Melatonin inhibits matrix metalloproteinase-9 (MMP-9) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 and BV2 cells and a mouse model of meningitis

Abstract: We explored anti‐inflammatory potential of melatonin against the lipopolysaccharide (LPS)‐induced inflammation in vivo and in vitro. RAW 264.7 and BV2 cells were stimulated by LPS, followed by the treatment with melatonin or vehicle at various time intervals. In a mouse model of meningitis induced by LPS, melatonin (5 mg/kg) or vehicle was intravenously injected at 30 min postinsult. The activity of matrix metalloproteinase‐2 (MMP‐2) and metalloproteinase‐9 (MMP‐9) was determined by gelatin zymography. Nuclear factor‐kappa B (NFκB) translocation and binding activity were determined by immunocytochemistry and electrophoretic mobility shift assay (EMSA). Our results showed that either pretreatment or cotreatment with melatonin at 50–500 μm effectively inhibited the LPS‐induced proMMP‐9 activation in the RAW 264.7 and BV2 cells, respectively (P < 0.05). This melatonin‐induced proMMP‐9 inhibition remained effective when treatment was delayed up to 2 and 6 hr postinsult for RAW 264.7 and BV2 cells, respectively (P < 0.05 for both groups). Additionally, melatonin significantly attenuated the rises of circulatory and cerebral MMP‐9 activity, respectively (P < 0.05) and reduced the loss of body weight (P < 0.05) in mice with meningitis. Moreover, melatonin (50 μm ) effectively inhibited nuclear factor‐kappa B (NFκB) translocation and binding activity in the LPS‐treated RAW 264.7 and BV2 cells, respectively (P < 0.05). These results demonstrate direct inhibitory actions of melatonin against postinflammatory NFκB translocation and MMP‐9 activation and highlight its ability to inhibit systemic and cerebral MMP‐9 activation following brain inflammation.     

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.     

The environmental neurotoxin β‐N‐methylamino‐L‐alanine inhibits melatonin synthesis in primary pinealocytes and a rat model

The environmental neurotoxin β‐N‐methylamino‐L‐alanine (BMAA) is a glutamate receptor agonist that can induce oxidative stress and has been implicated as a possible risk factor for neurodegenerative disease. Detection of BMAA in mussels, crustaceans, and fish illustrates that the sources of human exposure to this toxin are more abundant than previously anticipated. The aim of this study was to determine uptake of BMAA in the pineal gland and subsequent effects on melatonin production in primary pinealocyte cultures and a rat model. Autoradiographic imaging of 10‐day‐old male rats revealed a high and selective uptake in the pineal gland at 30 minutes to 24 hours after ¹⁴C‐L‐BMAA administration (0.68 mg/kg). Primary pinealocyte cultures exposed to 0.05‐3 mmol/L BMAA showed a 57%‐93% decrease in melatonin synthesis in vitro. Both the metabotropic glutamate receptor 3 (mGluR3) antagonist Ly341495 and the protein kinase C (PKC) activator phorbol‐12‐myristate‐13‐acetate prevented the decrease in melatonin secretion, suggesting that BMAA inhibits melatonin synthesis by mGluR3 activation and PKC inhibition. Serum analysis revealed a 45% decrease in melatonin concentration in neonatal rats assessed 2 weeks after BMAA administration (460 mg/kg) and confirmed an inhibition of melatonin synthesis in vivo. Given that melatonin is a most important neuroprotective molecule in the brain, the etiology of BMAA‐induced neurodegeneration may include mechanisms beyond direct excitotoxicity and oxidative stress.     

Matrix metalloproteinase (MMP)‐2 and MMP‐9 as inflammation markers of Trichinella spiralis and Trichinella pseudospiralis infections in mice

Trichinella spiralis and Trichinella pseudospiralis exhibit differences in the host‐parasite relationship such as the inflammatory response in parasitized muscles. Several studies indicate that matrix metalloproteinases (MMPs) represent a marker of inflammation since they regulate inflammation and immunity. The aim of this study was to evaluate the serum levels of gelatinases (MMP‐9 and MMP‐2) in mice experimentally infected with T. spiralis or T. pseudospiralis, to elucidate the involvement of these molecules during the inflammatory response to these parasites. Gelatin zymography on SDS polyacrilamide gels was used to assess the serum levels and in situ zymography on muscle histological sections to show the gelatinase‐positive cells. In T. spiralis infected mice, the total MMP‐9 serum level increased 6 days post‐infection whereas, the total MMP‐2 serum level increased onward. A similar trend was observed in T. pseudospiralis infected mice but the MMP‐9 level was lower than that detected in T. spiralis infected mice. Significant differences were also observed in MMP‐2 levels between the two experimental groups. The number of gelatinase positive cells was higher in T. spiralis than in T. pseudospiralis infected muscles. We conclude that MMP‐9 and MMP‐2 are markers of the inflammatory response for both T. spiralis and T. pseudospiralis infections.     

