Protective effect of melatonin against Opisthorchis viverrini‐induced oxidative and nitrosative DNA damage and liver injury in hamsters

Abstract: The liver fluke, Opisthorchis viverrini, is the risk factor of cholangiocarcinoma, which is a major health problem in northeastern Thailand. Production of reactive oxygen and nitrogen species during the host’s response leads to oxidative and nitrosative stress contributing to carcinogenesis. We investigated the protective effect of melatonin against O. viverrini‐induced oxidative and nitrosative stress and liver injury. Hamsters were infected with O. viverrini followed by oral administration of various doses of melatonin (5, 10, and 20 mg/kg body weight) for 30 days. Uninfected hamsters served as controls. Compared to the levels in O. viverrini‐infected hamsters without melatonin treatment, the indoleamine decreased the formation of oxidative and nitrosative DNA lesions, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine and 8‐nitroguanine, in the nucleus of bile duct epithelium and inflammatory cells, in parallel with a reduction in 3‐nitrotyrosine. Melatonin also reduced the expression of heme oxygenase‐1 and cytokeratin 19, nitrate/nitrite levels, and bile duct proliferation in the liver. Alanine transaminase activity and the levels of 8‐isoprostane and vitamin E were also dose dependently decreased in the plasma of melatonin‐treated hamsters. Melatonin reduced the mRNA expression of oxidant‐generating genes [inducible nitric oxide synthase, nuclear factor‐kappa B (NF‐κB), and cyclooxygenase‐2] and proinflammatory cytokines (TNF‐α and IL‐1β), accompanied by an increase in the expression of antioxidant genes [nuclear erythroid 2‐related factor 2 (Nrf2) and manganese superoxide dismutase]. Thus, melatonin may be an effective chemopreventive agent against O. viverrini‐induced cholangiocarcinoma by reducing oxidative and nitrosative DNA damage via induction of Nrf2 and inhibition of NF‐κB‐mediated pathways.     

Melatonin alleviates cadmium‐induced liver injury by inhibiting the TXNIP‐NLRP3 inflammasome

Cadmium (Cd) is a persistent environmental and occupational contaminant that accumulates in the liver and induces oxidative stress and inflammation. Melatonin possesses potent hepatoprotective properties against the development and progression of acute and chronic liver injury. Nevertheless, the molecular mechanism underlying the protective effects of melatonin against Cd‐induced hepatotoxicity remains obscure. In this study, we aimed to investigate the effects of melatonin on Cd‐induced liver inflammation and hepatocyte death. Male C57BL/6 mice were intraperitoneally injected with melatonin (10 mg/kg) once a day for 3 days before exposure to CdCl₂ (2.0 mg/kg). We found that Cd induced hepatocellular damage and inflammatory infiltration as well as increased serum ALT/AST enzymes. In addition, we showed that Cd triggered an inflammatory cell death, which is mediated by the NOD‐like receptor pyrin domain containing 3 (NLRP3) inflammasome. Moreover, melatonin treatment significantly alleviated Cd‐induced liver injury by decreasing serum ALT/AST levels, suppressing pro‐inflammatory cytokine production, inhibiting NLRP3 inflammasome activation, ameliorating oxidative stress, and attenuating hepatocyte death. Most importantly, melatonin markedly abrogated Cd‐induced TXNIP overexpression and decreased the interaction between TXNIP and NLRP3 in vivo and in vitro. However, treatment with siRNA targeting TXNIP blocked the protective effects of melatonin in Cd‐treated primary hepatocytes. Collectively, our results suggest that melatonin confers protection against Cd‐induced liver inflammation and hepatocyte death via inhibition of the TXNIP‐NLRP3 inflammasome pathway.     

Melatonin‐enhanced autophagy protects against neural apoptosis via a mitochondrial pathway in early brain injury following a subarachnoid hemorrhage

Melatonin is a strong antioxidant that has beneficial effects against early brain injury (EBI) following a subarachnoid hemorrhage (SAH) in rats; protection includes reduced mortality and brain water content. The molecular mechanisms underlying these clinical effects in the SAH model, however, have not been clearly identified. This study was undertaken to determine the influence of melatonin on neural apoptosis and the potential mechanism of these effects in EBI following SAH using the filament perforation model of SAH in male Sprague Dawley rats. Melatonin (150 mg/kg) or vehicle was given via an intraperitoneal injection 2 hr after SAH induction. Brain samples were extracted 24 hr after SAH. The results show that melatonin treatment markedly reduced caspase‐3 activity and the number of TUNEL‐positive cells, while the treatment increased the LC3‐II/LC3‐I, an autophagy marker, which indicated that melatonin‐enhanced autophagy ameliorated apoptotic cell death in rats subjected to SAH. To further identify the mechanism of autophagy protection, we demonstrated that melatonin administration reduced Bax translocation to the mitochondria and the release of cytochrome c into the cytosol. Taken together, this report demonstrates that melatonin improved the neurological outcome in rats by protecting against neural apoptosis after the induction of filament perforation SAH; moreover, the mechanism of these antiapoptosis effects was related to the enhancement of autophagy, which ameliorated cell apoptosis via a mitochondrial pathway.     

