The melatonin metabolite N1‐acetyl‐5‐methoxykynuramine facilitates long‐term object memory in young and aging mice

Melatonin (MEL) has been reported to enhance cognitive processes, making it a potential treatment for cognitive decline. However, the role of MEL’s metabolites, N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK) and N1‐acetyl‐5‐methoxykynuramine (AMK), in these effects are unknown. The current study directly investigated the acute effects of systemic MEL, AFMK, and AMK on novel object recognition. We also analyzed MEL, AFMK, and AMK levels in hippocampus and temporal lobe containing the perirhinal cortex following systemic MEL and AMK treatment. AMK administered post‐training had a more potent effect on object memory than MEL and AFMK. AMK was also able to rescue age‐associated declines in memory impairments when object memory was tested up to 4 days following training. Results from administering AMK at varying times around the training trial and the metabolism time course in brain tissue suggest that AMK’s memory‐enhancing effects reflect memory consolidation. Furthermore, inhibiting the MEL‐to‐AMK metabolic pathway disrupted object memory at 24 hours post‐training, suggesting that endogenous AMK might play an important role in long‐term memory formation. This is the first study to report that AMK facilitates long‐term object memory performance in mice, and that MEL crosses the blood‐brain barrier and is immediately converted to AMK in brain tissue. Overall, these results support AMK as a potential therapeutic agent to improve or prevent memory decline.     

On the free radical scavenging activities of melatonin's metabolites,AFMK and AMK

The reactions of N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK) and N¹‐acetyl‐5‐methoxykynuramine (AMK) with ^?OH, ^?OOH, and ?OOCCl₃ radicals have been studied using the density functional theory. Three mechanisms of reaction have been considered: radical adduct formation (RAF), hydrogen transfer (HT), and single electron transfer (SET). Their relative importance for the free radical scavenging activity of AFMK and AMK has been assessed. It was found that AFMK and AMK react with ?OH at diffusion‐limited rates, regardless of the polarity of the environment, which supports their excellent ?OH radical scavenging activity. Both compounds were found to be also very efficient for scavenging ?OOCCl₃, but rather ineffective for scavenging ?OOH. Regarding their relative activity, it was found that AFMK systematically is a poorer scavenger than AMK and melatonin. In aqueous solution, AMK was found to react faster than melatonin with all the studied free radicals, while in nonpolar environments, the relative efficiency of AMK and melatonin as free radical scavengers depends on the radical with which they are reacting. Under such conditions, melatonin is predicted to be a better ?OOH and ?OOCCl₃ scavenger than AMK, while AMK is predicted to be slightly better than melatonin for scavenging ?OH. Accordingly it seems that melatonin and its metabolite AMK constitute an efficient team of scavengers able of deactivating a wide variety of reactive oxygen species, under different conditions. Thus, the presented results support the continuous protection exerted by melatonin, through the free radical scavenging cascade.     

Melatonin and its metabolites ameliorate ultraviolet B‐induced damage in human epidermal keratinocytes

We investigated the protective effects of melatonin and its metabolites: 6‐hydroxymelatonin (6‐OHM), N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK), N‐acetylserotonin (NAS), and 5‐methoxytryptamine (5‐MT) in human keratinocytes against a range of doses (25, 50, and 75 mJ/cm²) of ultraviolet B (UVB) radiation. There was significant reduction in the generation of reactive oxygen species (50–60%) when UVB‐exposed keratinocytes were treated with melatonin or its derivatives. Similarly, melatonin and its metabolites reduced the nitrite and hydrogen peroxide levels that were induced by UVB as early as 30 min after the exposure. Moreover, melatonin and its metabolites enhanced levels of reduced glutathione in keratinocytes within 1 hr after UVB exposure in comparison with control cells. Using proliferation assay, we observed a dose‐dependent increase in viability of UVB‐irradiated keratinocytes that were treated with melatonin or its derivatives after 48 hr. Using the dot‐blot technique and immunofluorescent staining we also observed that melatonin and its metabolites enhanced the DNA repair capacity of UVB‐induced pyrimidine photoproducts (6‐4)or cyclobutane pyrimidine dimers generation in human keratinocytes. Additional evidence for induction of DNA repair in cells exposed to UVB and treated with the indole compounds was shown using the Comet assay. Finally, melatonin and its metabolites further enhanced expression of p53 phosphorylated at Ser‐15 but not at Ser‐46 or its nonphosphorylated form. In conclusion, melatonin, its precursor NAS, and its metabolites 6‐OHM, AFMK, 5‐MT, which are endogenously produced in keratinocytes, protect these cells against UVB‐induced oxidative stress and DNA damage.     

