Additional benefit of combined therapy with melatonin and apoptotic adipose‐derived mesenchymal stem cell against sepsis‐induced kidney injury

This study tested whether combined therapy with melatonin and apoptotic adipose‐derived mesenchymal stem cells (A‐ADMSCs) offered additional benefit in ameliorating sepsis‐induced acute kidney injury. Adult male Sprague–Dawley rats (n = 65) were randomized equally into five groups: Sham controls (SC), sepsis induced by cecal‐ligation and puncture (CLP), CLP‐melatonin, CLP‐A‐ADMSC, and CLP‐melatonin‐A‐ADMSC. Circulating TNF‐α level at post‐CLP 6 hr was highest in CLP and lowest in SC groups, higher in CLP‐melatonin than in CLP‐A‐ADMSC and CLP‐melatonin‐A‐ADMSC groups (all P < 0.001). Immune reactivity as reflected in the number of splenic helper‐, cytoxic‐, and regulatory‐T cells at post‐CLP 72 hr exhibited the same pattern as that of circulating TNF‐α among all groups (P < 0.001). The histological scoring of kidney injury and the number of F4/80+ and CD14+ cells in kidney were highest in CLP and lowest in SC groups, higher in CLP‐melatonin than in CLP‐A‐ADMSC and CLP‐melatonin‐A‐ADMSC groups, and higher in CLP‐A‐ADMSC than in CLP‐melatonin‐A‐ADMSC groups (all P < 0.001). Changes in protein expressions of inflammatory (RANTES, TNF‐1α, NF‐κB, MMP‐9, MIP‐1, IL‐1β), apoptotic (cleaved caspase 3 and PARP, mitochondrial Bax), fibrotic (Smad3, TGF‐β) markers, reactive‐oxygen‐species (NOX‐1, NOX‐2), and oxidative stress displayed a pattern identical to that of kidney injury score among the five groups (all P < 0.001). Expressions of antioxidants (GR+, GPx+, HO‐1, NQO‐1+) were lowest in SC group and highest in CLP‐melatonin‐A‐ADMSC group, lower in CLP than in CLP‐melatonin and CLP‐A‐ADMSC groups, and lower in CLP‐melatonin‐ than in CLP‐A‐ADMSC‐tretaed animals (all P < 0.001). In conclusion, combined treatment with melatonin and A‐ADMSC was superior to A‐ADMSC alone in protecting the kidneys from sepsis‐induced injury.     

Melatonin pretreatment enhances the therapeutic effects of exogenous mitochondria against hepatic ischemia–reperfusion injury in rats through suppression of mitochondrial permeability transition

We tested the hypothesis that melatonin (Mel) enhances exogenous mitochondria (Mito) treatment against rodent hepatic ischemia–reperfusion (IR) injury. In vitro study utilized three groups of hepatocytes (i.e. nontreatment, menadione, and menadione–melatonin treatment, 4.0 × 10⁵ each), while in vivo study used adult male Sprague Dawley rats (n = 40) equally divided into sham‐control (SC), IR (60‐min left‐lobe ischemia + 72‐hr reperfusion), IR‐Mel (melatonin at 30 min/6/8 hr after reperfusion), IR‐Mito (mitochondria 15,000 μg/rat 30 min after reperfusion), and IR‐Mel‐Mito. Following menadione treatment in vitro, oxidative stress (NOX‐1/NOX‐2/oxidized protein), apoptotic (cleaved caspase‐3/PARP), DNA damage (γ‐H2AX/CD90/XRCC1), mitochondria damage (cytosolic cytochrome c) biomarkers, and mitochondrial permeability transition were found to be lower, whereas mitochondrial cytochrome c were found to be higher in hepatocytes with melatonin treatment compared to those without (all P < 0.001). In vivo study demonstrated highest liver injury score and serum AST in IR group, but lowest in SC group and higher in IR‐Mito group than that in groups IR‐Mel and IR‐Mel‐Mito, and higher in IR‐Mel group than that in IR‐Mel‐Mito group after 72‐hr reperfusion (all P < 0.003). Protein expressions of inflammatory (TNF‐α/NF‐κB/IL‐1β/MMP‐9), oxidative stress (NOX‐1/NOX‐2/oxidized protein), apoptotic (caspase‐3/PARP/Bax), and mitochondria damage (cytosolic cytochrome c) biomarkers displayed an identical pattern, whereas mitochondria integrity marker (mitochondrial cytochrome c) showed an opposite pattern compared to that of liver injury score (all P < 0.001) among five groups. Microscopically, expressions of apoptotic nuclei, inflammatory (MPO ⁺ /CD68 ⁺ /CD14 ⁺ cells), and DNA damage (γ‐H2AX ⁺ cells) biomarkers exhibited an identical pattern compared to that of liver injury score (all P < 0.001) among five groups. Melatonin‐supported mitochondria treatment offered an additional benefit of alleviating hepatic IR injury.     