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.     

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 limits paclitaxel‐induced mitochondrial dysfunction in vitro and protects against paclitaxel‐induced neuropathic pain in the rat

Chemotherapy‐induced neuropathic pain is a debilitating and common side effect of cancer treatment. Mitochondrial dysfunction associated with oxidative stress in peripheral nerves has been implicated in the underlying mechanism. We investigated the potential of melatonin, a potent antioxidant that preferentially acts within mitochondria, to reduce mitochondrial damage and neuropathic pain resulting from the chemotherapeutic drug paclitaxel. In vitro, paclitaxel caused a 50% reduction in mitochondrial membrane potential and metabolic rate, independent of concentration (20‐100 μmol/L). Mitochondrial volume was increased dose‐dependently by paclitaxel (200% increase at 100 μmol/L). These effects were prevented by co‐treatment with 1 μmol/L melatonin. Paclitaxel cytotoxicity against cancer cells was not affected by co‐exposure to 1 μmol/L melatonin of either the breast cancer cell line MCF‐7 or the ovarian carcinoma cell line A2780. In a rat model of paclitaxel‐induced painful peripheral neuropathy, pretreatment with oral melatonin (5/10/50 mg/kg), given as a daily bolus dose, was protective, dose‐dependently limiting development of mechanical hypersensitivity (19/43/47% difference from paclitaxel control, respectively). Melatonin (10 mg/kg/day) was similarly effective when administered continuously in drinking water (39% difference). Melatonin also reduced paclitaxel‐induced elevated 8‐isoprostane F₂α levels in peripheral nerves (by 22% in sciatic; 41% in saphenous) and limited paclitaxel‐induced reduction in C‐fibre activity‐dependent slowing (by 64%). Notably, melatonin limited the development of mechanical hypersensitivity in both male and female animals (by 50/41%, respectively), and an additive effect was found when melatonin was given with the current treatment, duloxetine (75/62% difference, respectively). Melatonin is therefore a potential treatment to limit the development of painful neuropathy resulting from chemotherapy treatment.     

Melatonin enhances hyperthermia‐induced apoptotic cell death in human leukemia cells

Melatonin is an endogenous indoleamine with a wide range of biological functions. In addition to modulating circadian rhythms, it plays important roles in the health as an antioxidant. Melatonin has also the ability to induce apoptosis in cancer cells and to enhance the antitumoral activity of chemotherapeutic agents. In this study, the effect of melatonin on hyperthermia‐induced apoptosis was explored using human leukemia cells. The results demonstrate that melatonin greatly improved the cytotoxicity of hyperthermia in U937 cells. The potentiation of cell death was achieved with 1 mmol/L concentrations of the indoleamine but not with concentrations close to physiological levels in blood (1 nmol/L). This effect was associated to an enhancement of the apoptotic response, revealed by an increase in cells with hypodiploid DNA content and activation of multiple caspases (caspase‐2, caspase‐3, caspase‐8, and caspase‐9). Melatonin also increased hyperthermia‐induced Bid activation as well as translocation of Bax from the cytosol to mitochondria and cytochrome c release. Hyperthermia‐provoked apoptosis and potentiation by melatonin were abrogated by a broad‐spectrum caspase inhibitor (z‐VAD‐fmk) as well as by specific inhibitors against caspase‐8 or caspase‐3. In contrast, blocking of the mitochondrial pathway of apoptosis either with a caspase‐9 inhibitor or overexpressing the anti‐apoptotic protein Bcl‐2 (U937/Bcl‐2) reduced the number of apoptotic cells in response to hyperthermia but it was unable to suppress melatonin enhancement. Melatonin appears to modulate the apoptotic response triggered by hyperthermia in a cell type‐specific manner as similar results were observed in HL‐60 but not in K562 or MOLT‐3 cells.     

