selective melatonin mt2 receptor antagonist  (Tocris)

Journal: Redox Biology

Article Title: Melatonin attenuates atherosclerotic plaque vulnerability through SIRT6-dependent regulation of vascular smooth muscle cells senescence

doi: 10.1016/j.redox.2025.103939

Figure Lengend Snippet: Melatonin enhances the stability of vulnerable plaques via an MT membrane receptor-dependent manner in vivo. (A) Experimental design: ApoE −/− mice were administered either saline (vehicle), melatonin or melatonin combined with luzindole for eight weeks following the induction of vulnerable carotid plaques. (B) Representative longitudinal micro-ultrasound images of the left common carotid arteries in indicated groups. (C–E) Quantification analyses of IMT, EI and Vmax (n = 8 per group). (F) Representative macroscopic views of the left common carotid arterial segments in indicated groups (scale bar = 1 mm). (G) Representative cross‐sections of carotid plaques stained with H&E, Masson's trichrome, and Oil Red O in indicated groups (scale bar = 50 μm). (H–M) Quantitative analyses of fibrous cap-to-plaque area ratio, the core-to-plaque area ratio, fibrous cap-to-core area ratio, collagen content, lipid content, and collagen‐to‐lipid ratio (n = 7–8 per group). (N) Representative immunohistochemical staining of melatonin membrane receptors (MTNR1A/MTNR1B; brown) in carotid plaques in indicated groups (scale bar = 50 μm). (O–P) Quantification of positively stained area (n = 6–8 per group). ∗∗ P < 0.01 or ∗∗∗ P < 0.001 versus ApoE −/− + V; # P < 0.05, ## P < 0.01 or ### P < 0.001 versus ApoE −/− + Mel + Luz. EI, eccentric index; HE, hematoxylin and eosin; IMT, intimal‐medial thickness; Luz, luzindole; Mel, melatonin; V, vehicle; Vmax, maximal systolic velocity.

Article Snippet: Melatonin (HY–B0075, MCE, USA) and the melatonin receptor antagonist luzindole (HY-101254, MCE, USA) were first dissolved in dimethyl sulfoxide (DMSO; ID9010, Solarbio) and then diluted in a mixture of PEG300 (HY–Y0873, MCE), Tween 80 (HY–Y1891, MCE), and saline to obtain the working solutions.

Techniques: Membrane, In Vivo, Saline, Micro-Ultrasound, Staining, Immunohistochemical staining

Journal: Redox Biology

Article Title: Melatonin attenuates atherosclerotic plaque vulnerability through SIRT6-dependent regulation of vascular smooth muscle cells senescence

doi: 10.1016/j.redox.2025.103939

Figure Lengend Snippet: Melatonin attenuates VSMC senescence within plaques via an MT membrane receptor-dependent manner in vivo. (A) Representative co-staining of SA-β-gal activity (green) and α-SMA (brown) in carotid plaques in indicated groups (scale bar = 50 μm). (B) Quantitation of dual-positive staining areas (n = 5–6 per group). (C) Representative immunofluorescence staining of P16 (green), α-SMA (red) and DAPI (blue) in plaques in indicated groups (scale bar = 50 μm). (D) Proportion of P16 + cells among α-SMA + cells (n = 5–6 per group). (E) Representative immunofluorescence staining of P21 (green), α-SMA (red) and DAPI (blue) in plaques in indicated groups (scale bar = 50 μm). (F) Proportion of P21 + cells among α-SMA + cells (n = 5–6 per group). (G) Representative immunohistochemistry staining of IL‐1β, TNF‐α and MMP-2 (brown) in plaques in indicated groups (scale bar = 50 μm). (H–J) Quantification of positive staining areas (n = 6–7 per group). ∗ P < 0.05 or ∗∗∗ P < 0.001 versus ApoE −/− + V; # P < 0.05, ## P < 0.01 or ### P < 0.001 versus ApoE −/− + Mel + Luz. α-SMA, alpha-smooth muscle actin; DAPI, 4′6-diamidino-2-phenylindole; Luz, luzindole; Mel, melatonin; SA-β-gal, senescence-associated β-galactosidase; V, vehicle; VSMC, vascular smooth muscle cell.

