Chronic wounds often occur in patients with diabetes mellitus due to the impairment of wound healing. This has negative consequences for both the patient and the medical system and considering the growing prevalence of diabetes, it will be a significant medical, social, and economic burden in the near future. Hence, the need for therapeutic alternatives to the current available treatments that, although various, do not guarantee a rapid and definite reparative process, appears necessary. We here analyzed current treatments for wound healing, but mainly focused the attention on few classes of drugs that are already in the market with different indications, but that have shown in preclinical and few clinical trials the potentiality to be used in the treatment of impaired wound healing. In particular, repurposing of the antiglycemic agents dipeptidylpeptidase 4 (DPP4) inhibitors and metformin, but also, statins and phenyotin have been analyzed. All show encouraging results in the treatment of chronic wounds, but additional, well designed studies are needed to allow these drugs access to the clinics in the therapy of impaired wound healing.

Melatonin exerts direct neuroprotection against cerebral hypoxic damage, but the mechanisms of its action on microglia have been less characterized. Using both in vitro and in vivo models of hypoxia, we here focused on the role played by silent mating type information regulation 2 homolog 1 (SIRT1) in melatonin’s effects on microglia. Viability of rat primary microglia or microglial BV2 cells and SH-SY5Y neurons was significantly reduced after chemical hypoxia with CoCl2 (250 μM for 24 h). Melatonin (1 μM) significantly attenuated CoCl2 toxicity on microglia, an effect prevented by selective SIRT1 inhibitor EX527 (5 μM) and AMP-activated protein kinase (AMPK) inhibitor BML-275 (2 μM). CoCl2 did not modify SIRT1 expression, but prevented nuclear localization, while melatonin appeared to restore it. CoCl2 induced nuclear localization of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-kB), an effect contrasted by melatonin in an EX527-dependent fashion. Treatment of microglia with melatonin attenuated potentiation of neurotoxicity. Common carotid occlusion was performed in p7 rats, followed by intraperitoneal injection of melatonin (10 mg/kg). After 24 h, the number of Iba1+ microglia in the hippocampus of hypoxic rats was significantly increased, an effect not prevented by melatonin. At this time, SIRT1 was only detectable in the amoeboid, Iba1+ microglial population selectively localized in the corpus callosum. In these cells, nuclear localization of SIRT1 was significantly lower in hypoxic animals, an effect prevented by melatonin. NF-kB showed an opposite expression pattern, where nuclear localization in Iba1+ cells was significantly higher in hypoxic, but not in melatonin-treated animals. Our findings provide new evidence for a direct effect of melatonin on hypoxic microglia through SIRT1, which appears as a potential pharmacological target against hypoxic-derived neuronal damage.

Microglia represent a first-line defense in the brain. However, in pathological conditions such as Alzheimer’s disease (AD), a pro-inflammatory switch may occur, leading to loss of protective functions. Using the human microglial cell line HMC3, we showed that exposure to low concentrations of β-amyloid peptide 1-42 (Aβ42; 0.2 μM) initially (6 h) upregulated anti-inflammatory markers interleukin (IL)-4, IL-13, and brain-derived neurotrophic factor (BDNF). BDNF increase was prevented by selective inhibition of SIRT1 with EX527 (2 μM). Accordingly, these early effects were accompanied by a significant Aβ42-induced increase of SIRT1 expression, nuclear localization, and activity. SIRT1 modulation involved adenosine monophosphate-regulated kinase (AMPK), which was promptly (30 min) phosphorylated by Aβ42, while the AMPK inhibitor BML-275 (2 μM) attenuated Aβ42-induced SIRT1 increase. Initially observed microglial responses appeared transient, as microglial features changed when exposure to Aβ42 was prolonged (0.2 μM for 72 h). While SIRT1 and BDNF levels were reduced, the expression of inflammatory markers IL-1β and tumor necrosis factor (TNF)-α increased. This coincided with a rise in NF-kB nuclear localization. The effects of melatonin (1 μM) on prolonged microglial exposure to Aβ42 were analyzed for their protective potential. Melatonin was able to prolong SIRT1 and BDNF upregulation, as well as to prevent NF-kB nuclear translocation and acetylation. These effects were sensitive to the melatonin receptor antagonist, luzindole (25 μM). In conclusion, our data define an early microglial defensive response to Aβ42, featuring SIRT1-mediated BDNF upregulation that can be exogenously modulated by melatonin, thus identifying an important target for neuroprotection.