Dynamin‐related protein 1‐mediated mitochondrial fission contributes to post‐traumatic cardiac dysfunction in rats and the protective effect of melatonin

Mechanical trauma (MT ) causes myocardial injury and cardiac dysfunction. However, the underlying mechanism remains largely unclear. This study investigated the role of mitochondrial dynamics in post‐traumatic cardiac dysfunction and the protective effects of melatonin. Adult male Sprague Dawley rats were subjected to 5‐minute rotations (200 revolutions at a rate of 40 rpm) to induce MT model. Melatonin was administrated intraperitoneally 5 minute after MT . Mitochondrial morphology, myocardial injury, and cardiac function were determined in vivo. There was smaller size of mitochondria and increased number of mitochondria per μm² in the hearts after MT when the secondary myocardial injury was induced. Melatonin treatment at the dose of 30 mg/kg reduced serine 616 phosphorylation of Drp1 and inhibited mitochondrial Drp1 translocation and mitochondrial fission in the hearts of rats subjected to MT , which contributed to the reduction of myocardial injury and the improvement of cardiac function. In vitro, H9c2 cells cultured in 20% traumatic plasma (TP ) for 12 hour showed enhanced mitochondrial fission, mitochondrial membrane potential (?Ψm) loss, mitochondrial cytochrome c release, and decreased mitochondrial complex I‐IV activities. Pretreatment with melatonin (100 μmol/L) efficiently inhibited TP ‐induced mitochondrial fission, ?Ψm loss, cytochrome c release, and improved mitochondrial function. Melatonin's protective effects were attributed to its role in suppressing plasma TNF ‐α overproduction, which was responsible for Drp1‐mediated mitochondrial fission. Taken together, our results demonstrate for the first time that abnormal mitochondrial dynamics is involved in post‐traumatic cardiac dysfunction. Melatonin has significant pharmacological potential in protecting against MT ‐induced cardiac dysfunction by preventing excessive mitochondrial fission.     

Inhibition of SERPINA3N‐dependent neuroinflammation is essential for melatonin to ameliorate trimethyltin chloride–induced neurotoxicity

Trimethyltin chloride (TMT) is a potent neurotoxin that causes neuroinflammation and neuronal cell death. Melatonin is a well‐known anti‐inflammatory agent with significant neuroprotective activity. Male C57BL/6J mice were intraperitoneally injected with a single dose of melatonin (10 mg/kg) before exposure to TMT (2.8 mg/kg, ip). Thereafter, the mice received melatonin (10 mg/kg, ip) once a day for another three consecutive days. Melatonin dramatically alleviated TMT‐induced neurotoxicity in mice by attenuating hippocampal neuron loss, inhibiting epilepsy‐like seizures, and ameliorating memory deficits. Moreover, melatonin markedly suppressed TMT‐induced neuroinflammatory responses and astrocyte activation, as shown by a decrease in inflammatory cytokine production as well as the downregulation of neurotoxic reactive astrocyte phenotype markers. Mechanistically, serine peptidase inhibitor clade A member 3N (SERPINA3N) was identified as playing a central role in the protective effects of melatonin based on quantitative proteome and bioinformatics analysis. Most importantly, melatonin significantly suppressed TMT‐induced SERPINA3N upregulation at both the mRNA and protein levels. The overexpression of Serpina3n in the mouse hippocampus abolished the protective effects of melatonin on TMT‐induced neuroinflammation and neurotoxicity. Melatonin protected cells against TMT‐induced neurotoxicity by inhibiting SERPINA3N‐mediated neuroinflammation. Melatonin may be a promising and practical agent for reducing TMT‐induced neurotoxicity in clinical practice.     

Melatonin attenuates D‐galactose‐induced memory impairment,neuroinflammation and neurodegeneration via RAGE/NF‐KB/JNK signaling pathway in aging mouse model

Melatonin acts as a pleiotropic agent in various age‐related neurodegenerative diseases. In this study, we examined the underlying neuroprotective mechanism of melatonin against D‐galactose‐induced memory and synaptic dysfunction, elevated reactive oxygen species (ROS), neuroinflammation and neurodegeneration. D‐galactose was administered (100 mg/kg intraperitoneally (i.p.)) for 60 days. After 30 days of D‐galactose administration, vehicle (same volume) or melatonin (10 mg/kg, i.p.) was administered for 30 days. Our behavioral (Morris water maze and Y‐maze test) results revealed that chronic melatonin treatment alleviated D‐galactose‐induced memory impairment. Additionally, melatonin treatment reversed D‐galactose‐induced synaptic disorder via increasing the level of memory‐related pre‐and postsynaptic protein markers. We also determined that melatonin enhances memory function in the D‐galactose‐treated mice possibly via reduction of elevated ROS and receptor for advanced glycation end products (RAGE). Furthermore, Western blot and morphological results showed that melatonin treatment significantly reduced D‐galactose‐induced neuroinflammation through inhibition of microgliosis (Iba‐1) and astrocytosis (GFAP), and downregulating other inflammatory mediators such as p‐IKKβ, p‐NF‐_KB65, COX2, NOS2, IL‐1β, and TNFα. Moreover, melatonin lowered the oxidative stress kinase p‐JNK which suppressed various apoptotic markers, that is, cytochrome C, caspase‐9, caspase‐3 and PARP‐1, and prevent neurodegeneration. Hence, melatonin attenuated the D‐galactose‐induced memory impairment, neuroinflammation and neurodegeneration possibly through RAGE/NF‐_KB/JNK pathway. Taken together, our data suggest that melatonin could be a promising, safe and endogenous compatible antioxidant candidate for age‐related neurodegenerative diseases such as Alzheimer's disease (AD).     

