Melatonin promotes hepatic differentiation of human dental pulp stem cells: clinical implications for the prevention of liver fibrosis

Melatonin's effect on hepatic differentiation of stem cells remains unclear. The aim of this study was to investigate the action of melatonin on hepatic differentiation as well as its related signaling pathways of human dental pulp stem cells (hDPSCs) and to examine the therapeutic effects of a combination of melatonin and hDPSC transplantation on carbon tetrachloride (CCl₄)‐induced liver fibrosis in mice. In vitro hepatic differentiation was assessed by periodic acid‐Schiff (PAS) staining and mRNA expression for hepatocyte markers. Liver fibrosis model was established by injecting 0.5 mL/kg CCl₄ followed by treatment with melatonin (5 mg/kg, twice a week) and hDPSCs. In vivo therapeutic effects were evaluated by histopathology and by means of liver function tests including measurement of alanine transaminase (ALT), aspartate transaminase (AST), and ammonia levels. Melatonin promoted hepatic differentiation based on mRNA expression of differentiation markers and PAS‐stained glycogen‐laden cells. In addition, melatonin increased bone morphogenic protein (BMP)‐2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. Furthermore, melatonin activated p38, extracellular signal‐regulated kinase (ERK), and nuclear factor‐κB (NF‐κB) in hDPSCs. Melatonin‐induced hepatic differentiation was attenuated by inhibitors of BMP, p38, ERK, and NF‐κB. Compared to treatment of CCl₄‐injured mice with either melatonin or hDPSC transplantation alone, the combination of melatonin and hDPSC significantly suppressed liver fibrosis and restored ALT, AST, and ammonia levels. For the first time, this study demonstrates that melatonin promotes hepatic differentiation of hDPSCs by modulating the BMP, p38, ERK, and NF‐κB pathway. Combined treatment of grafted hDPSCs and melatonin could be a viable approach for the treatment of liver cirrhosis.     

Melatonin attenuates diabetic cardiomyopathy and reduces myocardial vulnerability to ischemia‐reperfusion injury by improving mitochondrial quality control: Role of SIRT6

Targeting mitochondrial quality control with melatonin has been found promising for attenuating diabetic cardiomyopathy (DCM), although the underlying mechanisms remain largely undefined. Activation of SIRT6 and melatonin membrane receptors exerts cardioprotective effects while little is known about their roles during DCM. Using high‐fat diet‐streptozotocin‐induced diabetic rat model, we found that prolonged diabetes significantly decreased nocturnal circulatory melatonin and heart melatonin levels, reduced the expressions of cardiac melatonin membrane receptors, and decreased myocardial SIRT6 and AMPK‐PGC‐1α‐AKT signaling. 16 weeks of melatonin treatment inhibited the progression of DCM and the following myocardial ischemia‐reperfusion (MI/R) injury by reducing mitochondrial fission, enhancing mitochondrial biogenesis and mitophagy via re‐activating SIRT6 and AMPK‐PGC‐1α‐AKT signaling. After the induction of diabetes, adeno‐associated virus carrying SIRT6‐specific small hairpin RNA or luzindole was delivered to the animals. We showed that SIRT6 knockdown or antagonizing melatonin receptors abolished the protective effects of melatonin against mitochondrial dysfunction as evidenced by aggravated mitochondrial fission and reduced mitochondrial biogenesis and mitophagy. Additionally, SIRT6 shRNA or luzindole inhibited melatonin‐induced AMPK‐PGC‐1α‐AKT activation as well as its cardioprotective actions. Collectively, we demonstrated that long‐term melatonin treatment attenuated the progression of DCM and reduced myocardial vulnerability to MI/R injury through preserving mitochondrial quality control. Melatonin membrane receptor‐mediated SIRT6‐AMPK‐PGC‐1α‐AKT axis played a key role in this process. Targeting SIRT6 with melatonin treatment may be a promising strategy for attenuating DCM and reducing myocardial vulnerability to ischemia‐reperfusion injury in diabetic patients.     