Protective effect of melatonin‐supported adipose‐derived mesenchymal stem cells against small bowel ischemia‐reperfusion injury in rat

We tested the hypothesis that combined melatonin and autologous adipose‐derived mesenchymal stem cells (ADMSC) was superior to either alone against small bowel ischemia‐reperfusion (SBIR) injury induced by superior mesenteric artery clamping for 30 min followed by reperfusion for 72 hr. Male adult Sprague Dawley rats (n = 50) were equally categorized into sham‐operated controls SC, SBIR, SBIR‐ADMSC (1.0 × 10⁶ intravenous and 1.0 × 10⁶ intrajejunal injection), SBIR‐melatonin (intraperitoneal 20 mg/kg at 30 min after SI ischemia and 50 mg/kg at 6 and 18 hr after SI reperfusion), and SBIR‐ADMSC‐melatonin groups. The results demonstrated that the circulating levels of TNF‐α, MPO, LyG6+ cells, CD68+ cells, WBC count, and gut permeability were highest in SBIR and lowest in SC, significantly higher in SBIR‐ADMSC group and further increased in SBIR‐melatonin group than in the combined therapy group (all P < 0.001). The ischemic mucosal damage score, the protein expressions of inflammation (TNF‐α, NF‐κB, MMP‐9, MPO, and iNOS), oxidative stress (NOX‐1, NOX‐2, and oxidized protein), apoptosis (APAF‐1, mitochondrial Bax, cleaved caspase‐3 and PARP), mitochondrial damage (cytosolic cytochrome C) and DNA damage (γ‐H2AX) markers, as well as cellular expressions of proliferation (PCNA), apoptosis (caspase‐3, TUNEL assay), and DNA damage (γ‐H2AX) showed an identical pattern, whereas mitochondrial cytochrome C exhibited an opposite pattern compared to that of inflammation among all groups (all P < 0.001). Besides, antioxidant expressions at protein (NQO‐1, GR, and GPx) and cellular (HO‐1) levels progressively increased from SC to the combined treatment group (all P < 0.001). In conclusion, combined melatonin‐ADMSC treatment offered additive beneficial effect against SBIR injury.     

Melatonin treatment improves adipose‐derived mesenchymal stem cell therapy for acute lung ischemia–reperfusion injury

This study investigated whether melatonin‐treated adipose‐derived mesenchymal stem cells (ADMSC) offered superior protection against acute lung ischemia–reperfusion (IR) injury. Adult male Sprague‐Dawley rats (n = 30) were randomized equally into five groups: sham controls, lung IR–saline, lung IR–melatonin, lung IR–melatonin–normal ADMSC, and lung IR–melatonin–apoptotic ADMSC. Arterial oxygen saturation was lowest in lung IR–saline; lower in lung IR–melatonin than sham controls, lung IR–melatonin–normal ADMSC, and lung IR–melatonin–apoptotic ADMSC; lower in lung IR–melatonin–normal ADMSC than sham controls and lung IR–melatonin–apoptotic ADMSC; lower in lung IR–melatonin–apoptotic ADMSC than sham controls (P < 0.0001 in each case). Right ventricular systolic blood pressure (RVSBP) showed a reversed pattern among all groups (all P < 0.0001). Changes in histological scoring of lung parenchymal damage and CD68+ cells showed a similar pattern compared with RVSBP in all groups (all P < 0.001). Changes in inflammatory protein expressions such as VCAM‐1, ICAM‐1, oxidative stress, TNF‐α, NF‐κB, PDGF, and angiotensin II receptor, and changes in apoptotic protein expressions of cleaved caspase 3 and PARP, and mitochondrial Bax, displayed identical patterns compared with RVSBP in all groups (all P < 0.001). Numbers of antioxidant (GR+, GPx+, NQO‐1+) and endothelial cell biomarkers (CD31+ and vWF+) were lower in sham controls, lung IR–saline, and lung IR–melatonin than lung IR–melatonin–normal ADMSC and lung IR–melatonin–apoptotic ADMSC, and lower in lung IR–melatonin–normal ADMSC than lung IR–melatonin–apoptotic ADMSC (P < 0.001 in each case). In conclusion, when the animals were treated with melatonin, the apoptotic ADMSC were superior to normal ADMSC for protection of lung from acute IR injury.     

The cardioprotective effect of melatonin and exendin‐4 treatment in a rat model of cardiorenal syndrome