Melatonin reduces median nerve injury‐induced mechanical hypersensitivity via inhibition of microglial p38 mitogen‐activated protein kinase activation in rat cuneate nucleus

In this study, we examined the relationships between p38 mitogen‐activated protein kinase (MAPK) activation in the cuneate nucleus (CN) and behavioral hypersensitivity after chronic constriction injury (CCI) of the median nerve. We further investigated effects of melatonin administration and pinealectomy on p38 MAPK activation and development of hypersensitivity. Using immunohistochemistry and immunoblotting, low levels of phosphorylated p38 (p‐p38) MAPK were detected in CN of normal rats. As early as 1 day after CCI, p‐p38 MAPK levels in the ipsilateral CN were significantly increased (1.4 ± 0.2‐fold, P < 0.05), which reached a maximum at 7 days (5.1 ± 0.4‐fold, P < 0.001). Double immunofluorescence labeling with cell‐specific markers showed that p‐p38 MAPK immunoreactive cells co‐expressed OX‐42, a microglia activation maker, suggesting the expression of p‐p38 MAPK in microglia. Microinjection of SB203580, a p38 MAPK inhibitor, into the CN 1 day after CCI attenuated injury‐induced behavioral hypersensitivity in a dose‐dependent manner. Furthermore, animals received melatonin treatment at daily doses of 37.5, 75, 150, or 300 mg/kg from 30 min before until 3 days after CCI. Melatonin treatment dose‐dependently attenuated p‐p38 MAPK levels, release of pro‐inflammatory cytokines, and behavioral hypersensitivity following CCI; conversely, pinealectomy that resulted in a reduction in endogenous melatonin levels exacerbated these effects. In conclusion, median nerve injury‐induced microglial p38 MAPK activation in the CN modulated development of behavioral hypersensitivity. Melatonin supplementation eased neuropathic pain via inhibition of p38 MAPK signaling pathway; contrarily, reducing endogenous blood melatonin levels by pinealectomy promoted phosphorylation of p38 MAPK and made rats more vulnerable to nerve injury‐induced neuropathic pain.     

Melatonin inhibits the sphingosine kinase 1/sphingosine‐1‐phosphate signaling pathway in rabbits with fulminant hepatitis of viral origin

The sphingosine kinase (SphK)1/sphingosine‐1‐phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10⁴ hemagglutination units of a RHDV isolate and received 20 mg/kg of melatonin at 0, 12, and 24 hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV‐infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll‐like receptor (TLR)4, tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐6, nuclear factor‐kappa B (NF‐κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.     

Melatonin prevents cell death and mitochondrial dysfunction via a SIRT1‐dependent mechanism during ischemic‐stroke in mice

Silent information regulator 1 (SIRT1), a type of histone deacetylase, is a highly effective therapeutic target for protection against ischemia reperfusion (IR) injury (IRI). Previous studies showed that melatonin preserves SIRT1 expression in neuronal cells of newborn rats after hypoxia–ischemia. However, the definite role of SIRT1 in the protective effect of melatonin against cerebral IRI in adult has not been explored. In this study, the brain of adult mice was subjected to IRI. Prior to this procedure, the mice were given intraperitoneal with or without the SIRT1 inhibitor, EX527. Melatonin conferred a cerebral‐protective effect, as shown by reduced infarct volume, lowered brain edema, and increased neurological scores. The melatonin‐induced upregulation of SIRT1 was also associated with an increase in the anti‐apoptotic factor, Bcl2, and a reduction in the pro‐apoptotic factor Bax. Moreover, melatonin resulted in a well‐preserved mitochondrial membrane potential, mitochondrial Complex I activity, and mitochondrial cytochrome c level while it reduced cytosolic cytochrome c level. However, the melatonin‐elevated mitochondrial function was reversed by EX527 treatment. In summary, our results demonstrate that melatonin treatment attenuates cerebral IRI by reducing IR‐induced mitochondrial dysfunction through the activation of SIRT1 signaling.     