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.     

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.     

Neuroprotective effect of melatonin against ischemia is partially mediated by alpha‐7 nicotinic receptor modulation and HO‐1 overexpression

Melatonin has been widely studied as a protective agent against oxidative stress. However, the molecular mechanisms underlying neuroprotection in neurodegeneration and ischemic stroke are not yet well understood. In this study, we evaluated the neuroprotective/antioxidant mechanism of action of melatonin in organotypic hippocampal cultures (OHCs) as well as in photothrombotic stroke model in vivo. Melatonin (0.1, 1, and 10 μm ) incubated postoxygen and glucose deprivation (OGD) showed a concentration‐dependent protection; maximum protection was achieved at 10 μm (90% protection). Next, OHCs were exposed to 10 μm melatonin at different post‐OGD times; the protective effect of melatonin was maintained at 0, 1, and 2 hr post‐OGD treatment, but it was lost at 6 hr post‐OGD. The protective effect of melatonin and the reduction in OGD‐induced ROS were prevented by luzindole (melatonin antagonist) and α‐bungarotoxin (α‐Bgt, a selective α7 nAChR antagonist). In Nrf2 knockout mice, the protective effect of melatonin was reduced by 40% compared with controls. Melatonin, incubated 0, 1, and 2 hr post‐OGD, increased the expression of heme oxygenase‐1 (HO‐1), and this overexpression was prevented by luzindole and α‐bungarotoxin. Finally, administration of 15 mg/kg melatonin following the induction of photothrombotic stroke in vivo, reduced infarct size (50%), and improved motor skills; this effect was partially lost in 0.1 mg/kg methyllycaconitine (MLA, selective α7 nAChR antagonist)‐treated mice. Taken together, these results demonstrate that postincubation of melatonin provides a protective effect that, at least in part, depends on nicotinic receptor activation and overexpression of HO‐1.     

Melatonin enhances L‐DOPA therapeutic effects,helps to reduce its dose,and protects dopaminergic neurons in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐induced parkinsonism in mice

L‐3,4‐dihydroxyphenylalanine (L‐DOPA) reduces symptoms of Parkinson's disease (PD), but suffers from serious side effects on long‐term use. Melatonin (10–30 mg/kg, 6 doses at 10 hr intervals) was investigated to potentiate L‐DOPA therapeutic effects in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced parkinsonism in mice. Striatal tyrosine hydroxylase (TH) immunoreactivity, TH, and phosphorylated ser 40 TH (p‐TH) protein levels were assayed on 7th day. Nigral TH‐positive neurons stereology was conducted on serial sections 2.8 mm from bregma rostrally to 3.74 mm caudally. MPTP caused 39% and 58% decrease, respectively, in striatal fibers and TH protein levels, but 2.5‐fold increase in p‐TH levels. About 35% TH neurons were lost between 360 and 600 μm from 940 μm of the entire nigra analyzed, but no neurons were lost between 250 μm rostrally and 220 μm caudally. When L‐DOPA in small doses (5–8 mg/kg) failed to affect MPTP‐induced akinesia or catalepsy, co‐administration of melatonin with L‐DOPA attenuated these behaviors. Melatonin administration significantly attenuated MPTP‐induced loss in striatal TH fibers (82%), TH (62%) and p‐TH protein (100%) levels, and nigral neurons (87–100%). Melatonin failed to attenuate MPTP‐induced striatal dopamine depletion. L‐DOPA administration (5 mg/kg, once 40 min prior to sacrifice, p.o.) in MPTP‐ and melatonin‐treated mice caused significant increase in striatal dopamine (31%), as compared to L‐DOPA and MPTP‐treated mice. This was equivalent to 8 mg/kg L‐DOPA administration in parkinsonian mouse. Therefore, prolonged, effective use of L‐DOPA in PD with lesser side effects could be achieved by treating with 60% lower doses of L‐DOPA along with melatonin.     

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.     