Melatonin suppresses activation of hepatic stellate cells through RORα‐mediated inhibition of 5‐lipoxygenase

Liver fibrosis is scar tissue resulting from an uncontrolled wound‐healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture‐activated rat HSC. Melatonin dose‐dependently suppressed the expression of HSC activation markers Col1a1 and alpha‐smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor‐related orphan receptor‐alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5‐lipoxygenase (5‐LO). The pharmacological 5‐LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα‐mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.     

Melatonin protects cardiac microvasculature against ischemia/reperfusion injury via suppression of mitochondrial fission‐VDAC1‐HK2‐mPTP‐mitophagy axis

The cardiac microvascular system, which is primarily composed of monolayer endothelial cells, is the site of blood supply and nutrient exchange to cardiomyocytes. However, microvascular ischemia/reperfusion injury (IRI) following percutaneous coronary intervention is a woefully neglected topic, and few strategies are available to reverse such pathologies. Here, we studied the effects of melatonin on microcirculation IRI and elucidated the underlying mechanism. Melatonin markedly reduced infarcted area, improved cardiac function, restored blood flow, and lower microcirculation perfusion defects. Histological analysis showed that cardiac microcirculation endothelial cells (CMEC) in melatonin‐treated mice had an unbroken endothelial barrier, increased endothelial nitric oxide synthase expression, unobstructed lumen, reduced inflammatory cell infiltration, and less endothelial damage. In contrast, AMP‐activated protein kinase α (AMPKα) deficiency abolished the beneficial effects of melatonin on microvasculature. In vitro, IRI activated dynamin‐related protein 1 (Drp1)‐dependent mitochondrial fission, which subsequently induced voltage‐dependent anion channel 1 (VDAC1) oligomerization, hexokinase 2 (HK2) liberation, mitochondrial permeability transition pore (mPTP) opening, PINK1/Parkin upregulation, and ultimately mitophagy‐mediated CMEC death. However, melatonin strengthened CMEC survival via activation of AMPKα, followed by p‐Drp1^S616 downregulation and p‐Drp1^S37 upregulation, which blunted Drp1‐dependent mitochondrial fission. Suppression of mitochondrial fission by melatonin recovered VDAC1‐HK2 interaction that prevented mPTP opening and PINK1/Parkin activation, eventually blocking mitophagy‐mediated cellular death. In summary, this study confirmed that melatonin protects cardiac microvasculature against IRI. The underlying mechanism may be attributed to the inhibitory effects of melatonin on mitochondrial fission‐VDAC1‐HK2‐mPTP‐mitophagy axis via activation of AMPKα.     

Melatonin ameliorates PM2.5‐induced cardiac perivascular fibrosis through regulating mitochondrial redox homeostasis

Fine particulate matter (PM_2.5) exposure is correlated with the risk of developing cardiac fibrosis. Melatonin is a major secretory product of the pineal gland that has been reported to prevent fibrosis. However, whether melatonin affects the adverse health effects of PM_2.5 exposure has not been investigated. Thus, this study was aimed to investigate the protective effect of melatonin against PM_2.5‐accelerated cardiac fibrosis. The echocardiography revealed that PM_2.5 had impaired both systolic and diastolic cardiac function in ApoE^?/? mice. Histopathological analysis demonstrated that PM_2.5 induced cardiomyocyte hypertrophy and fibrosis, particularly perivascular fibrosis, while the melatonin administration was effective in alleviating PM_2.5‐induced cardiac dysfunction and fibrosis in mice. Results of electron microscopy and confocal scanning laser microscope confirmed that melatonin had restorative effects against impaired mitochondrial ultrastructure and augmented mitochondrial ROS generation in PM_2.5‐treated group. Further investigation revealed melatonin administration could significantly reverse the PM_2.5‐induced phenotypic modulation of cardiac fibroblasts into myofibroblasts. For the first time, our study found that melatonin effectively alleviates PM_2.5‐induced cardiac dysfunction and fibrosis via inhibiting mitochondrial oxidative injury and regulating SIRT3‐mediated SOD2 deacetylation. Our findings indicate that melatonin could be a therapy medicine for prevention and treatment of air pollution‐associated cardiac diseases.     