Amyloid-β (Aβ) has long been shown to be critical in Alzheimer’s disease pathophysiology. Microglia contributes to the earliest responses to Aβ buildup, by direct interaction through multiple receptors. Microglial cells operate Aβ clearance and trigger inflammatory/regenerative processes that take place in the long years of silent disease progression that precede symptomatic appearance. But in time and with aging, the fine balance between pro- and anti-inflammatory activity of microglia deranges, negatively impacting its Aβ-clearing ability. Furthermore, in recent years, microglial activation has proven to be much more complex than the mere dichotomic pro/antiinflammatory polarization previously accepted. Microglia can display a wide spectrum of phenotypes, which can even be mixed. On these bases, it is evident that while pharmacological intervention aiding microglia to prolong its ability to cope with Aβ buildup could be extremely relevant, its feasibility is hampered by such high complexity, which still needs to be completely understood.

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Melatonin (MEL) is a neurohormone endowed with neuroprotective activity, exerted both directly on neuronal cells and indirectly through modulation of responsive glial cells. In particular, MEL’s effects on microglia are receptor-mediated and in part dependent on SIRT1 activation. In the present study, we exploited the highly preserved cytoarchitecture of organotypic brain cultures (OC) to explore the effects of MEL on hippocampal microglia in a 3D context as compared to a single cell type context represented by the human HMC3 cell line. We first evaluated the expression of MEL receptor MT1 and SIRT1 and then investigated MEL action against an inflammatory stimulation with LPS: OCs were cultured for a total of 2 weeks and during this time exposed to 0.1 μg/mL of LPS for 24 h either on day 1 (LPS 1°) or on day 11 (LPS 11°). MEL was added immediately after plating and kept for the entire experiment. Under these conditions, both MEL and LPS induced amoeboid microglia. However, the same round phenotype matched different polarization features. LPS increased the number of nuclear-NF-kB+ round cells and MEL alone or in combination with LPS increased BDNF+ round microglia. In addition, MEL contrasted LPS effects on NF-kB expression. Data from HMC3 microglia confirmed MEL’s anti-inflammatory effects against LPS in terms of CASP1 induction and BDNF release, identifying SIRT1 as a mediator. However, no effects were evident for MEL alone on HMC3 microglia. Overall, our results point to the importance of the multicellular context for full MEL activity, especially in a preventive view, and support the use of OCs as a favorable model to explore inflammatory responses.

The nucleus basalis of Meynert (nBM) is the major source of cholinergic neurons in the basal forebrain, which require nerve growth factor (NGF) for their survival. Melatonin, a pleiotropic hormone, has been shown to exert neuroprotection in several experimental models, but its effect on nBM neurons is not well known. Thus, the aim of this study is to evaluate the effect of melatonin in organotypic brain slices of the nBM. Organotypic nBM slices were incubated for 2 weeks without (control) or with 100 ng/mL NGF, 1 μM melatonin, or a combination of both. Cholinergic neurons were immunohistochemically stained for choline acetyltransferase (ChAT) and subjected to a co-localization study with <i>silent information regulator 1</i> (SIRT1) and melatonin receptor 1A (MT1A), both potentially involved in melatonin neuroprotection. Counting of ChAT-positive neurons in nBM slices showed that melatonin and NGF significantly increased the number of ChAT-positive neurons compared to the control in a dose-dependent manner (1–10 μM). In co-treatment with NGF, melatonin did not potentiate the maximal NGF-mediated effect. Immunohistochemical analysis proved that cholinergic nBM neurons co-localized with SIRT1 and MT1A receptor. Our data show that melatonin improves the survival of cholinergic nBM neurons and confirm that they express SIRT1 and MT1A.