Oxidation of melatonin and its catabolites, N1-acetyl-N2 -formyl-5-methoxykynuramine and N1-acetyl-5-methoxykynuramine, by activated leukocytes

N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxykynuramine (AMK), two melatonin catabolites, have been described as potent antioxidants. We aimed to follow the kinetics of AFMK and AMK formation when melatonin is oxidized by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS)-activated leukocytes. An HPLC-based method was used for AFMK and AMK determination in neutrophil and peripheral blood mononuclear cell cultures supernatants. Samples were separated isocratically on a C18 reverse-phase column using acetonitrile/H(2)O (25:75) as the mobile phase. AFMK was detected by fluorescence (excitation 340 nm and emission 460 nm) and AMK by UV-VIS absorbance (254 nm). Activation of neutrophils and mononuclear cells with PMA produces larger amounts of AFMK than activation with LPS, probably due to the lower levels of reactive oxygen species formation and myeloperoxidase (MPO) degranulation that occurs when cells are stimulated with LPS. The concentration of AMK found in the supernatant was about 5-10% (from 18-hr cultures) compared with AFMK. This result may reflect its reactivity. Indeed AMK, but not AFMK, is easily oxidized by activated neutrophils in a MPO and hydrogen peroxide-dependent reaction. In conclusion, we defined a simple procedure for the determination of AFMK and AMK in biological samples and demonstrated the capacity of leukocytes to oxidize melatonin and AMK.     

Novel pathway for N1-acetyl-5-methoxykynuramine: UVB-induced liberation of carbon monoxide from precursor N1-acetyl-N2-formyl-5-methoxykynuramine

Abstract:  Irradiation of the melatonin metabolite N ¹-acetyl- N ²-formyl-5-methoxykynuramine (AFMK) with UV light of 254 nm causes the release of carbon monoxide (CO) and, thus, deformylation to N ¹-acetyl-5-methoxykynuramine (AMK). Liberation of CO was demonstrated by reduction of PdCl₂ to metallic palladium, under avoidance of actions by other reductants. Photochemical AMK formation was not due to UV-induced hydroxyl radicals, because the reaction also took place with high efficiency in ethanol and 2-propanol. Moreover, AMK was generated from AFMK by UVB on a dry thin layer chromatographic plate. Although AMK seems to be the major primary product generated by UVB radiation, prolonged exposure of AFMK led to various other products, especially formed by destruction of AMK, as shown by irradiation of this latter compound. With regard to the demonstration of melatonin in skin and substantial amounts of AFMK in keratinocytes, these findings may be of dermatologic relevance.     

Melatonin and its metabolites as copper chelating agents and their role in inhibiting oxidative stress: a physicochemical analysis

The copper sequestering ability of melatonin and its metabolites cyclic 3‐hydroxymelatonin (3OHM), N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK), and N¹‐acetyl‐5‐methoxykynuramine (AMK) was investigated within the frame of the Density Functional Theory. It was demonstrated that these compounds are capable of chelating copper ions, yielding stable complexes. The most likely chelation sites were identified. Two different mechanisms were modeled, the direct‐chelation mechanism (DCM) and the coupled‐deprotonation‐chelation mechanism (CDCM). It is proposed that, under physiological conditions, CDCM would be the main chelation route for Cu(II). It was found that melatonin and its metabolites fully inhibited the oxidative stress induced by Cu(II)‐ascorbate mixtures, via Cu(II) chelation. In the same way, melatonin, AFMK, and 3OHM also prevented the first step of the Haber–Weiss reaction, consequently turning off the ˙OH production via the Fenton reaction. Therefore, it is proposed that, in addition to the previously reported free radical scavenging cascade, melatonin is also involved in a concurrent ‘chelating cascade’, thereby contributing to a reduction in oxidative stress. 3OHM was identified as the most efficient of the studied compounds for that purpose, supporting the important role of this metabolite in the beneficial effects of melatonin against oxidative stress.     

