***P<0.001 vs. Sham; ###P<0.001 vs. Cuff+Vel; n=8 for each group. (B) Analysis of the inflammatory cytokines TNF-α and IL-1β by ELISA in different DRG treatment groups as above (***P<0.001 vs. Sham; ###P<0.001 vs. Cuff+Vel; n=8 for each group).

Article Snippet: Mel, luzindole (MT1 and MT2 antagonist), ramelteon (MT1 and MT2 agonist), 8-methoxy-2-propionamidotetralin (8MP, MT2 agonist) 4-phenyl-2-propionamidotetralin (4PP, MT2 antagonist), CGP52608 (RAR-related orphan receptor alpha [RORα] receptor agonist), and ML-176 (RORα receptor inverse agonist) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Injection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Melatonin Inhibits NF-κB/CREB/Runx2 Signaling and Alleviates Aortic Valve Calcification

doi: 10.3389/fcvm.2022.885293

Figure Lengend Snippet: Effect of MT1 neutralized antibody on melatonin-attenuated VIC calcification. The left panel depicts representative Alizarin red S staining of VICs with or without melatonin (10 nmol/L) or with or without MT1 neutralized antibody (MT1 Ab, 0.5, 1 and 2 μg/mL) or control IgG (0.5, 1 and 2 μg/mL). We quantified the positive stain area (red color) per field (three fields per treatment) in the VICs ( n = 4). The right panel presents the summary data of the percentage of calcification area ( n = 4). Control (Con) vs OST: P = 0.002; OST vs. OST + Mel 10nM: P = 0.003; OST + Mel 10nM vs. OST + Mel 10nM + MT1 Ab 1 μg/mL: P = 0.027; Con vs. OST + Mel 10nM + MT1 Ab 1 μg/mL: 0.016; OST + Mel 10nM vs. OST + Mel 10nM + MT1 Ab 2 μg/mL: P = 0.012; Con vs. OST + Mel 10nM + MT1 Ab 2 μg/mL. P = 0.007. OST, osteogenic medium; Mel, melatonin; MT1, Melatonin receptor 1. * P < 0.05, ** P < 0.01, *** P < 0.005.

Article Snippet: Aortic VICs that reached 80% confluence were cultured in an osteogenic (OST) medium with a DMEM/F12 complete medium containing 50-mg/mL ascorbic acid, 10-mmol/L β-glycerophosphate, and 10-nmol/L dexamethasone for 5 days with or without melatonin (0.1–10 nmol/L), SC75741 (NF-κB inhibitor, 1 μmol/L), Luzindole (MT1/MT2 receptor inhibitor, 1 μmol/L), 4-P-PDOT (MT2 melatonin receptor antagonist, 1 μmol/L), and anti-MT1 antibody (0.5, 1, 2 μg/mL; Mel-1A-R; 1:5000; Cat# sc-390328, Santa Cruz) or nonspecific immunoglobulin (control IgG, 1 h prior to OST or melatonin treatment) as previously described ( ).

Techniques: Staining, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Melatonin Inhibits NF-κB/CREB/Runx2 Signaling and Alleviates Aortic Valve Calcification

doi: 10.3389/fcvm.2022.885293

Figure Lengend Snippet: Schematic of the potential mechanisms for the therapeutic effects of melatonin on osteogenesis in VICs. The melatonin/MT1 axis may downregulate Runx2 expression through inactivation of NF-κB/CREB signaling in OST medium-treated VICs. Conversely, activation of the NF-κB/CREB complex may bind to the Runx2 promoter to increase Runx2 gene transcription through VIC osteogenesis.

Article Snippet: Aortic VICs that reached 80% confluence were cultured in an osteogenic (OST) medium with a DMEM/F12 complete medium containing 50-mg/mL ascorbic acid, 10-mmol/L β-glycerophosphate, and 10-nmol/L dexamethasone for 5 days with or without melatonin (0.1–10 nmol/L), SC75741 (NF-κB inhibitor, 1 μmol/L), Luzindole (MT1/MT2 receptor inhibitor, 1 μmol/L), 4-P-PDOT (MT2 melatonin receptor antagonist, 1 μmol/L), and anti-MT1 antibody (0.5, 1, 2 μg/mL; Mel-1A-R; 1:5000; Cat# sc-390328, Santa Cruz) or nonspecific immunoglobulin (control IgG, 1 h prior to OST or melatonin treatment) as previously described ( ).

Techniques: Expressing, Activation Assay