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.     

Risk biomarkers for assessment and chemoprevention of liver fluke‐associated cholangiocarcinoma

Human liver fluke, Opisthorchis viverrini (Ov), is the major risk factor of cholangiocarcinoma (CCA) in northeastern Thailand. Our approach focuses on genetic progression and molecular changes in the carcinogenic pathway of liver fluke‐associated CCA aimed at assessing patients at risk of CCA and using chemoprevention as the secondary cancer prevention to reduce the incidence of CCA. This review summarizes altered gene expressions, biomolecules and their modification, i.e. DNA adducts, oxidized proteins, oxysterols and fibrotic markers in hamster‐ and human‐CCA. Potential risk biomarker(s) and chemopreventive agent(s) criteria and selection were based on results from experimental and epidemiological studies identifying hepatobiliary disease, including CCA. Laboratory results reveal that oxidative stress induced by Ov infection leads to bimolecular damage, tissue remodeling especially periductal fibrosis and alteration of gene expressions, which could be involved in all steps of CCA carcinogenesis. Some of these molecules are reported to change their levels in opisthorchiasis, periductal fibrosis diagnosed by ultrasonography and CCA. Chemoprevention in experimental CCA tumorigenesis is discussed. These multiple risk biomarkers could now be explored for screening including chemopreventive intervention of subjects living in endemic areas where the prevalence of opisthorchiasis remains high.     

Melatonin attenuates inflammatory response‐induced brain edema in early brain injury following a subarachnoid hemorrhage: a possible role for the regulation of pro‐inflammatory cytokines

Melatonin is a strong anti‐oxidant that has beneficial effects against early brain injury (EBI) following a subarachnoid hemorrhage (SAH) in rats; protection includes the reduction of both mortality and neurological deficits. The molecular mechanisms underlying these clinical effects in the SAH model have not been clearly identified. This study examined the influence of melatonin on brain edema secondary to disruption of the blood–brain barrier (BBB) and the relationship between these effects and pro‐inflammatory cytokines in EBI following SAH using the filament perforation model of SAH in male Sprague–Dawley rats. Melatonin (150 mg/kg) or vehicle was given via an intraperitoneal injection 2 hr after SAH induction. Brain samples were extracted 24 hr after SAH. Melatonin treatment markedly attenuated brain edema secondary to BBB dysfunctions by preventing the disruption of tight junction protein expression (ZO‐1, occludin, and claudin‐5). Melatonin treatment also repressed cortical levels of pro‐inflammatory cytokines (IL‐1β, IL‐6, and TNF‐α), which were increased in EBI 24 hr after SAH. To further identify the mechanism of this protection, we demonstrated that administration of melatonin attenuated matrix metallopeptidase 9 expression/activity and vascular endothelial growth factor expression, which are related to the inflammatory response and BBB disruption in EBI after SAH. Taken together, this report shows that melatonin prevents disruption of tight junction proteins which might play a role in attenuating brain edema secondary to BBB dysfunctions by repressing the inflammatory response in EBI after SAH, possibly associated with regulation of pro‐inflammatory cytokines.     

Melatonin regulates the calcium‐buffering proteins,parvalbumin and hippocalcin,in ischemic brain injury

Abstract: Melatonin has anti‐oxidant activity and it exerts a neuroprotective effects during ischemic brain injury. Calcium‐buffering proteins including parvalbumin and hippocalcin are involved in neuronal differentiation and maturation through calcium signaling. This study investigated whether melatonin moderates parvalbumin and hippocalcin expression in cerebral ischemia and glutamate toxicity‐induced neuronal cell death. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Male Sprague‐Dawley rats were treated with vehicle or melatonin (5 mg/kg) prior to MCAO, and cerebral cortical tissues were collected 24 hr after MCAO. Parvalbumin and hippocalcin levels were decreased in vehicle‐treated animal with MCAO, whereas melatonin prevented the ischemic injury‐induced reduction in these proteins. In cultured hippocampal cells, glutamate toxicity decreased parvalbumin and hippocalcin levels, while melatonin treatment prevented the glutamate exposure‐induced diminished in these proteins levels. Melatonin also attenuated the glutamate toxicity‐induced increase in intracellular Ca²⁺ levels. These results suggest that the maintenance of parvalbumin and hippocalcin levels by melatonin in ischemic injury contributes to the neuroprotective effect of melatonin against neuronal cell damage.