We investigated the cardioprotective effect of melatonin (Mel) and exendin‐4 (Ex4) treatment in a rat model of cardiorenal syndrome (CRS). Adult male SD rats (n=48) were randomly and equally divided into sham control (SC), dilated cardiomyopathy (DCM) (doxorubicin 7 mg/kg i.p. every five days/4 doses), CRS (defined as DCM+CKD) only, CRS‐Mel (20 mg/kg/d), CRS‐Ex4 (10 μg/kg/d), and CRS‐Mel‐Ex4 groups. In vitro results showed protein expressions of oxidative stress (NOX‐1/NOX‐2/oxidized protein), DNA/mitochondrial damage (γ‐H2AX/cytosolic cytochrome c), apoptosis (cleaved caspase‐3/PARP), and senescence (β‐galactosidase cells) biomarkers were upregulated, whereas mitochondrial ATP level was decreased in doxorubicin/p‐cresol‐treated H9c2 cells that were revised by Mel and Ex4 treatments (all P<.001). By day 60, LVEF was highest in the SC and lowest in the CRS, significantly lower in the DCM than in other treatment groups, lower in the CRS‐Mel and CRS‐Ex4 than in the CRS‐Mel‐Ex4, and lower in the CRS‐Mel than in the CRS‐Ex4, whereas LV chamber size and histopathology score showed a pattern opposite to that of LVEF among all groups (all P<.001). Plasma creatinine level was highest in the CRS and lowest in the SC and progressively decreased from the CRS‐Mel, CRS‐Ex4, CRS‐Mel‐Ex4 to DCM (P<.0001). Protein expressions of inflammation (TNF‐α/NF‐κB/MMP‐2/MMP‐9/IL‐1β), apoptosis/DNA damage (Bax/c‐caspase‐3/c‐PARP/γ‐H2AX), fibrosis (Smad3/TGF‐β), oxidative stress (NOX‐1/NOX‐2/NOX‐4/oxidized protein), cardiac hypertrophy/pressure overload (BNP/β‐MHC), and cardiac integrity (Cx43/α‐MHC) biomarkers in LV myocardium showed an opposite pattern compared to that of LVEF among all groups (all P<.001). Fibrotic area, DNA damage (γ‐H2AX⁺/53BP1⁺CD90⁺/XRCC1⁺CD90⁺), and inflammation (CD14⁺/CD68⁺) biomarkers in LV myocardium displayed a pattern opposite to that of LVEF among all groups (all P<.001). Combined melatonin and exendin‐4 treatment suppressed CRS‐induced deterioration of LVEF and LV remodeling.     

Melatonin treatment further improves adipose‐derived mesenchymal stem cell therapy for acute interstitial cystitis in rat

This study tests the hypothesis that combined melatonin and adipose‐derived mesenchymal stem cell (ADMSC, 1.2 × 10⁶ given intravenously) treatment offer superior protection against cyclophosphamide (CYP 150 mg/kg)‐induced acute interstitial cystitis (AIC) in rats. Male adult Sprague‐Dawley rats were treated as follows: sham controls, AIC alone, AIC + melatonin, AIC + ADMSC, and AIC + melatonin +ADMSC. When melatonin was used, it was given as follows: 20 mg/kg at 30 min after CYP and 50 mg/kg at 6 and 18 hr after CYP. Twenty‐four‐hour urine volume, urine albumin level, and severity of hematuria were highest in AIC rats and lowest in the controls; likewise urine volume was higher in AIC + melatonin rats than in AIC + ADMSC and AIC + melatonin + ADMSC treated rats; in all cases, P < 0.001. The numbers of CD14+, CD74+, CD68+, MIP+, Cox‐2+, substance P+, cells and protein expression of IL‐6, IL‐12, RANTES, TNF‐α, NF‐κB, MMP‐9, iNOS (i.e. inflammatory biomarkers), glycosaminoglycan level, expression of oxidized protein, and protein expression of reactive oxygen species (NOX‐1, NOX‐2, NOX‐4) in the bladder tissue exhibited an identical pattern compared with that of hematuria among the five groups (all P < 0.0001). The integrity of epithelial layer and area of collagen deposition displayed an opposite pattern compared to that of hematuria among all groups (P < 0.0001). The cellular expressions of antioxidants (GR, GPx, HO‐1, NQO 1) showed a significant progressive increase form controls to AIC + melatonin + ADMSC (all P < 0.0001). Combined regimen of melatonin and ADMSC was superior to either alone in protecting against CYP‐induced AIC.     

Melatonin reduces hepatic mitochondrial dysfunction in diabetic obese rats

Hepatic mitochondrial dysfunction is thought to play a role in the development of liver steatosis and insulin resistance, which are both common characteristics of obesity and type 2 diabetes mellitus (T2DM). It was hypothesized that the antioxidant properties of melatonin could potentially improve the impaired functions of hepatic mitochondria in diabetic obese animals. Male Zucker diabetic fatty (ZDF) rats and lean littermates (ZL) were given either melatonin (10 mg/kg BW/day) orally for 6 wk (M‐ZDF and M‐ZL) or vehicle as control groups (C‐ZDF and C‐ZL). Hepatic function was evaluated by measurement of serum alanine transaminase and aspartate transaminase levels, liver histopathology and electron microscopy, and hepatic mitochondrial functions. Several impaired functions of hepatic mitochondria were observed in C‐ZDF in comparison with C‐ZL rats. Melatonin treatment to ZDF rats decreases serum levels of ALT (P < 0.001), alleviates liver steatosis and vacuolation, and also mitigates diabetic‐induced mitochondrial abnormalities, glycogen, and lipid accumulation. Melatonin improves mitochondrial dysfunction in M‐ZDF rats by increasing activities of mitochondrial citrate synthase (P < 0.001) and complex IV of electron transfer chain (P < 0.05) and enhances state 3 respiration (P < 0.001), respiratory control index (RCR) (P < 0.01), and phosphorylation coefficient (ADP/O ratio) (P < 0.05). Also melatonin augments ATP production (P < 0.05) and diminishes uncoupling protein 2 levels (P < 0.001). These results demonstrate that chronic oral melatonin reduces liver steatosis and mitochondria dysfunction in ZDF rats. Therefore, it may be beneficial in the treatment of diabesity.     