N-methyl-4-isoleucine cyclosporine attenuates CCl -induced liver fibrosis in rats by interacting with cyclophilin B and D

Background and Aim: N‐methyl‐4‐isoleucine cyclosporine (NIM811), a new analogue of cyclosporine A, can inhibit collagen deposition in vitro and reduce liver necrosis in a bile‐duct‐ligation animal model. However, whether NIM811 effects on CCl₄‐induced rat liver fibrosis, and the related mechanism has not been determined. Methods: A liver fibrosis model was induced in Wistar rats using CCl₄ for 6 weeks. Meanwhile, two different doses of NIM811 (low‐dose 10 mg/kg and high‐dose 20 mg/kg) were given to the CCl₄‐treated rats. Liver fibrosis was then evaluated according to histopathological scoring and liver hydroxyproline content. Serum alanine aminotransferase, aspartate aminotransferase and albumin levels, expression of matrix metalloproteinase‐13, tissue inhibitor of metalloproteinase‐1, α‐smooth muscle actin and cyclophilin B and D in liver tissue were determined. Cyclophilin B and D were also studied in an hepatic stellate cell line. Results: Hydroxyproline content was decreased in both NIM811 groups compared with the model (P < 0.05). Liver necrosis and fibrosis were also attenuated in the NIM811 groups. NIM811 suppressed the expression of tissue inhibitor of metalloproteinase‐1, transforming growth factor beta mRNA and α‐smooth muscle actin protein in liver tissue. Expression of cyclophilin B in the fibrosis model was increased compared with the normal group (P < 0.05), and was decreased significantly in the low‐dose NIM811 treatment group (P < 0.05), which indicated that cyclophilin B might have a profibrotic effect. In vitro studies revealed that cyclophilin B and/or D knockout were associated with collagen inhibition. Conclusions: NIM811 attenuates liver fibrosis in a CCl₄‐induced rat liver fibrosis model, which may be related to binding with cyclophilin B and D.     

Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride‐induced chronic liver injury

Background/Aims: Hepatocyte growth factor (HGF) inhibits liver fibrosis induced by carbon tetrachloride (CCl₄) in animal models. NK2 is a natural splice variant of HGF, but its in vivo function remains to be elucidated. We investigated the in vivo effects of NK2 on CCl₄‐induced liver fibrosis. Methods: NK2 transgenic mice and wild‐type (WT) mice were injected intraperitoneally with CCl₄ twice a week. The extent of hepatic fibrosis was evaluated by Azan–Mallory staining. Expression levels of mRNAs of transforming growth factor‐β1 (TGF‐β1) and matrix metalloproteinase‐13 (MMP‐13) were examined by real‐time polymerase chain reaction. The protein levels of α‐smooth muscle actin (α‐SMA), c‐Met and its phosphorylation were determined by Western blot analysis. Results: Liver fibrosis was significantly more severe in NK2 transgenic mice than in WT mice. CCl₄ administration increased the expression levels of TGF‐β1 mRNA and α‐SMA protein, and decreased the expression of MMP‐13 mRNA in livers of NK2 transgenic mice compared with those of WT mice. c‐Met protein expression in the liver was compatible with the degree of fibrosis. As for c‐Met activation, no difference was found between NK2 and WT livers. Conclusion: Overexpression of NK2 acts as an antagonist of HGF and promotes liver fibrosis in CCl₄‐induced chronic liver injury.     

