Autophagy as a Molecular Target of Flavonoids Underlying their Protective Effects in Human Disease.

BACKGROUND: Autophagy is a cellular pathway with the ability to maintain cell homeostasis through the elimination of damaged or useless cellular components, and its deregulation may initiate or aggravate different human diseases. Flavonoids, a group of plant metabolites, are able to modulate different molecular and cellular processes including autophagy. OBJECTIVE: To review the effects of flavonoids on autophagy pathway in both invasive and noninvasive human diseases, focusing on the global outcomes in their progression. Moreover, the efficacy of the combination of flavonoids with drugs or other natural nontoxic compounds was also reviewed. METHODS: A literature search was performed to identify and analyze peer-reviewed publications containing in vitro and in vivo studies focused on autophagy deregulation in different proliferative and non-proliferative pathologies and the potential protective effects of flavonoids. RESULTS: Analyzed publications indicated that imbalance between cell death and survival induced by changes in autophagy play an important role in the pathophysiology of a number of human diseases. The use of different flavonoids as autophagy modulators, alone or in combination with other molecules, might be a worthy strategy in the treatment of cancer, neurodegenerative disorders, cardiovascular diseases, hepatic diseases, leishmaniasis, influenza, gastric ulcers produced by Helicobacter pylori infection, diabetes, asthma, age-related macular degeneration or osteoporosis. CONCLUSION: Flavonoids could potentially constitute important adjuvant agents of conventional therapies in the treatment of autophagy deregulation-related diseases. Moreover, combined therapy may help to diminish the doses of those conventional treatments, leading to reduced drug-derivative side effects and to improved patients' survival.

Modulation of PI3K-LXRα-dependent lipogenesis mediated by oxidative/nitrosative stress contributes to inhibition of HCV replication by quercetin.

There is experimental evidence that some antioxidant flavonoids show therapeutic potential in the treatment of hepatitis C through inhibition of hepatitis C virus (HCV) replication. We examined the effect of treatment with the flavonols quercetin and kaempferol, the flavanone taxifolin and the flavone apigenin on HCV replication efficiency in an in vitro model. While all flavonoids studied were able to reduce viral replication at very low concentrations (ranging from 0.1 to 5 µM), quercetin appeared to be the most effective inhibitor of HCV replication, showing a marked anti-HCV activity in replicon-containing cells when combined with interferon (IFN)α. The contribution of oxidative/nitrosative stress and lipogenesis modulation to inhibition of HCV replication by quercetin was also examined. As expected, quercetin decreased HCV-induced reactive oxygen and nitrogen species (ROS/RNS) generation and lipoperoxidation in replicating cells. Quercetin also inhibited liver X receptor (LXR)α-induced lipid accumulation in LXRα-overexpressing and replicon-containing Huh7 cells. The mechanism underlying the LXRα-dependent lipogenesis modulatory effect of quercetin in HCV-replicating cells seems to involve phosphatidylinositol 3-kinase (PI3K)/AKT pathway inactivation. Thus, inhibition of the PI3K pathway by LY294002 attenuated LXRα upregulation and HCV replication mediated by lipid accumulation, showing an additive effect when combined with quercetin. Inactivation of the PI3K pathway by quercetin may contribute to the repression of LXRα-dependent lipogenesis and to the inhibition of viral replication induced by the flavonol. Combined, our data suggest that oxidative/nitrosative stress blockage and subsequent modulation of PI3K-LXRα-mediated lipogenesis might contribute to the inhibitory effect of quercetin on HCV replication.

Deleterious effect of human umbilical cord blood mononuclear cell transplantation on thioacetamide-induced chronic liver damage in rats.

Our research group investigates whether human mononuclear cells isolated from umbilical cord blood (HUCBM cells) might be valuable in hepatic regenerative medicine. We recently demonstrated that HUCBM cell transplantation improves histological alterations and function of the liver in rats with acute liver damage induced by D-galactosamine. In the present study, HUCBM cells were transplanted into rats with thioacetamide (TAA)-induced liver cirrhosis, an experimental model that generates an intense fibrosis and mimics the histological and biochemical alterations found in the human disease. HUCBM transplantation had no effect on hepatic histology of cirrhotic animals. In contrast, analysis of plasma albumin and total bilirubin, liver damage markers, revealed a harmful effect of HUCBM cell transplantation in our experimental model of liver cirrhosis. Significantly higher plasma urea concentrations, marker of renal function, were observed in the cirrhotic and control rats intraportally injected with HUCBM cells than in those not receiving this therapy. Histological study revealed tubular and glomerular lesions in kidneys of cirrhotic animals transplanted with HUCBM cells. The glomeruli appeared ischemic, and the tubules showed a severe involvement that included peripheral asymmetric vacuolization and disappearance of the tubular lumen. Taken together, the histological and biochemical data suggest that the cirrhotic rats subjected to HUCBM cell therapy developed a hepatorenal syndrome.