Combined melatonin and exendin‐4 therapy preserves renal ultrastructural integrity after ischemia–reperfusion injury in the male rat

We tested whether combined melatonin (Mel) and exendin‐4 (Ex4) treatment can better preserve glomerular structural integrity after ischemia–reperfusion (IR) injury compared with either alone. Adult male Sprague Dawley rats (n = 50) were equally divided into sham control (SC), IR, IR‐Ex4 (10 μg/kg subcutaneously 30 min after reperfusion and daily for 5 days), IR‐Mel (20 mg/kg intraperitoneally at 30 min postreperfusion and 50 mg/kg at 6 and 18 hr), and IR‐Ex4‐Mel were euthanized at day 14. Serum creatinine level and urine protein‐to‐creatinine ratio at days 3 and 14 were highest in IR group and lowest in SC, significantly higher in IR‐Ex4 and IR‐Mel groups than in IR‐Ex4‐Mel group (all P < 0.001) without significant difference between IR‐Ex4 and IR‐Mel groups. Changes in podocyte injury score (PIS) and kidney injury score were highest in IR group and lowest in SC, significantly higher in IR‐Ex4 and IR‐Mel groups than in IR‐Ex4‐Mel, and significantly higher in IR‐Mel group than in IR‐Ex4 group (all P < 0.001). Immunohistochemical microscopic findings of the expressions of FSP‐1 and WT‐1 (two glomerular damage indicators) and KIM‐1 and snail (two renal tubular‐damaged indicators) showed an identical pattern, whereas the expressions of ZO‐1, p‐cadherin, podocin, dystroglycan, fibronectin, and synaptopodin (six indices of glomerular integrity) demonstrated an opposite pattern compared to that of PIS among five groups (all P < 0.001). Protein expressions of inflammatory (TNF‐α/NF‐κB/MMP‐9) and oxidative stress (NOX‐1, NOX‐2, oxidized protein) biomarkers exhibited an identical pattern to that of PIS among five groups (all P < 0.001). Combined melatonin–exednin‐4 therapy further protected glomerulus from IR injury.     

Melatonin ameliorates low‐grade inflammation and oxidative stress in young Zucker diabetic fatty rats

The aim of this study was to investigate the effects of melatonin on low‐grade inflammation and oxidative stress in young male Zucker diabetic fatty (ZDF) rats, an experimental model of metabolic syndrome and type 2 diabetes mellitus (T2DM). ZDF rats (n = 30) and lean littermates (ZL) (n = 30) were used. At 6 wk of age, both lean and fatty animals were subdivided into three groups, each composed of 10 rats: naive (N), vehicle treated (V), and melatonin treated (M) (10 mg/kg/day) for 6 wk. Vehicle and melatonin were added to the drinking water. Pro‐inflammatory state was evaluated by plasma levels of interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), and C‐reactive protein (CRP). Also, oxidative stress was assessed by plasma lipid peroxidation (LPO), both basal and after Fe²⁺/H₂O₂ inducement. ZDF rats exhibited higher levels of IL‐6 (112.4 ± 1.5 pg/mL), TNF‐α (11.0 ± 0.1 pg/mL) and CRP (828 ± 16.0 µg/mL) compared with lean rats (IL‐6, 89.9 ± 1.0, P < 0.01; TNF‐α, 9.7 ± 0.4, P < 0.01; CRP, 508 ± 21.5, P < 0.001). Melatonin lowered IL‐6 (10%, P < 0.05), TNF‐α (10%, P < 0.05), and CRP (21%, P < 0.01). Basal and Fe²⁺/H₂O₂‐induced LPO, expressed as malondialdehyde equivalents (µmol/L), were higher in ZDF rats (basal, 3.2 ± 0.1 versus 2.5 ± 0.1 in ZL, P < 0.01; Fe²⁺/H₂O₂‐induced, 8.7 ± 0.2 versus 5.5 ± 0.3 in ZL; P < 0.001). Melatonin improved basal LPO (15%, P < 0.05) in ZDF rats, and Fe²⁺/H₂O₂‐ induced LPO in both ZL (15.2%, P < 0.01) and ZDF rats (39%, P < 0.001). These results demonstrated that oral melatonin administration ameliorates the pro‐inflammatory state and oxidative stress, which underlie the development of insulin resistance and their consequences, metabolic syndrome, diabetes, and cardiovascular disease.     