Melatonin and its derivatives counteract the ultraviolet B radiation‐induced damage in human and porcine skin ex vivo

Melatonin and its derivatives (N¹‐acetyl‐N²‐formyl‐5‐methoxykynurenine [AFMK] and N‐acetyl serotonin [NAS]) have broad‐spectrum protective effects against photocarcinogenesis, including both direct and indirect antioxidative actions, regulation of apoptosis and DNA damage repair; these data were primarily derived from in vitro models. This study evaluates possible beneficial effects of melatonin and its active derivatives against ultraviolet B (UVB)‐induced harm to human and porcine skin ex vivo and to cultured HaCaT cells. The topical application of melatonin, AFMK, or NAS protected epidermal cells against UVB‐induced 8‐OHdG formation and apoptosis with a further increase in p53^ser15 expression, especially after application of melatonin or AFMK but not after NAS use. The photoprotective action was observed in pre‐ and post‐UVB treatment in both human and porcine models. Melatonin along with its derivatives upregulated also the expression of antioxidative enzymes after UVB radiation of HaCaT cells. The exogenous application of melatonin or its derivatives represents a potent and promising tool for preventing UVB‐induced oxidative stress and DNA damage. This protection results in improved genomic, cellular, and tissue integrity against UVB‐induced carcinogenesis, especially when applied prior to UV exposure. In addition, our ex vivo experiments provide fundamental justification for further testing the clinical utility of melatonin and metabolites as protectors again UVB in human subjects. Our ex vivo data constitute the bridge between vitro to vivo translation and thus justifies the pursue for further clinical utility of melatonin in maintaining skin homeostasis.     

Metabolism of melatonin by cytochrome P450s in rat liver mitochondria and microsomes

Abstract: In the present study we provide direct evidence for the involvement of rat microsomal cytochrome P450s in melatonin O‐demethylation and hydroxylation at two different positions: 2 and 6, as well as generation of N¹‐acetyl‐N²‐formyl‐5‐methoxy‐kynuramine (AFMK) and two unknown products. Moreover, we found that mitochondrial cytochrome P450s also converts melatonin into AFMK, N‐acetylserotonin, 2‐hydroxymelatonin, 6‐hydroxymelatonin and the same two unknown products. Eadie–Hofstee plots for 6‐hydroxylation and O‐demethylation reactions were curvilinear for all tested fractions, suggestive of involvement of at least two components, one with a high affinity and low capacity, and another with a low affinity and high capacity. Mitochondrial cytochrome P450s exhibited higher affinity (suggesting lower K_m value) and higher V_max for melatonin 6‐hydroxylation and O‐demethylation for both high‐affinity and low‐affinity components as compared with microsomal enzymes. The intrinsic clearance for melatonin hydroxylation by high‐ and low‐affinity components displayed the highest values in all tested fractions, indicating that both mitochondrial and microsomal cytochrome P450s metabolize melatonin principally by 6‐hydroxylation, with O‐demethylation representing a minor metabolic pathway.     

Optimized synthesis of the melatonin metabolite N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK)

N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) is the product of oxidative pyrrole ring cleavage of melatonin. AFMK and its deformylated derivative N(1)-acetyl-5-methoxykynuramine (AMK) are compounds for which there are increasing demands because of their antioxidant, immunomodulatory and anti-inflammatory properties. Here, we sought to determine the best reaction conditions for preparation of AFMK using chlorpromazine (CPZ) as a co-catalyst in the peroxidase-mediated oxidation of melatonin. The parameters studied were pH, identity and concentration of buffers, hydrogen peroxide (H(2)O(2)) and CPZ concentrations and the presence or absence of dissolved molecular oxygen in the reaction medium. The rate and efficiency of AFMK production were compared with a noncatalyzed method which uses a high concentration of H(2)O(2). We found that by using CPZ and bubbling molecular oxygen during the course of the reaction, the yield of AFMK was significantly increased (about 60%) and the reaction time decreased (about 30 min), as compared with the noncatalyzed reaction (yield 32% and reaction time 4 hr). Based on these data, we suggest that this could be a new, easily performed and efficient route for AFMK preparation. Additionally, we provide evidence that a radical chain reaction could be responsible for the formation of AFMK.     