The interferon‐gamma (IFN‐γ) +874T allele reduces the risk of hepatitis B infection in an Asian population

Increasing evidence suggests that polymorphism of the interferon‐gamma (IFN‐γ) gene in the first intron at position +874 may be associated with chronic hepatitis B virus (HBV) infection and/or HBV clearance. However, the results of relevant studies have been inconsistent. To derive a more precise estimation of the association, we performed a meta‐analysis. In total, 10 independent studies including 1661 chronic HBV‐infected patients and 1142 controls were included in this meta‐analysis. In studies following Hardy–Weinberg equilibrium (HWE), a significantly decreased risk of chronic HBV infection was associated with the IFN‐γ + 874TT genotype in the overall population (TT vs AA: odds ratio (OR) = 0.714, 95% confidence interval (CI) = 0.526–0.969, P = 0.031) when compared with a spontaneously recovered population. Subgroup analysis by ethnicity revealed a similar association in Asian individuals (TT vs AA: OR = 0.706, 95% CI = 0.518–0.962, P = 0.028). Moreover, when compared with a healthy control group, the 874T allele was associated with a significant lower risk of chronic HBV infection in the overall populations (TA vs AA: OR = 0.439, 95% CI = 0.193–0.997, P = 0.049; TT + TA vs AA: OR = 0.475, 95% CI = 0.271–0.832, P = 0.009) and in Asian individuals (TA vs AA: OR = 0.862, 95% CI = 0.744–0.999, P = 0.048). In conclusion, the IFN‐γ + 874TT genotype and 874T allele reduce the risk of chronic HBV infection in Asian individuals.     

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.     

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.     

Melatonin potentiates running wheel‐induced neurogenesis in the dentate gyrus of adult C3H/HeN mice hippocampus

This study assessed the role of melatonin in modulating running wheel(RW)‐induced hippocampal neurogenesis in adult C3H/HeN mice. Chronic melatonin (0.02 mg/mL, oral for 12 days) treatment did not affect cell proliferation or cell survival determined by the number of BrdU‐positive cells in dentate gyrus of mice with access to fixed wheel (FW). RW activity significantly increased cell proliferation [RW (n = 8) versus FW (n = 6): dorsal, 199 ± 18 versus 125 ± 12, P < 0.01; ventral, 211 ± 15 versus 123 ± 13, P < 0.01] and newborn cell survival [RW (n = 7) versus FW (n = 8): dorsal, 45 ± 8.5 versus 15 ± 1.8, P < 0.01; ventral, 48 ± 8.1 versus 15 ± 1.4)] in the dorsal and ventral dentate gyrus. Oral melatonin treatment further potentiated RW activity‐induced cell survival in both areas of the dentate gyrus [melatonin (n = 10) versus vehicle (n = 7): dorsal, 63 ± 5.4 versus 45 ± 8.5 P < 0.05; ventral, 75 ± 7.9 versus 48 ± 8.1, P < 0.01] and neurogenesis [melatonin (n = 8) versus vehicle (n = 8): dorsal, 46 ± 3.4, versus 34 ± 4.5, P < 0.05; ventral, 41 ± 3.4 versus 25 ± 2.4, P < 0.01]. We conclude that melatonin potentiates RW‐induced hippocampal neurogenesis by enhancing neuronal survival suggesting that the combination of physical exercise and melatonin may be an effective treatment for diseases affecting the hippocampus neurogenesis.     

Melatonin protects embryonic development and maintains sleep/wake behaviors from the deleterious effects of fluorene‐9‐bisphenol in zebrafish (Danio rerio)

Environmental endocrine chemicals have various adverse effects on the development of vertebrates. Fluorene‐9‐bisphenol (BHPF), a substitute of bisphenol A (BPA), is widely used in commercial production. The effects of BHPF on development and behavior are unclear. Melatonin plays a protective role under many unfavorable conditions. In this study, we investigated the effects of BHPF on the development and behaviors of zebrafish and whether melatonin reverses effects induced by BHPF. Zebrafish embryos were exposed to 0.1, 10, or 1000 nmol/L BHPF with or without 1 μmol/L melatonin from 2 hours postfertilization to 6 days postfertilization. The results showed that 0.1 and 10 nmol/L BHPF had little effect on development. High‐dose BHPF (1000 nmol/L) delayed the development, increased mortality and surface tension of embryonic chorions, caused aberrant expression of the key genes (ntl, shh, krox20, pax2, cmlc2) in early development detected by in situ hybridization, and damaged the CaP motor neurons, which were associated with locomotion ability detected by immunofluorescence. Melatonin addition reversed or weakened these adverse effects of BHPF on development, and melatonin alone increased surface tension as the effects of high‐dose BHPF. However, all groups of BHPF exposure triggered insomnia‐like behaviors, with increased waking activity and decreased rest behaviors. BHPF acted on the hypocretin (hcrt) system and upregulated the expression of sleep/wake regulators such as hcrt, hcrt receptor (hcrtr), arylalkylamine N‐acetyltransferase‐2 (aanat2). Melatonin recovered the alternation of sleep/wake behaviors induced by BHPF and restored abnormal gene expression to normal levels. This study showed that high‐dose BHPF had adverse effects on early development and induced behavioral alternations. However, melatonin prevented BHPF‐induced aberrant development and sleep/wake behaviors.     