All‐trans‐retinoic acid ameliorates carbon tetrachloride‐induced liver fibrosis in mice through modulating cytokine production

Background/Aims: Liver fibrosis with any aetiology, induced by the transdifferentiation and proliferation of hepatic stellate cells (HSCs) to produce collagen, is characterized by progressive worsening in liver function, leading to a high incidence of death. We have recently reported that all‐trans‐retinoic acid (ATRA) suppresses the transdifferentiation and proliferation of lung fibroblasts and prevents radiation‐ or bleomycin‐induced lung fibrosis. Methods: We examined the impact of ATRA on carbon tetrachloride (CCl₄)‐induced liver fibrosis. We performed histological examinations and quantitative measurements of transforming growth factor (TGF)‐β1 and interleukin (IL)‐6 in CCl₄‐treated mouse liver tissues with or without the administration of ATRA, and investigated the effect of ATRA on the production of the cytokines in quiescent and activated HSCs. Results: CCl₄‐induced liver fibrosis was attenuated in histology by intraperitoneal administration of ATRA, and the overall survival rate at 12 weeks was 26.5% without ATRA (n=25), whereas it was 75.0% (n=24) in the treatment group (P=0.0187). In vitro studies disclosed that the administration of ATRA reduced (i) the production of TGF‐β1, IL‐6 and collagen from HSCs, (ii) TGF‐β‐dependent transdifferentiation of the cells and IL‐6‐dependent cell proliferation and (iii) the activities of nuclear factor‐κB p65 and p38mitogen‐activated protein kinase, which stimulate the production of TGF‐β1 and IL‐6, which could be the mechanism underlying the preventive effect of ATRA on liver fibrosis. Conclusions: Our findings indicate that ATRA ameliorates liver fibrosis. As the oral administration of the drug results in good compliance, ATRA could be a novel approach in the treatment of liver fibrosis.     

Melatonin induces apoptosis of colorectal cancer cells through HDAC4 nuclear import mediated by CaMKII inactivation

Melatonin induces apoptosis in many different cancer cell lines, including colorectal cancer. However, the precise mechanisms involved remain largely unresolved. In this study, we provide evidence to reveal a new mechanism by which melatonin induces apoptosis of colorectal cancer LoVo cells. Melatonin at pharmacological concentrations significantly suppressed cell proliferation and induced apoptosis in a dose‐dependent manner. The observed apoptosis was accompanied by the melatonin‐induced dephosphorylation and nuclear import of histone deacetylase 4 (HDAC4). Pretreatment with a HDAC4‐specific siRNA effectively attenuated the melatonin‐induced apoptosis, indicating that nuclear localization of HDAC4 is required for melatonin‐induced apoptosis. Moreover, constitutively active Ca²⁺/calmodulin‐dependent protein kinase II alpha (CaMKIIα) abrogated the melatonin‐induced HDAC4 nuclear import and apoptosis of LoVo cells. Furthermore, melatonin decreased H3 acetylation on bcl‐2 promoter, leading to a reduction of bcl‐2 expression, whereas constitutively active CaMKIIα(T286D) or HDAC4‐specific siRNA abrogated the effect of melatonin. In conclusion, the present study provides evidence that melatonin‐induced apoptosis in colorectal cancer LoVo cells largely depends on the nuclear import of HDAC4 and subsequent H3 deacetylation via the inactivation of CaMKIIα.     

In vivo hepatic oxidative stress because of carbon tetrachloride toxicity: protection by melatonin and pinoline

