Beneficial effects of exercise on gut microbiota functionality and barrier integrity, and gut-liver crosstalk in an in vivo model of early obesity and non-alcoholic fatty liver disease.

Childhood obesity has reached epidemic levels, representing one of the most serious public health concerns associated with metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). There is limited clinical experience concerning pediatric NAFLD patients, and thus the therapeutic options are scarce. The aim of this study was to evaluate the benefits of exercise on gut microbiota composition and functionality balance, and consequent effects on early obesity and NAFLD onset in an in vivo model. Juvenile (21-day-old) male Wistar rats fed a control diet or a high-fat diet (HFD) were subjected to a combined aerobic and resistance training protocol. Fecal microbiota was sequenced by an Illumina MiSeq system, and parameters related to metabolic syndrome, fecal metabolome, intestinal barrier integrity, bile acid metabolism and transport, and alteration of the gut-liver axis were measured. Exercise decreased HFD-induced body weight gain, metabolic syndrome and hepatic steatosis, as a result of its lipid metabolism modulatory capacity. Gut microbiota composition and functionality were substantially modified as a consequence of diet, age and exercise intervention. In addition, the training protocol increased Parabacteroides, Bacteroides and Flavobacterium genera, correlating with a beneficial metabolomic profile, whereas Blautia, Dysgonomonas and Porphyromonas showed an opposite pattern. Exercise effectively counteracted HFD-induced microbial imbalance, leading to intestinal barrier preservation, which, in turn, prevented deregulation of the gut-liver axis and improved bile acid homeostasis, determining the clinical outcomes of NAFLD. In conclusion, we provide scientific evidence highlighting the benefits of gut microbiota composition and functionality modulation by physical exercise protocols in the management of early obesity and NAFLD development.

Melatonin enhances sorafenib actions in human hepatocarcinoma cells by inhibiting mTORC1/p70S6K/HIF-1α and hypoxia-mediated mitophagy.

The antiangiogenic effects of sustained sorafenib treatment in hepatocellular carcinoma (HCC) lead to the occurrence of hypoxia-mediated drug resistance resulting in low therapy efficiency and negative outcomes. Combined treatments with coadjuvant compounds to target the hypoxia-inducible factor-1α (HIF-1α) represent a promising therapeutic approach through which sorafenib efficiency may be improved. Melatonin is a well-documented oncostatic agent against different cancer types. Here, we evaluated whether melatonin could enhance sorafenib cytotoxicity and overcome the hypoxia-mediated resistance mechanisms in HCC. The pharmacological melatonin concentration (2 mM) potentiated the oncostatic effects of sorafenib (5 µM) on Hep3B cells even under hypoxia. Melatonin downregulated the HIF-1α protein synthesis through the inhibition of the mammalian target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase beta-1 (p70S6K)/ribosomal protein S6 (RP-S6) pathway, although the indole enhanced Akt phosphorylation by the mTORC1/C2 negative feedback. Furthermore, melatonin and sorafenib coadministration reduced the HIF-1α-mitophagy targets expression, impaired autophagosome formation and subsequent mitochondria and lysosomes colocalization. Together, our results indicate that melatonin improves the Hep3B sensitivity to sorafenib, preventing HIF-1α synthesis to block the cytoprotective mitophagy induced by the hypoxic microenvironment, an important element of the multifactorial mechanisms responsible for the chemotherapy failure.

Inhibition of matrix metalloproteinase-9 and nuclear factor kappa B contribute to melatonin prevention of motility and invasiveness in HepG2 liver cancer cells.

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide because of its high incidence and its metastatic potential. Extracellular matrix degradation by matrix metalloproteinases (MMPs) has been connected with cancer cell invasion, and it has been suggested that inhibition of MMPs by synthetic and natural inhibitors may be of great importance in the HCC therapies. Melatonin, the main product of the pineal gland, exerts antiproliferative, proapoptotic, and antiangiogenic properties in HepG2 human hepatocellular cells, and exhibits anti-invasive and antimetastatic activities by suppressing the enzymatic activity of MMP-9 in different tumor types. However, the underlying mechanism of anti-invasive activity in HCC models has not been fully elucidated. Here, we demonstrate that 1 mm melatonin dosage reduced in IL-1ß-induced HepG2 cells MMP-9 gelatinase activity and inhibited cell invasion and motility through downregulation of MMP-9 gene expression and upregulation of the MMP-9-specific inhibitor tissue inhibitor of metalloproteinases (TIMP)-1. No significant changes were observed in the expression and activity of MMP-2, the other proteinase implicated in matrix collagen degradation, and its tissue inhibitor, TIMP-2. Also, melatonin significantly suppressed IL-1ß-induced nuclear factor-kappaB (NF-κB) translocation and transcriptional activity. In summary, we demonstrate that melatonin modulates motility and invasiveness of HepG2 cell in vitro through a molecular mechanism that involves TIMP-1 upregulation and attenuation of MMP-9 expression and activity via NF-κB signal pathway inhibition.

New therapeutic approach: diphenyl diselenide reduces mitochondrial dysfunction in acetaminophen-induced acute liver failure.