Systemic combined melatonin–mitochondria treatment improves acute respiratory distress syndrome in the rat

Despite high in‐hospital mortality associated with acute respiratory distress syndrome (ARDS), there is no effective therapeutic strategy. We tested the hypothesis that combined melatonin–mitochondria treatment ameliorates 100% oxygen‐induced ARDS in rats. Adult male Sprague‐Dawley rats (n = 40) were equally categorized into normal controls, ARDS, ARDS‐melatonin, ARDS with intravenous liver‐derived mitochondria (1500 μg per rat 6 hr after ARDS induction), and ARDS receiving combined melatonin–mitochondria. The results showed that 22 hr after ARDS induction, oxygen saturation (saO₂) was lowest in the ARDS group and highest in normal controls, significantly lower in ARDS‐melatonin and ARDS‐mitochondria than in combined melatonin–mitochondria group, and significantly lower in ARDS‐mitochondria than in ARDS‐melatonin group. Conversely, right ventricular systolic blood pressure and lung weight showed an opposite pattern compared with saO₂ among all groups (all P < 0.001). Histological integrity of alveolar sacs showed a pattern identical to saO₂, whereas lung crowding score exhibited an opposite pattern (all P < 0.001). Albumin level and inflammatory cells (MPO+, CD40+, CD11b/c+) from bronchoalveolar lavage fluid showed a pattern opposite to saO₂ (all P < 0.001). Protein expression of indices of inflammation (MMP‐9, TNF‐α, NF‐κB), oxidative stress (oxidized protein, NO‐1, NOX‐2, NOX‐4), apoptosis (mitochondrial Bax, cleaved caspase‐3, and PARP), fibrosis (Smad3, TGF‐β), mitochondrial damage (cytochrome C), and DNA damage (γ‐H2AX+) exhibited an opposite pattern compared to saO₂ in all groups, whereas protein (HO‐1, NQO‐1, GR, GPx) and cellular (HO‐1+) expressions of antioxidants exhibited a progressively increased pattern from normal controls to ARDS combined melatonin–mitochondria group (all P < 0.001). In conclusion, combined melatonin–mitochondrial was superior to either treatment alone in attenuating ARDS in this rat model.     

Association between daily salt intake of 3‐year‐old children and that of their mothers: A cross‐sectional study

This study investigated the association between the daily salt intake of 3‐year‐old children and that of their mothers. A total of 641 children were studied. The daily salt intake of the children and their mothers was estimated by morning and spot urine methods, respectively. In the multivariable analysis, a 1 g higher maternal daily salt intake was associated with a 0.14 g (95% confidence interval [CI], 0.07‐0.22, P < .001) higher salt intake of her children. In the secondary analysis, the odds ratios for excess salt intake of children were 1.61 (95% CI, 1.01‐2.55, P = .045) and 1.81 (95% CI, 1.12‐2.91, P = .015) for 9.7‐11.5 g and 11.5 g or more of maternal daily salt intake, respectively. Our findings could help to convince mothers of the importance of appropriate salt intake, not only for themselves but also for their children.     

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.     

Meta‐analysis of association between cytokine gene polymorphisms and Behcet's disease risk

