The purpose of this study is to evaluate the association between polyphenol consumption and risk of nonalcoholic fatty liver disease NAFLD and its related conditions such as impaired lipid profile and elevated hepatic enzymes. A total of NAFLD patients and controls were enrolled in this case-control study.

Biochemical and anthropometric measurements as well as polyphenol consumption during the previous year were assessed. In unadjusted model, participants who consumed the third and fourth quartiles of polyphenols intake were less likely to have NAFLD in comparison to those who consumed the first quartile [odds ratio, OR: 0.

In separate analysis of genders, this effect was seen only in male participants [OR: 0. Higher total polyphenol intake is associated with a decreased risk of NAFLD. Prospective studies are needed to confirm these results. Nikkhah-Bodaghi, M. and Hekmatdoost, A.

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Musial C, Kuban-Jankowska A. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

There is a need for continued drug development for nonalcoholic steatohepatitis NASH. Bergamot is a plant whose fruit juice is enriched with flavonoids and phenolic compounds which improves dyslipidemia and markers of systemic inflammation in patients with Metabolic Syndrome.

A disease reversal study was performed in the diet-induced animal model of NAFLD DIAMOND. Groups of 8 weeks old mice were randomly assigned to receive chow diet, high fat diet with sugar in drinking water Western diet- WD.

Mice were euthanized after 11 additional weeks. The primary endpoint was resolution of NASH. Secondary endpoints included changes in individual histological features, body weight, liver enzymes, dyslipidemia, markers of oxidative stress and molecular markers of disease activity and fibrosis.

The results showed that BPF99 reduced ALT mean BPF99 reduced markers of oxidative stress, along with reduced JNK and p38 MAP kinase activity.

Collectively our results showed that BPF99 resolves NASH and ameliorates key histological and pathophysiological features of NASH along with improvement in ALT and dyslipidemia in the DIAMOND mice.

Nonalcoholic fatty liver disease NAFLD is a major cause of liver-related morbidity and mortality for which there are no approved therapies 1. The clinical-histological spectrum of NAFLD extends from a nonalcoholic fatty liver NAFL to nonalcoholic steatohepatitis NASH 2.

NASH is a more aggressive phenotype of NAFLD and is more likely to progress to cirrhosis 3 , 4. Excess liver-related mortality from NASH is mainly due to progression to cirrhosis and development of hepatocellular cancer. Evidence exists that, in the early steps of NAFLD, accumulation of fat in liver tissue occurs as a consequence of the imbalance between overconsumption of high-fat diet and increased de novo lipogenesis 5 , on one hand, and decreased lipid disposal, mainly through free fatty acid FFA oxidation 6 , on the other.

The cellular stress produced either directly by lipotoxicity or the cellular response to metabolic overload drives cell-death and inflammatory signaling via activation of the innate immune system 7.

In this scenario, some pathways including oxidative stress and the unfolded protein response 8 , 9 contribute to the development of chronic inflammation, inducing a fibrogenic response and progressive fibrosis leading to cirrhosis.

Thus, a better understanding of the steps involved in regulating lipid accumulation, inflammation and oxidative stress might provide a new therapeutic strategy for NAFLD prevention and treatment. Many therapies are directed towards targets that are expected to reduce disease activity e.

pioglitazone and glucagon like peptide-1receptor agonists and thus eventually translate into decreased disease progression whereas others directly target fibrogenic pathways.

In the long-term, drugs reducing disease activity should reduce progression to cirrhosis. In particular, it has been shown that polyphenols, which are found ubiquitously in plants, and their regular consumption are associated with a reduction in the risk of a number of metabolic diseases, including NAFLD Bergamot Citrus bergamia Risso et Poiteau is an endemic plant growing in Calabria Southern Italy , which has a particular composition and high content of glycosylated flavanones and flavones A particular feature of certain polyphenols, abundant in BPF99, is the potential protective cellular activity as demonstrated by several in vitro experiments.

Particularly, naringine is known to exert a protective action on oxidative damage in tert-butyl hydroperoxide-induced HepG2 injury 15 and in rat hepatocytes against toxin-induced overphosphorylation and disruption of the keratin cytoskeletal network, as well as against toxin-induced apoptotic cell death 16 ; in addition it has shown anti-inflammatory, antioxidant and antiapoptotic activities on H9C2 cells 17 , 18 and on RAW A peculiarity of bruteridin and melitidin is the ability to bind the catalytic site of HMG-CoA reductase and inhibit cholesterol synthesis by replacing its endogenous substrate HMG-CoA Recently, it has ben reported that BPF99 phytocomplex was able to modulate autophagy in HepG2 cells in a time- and dose-dependent fashion and the effect was amplified in cells loaded with palmitic acid Lastly, it has been shown that an analogue bergamot whole-fruit powder extract exerts antioxidant activity, in vitro , showing a selective inhibition against pathogenic strains and a growth stimulation effect on some beneficial gut bacteria.