Hepatitis C virus NS5A and core proteins induce oxidative stress-mediated calcium signalling alterations in hepatocytes.

BACKGROUND/AIMS: The hepatitis C virus (HCV) structural core and non-structural NS5A proteins induce in liver cells a series of intracellular events, including elevation of reactive oxygen and nitrogen species (ROS/RNS). Since oxidative stress is associated to altered intracellular Ca(2+) homeostasis, we aimed to investigate the effect of these proteins on Ca(2+) mobilization in human hepatocyte-derived transfected cells, and the protective effect of quercetin treatment. METHODS: Ca(2+) mobilization and actin reorganization were determined by spectrofluorimetry. Production of ROS/RNS was determined by flow cytometry. RESULTS: Cells transfected with NS5A and core proteins showed enhanced ROS/RNS production and resting cytosolic Ca(2+) concentration, and reduced Ca(2+) concentration into the stores. Phenylephrine-evoked Ca(2+) release, Ca(2+) entry and extrusion by the plasma membrane Ca(2+)-ATPase were significantly reduced in transfected cells. Similar effects were observed in cytokine-activated cells. Phenylephrine-evoked actin reorganization was reduced in the presence of core and NS5A proteins. These effects were significantly prevented by quercetin. Altered Ca(2+) mobilization and increased calpain activation were observed in replicon-containing cells. CONCLUSIONS: NS5A and core proteins induce oxidative stress-mediated Ca(2+) homeostasis alterations in human hepatocyte-derived cells, which might underlie the effects of both proteins in the pathogenesis of liver disorders associated to HCV infection.

Xenotransplantation of human umbilical cord blood mononuclear cells to rats with D-galactosamine-induced hepatitis.

Cord blood is an attractive cell source in regenerative medicine and represents an alternative to bone marrow. The aim of this study was to investigate whether human umbilical cord blood mononuclear (HUCBM) cells might be valuable in hepatic regenerative medicine. HUCBM cells differentiated in vitro into hepatocytes, as suggested by expression of albumin, cytokeratin-18, glutamine synthetase, alpha-fetoprotein, and cytochrome P450 3A4 at both mRNA and protein levels in a time-dependent fashion. In contrast, the hematopoietic phenotype was gradually lost, as demonstrated by disappearance of CD45 expression. The regenerative potential of HUCBM cells was tested by using a human-to-rat xenotransplant model in which HUCBM cells were intraportally injected into rats with D-galactosamine-induced hepatitis. Liver histology and biochemical markers of hepatic damage were determined. Presence of human cells was detected in blood and liver of both control and D-galactosamine-treated animals. Cell transplantation produced an improvement in both the histological damage and liver function, as demonstrated by plasma values of alkaline phosphatase, gamma-glutamyl transferase, lactate dehydrogenase, and total and direct bilirubins. Results obtained suggest that HUCBM cells are capable of hepatic engraftment in this human-to-rat xenotransplant model and that transplantation of HUCBM cells may be a suitable therapy for liver disease.

A comparison of the effects of kaempferol and quercetin on cytokine-induced pro-inflammatory status of cultured human endothelial cells.

We investigated the effects of the flavonols kaempferol and quercetin on the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), endothelial cell selectin (E-selectin), inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2), and on the activation of the signalling molecules NF-kappaB and activator protein-1 (AP-1), induced by a cytokine mixture in cultured human umbilical vein endothelial cells. Inhibition of reactive oxygen and nitrogen species generation did not differ among both flavonols at 1 micromol/l but was significantly stronger for kaempferol at 5-50 micromol/l. Supplementation with increasing concentrations of kaempferol substantially attenuated the increase induced by the cytokine mixture in VCAM-1 (10-50 micromol/l), ICAM-1 (50 micromol/l) and E-selectin (5-50 micromol/l) expression. A significantly inhibitory effect of quercetin on VCAM-1 (10-50 micromol/l), ICAM-1 (50 micromol/l) and E-selectin (50 micromol/l) expression was also observed. Expression of adhesion molecules was always more strongly inhibited in kaempferol-treated than in quercetin-treated cells. The inhibitory effect on iNOS and COX-2 protein level was stronger for quercetin at 5-50 micromol/l. The effect of kaempferol on NF-kappaB and AP-1 binding activity was weaker at high concentrations (50 micromol/l) as compared with quercetin. The present study indicates that differences exist in the modulation of pro-inflammatory genes and in the blockade of NF-kappaB and AP-1 by kaempferol and quercetin. The minor structural differences between both flavonols determine differences in their anti-inflammatory properties and in their efficiency in inhibiting signalling molecules.