Abstract: The protective in vivo effects of melatonin or pinoline on carbon tetrachloride (CCl₄)‐induced oxidative damage were investigated in liver of rats and compared to rats injected only with CCl₄ (5 mL/kg body weight). Hepatic cell membrane fluidity, monitored using fluorescence spectroscopy, exhibited a significant decrease in animals exposed to CCl₄ compared to control rats. Increases in lipid and protein oxidation, as assessed by concentrations of malondialdehyde (MDA) and 4‐hydroxyalkenals (4‐HDA), and protein carbonylation, respectively, were also seen in hepatic homogenates of animals exposed to CCl₄. The administration of melatonin (10 mg/kg body weight) or pinoline injected 30 min before and 1 hr after CCl₄, fully prevented membrane rigidity and protein oxidation. However, treatment with melatonin was more effective in terms of reducing lipid peroxidation than pinoline, as the increases in MDA+4‐HDA levels because of CCl₄ were reduced by 93.4% and 34.4% for melatonin or pinoline, respectively. Livers from CCl₄‐injected rats showed several histopathological alterations; above all, there were signs of necrosis and ballooning degeneration. The concurrent administration of melatonin or pinoline reduced the severity of these morphological changes. On the basis of the biochemical and histopathological findings, we conclude that both melatonin and pinoline were highly effective in protecting the liver against oxidative damage and membrane rigidity because of CCl₄. Therefore, these indoles may be useful as cotreatments for patients with hepatic intoxication induced by CCl₄.     

Melatonin inhibits cholangiocarcinoma and reduces liver injury in Opisthorchis viverrini‐infected and N‐nitrosodimethylamine‐treated hamsters

The human liver fluke Opisthorchis viverrini infection and N‐nitrosodimethylamine (NDMA) administration induce cholangiocarcinoma (CCA) and liver injury in hamsters. Melatonin protects against liver injury and reduces the alteration of mitochondrial structure, mitochondrial membrane potential, and mitochondrial pro‐ and anti‐apoptotic pathways in various cancer types. To investigate the chemopreventive effect of melatonin on CCA genesis and liver injury, hamsters were treated with a combination of O. viverrini infection and NDMA concurrently administered with melatonin (10 mg/kg and 50 mg/kg) for 120 days. Melatonin treatment at 50 mg/kg caused a significant reduction in liver/body weight ratios and decreased tumor volumes leading to an increase in the survival of animals. In the tumorous tissues, the high‐dose melatonin reduced DNA fragmentation and mitochondrial apoptosis by inducing anti‐apoptotic protein (Bcl‐2) in the mitochondrial fraction and down‐regulating cytochrome c, pro‐apoptotic protein (Bax), and caspase‐3 in tumor cytosol. Moreover, a high‐dose melatonin treatment significantly increased mitochondrial antioxidant enzymes and prevented mitochondrial ultrastructure changes in the tumor. Overall, melatonin has potent chemopreventive effects in inhibiting CCA genesis and also reduces liver injury in hamster CCA, which, in part, might involve in the suppression of CCA by reducing tumor mitochondria alteration.     

(Z)2-(5-(4-methoxybenzylidene)-2, 4-dioxothiazolidin-3-yl) acetic acid protects rats from CCl(4) -induced liver injury

Background and Aim: (Z)2‐(5‐(4‐methoxybenzylidene)‐2, 4‐dioxothiazolidin‐3‐yl) acetic acid (MDA) is an aldose reductase (AR) inhibitor. Recent studies suggest that AR contributes to the pathogenesis of inflammation by affecting the nuclear factor κB (NF‐κB)‐dependent expression of cytokines and chemokines and therefore could be a novel therapeutic target for inflammatory pathology. The current study evaluated the in vivo role of MDA in protecting the liver against injury and fibrogenesis caused by CCl₄ in rats, and the underlying mechanisms. Methods: A single injection of CCl₄ induced acute hepatitis, and repeated injections were used to induce hepatic fibrosis in rats. Therapeutic efficacy was assessed by comparison of the severity of hepatic injury and fibrosis in MDA ‐ treated rats versus untreated controls. Results: MDA significantly protected the liver from injury by reducing the activity of serum alanine aminotransferase, and improving the histological architecture of the liver. MDA modulated NF‐κB‐dependent activation of inflammatory cytokines by reducing hepatic mRNA levels of tumor necrosis factor‐α, interleukin‐1β, inducible nitric oxide (NO) synthase and transforming growth factor‐β. In addition, MDA attenuated oxidative stress by increasing the content of hepatic glutathione. These favorable changes were associated with suppressed hepatic NF‐κB activation by MDA. MDA treatment improved liver fibrosis in rats that received repeated CCl₄ injections. In vitro, MDA attenuated phosphorylation of IκB and activation of NF‐κB, and thus prevented biosynthesis of NO in lipopolysaccharide‐activated RAW264.7 cells. Conclusions: The present study suggests that AR is a novel therapeutic anti‐inflammatory target for the treatment of hepatitis and liver fibrosis.     