Protective stabilization of mitochondrial permeability transition and mitochondrial oxidation during mitochondrial Ca2+ stress by melatonin's cascade metabolites C3‐OHM and AFMK in RBA1 astrocytes

Cyclic 3‐hydroxymelatonin (C3‐OHM) and N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK) are two major cascade metabolites of melatonin. We previously showed melatonin provides multiple levels of mitochondria‐targeted protection beyond as a mitochondrial antioxidant during ionomycin‐induced mitochondrial Ca²⁺ (mCa²⁺) stress in RBA1 astrocytes. Using noninvasive laser scanning fluorescence coupled time‐lapse digital imaging microscopy, this study investigated whether C3‐OHM and AFMK also provide mitochondrial levels of protection during ionomycin‐induced mCa²⁺ stress in RBA1 astrocytes. Interestingly, precise temporal and spatial dynamic live mitochondrial images revealed that C3‐OHM and AFMK prevented specifically mCa²⁺‐mediated mitochondrial reactive oxygen species (mROS) formation and hence mROS‐mediated depolarization of mitochondrial membrane potential (△Ψ_m) and permanent lethal opening of the MPT (p‐MPT). The antioxidative effects of AFMK, however, were less potent than that of C3‐OHM. Whether C3‐OHM and AFMK targeted directly the MPT was investigated under a condition of “oxidation free‐Ca²⁺ stress” using a classic antioxidant vitamin E to remove mCa²⁺‐mediated mROS stress and the potential antioxidative effects of C3‐OHM and AFMK. Intriguingly, two compounds still effectively postponed “oxidation free‐Ca²⁺ stress”‐mediated depolarization of △Ψ_m and p‐MPT. Measurements using a MPT pore‐specific indicator Calcein further identified that C3‐OHM and AFMK, rather than inhibiting, stabilized the MPT in its transient protective opening mode (t‐MPT), a critical mechanism to reduce overloaded mROS and mCa²⁺. These multiple layers of mitochondrial protection provided by C3‐OHM and AFMK thus crucially allow melatonin to extend its metabolic cascades of mitochondrial protection during mROS‐ and mCa²⁺‐mediated MPT‐associated apoptotic stresses and may provide therapeutic benefits against astrocyte‐mediated neurodegeneration in the CNS.     

Melatonin and its derivatives cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine and 6-methoxymelatonin reduce oxidative DNA damage induced by Fenton reagents

Abstract: Free radicals are generated in vivo and they oxidatively damage DNA because of their high reactivities. In the last several years, hundreds of publications have confirmed that melatonin is a potent endogenous free radical scavenger. Some of the metabolites produced as a result of these scavenging actions have been identified using pure chemical systems. This is the case with both N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK), identified as a product of the scavenging reaction of H₂O₂ by melatonin, and cyclic 3‐hydroxymelatonin (C‐3‐OHM) which results when melatonin detoxifies two hydroxyl radicals (?OH). In the present in vitro study, we investigated the potential of two different derivatives of melatonin to scavenger free radicals. One of these derivatives is C‐3‐OHM, while the other is 6‐methoxymelatonin (6‐MthM). We also examined the effect of two solvents, i.e., methanol and acetonitrile, in this model system. As an endpoint, using high‐performance liquid chromatography we measured the formation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OH‐dG) in purified calf thymus DNA treated with the Fenton reagents, chromium(III) [Cr(III)] plus H₂O₂, in the presence and in the absence of these molecules. The 8‐OH‐dG is considered a biomarker of oxidative DNA damage. Increasing concentrations of Cr(III) (as CrCl₃) and H₂O₂ was earlier found to induce progressively greater levels of 8‐OH‐dG in isolated calf thymus DNA because of the generation of ?OH via the Fenton‐type reaction. We found that C‐3‐OHM reduces ?OH‐mediated damage in a dose‐dependent manner, with an IC₅₀ = 5.0 ± 0.2 μm ; melatonin has an IC₅₀ = 3.6 ± 0.1 μm . These values differ statistically significantly with P < 0.05. In these studies, AFMK had an IC₅₀ = 17.8 ± 0.7 μm (P < 0.01). The 6‐MthM also reduced DNA damage in a dose‐dependent manner, with an IC₅₀ = 4.2 ± 0.2 μm ; this value does not differ from the ICs for melatonin and C‐3‐OHM. We propose a hypothetical reaction pathway in which a mole of C‐3‐OHM scavenges 2 mol of ?OH yielding AFMK as a final product. As AFMK is also a free radical scavenger, the action of melatonin as a free radical scavenger is a sequence of scavenging reactions in which the products are themselves scavengers, resulting in a cascade of protective reactions.     