The aim of this study was to perform a meta‐analysis of eligible studies to derive precise estimation of the association of interleukin‐1 (IL‐1), IL‐10 and tumor necrosis factor (TNF)‐α polymorphisms with Behcet's disease (BD). Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association. A total of 4003 cases and 4748 controls in 19 eligible studies were included in the meta‐analysis. We examined the relationship between seven single nucleotide polymorphisms (SNPs) in the above‐mentioned three cytokine genes and susceptibility to BD. Meta‐analysis indicated the association between the cytokine gene polymorphisms in all study subjects in the allelic model (TNF‐α ‐308A/G: OR = 0.73, 95% CI: 0.61–0.88, P = 0.001; IL‐10 ‐819C/T: OR = 0.72, 95% CI: 0.66–0.78, P < 0.001; IL‐10 ‐592C/A: OR = 0.74, 95% CI: 0.64–0.86, P < 0.001); the dominant model (TNF‐α ‐308A/G: OR = 0.77, 95% CI: 0.64–0.92, P = 0.004; IL‐10 ‐1082G/A: OR = 1.64, 95% CI: 1.10–2.44, P = 0.014); the recessive model (TNF‐α ‐308A/G: OR = 0.27, 95% CI: 0.12–0.65, P = 0.003; IL‐10 ‐819C/T: OR = 0.71, 95% CI: 0.57–0.90, P = 0.004). However, no significant evidence for the associations of IL‐1α ‐889C/T, IL‐1β ‐551C/T, IL‐1β ‐3962C/T polymorphisms with BD susceptibility was detected. The present study might suggest that TNF‐α ‐308A/G, IL‐10 ‐1082G/A, ‐819C/T, ‐592C/A polymorphisms are associated with BD susceptibility.     

Tumour Necrosis Factor‐alpha (TNF‐α) and its soluble receptor type 1 (sTNFR I) in human active and healed leishmaniases

The role of tumour necrosis factor‐alpha (TNF‐α) is not fully understood in human leishmaniasis. We analysed the alterations in the levels of TNF‐α, soluble TNF receptor type 1 (sTNFR I), IL‐17 and IL‐22 productions in active and healed leishmaniases. Blood samples were collected from volunteers with active cutaneous leishmaniasis (ACL), the same subjects after lesion healing (healed CL = HCL), volunteers with active visceral leishmaniasis (AVL), healed VL (HVL) and healthy controls. Levels of cytokines were titrated on Leishmania Ag‐stimulated PBMC culture. The mean level of TNF‐α production from stimulated cells was significantly higher in ACL than controls (P < 0·001) and significantly reduced after treatment in HCL volunteers (P < 0·05). The mean level of sTNFR I production was significantly higher in ACL than controls (P < 0·001) and significantly reduced after treatment in HCL volunteers (P < 0·05). The mean level of IL‐22 production in AVL was significantly higher than controls (P < 0·05) and was significantly lower in HVL compared with AVL (P < 0·001) and controls (P < 0·05). The levels of TNF‐α (P = 0·0025) and sTNFR I (P < 0·01) productions from PBMCs showed significant decreasing trend after treatment in each CL volunteer. Reduction in TNF‐α is associated with clinical response to treatment and healing of CL lesions due to L. major.     

Melatonin receptor activation protects against low potassium‐induced ventricular fibrillation by preserving action potentials and connexin‐43 topology in isolated rat hearts

Hypokalemia prolongs the QRS and QT intervals, deteriorates intercellular coupling, and increases the risk for arrhythmia. Melatonin preserves gap junctions and shortens action potential as potential antiarrhythmic mechanisms, but its properties under hypokalemia remain unknown. We hypothesized that melatonin protects against low potassium‐induced arrhythmias through the activation of its receptors, resulting in action potential shortening and connexin‐43 preservation. After stabilization in Krebs‐Henseleit solution (4.5 mEq/L K⁺), isolated hearts from Wistar rats underwent perfusion with low‐potassium (1 mEq/L) solution and melatonin (100 μmol/L), a melatonin receptor blocker (luzindole, 5 μmol/L), melatonin + luzindole or vehicle. The primary endpoint of the study was the prevention of ventricular fibrillation. Electrocardiography was used, and epicardial action potentials and heart function were measured and analyzed. The ventricular expression, dephosphorylation, and distribution of connexin‐43 were examined. Melatonin reduced the incidence of low potassium‐induced ventricular fibrillation from 100% to 59%, delayed the occurrence of ventricular fibrillation and induced a faster recovery of sinus rhythm during potassium restitution. Melatonin prevented QRS widening, action potential activation delay, and the prolongation of action potential duration at 50% of repolarization. Other ECG and action potential parameters, the left ventricular developed pressure, and nonsustained ventricular arrhythmias did not differ among groups. Melatonin prevented connexin‐43 dephosphorylation and its abnormal topology (lateralization). Luzindole abrogated the protective effects of melatonin on electrophysiological properties and connexin‐43 misdistribution. Our results indicate that melatonin receptor activation protects against low potassium‐induced ventricular fibrillation, shortens action potential duration, preserves ventricular electrical activation, and prevents acute changes in connexin‐43 distribution. All of these properties make melatonin a remarkable antifibrillatory agent.