The acute liver failure (ALF) induced by acetaminophen (APAP) is closely related to oxidative damage and depletion of hepatic glutathione, consequently changes in cell energy metabolism and mitochondrial dysfunction have been observed after APAP overdose. Diphenyl diselenide [(PhSe)2], a simple organoselenium compound with antioxidant properties, previously demonstrated to confer hepatoprotection. However, little is known about the protective mechanism on mitochondria. The main objective of this study was to investigate the effects (PhSe)2 to reduce mitochondrial dysfunction and, secondly, compare in the liver homogenate the hepatoprotective effects of the (PhSe)2 to the N-acetylcysteine (NAC) during APAP-induced ALF to validate our model. Mice were injected intraperitoneal with APAP (600 mg/kg), (PhSe)2 (15.6 mg/kg), NAC (1200 mg/kg), APAP+(PhSe)2 or APAP+NAC, where the (PhSe)2 or NAC treatment were given 1 h following APAP. The liver was collected 4 h after overdose. The plasma alanine and aspartate aminotransferase activities increased after APAP administration. APAP caused a remarkable increase of oxidative stress markers (lipid peroxidation, reactive species and protein carbonylation) and decrease of the antioxidant defense in the liver homogenate and mitochondria. APAP caused a marked loss in the mitochondrial membrane potential, the mitochondrial ATPase activity, and the rate of mitochondrial oxygen consumption and increased the mitochondrial swelling. All these effects were significantly prevented by (PhSe)2. The effectiveness of (PhSe)2 was similar at a lower dose than NAC. In summary, (PhSe)2 provided a significant improvement to the mitochondrial redox homeostasis and the mitochondrial bioenergetics dysfunction caused by membrane permeability transition in the hepatotoxicity APAP-induced.

Reduction of acute hepatic damage induced by acetaminophen after treatment with diphenyl diselenide in mice.

In this study, the authors evaluated the ability of diphenyl diselenide (PhSe)(2) to reverse acute hepatic failure induced by acetaminophen (APAP) in mice. The animals received an APAP dose of 600 mg/kg intraperitoneally (i.p.), and then 1 hour later, they received 15.6 mg/kg i.p. of (PhSe)(2). Three hours after (PhSe)(2) administration, the animals were sacrificed and blood and liver samples were collected for analysis. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured. The levels of reduced glutathione (GSH) and oxidized glutathione (GSSG), thiobarbituric acid-reactive substances (TBARS), 2',7'-dichlorofluorescein (DFC), catalase activity (CAT), and myeloperoxidase (MPO) activity were determined in the liver. A methyl-tetrazolium reduction (MTT) assay was also performed on the liver. Histopathological studies were conducted in all groups. Exposure of animals to APAP induced oxidative stress, increased lipid peroxidation (LPO), and the generation of reactive species, reduced the levels of GSH, and caused an increase in the MPO activity. Treatment with (PhSe)(2) reduced LPO and the formation of reactive species and inhibited the processes of inflammation, reducing the hepatic damage induced by APAP. The results of this study show that (PhSe)(2) is a promising therapeutic option for the treatment of acute hepatic failure.

Melatonin modulation of intracellular signaling pathways in hepatocarcinoma HepG2 cell line: role of the MT1 receptor.

Melatonin reduces proliferation in many different cancer cell lines. However, studies on the oncostatic effects of melatonin in hepatocarcinoma are limited. We have previously demonstrated that melatonin administration induces cycle arrest, apoptosis, and changes in the expression of its specific receptors in HepG2 human hepatocarcinoma cells. In this study, we used the receptor antagonist luzindole to assess the contribution of MT1 melatonin membrane receptor to melatonin effects on cell viability, mitogen-activated protein kinase (MAPKs) activation, and cAMP levels. Additionally, effects of MT1 inhibition on mRNA levels of cytosolic quinone reductase type-2 (NQO2) receptor and nuclear retinoic acid-related orphan receptor alpha (RORα) were tested. Melatonin, at 1000 and 2500 µm, significantly reduced cell viability. Pre-incubation with luzindole partially inhibited the effects of melatonin on cell viability. Melatonin at 2500 µm significantly reduced cAMP levels, and this effect was partially blocked by luzindole. Both melatonin concentrations increased the expression of phosphorylated p38, ERK, and JNK. ERK activation was completely abolished in the presence of luzindole. NQO2 but not RORα mRNA level significantly increased in luzindole-treated cells. Results obtained provide evidence that the melatonin effects on cell viability and proliferation in HepG2 cells are partially mediated through the MT1 membrane receptor, which seems to be related also with melatonin modulation of cAMP and ERK activation. This study also highlights a possible interplay between MT1 and NQO2 melatonin receptors in liver cancer cells.

Changes in the expression of melatonin receptors induced by melatonin treatment in hepatocarcinoma HepG2 cells.

Hepatocellular carcinoma (HCC) is one of the most common cancers and its incidence is increasing worldwide. Melatonin, an indoleamine hormone, exerts anti-oxidant, immunomodulatory, anti-aging, and antitumor effects. Previous studies have shown that melatonin can act through specific receptors, including MT(1), MT(2), MT(3) receptors as well as a nuclear receptor belonging to the orphan nuclear receptor family. Recently, we have described their role in the oncostatic and pro-apoptotic effects of melatonin on HepG2 human HCC cells. However, the potential role of the different melatonin cellular receptors on its antiproliferative effects remains unknown. In the present study, we examined the effect of melatonin treatment on HepG2 human HCC cells, analyzing cell cycle arrest and melatonin receptor expression. Melatonin was administered for 2, 4, and 6 days at 1000 or 2500 microm. Melatonin induced a dose- and time-dependent inhibition on cell proliferation. This treatment caused an alteration in the cell cycle, with an increase in the number of cells in G(2)/M phase at both 1000 and 2500 microm melatonin concentrations, and a significant increase on S phase cell percentage by the highest dose. Furthermore, increases in protein expression of MT(1), MT(3), and retinoic acid-related orphan receptor-alpha were found after melatonin treatments. These increases were coincident with a significant induction in the expression of p21 protein, which negatively regulates cell cycle progression. Our results confirm the antitumor effect of melatonin in HCC cells, suggesting that its oncostatic properties are related, at least in part, to changes on the expression of their different subtypes of receptors.