Article Snippet: Melatonin (HY–B0075, MCE, USA) and the melatonin receptor antagonist luzindole (HY-101254, MCE, USA) were first dissolved in dimethyl sulfoxide (DMSO; ID9010, Solarbio) and then diluted in a mixture of PEG300 (HY–Y0873, MCE), Tween 80 (HY–Y1891, MCE), and saline to obtain the working solutions.

Techniques: Membrane, In Vivo, Staining, Activity Assay, Quantitation Assay, Immunofluorescence, Immunohistochemistry

Journal: Redox Biology

Article Title: Melatonin attenuates atherosclerotic plaque vulnerability through SIRT6-dependent regulation of vascular smooth muscle cells senescence

doi: 10.1016/j.redox.2025.103939

Figure Lengend Snippet: Melatonin attenuated VSMC senescence via an MT membrane receptor-dependent manner in vitro. (A) Representative SA-β-gal staining (green) of VSMCs treated for 72 h with vehicle, H 2 O 2 , H 2 O 2 + melatonin, or H 2 O 2 + melatonin + luzindole (scale bar = 100 μm). (B) Quantification of SA-β-gal-positive cells (n = 3 per group). (C) Western blot of P16 and P21 in VSMCs under the indicated treatments. (D–E) Quantification of protein levels (n = 3 per group). (F–H) ELISA measurements of IL-1β, TNF-α, and MMP-2 in culture supernatants of VSMCs under the indicated treatments (n = 8 per group). ∗∗ P < 0.01 or ∗∗∗ P < 0.001 versus control; ## P < 0.01 or ### P < 0.001 versus H 2 O 2 +Mel; @ P < 0.05, @@ P < 0.01 or @@@ P < 0.01 versus H 2 O 2 +Mel + Luz. H 2 O 2, hydrogen peroxide; Luz, luzindole; Mel, melatonin; SA-β-gal, senescence-associated β-galactosidase; VSMC, vascular smooth muscle cell.

Article Snippet: Melatonin (HY–B0075, MCE, USA) and the melatonin receptor antagonist luzindole (HY-101254, MCE, USA) were first dissolved in dimethyl sulfoxide (DMSO; ID9010, Solarbio) and then diluted in a mixture of PEG300 (HY–Y0873, MCE), Tween 80 (HY–Y1891, MCE), and saline to obtain the working solutions.

Techniques: Membrane, In Vitro, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Redox Biology

Article Title: Melatonin attenuates atherosclerotic plaque vulnerability through SIRT6-dependent regulation of vascular smooth muscle cells senescence

doi: 10.1016/j.redox.2025.103939

Figure Lengend Snippet: Melatonin upregulates SIRT6 expression via an MT membrane receptor-dependent manner. (A) Relative mRNA expression of sirtuin family members in carotid plaques from ApoE −/− mice treated with melatonin or saline, assessed by Nanostring transcriptomics (n = 5 per group). Data were normalized to housekeeping genes, log 2 -transformed, and analyzed using the Benjamini-Hochberg method. SIRT6 showed the most significant upregulation in melatonin-treated mice (adjusted P = 0.021; |log 2 (fold change)| ≥ 1). (B) Representative immunofluorescence staining of SIRT6 (green), α-SMA (red) and DAPI (blue) in carotid plaques from ApoE −/− mice treated as indicated (scale bar = 50 μm). (C) Proportion of SIRT6 + cells among α-SMA + cells (n = 6 per group). ∗∗∗ P < 0.001 versus ApoE −/− + V, ### P < 0.001versus ApoE −/− + Mel + Luz. (D) qRT-PCR analysis of SIRT6 mRNA in VSMCs treated for 72 h with vehicle, H 2 O 2 , H 2 O 2 + melatonin, or H 2 O 2 + melatonin + luzindole (n = 3 per group). (E) Western blot of SIRT6 in VSMCs under the indicated treatments. (F) Quantification of SIRT6 protein expression (n = 3 per group). ∗∗ P < 0.01 or ∗∗∗ P < 0.001 versus control; ## P < 0.01 or ### P < 0.001 versus H 2 O 2 +Mel; @ P < 0.05 or @@ P < 0.01 versus H 2 O 2 +Mel + Luz. α-SMA, alpha-smooth muscle actin; DAPI, 4′6-diamidino-2-phenylindole; H 2 O 2, hydrogen peroxide; Luz, luzindole; Mel, melatonin; SIRT6, sirtuin 6; V, vehicle; VSMC, vascular smooth muscle cell.