Moreover, the Citrus extract, rich in healthful phytochemical compounds, was able to protect human microvascular endothelial cells from LPS-induced activation and dysfunction, reducing the endoplasmic reticulum stress The pleiotropic effects of these flavonoids contained in BPF formulation can justify the anti-oxidative and anti-inflammatory properties observed in patients suffering from metabolic syndrome 24 , In this population, bergamot polyphenols reduce LDL-cholesterol LDL-C , and increases HDL-cholesterol HDL-C as well as markers of systemic inflammation linked to cardiovascular outcomes such as the hs-CRP 26 , 27 , Recently, it has also been shown to improve the results of the Steato-Test in this population Altogether, these discoveries provide a rationale to further evaluate its potential impact on underlying NASH.

Prior to engaging in human trials, the objective of this study was to provide preclinical proof of concept that BPF99 can improve the histological features of NASH in a preclinical model of NASH that has been validated to resemble most features of human disease.

The key cellular signalling pathways relevant for human NASH are also activated in this model and the evolution of the transcriptomic changes with disease evolution are concordant with human NASH Further, the lipidome of the DIAMOND mouse is also similar to human NASH We therefore tested the effect of BPF99 on the histological severity of NASH in the DIAMOND mouse.

Secondary analysis of the effects of BPF99 on key pathways related to lipogenesis, cell stress, apoptosis, inflammation and fibrosis were also assessed.

The protocol was further reviewed and approved by the IACUC at Eastern Virginia Medical School AALAC-certified animal research facility where the animal studies were performed.

Two mice were maintained in each cage and mouse handling and care was provided in accordance with recommendations from the mouse metabolic phenotyping facility. Water was used as a vehicle to dilute BPF99 to a final concentration of 0.

Gavage was given without anesthesia. Mice of 8—12 weeks of age and weight of The choice of diet was based on previously published studies Effect of BPF99 on body change over time.

One day before starting diet regimen, baseline body weight was assessed. Animals were weighed once a week till the end of the study. The day of the sacrifice, animals were exposed to inhaled isoflurane prior to being euthanized. Euthanasia was performed by cervical dislocation. The entire liver was removed from the abdominal cavity and weighed.

Liver histology was assessed from paraffin-embedded tissue sections stained with hematoxylin and eosin or picrosirius red. Histology was reviewed using the NASH-Clinical Research Network CRN criteria and fatty liver inhibition of progression FLIP algorithm by an expert liver pathologist Dr.

Pierre Bedossa , blinded to the treatment group. Given the similarity of the histological expression of disease in the DIAMOND mice vis-à-vis human NAFLD, the presence of steatohepatitis was diagnosed as described previously For each liver slide, the main histological lesions were assessed using the Steatosis-Activity-Fibrosis SAF score system 33 , Grade of activity 0—2 is given by the sum between the presence of balloning and inflammation.

Ballooning hepatocytes was graded as 0 none , 1 when few hepatocytes presented a rounded shaped, reticulated and pale cytoplasm, but with normal dimensions , and 2 when there is a cluster of prominent balooning hepatocytes.

The NAFLD activity score NAS was calculated by addition of grades of steatosis, inflammation and ballooning 2. Detection of collagen fibers was performed by picrosirius red staining and fibrosis stage was scored according to the NASH-CRN system 2.

Fibrosis was also quantified by morphometry and expressed as collagen proportionate area CPA Then, FLIP algorithm was used for classification of liver slides in steatosis-without-NASH and steatosis-with-NASH, in according to the SAF score Oxidative damage in the liver was assessed by immunohistochemical staining for 3-nitrotyrosine 3-NT , a biomarker of oxidative damage mediated by peroxynitrite Hematoxylin , Thermo Scientific, Waltham, Massachusetts, USA was used for nuclear counterstaining.

Positive staining was visualized by intense brown staining. In each case, a negative control without primary antibody was used. Images of liver tissue immunostained for 3-NT were captured using an Olympus microscope BX53 and high-resolution Olympus digital image camera XC Cellsens Dimension software was used to acquire the images.