Oxidation of melatonin by taurine chloramine

Abstract: Melatonin is widely known for its antioxidant, immunomodulatory, and anti‐inflammatory effects. Hypochlorous acid (HOCl) is one example of an endogenous oxidant that is promptly neutralized by melatonin. Melatonin also inhibits myeloperoxidase, the enzyme that catalyzes the oxidation of chloride to HOCl. Taurine is the most abundant free amino acid in leukocytes. In activated neutrophils, taurine is converted to taurine chloramine (Tau‐NHCl) through a reaction with HOCl. In addition, the related compound taurine bromamine (Tau‐NHBr) can be released by neutrophils and eosinophils. The aim of this study was to investigate the reactivity of Tau‐NHCl and Tau‐NHBr with melatonin. We found that melatonin can react with either Tau‐NHCl or Tau‐NHBr, leading to the production of 2‐hydroxymelatonin and N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK). The reaction was pH‐dependent, and it occurs more rapidly at a slightly acidic pH. Tau‐NHBr was significantly more reactive than Tau‐NHCl. Using Tau‐NHBr as the oxidizing agent, 1 mm melatonin was oxidized in less than 1 min. The pH dependence of the reaction with Tau‐NHCl and the increased reactivity of Tau‐NHBr can be explained by a mechanism based on the initial attack of chloronium (Cl⁺) or bromonium (Br⁺) ions on melatonin. We also found that the addition of iodide to the reaction medium increased the yield of AFMK. These findings could contribute to the establishment of new functions for melatonin in inflammatory and parasitic diseases, where the role of this indoleamine has been extensively investigated.     

A novel enzyme‐dependent melatonin metabolite in humans

Exogenous melatonin is widely used in humans for multiple pharmacologic purposes. The metabolic pathways of melatonin reflect the fate and functions of melatonin in vivo. This study was designed to re‐profile melatonin metabolism in humans using a metabolomic approach. In the urine of healthy subjects treated with 10 mg melatonin, sulfate‐ or glucuronide‐conjugated metabolites of melatonin were detected, including 6‐hydroxymelatonin sulfate, 6‐hydroxymelatonin glucuronide, N‐acetylserotonin glucuronide, N‐acetylserotonin sulfate, and an unknown sulfated metabolite (X). The molecular weight of metabolite X was 14 Da smaller than 6‐hydroxymelatonin sulfate, but 16 Da larger than N‐acetylserotonin sulfate. Further studies suggest that metabolite X was produced via O‐demethylation, 6‐hydroxylation, and sulfation. The antioxidant products of melatonin, N(1)‐acetyl‐N(2)‐formyl‐5‐methoxykynuramine and N(1)‐acetyl‐5‐methoxy‐kynuramine, were not detected in human urine. In summary, this study provided a global view of melatonin metabolism in humans and extended our knowledge of enzyme‐dependent pathways of melatonin metabolism.     

Urinary metabolites and antioxidant products of exogenous melatonin in the mouse

Exogenous melatonin is widely used for sleep disorders and has potential value in neuroprotection, cardioprotection and as an antioxidant. Here, a novel method is described for the determination of melatonin and six metabolites in mouse urine by use of LC-MS/MS and GC-MS. LC-MS/MS is used for the measurement of melatonin, N1-acetyl-5-methoxykynuramine (AMK), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and 6-hydroxymelatonin (6-HMEL), while GC/MS is used for the determination of N-[2-(5-methoxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-ethyl]-acetamide (2-OMEL) and cyclic 3-hydroxymelatonin (3-HMEL) with detection limits on column of 0.02-0.5 pmol, depending on the metabolite. Following oral administration of melatonin to mice, a 0-24 hr urine collection revealed the presence of melatonin (0.2% dose), 6-HMEL (37.1%) and NAS (3.1%) comprising >90% of the total metabolites; AMK and AFMK were also detected at 0.01% each; 2-OMEL was found at 2.2% of the dose, which is >100 times more than the AMK/AFMK pathway, and comprises >5% of the melatonin-related material detected in mouse urine. 3-HMEL was largely found as a sulfate conjugate. These studies establish sensitive assays for determination of six melatonin metabolites in mouse urine and confirm the potential for antioxidant activity of melatonin through the identification in vivo of AMK and AFMK, ring-opened metabolites with a high capacity for scavenging reactive oxygen species.     