Article Snippet: Melatonin (HY–B0075, MCE, USA) and the melatonin receptor antagonist luzindole (HY-101254, MCE, USA) were first dissolved in dimethyl sulfoxide (DMSO; ID9010, Solarbio) and then diluted in a mixture of PEG300 (HY–Y0873, MCE), Tween 80 (HY–Y1891, MCE), and saline to obtain the working solutions.

Techniques: Expressing, Membrane, Saline, Transformation Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Western Blot, Control

Journal: Redox Biology

Article Title: Melatonin attenuates atherosclerotic plaque vulnerability through SIRT6-dependent regulation of vascular smooth muscle cells senescence

doi: 10.1016/j.redox.2025.103939

Figure Lengend Snippet: Melatonin up-regulates Nrf2 expression and inhibits ROS production via an MT membrane receptor-dependent manner. (A) Representative immunofluorescence staining of Nrf2 (green), α-SMA (red) and DAPI (blue) in plaques from ApoE −/− mice in indicated groups (scale bar = 50 μm). (B) Proportion of Nrf2 + cells among α-SMA + cells (n = 6 per group). (C) Representative DHE staining in carotid plaques in indicated groups. (D) Quantification of ROS levels (n = 6 per group). ∗∗∗ P < 0.001 versus ApoE −/− + V, ## P < 0.01 or ### P < 0.001 versus ApoE −/− + Mel + Luz. (E) Western blot of Nrf2 in VSMCs treated for 72 h with vehicle, H 2 O 2 , H 2 O 2 + melatonin, or H 2 O 2 + melatonin + luzindole. (F) Quantification of Nrf2 protein expression (n = 3 per group). (G) Representative DHE staining in VSMCs under the indicated treatments (scale bar = 100 μm). (H) Quantification of ROS levels (n = 3 per group). ∗∗ P < 0.01 or ∗∗∗ P < 0.001 versus control; # P < 0.05 or ## P < 0.01 versus H 2 O 2 +Mel; @ P < 0.05 or @@ P < 0.01 versus H 2 O 2 +Mel + Luz. α-SMA, alpha-smooth muscle actin; DAPI, 4′6-diamidino-2-phenylindole; DHE, dihydroethidium; H 2 O 2, hydrogen peroxide; Luz, luzindole; Mel, melatonin; Nrf2, Nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; SA-β-gal, senescence-associated β-galactosidase; V, vehicle; VSMC, vascular smooth muscle cell.

Article Snippet: Melatonin (HY–B0075, MCE, USA) and the melatonin receptor antagonist luzindole (HY-101254, MCE, USA) were first dissolved in dimethyl sulfoxide (DMSO; ID9010, Solarbio) and then diluted in a mixture of PEG300 (HY–Y0873, MCE), Tween 80 (HY–Y1891, MCE), and saline to obtain the working solutions.