Melatonin improves mitochondrial function in inguinal white adipose tissue of Zücker diabetic fatty rats

Mitochondrial dysfunction in adipose tissue may contribute to obesity‐related metabolic derangements such as type 2 diabetes mellitus (T2DM). Because mitochondria are a target for melatonin action, the goal of this study was to investigate the effects of melatonin on mitochondrial function in white (WAT) and beige inguinal adipose tissue of Zücker diabetic fatty (ZDF) rats, a model of obesity‐related T2DM. In this experimental model, melatonin reduces obesity and improves the metabolic profile. At 6 wk of age, ZDF rats and lean littermates (ZL) were subdivided into two groups, each composed of four rats: control (C‐ZDF and C‐ZL) and treated with oral melatonin in the drinking water (10 mg/kg/day) for 6 wk (M‐ZDF and M‐ZL). After the treatment period, animals were sacrificed, tissues dissected, and mitochondrial function assessed in isolated organelles. Melatonin increased the respiratory control ratio (RCR) in mitochondria from white fat of both lean (by 26.5%, P < 0.01) and obese (by 34.5%, P < 0.01) rats mainly through a reduction of proton leaking component of respiration (state 4) (28% decrease in ZL, P < 0.01 and 35% in ZDF, P < 0.01). However, melatonin treatment lowered the RCR in beige mitochondria of both lean (by 7%, P < 0.05) and obese (by 13%, P < 0.05) rats by maintaining high rates of uncoupled respiration. Melatonin also lowered mitochondrial oxidative status by reducing nitrite levels and by increasing superoxide dismutase activity. Moreover, melatonin treatment also caused a profound inhibition of Ca‐induced opening of mPTP in isolated mitochondria from both types of fat, white and beige, in both lean and obese rats. These results demonstrate that chronic oral melatonin improves mitochondrial respiration and reduces the oxidative status and susceptibility to apoptosis in white and beige adipocytes. These melatonin effects help to prevent mitochondrial dysfunction and thereby to improve obesity‐related metabolic disorders such as diabetes and dyslipidemia of ZDF rats.     

Lower incidence of acute respiratory distress syndrome in community‐acquired pneumonia patients aged 85 years or older

Background and objective: The incidence of and risk factors for ARDS among elderly patients with community‐acquired pneumonia (CAP) have not been well characterized. Methods: The clinical details of 221 consecutive patients aged ≥65 years, who were admitted with CAP during the period April 2006 to June 2008, were investigated by review of patient charts. Clinical variables and laboratory data at admission for CAP were compared between patients with and without ARDS. Results: Eighteen patients (8.1%) developed ARDS 1–5 days after admission. The mortality rate was 44% in patients with ARDS and 10.3% in those without ARDS (P < 0.001). The incidence of ARDS was 8.5–20% among patients aged <85 years and 1.1% in patients aged ≥85 years (P < 0.001), while overall mortality rates were not significantly different among the age groups. Predictors for the development of ARDS included higher serum levels of CRP and glucose, lower PaO₂/fraction of inspired O₂ (FiO₂), PaCO₂ and HCO₃^‐, and the presence of systemic inflammatory response syndrome at admission. ARDS developed less frequently among patients with pneumonia associated with oropharyngeal aspiration (AP). Multivariate analysis indicated that lower age, serum glucose, pre‐existence of systemic inflammatory response syndrome and non‐oropharyngeal AP were significant risk factors for ARDS. The Pneumonia Severity Index and confusion, urea, respiratory rate, blood pressure, age ≥65 score were not correlated with the incidence of ARDS. Conclusions: Predictors for ARDS appeared to differ from the determinants of severity of CAP in the elderly. ARDS developed less frequently in patients aged ≥85 years and in those with oropharyngeal AP. It is important to identify subjects at high risk for ARDS upon admission and to observe them closely.     

Melatonin‐mediated downregulation of ZNF746 suppresses bladder tumorigenesis mainly through inhibiting the AKT‐MMP‐9 signaling pathway

There still lacking effective treatment for bladder cancer. This study investigated whether melatonin (Mel) can suppress the growth and invasion of bladder cancer cells. Male C57B/L6 mice were categorized into control group (ie, subcutaneous injection of HT1197 bladder cancer cell line at the back] and treatment group [subcutaneous HT1197 cells + intraperitoneal Mel (100 mg/kg/d) from day 8 to day 21 after tumor cell injection]. In vitro Mel suppressed cell growth of four bladder cancer cell lines (ie, T24, RT4, HT1197, HT1376), cell migration in HT1197/HT1376, mitochondrial membrane potential (MMP) in T24 and colony formation in RT4 cells as well as arrested the cell cycle at G0 phase and inhibited the mitotic phase of T24 cells (all P < 0.0001). Protein expression of ZNF746 in RT4/T24 cells and protein expression phosphorylated (p)‐AKT/MMP‐2/MMP‐9 in HT1197/HT1376 cells were reduced following Mel treatment (all P < 0.001). Transfection of T24 cells with plasmid‐based shRNA (ie, ZNF746‐silencing) downregulated the protein expression of MMP‐9, cell growth, and invasion and attachment to endothelial cells but upregulated the colony formation (all P < 0.001). Mel suppressed oxidative stress and MMP but upregulated mitochondria mass in ZNF746‐silenced T24 cells, whereas these parameters exhibited a similar patter to Mel treatment in ZNF746‐silenced T24 cells (all P < 0.0001). In vivo study demonstrated that Mel treatment significantly suppressed cellular expressions of MMP‐9/MMP‐2, protein expressions of ZNF746/p‐AKT, and tumor size (all P < 0.001). Mel treatment suppressed the growth, migration, and invasion of bladder carcinoma cells through downregulating ZNF746‐regulated MMP‐9/MMP‐2 signaling.     

