Fig 2: Table of Contents Image (TOCI): a schematic diagram depicting the cellular and molecular events associated with melatonin treatment in postnatal rats given LPS injection. The illustration follows two paths: the solid line shows that melatonin binds to its cognate receptor (MT1) on the microglia which activates the JAK2/STAT3 pathways and increases the expression of telomerase in the nucleus. This decreases M1 microglial production of proinflammatory cytokines, such as IL-1β, TNF-α and iNOS, and increases M2 production of anti-inflammatory cytokines, such as CD206 and TGF-β. Dash line denotes the effect of LPS on microglia. LPS stimulates toll-like receptor 4 (TLR4) which recruits its downstream NF-κB pathway, and ultimately mediates the production of proinflammatory mediators [59]. However, LPS inhibits the activation of JKA2/STAT3 pathway and reduces the expression p-JAK2 and p-STAT3 proteins expression, which is contrary to melatonin treatment. Production of proinflammatory cytokines by M1 microglia causes hypomyelination in the corpus callosum after LPS administration; while production of anti-inflammatory cytokines by M2 induced by melatonin improves hypomyelination

Fig 3: Expression profiles of MT1 and MT2 at different stages of BIP differentiation. (A) The mRNA expression of the MT1 and MT2 genes at 3, 8 and 12 d of BIPs differentiation; (B) proteins level of MT1 and MT2 were quantitated using densitometry and normalized to β-actin levels; (C) Effect of melatonin on the mRNA and protein expression of the MT1 and MT2 genes at 8 d of BIPs differentiation. The expression of MT1 and MT2 mRNA were normalized to housekeeping genes β-actin and GAPDH. Protein level of MT1 and MT2 were quantitated using densitometry and normalized to β-actin levels. Values are presented as the means ± S.E.M. “**”represents significant differences P < 0.01. MT1 = melatonin receptors 1; MT2 = melatonin receptors 2; BIPs = bovine intramuscular preadipocytes.

Fig 4: Melatonin reversed the low expression of anti-inflammatory mediators, TERT and melatonin receptor MT1 in primary microglia stimulated by LPS in vitro. Immunofluorescence images of cultured primary microglia show expression of lectin (green), CD206 (red) and DAPI (blue) (A–I) at 24 h after the LPS or melatonin treatment when compared with the corresponding control. Panel J shows CD206 (166 kDa), TGF-β (50 kDa), TERT (127 kDa), MT1 (47 kDa) and GAPDH (37 kDa) immunoreactive bands. Bar graphs in K-N show optical density changes of CD206, TGF-β, TERT, MT1 relative to GAPDH. Note LPS decreased the expression of CD206, TGF-β, TERT and MT1 protein expression in primary microglia. Melatonin treatment could significantly reverse the low expression of CD206, TGF-β, TERT and MT1 proteins induced by LPS. Of note, the effect of melatonin was blocked by luzindole. Scale bars: A–I 20 µm. *P < 0.05, n = 5 for each group

Fig 5: Melatonin administration on melatonin receptors expression levels in cerebral cortex and colon. (a) melatonin receptors expression level in the cerebral cortex; (b) melatonin receptors expression level in the colon. MT1—melatonin receptors 1; MT2—melatonin receptors 2. Data presented as mean ± standard error (n = 6). Different letters indicated significant differences among groups at p < 0.05 as per one-way analysis of variance.