Measurement of melatonin and its metabolites: importance for the evaluation of their biological roles

Many physiologic changes related to lightdark cycles and antioxidant effects have been related to melatonin (N-acetyl-5methoxytryptamine) and its metabolites, N ¹-acetyl-N ²-formyl-5methoxykynuramine AMK) and N ¹-acetyl-5methoxykynuramine AMK). In this review, we discuss some methodologies, in particular, those employing high-performance liquid chromatography tandem mass spectrometry (HP (MS/MS)assays to quantitatively determine melatonin, AMK, and AMK. These approaches offer a highly specific and an accurate quantification of melatonin and its metabolites. These characteristics are essential to point out correctly the biological effects of these compounds in physiological and pathological conditions.     

Melatonin impedes Tet1‐dependent mGluR5 promoter demethylation to relieve pain

Melatonin (N‐acetyl‐5‐methoxytryptamine)/MT2 receptor‐dependent epigenetic modification represents a novel pathway in the treatment of neuropathic pain. Because spinal ten‐eleven translocation methylcytosine dioxygenase 1 (Tet1)‐dependent epigenetic demethylation has recently been linked to pain hypersensitivity, we hypothesized that melatonin/MT2‐dependent analgesia involves spinal Tet1‐dependent demethylation. Here, we showed that spinal Tet1 gene transfer by intrathecal delivery of Tet1‐encoding vectors to naïve rats produced profound and long‐lasting nociceptive hypersensitivity. In addition, enhanced Tet1 expression, Tet1‐metabotropic glutamate receptor subtype 5 (mGluR5) promoter coupling, demethylation at the mGluR5 promoter, and mGluR5 expression in dorsal horn neurons were observed. Rats subjected to spinal nerve ligation and intraplantar complete Freund's adjuvant injection displayed tactile allodynia and behavioral hyperalgesia associated with similar changes in the dorsal horn. Notably, intrathecal melatonin injection reversed the protein expression, protein‐promoter coupling, promoter demethylation, and pain hypersensitivity induced by Tet1 gene transfer, spinal nerve ligation, and intraplantar complete Freund's adjuvant injection. All the effects caused by melatonin were blocked by pretreatment with a MT2 receptor‐selective antagonist. In conclusion, melatonin relieves pain by impeding Tet1‐dependent demethylation of mGluR5 in dorsal horn neurons through the MT2 receptor. Our findings link melatonin/MT2 signaling to Tet1‐dependent epigenetic demethylation of nociceptive genes for the first time and suggest melatonin as a promising therapy for the treatment of pain.     

Melatonin prevents Drp1‐mediated mitochondrial fission in diabetic hearts through SIRT1‐PGC1α pathway

Myocardial contractile dysfunction is associated with an increase in mitochondrial fission in patients with diabetes. However, whether mitochondrial fission directly promotes diabetes‐induced cardiac dysfunction is still unknown. Melatonin exerts a substantial influence on the regulation of mitochondrial fission/fusion. This study investigated whether melatonin protects against diabetes‐induced cardiac dysfunction via regulation of mitochondrial fission/fusion and explored its underlying mechanisms. Here, we show that melatonin prevented diabetes‐induced cardiac dysfunction by inhibiting dynamin‐related protein 1 (Drp1)‐mediated mitochondrial fission. Melatonin treatment decreased Drp1 expression, inhibited mitochondrial fragmentation, suppressed oxidative stress, reduced cardiomyocyte apoptosis, improved mitochondrial function and cardiac function in streptozotocin (STZ )‐induced diabetic mice, but not in SIRT 1^?/? diabetic mice. In high glucose‐exposed H9c2 cells, melatonin treatment increased the expression of SIRT 1 and PGC ‐1α and inhibited Drp1‐mediated mitochondrial fission and mitochondria‐derived superoxide production. In contrast, SIRT 1 or PGC ‐1α siRNA knockdown blunted the inhibitory effects of melatonin on Drp1 expression and mitochondrial fission. These data indicated that melatonin exerted its cardioprotective effects by reducing Drp1‐mediated mitochondrial fission in a SIRT 1/PGC ‐1α‐dependent manner. Moreover, chromatin immunoprecipitation analysis revealed that PGC ‐1α directly regulated the expression of Drp1 by binding to its promoter. Inhibition of mitochondrial fission with Drp1 inhibitor mdivi‐1 suppressed oxidative stress, alleviated mitochondrial dysfunction and cardiac dysfunction in diabetic mice. These findings show that melatonin attenuates the development of diabetes‐induced cardiac dysfunction by preventing mitochondrial fission through SIRT 1‐PGC 1α pathway, which negatively regulates the expression of Drp1 directly. Inhibition of mitochondrial fission may be a potential target for delaying cardiac complications in patients with diabetes.     