Techniques: Expressing, Membrane, Immunofluorescence, Staining, Western Blot, Control

Journal: Pharmacological research

Article Title: Mtnr1b deletion disrupts placental angiogenesis through the VEGF signaling pathway leading to fetal growth restriction.

doi: 10.1016/j.phrs.2024.107290

Figure Lengend Snippet: Fig. 1. Effects of Mtnr1b knockout in mice on placental functions. (a) Mtnr1b knockout murine construction strategy. (b) PCR results of Mtnr1b knockout at the genomic level. (c) The mRNA expression levels in placental tissues (n = 4). (d) The average litter size (n = 4). (e) Photograph of feta size at day 18 of gestation. (f) The average of fetal weight (n = 33). (g) The average of placental weight (n = 33). (h) The average of placental diameter (n = 33). (i) The average of placental efficiency (n = 33). Data are presented as means ± SEM. * p < 0.05. *** p < 0.001.

Article Snippet: The STAT3 activation inhibitor stattic (HY-13818) and melatonin receptor MTNR1B specific antagonist 4 P-PDOT (HY-100609) were purchased from the Chinese MCE company (Shanghai, China).

Techniques: Knock-Out, Expressing

Journal: Pharmacological research

Article Title: Mtnr1b deletion disrupts placental angiogenesis through the VEGF signaling pathway leading to fetal growth restriction.

doi: 10.1016/j.phrs.2024.107290

Figure Lengend Snippet: Fig. 2. Effects of Mtnr1b knockout in mice on placental apoptosis and antioxidant enzymes. (a) Placental tissue’s TUNEL staining. Scale Bar = 100 μm. (b) The average of fluorescence intensity (apoptotic staining) was statistically analyzed with Image J software (n = 3). (c, d) The expression levels of apoptosis related proteins Cleaved-caspase3 and Bcl-2 in placental tissues (n = 4). (e, f) The expression levels of antioxidant enzymes of Cat and HO-1 in placenta tissues (n = 4). Data are presented as means ± SEM. * p < 0.05. ** p < 0.01.

Article Snippet: The STAT3 activation inhibitor stattic (HY-13818) and melatonin receptor MTNR1B specific antagonist 4 P-PDOT (HY-100609) were purchased from the Chinese MCE company (Shanghai, China).

Techniques: Knock-Out, TUNEL Assay, Staining, Fluorescence, Software, Expressing

Journal: Pharmacological research

Article Title: Mtnr1b deletion disrupts placental angiogenesis through the VEGF signaling pathway leading to fetal growth restriction.

doi: 10.1016/j.phrs.2024.107290

Figure Lengend Snippet: Fig. 4. Effects of Mtnr1b knockout in mice on placental angiogenesis. (a) The mRNA levels of angiogenesis related factors (n = 4). (b) The mRNA levels of VEGF receptors (n = 4). (c-h) The protein expression levels of VEGFR2, p-AKT and eNOS3 in placenta tissues (n = 4). Data are presented as means ± SEM. * p < 0.05. ** p < 0.01.

Article Snippet: The STAT3 activation inhibitor stattic (HY-13818) and melatonin receptor MTNR1B specific antagonist 4 P-PDOT (HY-100609) were purchased from the Chinese MCE company (Shanghai, China).

Techniques: Knock-Out, Expressing

Journal: Pharmacological research

Article Title: Mtnr1b deletion disrupts placental angiogenesis through the VEGF signaling pathway leading to fetal growth restriction.

doi: 10.1016/j.phrs.2024.107290

Figure Lengend Snippet: Fig. 7. Effects of melatonergic system on STAT3/VEGFR2 pathway. (a) The mRNA expression levels of VEGFR2 upon STAT3 activation (n = 3). (b, c) Western blotting was used to detect the expression levels of VEGFR2 and p-STAT3 after STAT3 phosphorylation activation, (n = 3). (d) The mRNA expression levels of VEGFR2 upon inhibition of STAT3 activation (n = 3). (e, f) Western blotting was used to detect the expression levels of VEGFR2 and p-STAT3 after inhibition of STAT3 activation, (n = 3). (g, h) The protein level of VEGFR2 was detected by supplementing STAT3 after MTNR1B inhibition, (n = 3). (i, j) The expression level of p- STAT3 in mouse placenta was detected by Western blotting (n = 4). (k, l) Western blotting was used to detect the expression level of p-STAT3 in porcine placenta (n = 3). Data are presented as means ± SEM. * means p < 0.05. *** means p < 0.001.