Daily melatonin protects the endothelial lineage and functional integrity against the aging process,oxidative stress,and toxic environment and restores blood flow in critical limb ischemia area in mice

We tested the hypothesis that daily melatonin treatment protects endothelial lineage and functional integrity against the aging process, oxidative stress/endothelial denudation (ED), and toxic environment and restored blood flow in murine critical limb ischemia (CLI). In vitro study using HUVECs, in vivo models (ie, CLI through left femoral artery ligation and ED through carotid artery wire injury), and model of lipopolysaccharide‐induced aortic injury in young (3 months old) and aged (8 months old) mice were used to elucidate effects of melatonin treatment on vascular endothelial integrity. In vitro study showed that menadione‐induced oxidative stress (NOX‐1/NOX‐2), inflammation (TNF‐α/NF‐kB), apoptosis (cleaved caspase‐3/PARP), and mitochondrial damage (cytosolic cytochrome c) in HUVECs were suppressed by melatonin but reversed by SIRT3‐siRNA (all P < .001). In vivo, reduced numbers of circulating endothelial progenitor cells (EPCs) (C‐kit/CD31+/Sca‐1/KDR+/CXCR4/CD34+), and angiogenesis (Matrigel assay of bone marrow‐derived EPC and ex vivo aortic ring cultures) in older (compared with younger) mice were significantly reversed through daily melatonin administration (20 mg/kg/d, ip) (all P < .001). Aortic vasorelaxation and nitric oxide release were impaired in older mice and reversed in age‐match mice receiving melatonin (all P < .01). ED‐induced intimal/medial hyperplasia, reduced blood flow to ischemic limb, and angiogenesis (reduced CD31+/vWF+ cells/small vessel number) were improved after daily melatonin treatment (all P < .0001). Lipopolysaccharide‐induced aortic endothelial cell detachment, which was more severe in aged mice, was also alleviated after daily melatonin treatment (P < .0001). Daily melatonin treatment protected both structural and functional integrity of vascular endothelium against aging‐, oxidative stress‐, lipopolysaccharide‐, and ischemia‐induced damage probably through upregulating the SIRT signaling pathway.     

Melatonin improves cerebrovascular function and decreases oxidative stress in chronically hypoxic lambs

Chronic hypoxia during gestation and delivery results in oxidative stress and cerebrovascular dysfunction in the neonate. We assessed whether melatonin, a potent antioxidant and potential vasodilator, improves the cerebral vascular function in chronically hypoxic neonatal lambs gestated and born in the highlands (3600 m). Six lambs received melatonin (1 mg/kg per day oral) and six received vehicle, once a day for 8 days. During treatment, biometry and hemodynamic variables were recorded. After treatment, lambs were submitted to a graded FiO₂ protocol to assess cardiovascular responses to oxygenation changes. At 12 days old, middle cerebral arteries (MCA) were collected for vascular reactivity, morphostructural, and immunostaining evaluation. Melatonin increased fractional growth at the beginning and improved carotid blood flow at all arterial PO₂ levels by the end of the treatment (P < 0.05). Further, melatonin treatment improved vascular responses to potassium, serotonin, methacholine, and melatonin itself (P < 0.05). In addition, melatonin enhanced the endothelial response via nitric oxide‐independent mechanisms in isolated arteries (162 ± 26 versus 266 ± 34 AUC, P < 0.05). Finally, nitrotyrosine staining as an oxidative stress marker decreased in the MCA media layer of melatonin‐treated animals (0.01357 ± 0.00089 versus 0.00837 ± 0.00164 pixels/μm², P < 0.05). All the melatonin‐induced changes were associated with no systemic cardiovascular alterations in vivo. In conclusion, oral treatment with melatonin modulates cerebral vascular function, resulting in a better cerebral perfusion and reduced oxidative stress in the neonatal period in chronically hypoxic lambs. Melatonin is a potential therapeutic agent for treating cerebrovascular dysfunction associated with oxidative stress and developmental hypoxia in neonates.     

Melatonin promotes seed germination under high salinity by regulating antioxidant systems,ABA and GA4 interaction in cucumber (Cucumis sativus L.)