JAK2/STAT3 activation by melatonin attenuates the mitochondrial oxidative damage induced by myocardial ischemia/reperfusion injury

Ischemia/reperfusion injury (IRI) is harmful to the cardiovascular system and causes mitochondrial oxidative stress. Numerous data indicate that the JAK2/STAT3 signaling pathway is specifically involved in preventing myocardial IRI. Melatonin has potent activity against IRI and may regulate JAK2/STAT3 signaling. This study investigated the protective effect of melatonin pretreatment on myocardial IRI and elucidated its potential mechanism. Perfused isolated rat hearts and cultured neonatal rat cardiomyocytes were exposed to melatonin in the absence or presence of the JAK2/STAT3 inhibitor AG490 or JAK2 siRNA and then subjected to IR. Melatonin conferred a cardio‐protective effect, as shown by improved postischemic cardiac function, decreased infarct size, reduced apoptotic index, diminished lactate dehydrogenase release, up‐regulation of the anti‐apoptotic protein Bcl2, and down‐regulation of the pro‐apoptotic protein Bax. AG490 or JAK2 siRNA blocked melatonin‐mediated cardio‐protection by inhibiting JAK2/STAT3 signaling. Melatonin exposure also resulted in a well‐preserved mitochondrial redox potential, significantly elevated mitochondrial superoxide dismutase (SOD) activity, and decreased formation of mitochondrial hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), which indicates that the IR‐induced mitochondrial oxidative damage was significantly attenuated. However, this melatonin‐induced effect on mitochondrial function was reversed by AG490 or JAK2 siRNA treatment. In summary, our results demonstrate that melatonin pretreatment can attenuate IRI by reducing IR‐induced mitochondrial oxidative damage via the activation of the JAK2/STAT3 signaling pathway.     

Antifibrotic effect of heparin on liver fibrosis model in rats

AIM: To evaluate the effect of chronic thrombin inhibition by heparin on experimentally induced chronic liver injury (liver fibrosis) in rats.METHODS: Chronic liver injury (liver fibrosis) was induced in Wistar rats by oral administration of carbon tetrachloride (CCl₄) for 7 wk, an animal model with persistent severe hepatic fibrosis. Intravenous administration of the thrombin antagonist (heparin) started 1 wk after the start of CCl₄ intoxication for 6 wk. After completion of treatment (7 wk), markers of hepatic dysfunction were measured and changes evaluated histopathologically.RESULTS: Higher serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), total, direct and indirect bilirubin levels, as well as lower fibrinogen levels, were found in CCl₄ intoxicated rats. Heparin, silymarin and combination of drug (heparin and silymarin) treatment for 6 wk prevented a rise in SGOT, SGPT, ALP, total, direct and indirect bilirubin levels and improved fibrinogen levels. Deterioration in hepatic function determined by the fibrosis area was retarded, as evident from hepatic histopathology. Total protein levels were not changed in all groups.CONCLUSION: Heparin, a thrombin antagonist, preserved hepatic function and reduced severity of hepatic dysfunction/fibrogenesis. Combination of heparin and silymarin produced additional benefits on liver fibrosis.     