The intensity of 3-NT staining was assessed using ImageJ NIH as described Positive brown staining was registered between 30 and intensity units while the background and nuclear staining were greater than intensity units. A total of 4 images 40X magnification from each slide were analyzed for the percent of pixels within the 30— intensity unit as measure of immunoreactivity.

To perform glucose tolerance test GTT and insulin tolerance test ITT , a small blood volume was collected simply puncturing the tail vein with a small gauge needle. Blood glucose levels were measured using a glucometer MultiCare In, Biochemical Systems International, Arezzo, Italy. For the GTT, mice were fasted overnight and baseline blood glucose levels were measured.

ITT was performed, three days later, on the same animals used for the GTT. Blood was collected via cardiac puncture from the heart of euthanized mice. Briefly, serum sample is first treated with trichloroacetic acid TCA for protein precipitation and then treated with thiobarbituric acid.

One molecule of MDA reacts with two molecules of thiobarbituric acid. Serum total antioxidant status TAS was measured spectrophotometrically using kit manufactured by Randox NX on a Randox RX Monza Randox Laboratories Ltd, Crumlin, UK.

Antioxidants in the added serum, suppress the production of the radical cation in a concentration dependent manner and the color intensity decreases proportionally.

Processed serum was analyzed by RP-HPLC analysis on a Thermo Scientific Ultimate Dionex UHPLC equipped with an Hypersil gold column C18 dim. The instrumentation performance, chromatograms, and initial data processing were carried out with using Thermo Scientific Chromeleon Chromatography Data System CDS software.

See Supplementary Materials and methods. Tissue lysates were prepared as previously described 38 , Bound antibody was visualized using the chemiluminescent kit ECL WB Detection, GE Healthcare RPN, Little Chalfont, United Kingdom , immunoblots scanning and analyses were performed using an imaging system UVITEC Imaging Systems, Cambridge, United Kingdom.

Quantification of the bands was performed using the ImageJ Software NIH, Bethesda, MD, USA. Data were analysed with GraphPad PRISM 6. Inter-group comparisons were made using analysis of variance ANOVA with post hoc Bonferroni correction for multiple comparisons as appropriate for normally distributed variables.

The primary endpoint was the proportion of mice with steatohepatitis at the end of the study. The presence of steatohepatitis was aligned with recent case definitions from the liver forum 33 and absence of steatohepatitis was defined by a ballooning score of 0 along with minimal grade1 or no lobular inflammation.

Additional analyses included comparison of the severity scores for the individual components of NAFLD and comparison of the composite NAFLD activity score NAS and Steatosis-Activity-Fibrosis SAF scores as well as the collagen proportional area. Other endpoints of interest included body weight, liver weight, measures of glucose tolerance, liver enzymes and functions, and lipids In additional specific markers related to disease pathophysiology were measured including: 1 Metabolic-acyl CoA carboxylase ACC , AMP kinase and their phosphorylated state; 2 Cell stress and apoptosis: PARP, caspase 3; 3 inflammation: MAP kinases, JNK, NFκB, and 4 Fibrosis-collagen 1 and 3.

These markers were measured by Western blots. A total of 25 mice was randomly divided to receive chow diet, Western diet with ad libitum sugar at 8 weeks of age from the same birth cohort of mice. All mice tolerated treatment and there were no deaths from study initiation to end of study.

BPF99 most abundant flavonoids were identified in serum sample Supplementary results. Body weight of mice fed a WD SW diet and treated with vehicle, increased significantly, compared to normal chow-fed controls Fig. The liver weight increased significantly in WD SW-fed positive control mice compared to negative controls and was not affected significantly by BPF99 administration.

Following glucose administration during a GTT, mice on WD SW had significantly elevated circulating glucose levels with both higher peak glucose and overall glucose area under the curve AUC Fig. These indicate that BPF99 improves glucose disposal following a glucose load.

Glucose tolerance test GTT and insulin tolerance test ITT. A Glucose levels were higher, following intraperitoneal glucose administration, in mice fed a WD SW diet treated with vehicle, compared to NC NW-fed mice. B The response to insulin to dispose glucose, was worsened in WD SW-fed mice treated with vehicle compared to CD NW-fed mice.

NC NW red line ; WD SW Vehicle orange line ; WD SW BPF99 green line. To test the effects of BPF99 on systemic insulin resistance, a key pathophysiological driver of NAFLD, an ITT was performed. Chow diet fed control mice decreased their glucose levels as expected following the insulin challenge.