Although previous studies have found that melatonin can promote seed germination, the mechanisms involved in perceiving and signaling melatonin remain poorly understood. In this study, it was found that melatonin was synthesized during cucumber seed germination with a peak in melatonin levels occurring 14 hr into germination. This is indicative of a correlation between melatonin synthesis and seed germination. Meanwhile, seeds pretreated with exogenous melatonin (1 μm ) showed enhanced germination rates under 150 mm NaCl stress compared to water‐pretreated seeds under salinity stress. There are two apparent mechanisms by which melatonin alleviated salinity‐induced inhibition of seed germination. Exogenous melatonin decreased oxidative damage induced by NaCl stress by enhancing gene expression of antioxidants. Under NaCl stress, compared to untreated control, the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were significantly increased by approximately 1.3–5.0‐fold, with a concomitant 1.4–2.0‐fold increase of CsCu‐ZnSOD, CsFe‐ZnSOD, CsCAT, and CsPOD in melatonin‐pretreated seeds. Melatonin also alleviated salinity stress by affecting abscisic acid (ABA) and gibberellin acid (GA) biosynthesis and catabolism during seed germination. Compared to NaCl treatment, melatonin significantly up‐regulated ABA catabolism genes (e.g., CsCYP707A1 and CsCYP707A2, 3.5 and 105‐fold higher than NaCl treatment at 16 hr, respectively) and down‐regulated ABA biosynthesis genes (e.g., CsNECD2, 0.29‐fold of CK2 at 16 hr), resulting in a rapid decrease of ABA content during the early stage of germination. At the same time, melatonin positively up‐regulated GA biosynthesis genes (e.g., GA20ox and GA3ox, 2.3 and 3.9‐fold higher than NaCl treatment at 0 and 12 hr, respectively), contributing to a significant increase of GA (especially GA₄) content. In this study, we provide new evidence suggesting that melatonin alleviates the inhibitory effects of NaCl stress on germination mainly by regulating the biosynthesis and catabolism of ABA and GA₄.     

Melatonin improves bone mineral density at the femoral neck in postmenopausal women with osteopenia: a randomized controlled trial

Melatonin is known for its regulation of circadian rhythm. Recently, studies have shown that melatonin may have a positive effect on the skeleton. By increasing age, the melatonin levels decrease, which may lead to a further imbalanced bone remodeling. We aimed to investigate whether treatment with melatonin could improve bone mass and integrity in humans. In a double‐blind RCT, we randomized 81 postmenopausal osteopenic women to 1‐yr nightly treatment with melatonin 1 mg (N = 20), 3 mg (N = 20), or placebo (N = 41). At baseline and after 1‐yr treatment, we measured bone mineral density (BMD) by dual X‐ray absorptiometry, quantitative computed tomography (QCT), and high‐resolution peripheral QCT (HR‐pQCT) and determined calciotropic hormones and bone markers. Mean age of the study subjects was 63 (range 56–73) yr. Compared to placebo, femoral neck BMD increased by 1.4% in response to melatonin (P < 0.05) in a dose‐dependent manner (P < 0.01), as BMD increased by 0.5% in the 1 mg/day group (P = 0.55) and by 2.3% (P < 0.01) in the 3 mg/day group. In the melatonin group, trabecular thickness in tibia increased by 2.2% (P = 0.04), and volumetric bone mineral density (vBMD) in the spine, by 3.6% (P = 0.04) in the 3 mg/day. Treatment did not significantly affect BMD at other sites or levels of bone turnover markers; however, 24‐hr urinary calcium was decreased in response to melatonin by 12.2% (P = 0.02). In conclusion, 1‐yr treatment with melatonin increased BMD at femoral neck in a dose‐dependent manner, while high‐dose melatonin increased vBMD in the spine. Further studies are needed to assess the mechanisms of action and whether the positive effect of nighttime melatonin will protect against fractures.     

Blood pressure means rather than nocturnal dipping pattern are related to complications in Type 2 diabetic patients

Aim To determine whether systolic and diastolic blood pressure (BP) means, during ambulatory BP monitoring (ABPM), are more strongly correlated with microvascular complications and echocardiographic structural alterations than night‐time/daytime (N/D) BP ratio. Methods A cross‐sectional study was conducted in 270 Type 2 diabetes mellitus (DM) outpatients who underwent clinical and laboratory investigations, urinary albumin excretion rate (UAER) determination, echocardiography, office and 24‐h ABPM (Spacelabs 90207). Results UAER, after multivariate adjustments, was associated with office BP (systolic: R²_a 0.162, P < 0.001; diastolic: R²_a 0.124, P < 0.001) and ABPM (24‐h systolic: R²_a 0.195, P < 0.001; 24‐h diastolic: R²_a 0.197, P < 0.001) but not with N/D BP ratios (systolic: R²_a 0.062, P = 0.080; diastolic: R²_a 0.063, P = 0.069). Similar results were observed for echocardiographic parameters. The presence of retinopathy was associated only with night‐time BP values [systolic means: odds ratio (OR) 1.13, 95% confidence interval (CI) 1.03–1.24 and diastolic means: OR 1.21, CI 1.04–1.40 and N/D diastolic BP ratio > 0.90, OR 3.21, CI 1.65–6.25]. Conclusions UAER and echocardiographic structural alterations had more consistent correlations of a greater magnitude with systolic BP means than with N/D BP ratios. The nocturnal BP values appear to be more relevant for diabetic retinopathy. BP measurement in patients with Type 2 DM should take into account the 24‐h period rather than focusing on a specific time span of BP homeostasis.     