Melatonin protects against diabetic cardiomyopathy through Mst1/Sirt3 signaling

This study investigated the effects of melatonin on diabetic cardiomyopathy (DCM) and determined the underlying mechanisms. Echocardiography indicated that melatonin notably mitigated the adverse left ventricle remodeling and alleviated cardiac dysfunction in DCM. The mechanisms were attributed to increased autophagy, reduced apoptosis, and alleviated mitochondrial dysfunction. Furthermore, melatonin inhibited Mst1 phosphorylation and promoted Sirt3 expression in DCM. These results indicated that melatonin may exert its effects through Mst1/Sirt3 signaling. To verify this hypothesis, a DCM model using Mst1 transgenic (Mst1 Tg) and Mst1 knockout (Mst1^?/?) mice was constructed. As expected, melatonin increased autophagy, reduced apoptosis and improved mitochondrial biogenesis in Mst1 Tg mice subjected to DCM injury, while it had no effects on Mst1^?/? mice. In addition, cultured neonatal mouse cardiomyocytes were subjected to simulated diabetes to probe the mechanisms involved. Melatonin administration promoted autophagic flux as demonstrated by elevated LC3‐II and lowered p62 expression in the presence of bafilomycin A1. The results suggest that melatonin alleviates cardiac remodeling and dysfunction in DCM by upregulating autophagy, limiting apoptosis, and modulating mitochondrial integrity and biogenesis. The mechanisms are associated with Mst1/Sirt3 signaling.     

Melatonin enhances mitochondrial biogenesis and protects against rotenone-induced mitochondrial deficiency in early porcine embryos

Melatonin, a major hormone of the pineal gland, exerts many beneficial effects on mitochondria. Several studies have shown that melatonin can protect against toxin-induced oocyte quality impairment during maturation. However, there is little information regarding the beneficial effects of melatonin on toxin-exposed early embryos, and the mechanisms underlying such effects have not been determined. Rotenone, a chemical widely used in agriculture, induces mitochondrial toxicity, therefore, damaging the reproductive system, impairing oocyte maturation, ovulation, and fertilization. We investigated whether melatonin attenuated rotenone exposure-induced impairment of embryo development by its mitochondrial protection effect. Activated oocytes were randomly assigned to four groups: the control, melatonin treatment, rotenone-exposed, and “rotenone + melatonin” groups. Treatment with melatonin abrogated rotenone-induced impairment of embryo development, mitochondrial dysfunction, and ATP deficiency, and significantly decreased oxidative stress and apoptosis. Melatonin also increased SIRT1 and PGC-1α expression, which promoted mitochondrial biogenesis. SIRT1 knockdown or pharmacological inhibition abolished melatonin's ability to revert rotenone-induced impairment. Thus, melatonin rescued rotenone-induced impairment of embryo development by reducing ROS production and promoting mitochondrial biogenesis. This study shows that melatonin rescues toxin-induced impairment of early porcine embryo development by promoting mitochondrial biogenesis.     

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.     

Melatonin attenuates hypoxic pulmonary hypertension by inhibiting the inflammation and the proliferation of pulmonary arterial smooth muscle cells

Hypoxia‐induced inflammation and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) play important roles in the pathological process of hypoxic pulmonary hypertension (HPH). Melatonin possesses anti‐inflammatory and antiproliferative properties. However, the effect of melatonin on HPH remains unclear. In this study, adult Sprague–Dawley rats were exposed to intermittent chronic hypoxia for 4 wk to mimic a severe HPH condition. Hemodynamic and pulmonary pathomorphology data showed that chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of the right ventricle/left ventricle plus septum (RV/LV+S) ratio, and median width of pulmonary arterioles. Melatonin attenuated the elevation of RVSP, RV/LV+S, and mitigated the pulmonary vascular structure remodeling. Melatonin also suppressed the hypoxia‐induced high expression of proliferating cell nuclear antigen (PCNA), hypoxia‐inducible factor‐1α (HIF‐1α), and nuclear factor‐κB (NF‐κB). In vitro, melatonin concentration‐dependently inhibited the proliferation of PASMCs and the levels of phosphorylation of Akt and extracellular signal‐regulated kinases1/2 (ERK1/2) caused by hypoxia. These results suggested that melatonin might potentially prevent HPH via anti‐inflammatory and antiproliferative mechanisms.