Mice of WD SW had an early significant impairment in insulin sensitivity with higher glucose levels than mice on chow diet after a similar insulin dose; however, over time, they were able to respond and bring their glucose levels down.

Administration of BPF99 reversed the early impairment of insulin-mediated hypoglycemia and returned the curve to the pattern seen in negative controls fed a chow diet only Fig.

WD SW diet also induced an increase in circulating triglycerides, total cholesterol and LDL-cholesterol in the fasted state. Although fasting hyperglycemia induced by WD SW diet was decreased by BPF99, this did not reach statistical significance.

None of the negative controls i. on chow diet developed steatohepatitis at the end of study. All of these mice had NAFL and none of the mice had total resolution of NAFLD. Effect of BPF99 on NASH development.

A Category. None of the negative controls developed steatohepatitis. In contrast, 8 out of 10 mice on a high fat diet with ad libitum sugar water administration had steatohepatitis. In the BPF99 group, 3 out of 10 mice developed steatohepatitis.

B Disease severity expressed in percentage. None of the negative controls developed NASH at the end of study. WD SW fed positive control mice had a mean steatosis grade of 2.

BPF99 did not significantly decrease the overall steatosis grade or the portion of steatotic cells. These differences were driven by a lower impact on steatosis grade which is part of the NAS. The mean fibrosis stage increased in positive control mice fed a WD SW compared to negative control mice on chow diet Fig.

Effect of BPF99 on liver weight and histological features associated with NASH. A High fat Western Diet was associated with a significant increased in terminal liver weight compared to CD NW-fed animals. B Liver histology showed no evidence of steatosis, NASH or sinusoidal fibrosis in mice fed a chow diet and tap water NC NW, a,d.

Steatosis, hepatocyte ballooning, inflammatory foci and clear sign of sinusoidal fibrosis were observed in WD SW-fed mice treated with vehicle b,e. Original magnification, x10—x Grey star: macrovesicular steatosis; Black arrow: hepatocyte ballooning; Grey arrow: inflammatory foci. ACC total and p-ACC and AMPK phosphorylation were increased Fig.

BPF99 did not have a significant effect on either ACC or AMPK, two key metabolic drivers of steatosis and its disposal in NAFLD In contrast, BPF99 had a highly significant effect on JNK and p38 MAPK phosphorylation returning them to levels seen in chow fed mice and lower than the levels seen in positive controls Fig.

These effects were specific with no impact of BPF99 on NF-kB activation Fig. Effects of BPF99 on inflammatory, metabolic and stress-related markers associated with NASH. A Immunoblot analysis were performed for NF-kB; phosphorylated and total JNK p54, p46 ; phosphorylated and total p38; phosphorylated and total AMPK; phosphorylated and total ACC, Caspase-3; COL1A1; PIIINP; PARP; cleaved Caspase-3 and cleaved PARP product p89 were not visualized despite long exposure times.

GAPDH and Tubulin were used as a control for protein loading. F NF-kB was increased in WD SW-fed mice treated with vehicle or BPF G Caspase-3 levels were similar in NASH animals treated with vehicle or BPF99 and higher compared to control animals.

H PARP protein levels were higher in NASH animals treated with vehicle, whereas BPF99 reduced PARP expression to control levels. I Procollagen-1 and L procollagen-3 levels were higher in WD SW-fed mice treated with vehicle compared to control animals.

BPF99 was able to reduce Procollagen I and III levels. Full-length blots are presented in Supplementary Fig. Next, the effect of BPF99 on fibrogenic activity was assessed by evaluating procollagen I expression in the liver of mice fed a chow diet, western diet with sugar water or a western diet with sugar water and concomitant admnistration of BPF Procollagen I protein levels were increased in the mice fed a Western diet compared to mice fed a chow diet Fig.

Effects of BPF99 on serum lipid peroxidation and total antioxidant status. Next, the effect of BPF99 on total antioxidant status was assessed. The mean serum TAS was significantly decreased in mice with steatohepatitis compared to negative control group Fig. BPF99 administration was associated with a significant increase in serum TAS in mice fed a western diet with sugar water Fig.

Animals fed a western diet and sugar water, treated with BPF99, exhibited the increases TAS value even in comparison with the mice fed a chow diet Fig. Assessment and quantification of protein nitrotyrosine adducts in the liver. A Formalin-fixed, paraffin-embedded sections were incubated with antinitrotyrosine antibody followed by anti-rabbit HRP coniugated antibody and 3,3-diaminobenzidine.

The nuclei were counterstained with hematoxylin. The degree of staining for 3-NT was significantly higher in the livers of mice fed a WD SW compared to negative control group fed a normal chow diet.