N‐acetyl‐L‐cysteine improves mesenchymal stem cell function in prolonged isolated thrombocytopenia post‐allotransplant

Prolonged isolated thrombocytopenia (PT) is a serious complication of allogeneic haematopoietic stem cell transplantation (allo‐HSCT). Murine studies and in vitro experiments suggest that mesenchymal stem cells (MSCs) can, not only to support haematopoiesis, but also preferentially support megakaryocytopoiesis in bone marrow (BM). However, little is known about the quantity and function of BM MSCs in PT patients. In a case‐control study, we found that BM MSCs from PT patients exhibited significantly reduced proliferative capacities, increased reactive oxygen species and senescence. Antioxidant (N‐acetyl‐L‐cysteine, NAC) treatment in vitro not only quantitatively and functionally improved BM MSCs derived from PT patients through down‐regulation of the p38 (also termed MAPK14) and p53 (also termed TP53) pathways but also partially rescued the impaired ability of BM MSCs to support megakaryocytopoiesis. Subsequently, a pilot study showed that the overall response of NAC treatment was obtained in 7 of the enrolled PT patients (N = 10) without significant side effects. Taken together, the results indicated that dysfunctional BM MSCs played a role in the pathogenesis of PT and the impaired BM MSCs could be improved by NAC in vitro. Although requiring further validation, our data indicate that NAC might be a potential therapeutic approach for PT patients after allo‐HSCT.     

Melatonin prevents the dynamin‐related protein 1‐dependent mitochondrial fission and oxidative insult in the cortical neurons after 1‐methyl‐4‐phenylpyridinium treatment

Mitochondrial dysfunction and oxidative stress are involved in the pathogenesis of Parkinson's disease (PD). Mitochondrial morphology is dynamic and precisely regulated by the mitochondrial fission and fusion machinery. Aberrant mitochondrial fragmentation controlled by the mitochondrial fission protein, dynamin‐related protein 1 (Drp1), may result in cell death. Our previous results showed that melatonin protected neurons by inhibiting oxidative stress in a 1‐methyl‐4‐phenylpyridinium (MPP⁺)‐induced PD model. However, the effect of melatonin on mitochondrial dynamics remains uncharacterized. Herein, we investigated the effect of melatonin and the role of Drp1 on MPP⁺‐induced mitochondrial fission in rat primary cortical neurons. We found that MPP⁺ induced a rapid increase in the ratio of GSSG:total glutathione (a marker of oxidative stress) and mitochondrial fragmentation, Drp1 upregulation within 4 hours, and finally resulted in neuron loss 48 hours after the treatment. Neurons overexpressing wild‐type Drp1 promoted mitochondrial and nuclear fragmentation; however, neurons overexpressing dominant‐negative Drp1^K38A or cotreated with melatonin exhibited significantly reduced MPP⁺‐induced mitochondrial fragmentation and neuron death. Moreover, melatonin cotreatment prevented an MPP⁺‐induced high ratio of GSSG and mitochondrial Drp1 upregulation. The prevention of mitochondrial fission by melatonin was not found in neurons transfected with wild‐type Drp1. These results provide a new insight that the neuroprotective effect of melatonin against MPP⁺ toxicity is mediated by inhibiting the oxidative stress and Drp1‐mediated mitochondrial fragmentation.