Differential effects of dietary flavonoids on reactive oxygen and nitrogen species generation and changes in antioxidant enzyme expression induced by proinflammatory cytokines in Chang Liver cells.

This study was aimed to investigate the differential protective effect of dietary flavonoids against oxidative stress induced by proinflammatory stimuli in parenchymal liver cells. Chang Liver cells were incubated with a cytokine mixture (CM) supplemented with the flavonols quercetin and kaempferol, the flavanone taxifolin and the flavone apigenin (5-50 microM). Concentrations of oxidised and reduced glutathione, generation of different ROS/RNS, and expression of antioxidant enzymes were measured. Oxidised glutathione concentration and the oxidised/reduced glutathione ratio were increased by the CM. These effects were significantly prevented by quercetin, kaempferol and taxifolin at all tested concentrations. Effects of apigenin reached a lesser extent and were not significant at 25 microM. Treatment with quercetin and kaempferol prevented the production of peroxides, superoxide anion and nitric oxide induced by CM. Taxifolin 50 microM and apigenin 25-50 microM caused a significant increase in peroxides and nitric oxide generation. Protein concentration of the different antioxidant enzymes was generally reduced by kaempferol and quercetin in comparison to CM, although quercetin 25 and 50 microM increased Mn SOD protein concentration. GPx protein level was significantly increased by apigenin 25 and 50 microM. Changes in mRNA tended to be parallel to those in protein concentration. Our study reveals that important differences exist between flavonoids with different structural features in their capacity to abrogate the generation of different ROS/RNS, and suggests that the modulation of antioxidant enzymes by flavonoids may be also important in their antioxidant effects in liver cells.

Melatonin is able to reduce the apoptotic liver changes induced by aging via inhibition of the intrinsic pathway of apoptosis.

We examined the effect of daily melatonin supplementation on liver apoptosis induced by aging in rats. Young (3-month-old) and aged (24-month-old) Wistar rats were supplemented daily with melatonin in their drinking water (20 mg/L) for 4 weeks. Aged rats showed increases in the liver concentration of thiobarbituric acid-reactive substances and in the oxidized/reduced glutathione ratio. These increases were accompanied by apoptotic ultrastructural alterations and increases in cytochrome c mitochondrial release, Bax to Bcl-2 relative expression, and activity of caspase-3. No significant changes were observed in Fas-ligand (Fas-L) expression and caspase-8 activity. Melatonin administration was able to abrogate changes detected in aged rats. Data suggest that liver apoptotic cell death is induced by reactive oxygen species, via the intrinsic signalling pathway, and that the antiapoptotic action provided by melatonin is related to its antioxidant effect, with reduction of cytochrome c release by the modulation of Bcl-2 and Bax genes.

Melatonin prevents oxidative stress and changes in antioxidant enzyme expression and activity in the liver of aging rats.

This study compared the effects of melatonin supplementation on markers of oxidative stress, and on the activity and expression of antioxidant enzymes in the liver of young (3-month-old) and aging (24-month-old) rats. Animals were supplemented with melatonin in the drinking water (20 mg/L) for 4 wk. Liver concentration of thiobarbituric-reactive substances (TBARS), as an index of lipid peroxidation, and the oxidized to reduced glutathione ratio significantly increased in aged rats (+58%), while values did not significantly differ from the young in aged animals receiving melatonin. Significant decreases in the liver activities of Cu,Zn-superoxide dismutase (SOD) (-25%), cytosolic (-21%) and mitochondrial (-40%) glutathione peroxidase (GPx), and catalase (CAT) (-34%) were found in aged rats. Melatonin abolished these changes and also prevented the reduction of Cu,Zn-SOD (-33%), cytosolic GPx (-30%), and mitochondrial GPx (-47%) liver protein content as measured by Western blot. Reductions in Cu,Zn-SOD mRNA (-39%), and GPx mRNA (-86%) levels induced by aging were also abolished by melatonin. In summary, our data indicate that melatonin treatment abrogates oxidative stress in the liver of aged rats, and that prevention of the decreased activity of CAT and the downregulation of Cu,Zn-SOD and GPx gene expression contribute to this effect.