Article Snippet: The STAT3 activation inhibitor stattic (HY-13818) and melatonin receptor MTNR1B specific antagonist 4 P-PDOT (HY-100609) were purchased from the Chinese MCE company (Shanghai, China).

Techniques: Expressing, Activation Assay, Western Blot, Phospho-proteomics, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Melatonin modulates neuroinflammatory response and microglial activation in mice exposed to dim blue light at night

doi: 10.3389/fphar.2024.1416350

Figure Lengend Snippet: Melatonin inhibits the activation of BV2 through MT2 and reduces the occurrence of inflammation. (A–E) The mRNA levels of IL-1β, TNF-α, IL-10, Cd86, and Cd206 in BV2 cells treated with CORT, Meltonin, Luzindole, and 4-P-PDOT ( n = 6). The outcome is the mean ± standard error of the mean. Values (a,b,c and d) without a common superscript letter differ significantly at p < 0.05, but values (a,b and c) with the same letter do not differ significantly at p ≥ 0.05. CORT, corticosterone, Mel, Melatonin; Luzindole, MT1 receptor blockers; 4-P-PDOT, MT2 receptor blockers.

Article Snippet: The MT1-selective melatonin receptor antagonist Luzindole (HY-101254; MCE, China), the MT2-selective melatonin receptor antagonist 4P-PDOT (HY-100609; MCE, China), and the corticosterone (HY-B1618; MCE, China) were all individually dissolved in DMSO.

Techniques: Activation Assay

Journal: International journal of molecular sciences

Article Title: Melatonin Alleviates Lipopolysaccharide-Induced Abnormal Pregnancy through MTNR1B Regulation of m6A.

doi: 10.3390/ijms25020733

Figure Lengend Snippet: Figure 7. Melatonin plays a protective role through MTNR1B. (A) Cell proliferation assay by CCK8 method. n = 6 independent biological replicates. 4-P-PDOT: 4-phenyl-2-propionamidotetralin; SAH: S-adenosylhomocysteine; * Indicates a significant difference compared with the 4-P-PDOT/SAH 0 µM group; * p < 0.05; ** p < 0.01; *** p < 0.001; # Indicates a significant difference compared with the LPS added group alone Difference; # p < 0.05; ## p < 0.01; ### p < 0.001. (B) Global m6A levels of human endometrial stromal cells treated with 4PPDOT. (C) Western blot bands of inflammation- related proteins in human endometrial stromal cells. (D) Immunoblot analysis of p-RELA, RELA, ERK1/2. (E) Western blot bands of autophagy related proteins in human endometrial stromal cells. (F) Immunoblot analysis of LC3B, ATG5, ATG7. (G) Western blot bands of apoptosis related proteins in human endometrial stromal cells. (H) Immunoblot analysis of c-PARP, BAX, CASP1, c-CASP3. (I) Flow cytometry was used to detect apoptosis after adding the 4PPDOT and melatonin, and apoptosis cells were measured. n = 3 independent biological replicates. Veh: vehicle treatment group; LPS: LPS treatment group; Mel+LPS: melatonin and LPS co-treatment group; Mel: melatonin treatment group; The data are presented as the mean ± SD. Levels of statistical significance for all data were determined by one-way ANOVA and Tukey’s test (* Indicates significant difference between the two groups; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Once cells were attached, HESCs were starved for 12 h in DMEM/F12, cells were treated with 1 μM Melatonin and 50 μg/mL LPS for 48 h. For experiments involving inhibitors, cells were incubated with inhibitors for 48 h. The following specific inhibitors were used: melatonin receptor antagonist 4-P-PDOT (MedChemExpress, Monmouth Junction, NJ, USA), and the METTL3-METTL14 complex inhibitor SAH (MCE, Monmouth Junction, NJ, USA).

Techniques: Proliferation Assay, Western Blot, Flow Cytometry