Pan‐cancer genomic analyses reveal prognostic and immunogenic features of the tumor melatonergic microenvironment across 14 solid cancer types

We performed comprehensive genomic analyses of the melatonergic system within the tumor microenvironment and their clinical relevance across a broad spectrum of solid tumors. RNA‐seq data from The Cancer Genome Atlas (TCGA) of 14 solid tumors representing 6658 human samples were analyzed. The tumor melatonergic system was characterized by the rates of melatonin synthesis and metabolism using a two‐gene expression model (melatonin synthesis/metabolism Index). We calculated three indexes according to different melatonin metabolism isoenzymes (Index‐I [ASMT:CYP1A1], Index‐II [ASMT:CYP1A2], and Index‐III [ASMT:CYP1B1]). Samples of each cancer type were classified into two subgroups (high vs low) based on median values. Clinical outcomes, mutational burden, and neoepitope abundance were analyzed and compared. We found that the ability of the tumor microenvironment to synthesize and accumulate melatonin varied across cancer types and negatively correlated with tumor burden. Kaplan‐Meier survival analyses and multivariable modeling showed that the three indexes played different roles across different cancers and harbored prognostic values in breast cancer (adjusted hazard ratio [AHR]_Index‐II = 0.65 [0.44‐0.97]; P = 0.03), cervical cancer (AHR_Index‐I = 0.62 [0.39‐0.98]; P = 0.04), lung squamous cell carcinoma (AHR_Index‐III = 0.75 [0.56‐0.99]; P = 0.04), melanoma (AHR_Index‐I = 0.74 [0.55‐0.98]; P = 0.04), and stomach adenocarcinoma (AHR_Index‐III = 0.68 [0.41‐0.94]; P = 0.02). We further investigated its clinical relevance with tumor immunogenic features (mutational burden and neoantigen abundance), which may predict immunotherapy benefits. We observed significant negative correlations with mutational burden in the majority of tumors (P < 0.05), except cervical cancer, pancreatic adenocarcinoma, and thyroid carcinoma. Our study provides a systematic overview of the oncostatic values of the melatonergic system and highlights the utilization of this simple and promising gene signature as a prognosticator and potential predictor of response to immunotherapy.     

Plant mitochondria synthesize melatonin and enhance the tolerance of plants to drought stress

Synthesis of melatonin in mitochondria was reported in animals. However, there is no report on whether plant mitochondria also produce melatonin. Herein, we show that plant mitochondria are a major site for melatonin synthesis. In an in vitro study, isolated apple mitochondria had the capacity to generate melatonin. Subcellular localization analysis documented that an apple SNAT isoform, MzSNAT5, was localized in the mitochondria of both Arabidopsis protoplasts and apple callus cells. The kinetic analysis revealed that the recombinant MzSNAT5 protein exhibited high enzymatic activity to catalyze serotonin to N‐acetylserotonin with the K_m and V_max of 55 μmol/L and 0.909 pmol/min/mg protein at 35°C, respectively; this pathway functioned over a wide range of temperatures from 5 to 75°C. In an in vivo study, MzSNAT5 was drought inducible. The transgenic Arabidopsis ectopically expressing MzSNAT5 elevated the melatonin level and, hence, enhanced drought tolerance. The mechanistic study indicated that the ectopically expressing MzSNAT5 allows plant mitochondria to increase melatonin synthesis. As a potent free radical scavenger, melatonin reduces the oxidative stress caused by the elevated reactive oxygen species which are generated under drought stress in plants. Our findings provide evidence that engineered melatonin‐enriched plants exhibit enhanced oxidative tolerance.     

Melatonin increases brown adipose tissue mass and function in Zücker diabetic fatty rats: implications for obesity control

Melatonin limits obesity in rodents without affecting food intake and activity, suggesting a thermogenic effect. Previously we demonstrated that melatonin browns subcutaneous fat in Zücker diabetic fatty (ZDF) rats. Other works pointed to melatonin as a signal that increases brown adipose tissue (BAT) mass and function in rodents. However, direct proof of thermogenic properties (uncoupled mitochondria) of the newly recruited BAT in response to melatonin is still lacking. Therefore, in this work, we investigated if melatonin recruits thermogenic BAT in ZDF rats. Zücker lean (ZL) and ZDF animals were subdivided into two groups, control (C) and treated with oral melatonin (M) for 6 weeks. Mitochondrial mass, activity of citrate synthase (CS), and respiratory chain complexes I and IV were lower in C‐ZDF than in C‐ZL animals (P < .001). Melatonin treatment increased BAT weight in ZDF rats (P < .001). Also, it rose mitochondrial mass (P < .01) and activities of CS and complexes I and IV (P < .001) in both, ZDF and ZL rats. Uncoupling protein 1 (UCP1) mRNA and protein were 50% lower in BAT from obese rats. Also, guanosine diphosphate (GDP) binding was lower in ZDF than in lean rats (P < .01). Melatonin treatment of obese rats restored the expression of UCP1 and GDP binding to levels of lean rats and sensitized the thermogenic response to cold exposure. These data demonstrated that melatonin recruits thermogenic BAT in ZDF rats. This may contribute to melatonin's control of body weight and its metabolic benefits.