A less amount of staining for 3-NT was observed in the livers of mice fed a WD SW and treated with BPF Sections without antinitrotyrosine antibody were used as negative control.

DAB-positive pixels were in the 30—pixel intensity range in the blue channel of the RGB histogram, whereas background staining was greater than intensity units.

The percents of pixels in the 30— range in the blue channel are shown. NAFLD is the leading cause of liver-related morbidity and mortality. NASH is expected to become the leading etiology of end-stage liver disease and indication for liver transplantation As drug development efforts are currently focused on NASH and stage 2—3 fibrosis or cirrhosis 4 , the treatment of the greatest burden of disease which characterizes either NAFL or NASH early stage 0—1 remains an important unmet medical need because the patients still have excess cancer- and cardiometabolic risk Thus, the missed opportunity to treat the disease early when the fibrosis is potentially more easily reversible leaves some patients at risk of developing end stage further diseases despite current therapeutic development approaches.

There is thus a need for development of simple approaches to enhance diet and life style interventions for those with early stage disease. In the current study, we provide evidence of the potential benefits of BPF99 in a preclinical model of NASH.

This model has been validated to develop many of the features of human disease such as obesity, insulin resistance, systemic inflammation with crown-like bodies in adipose tissue, elevated IL-6 and decreased adiponectin It further sequentially develops steatosis followed by steatohepatitis and then increasing fibrosis which is associated with activation of cellular pathways also activated in human disease.

The transcriptomic signature of various phases of the course of NAFLD also resemble that seen in humans. While these studies focused on early stage disease in the mice to generate proof of concept, the similarities between this model and human disease increases the potential for translatability from mice to humans.

There are also additional indications that the beneficial effects of BPF99 may extend to more advanced stages of disease as well.

These facts demonstrate that resveratrol is useful not only in the prevention of liver steatosis but also in its treatment. Only two of all the studies reported used more than one dose in the same experiment. By contrast, in the case of Cho et al [ 34 ], the low dose 0.

Altogether data reported concerning the mechanisms of action of resveratrol underlying its liver anti-lipidogenic effect demonstrate that this polyphenol decreases de novo fatty acid synthesis, as well as triacylglycerol synthesis, and increases fatty acid oxidation.

Moreover, the reduction of oxidative stress also contributes to this positive effect. The activation of AMPK and SIRT1 mediates these changes. With regard to human beings, there is only one published study so far. Plasma ALT concentration was significantly lower after resveratrol treatment compared to the placebo group.

Intrahepatic lipid content was lower after 30 d of resveratrol supplementation in comparison to placebo. This was paralleled by lower plasma ALT value, both indicating improved liver function[ 44 ].

Quercetin is a natural polyphenol belonging to a group with a variable structure, known as flavonoids. It is found in onions, broccoli, tomatoes, apples and berries[ 45 ]. It has been reported that quercetin exhibits a wide range of biological functions, including antioxidant, anticarcinogenic and anti-inflammatory activities[ 46 - 50 ].

More recently, beneficial effects on blood pressure and heart disease have been described[ 50 - 52 ]. Since , several studies have shown the interesting properties of this flavonoid in the prevention of liver steatosis Tables 1 and 3.

Vidyashankar et al [ 53 ] carried out a study with HepG2 cells rendered steatosis by incubation with oleic acid-bovine serum albumin complex. Decreased triacylglycerol accumulation, insulin resistance and inflammatory cytokine secretion, and increased cellular antioxidants were observed.

The study suggested that quercetin was an effective molecule reversing the symptoms of NAFLD. As in the case of resveratrol, different animal models have been used in in vivo studies. The lowest dose was ineffective. Only the highest dose reduced liver collagen.

The rest of the studies reported were conducted in mice. When quercetin was added to the diet at 0. The same research group carried out another study by means of a different experimental design[ 56 ]. Treated mice showed a reduction in liver triacylglycerol accumulation.

Moreover, the increase induced by the diet in the expression of peroxisome proliferator activated receptor γ PPAR-γ , cluster of differentiation 36 CD36 , SREBP-1c and FAS, genes which promote lipid accumulation, was normalized.

Another interesting effect was the increase in the expression of peroxisome proliferator activated receptor α PPAR-α , a transcription factor which control fatty acid oxidation, reduced by high-fat feeding. Finally, oxidative stress was reduced by quercetin. The authors suggested that this effect was related to the down-regulation of NF-κB, a transcriptional factor that stimulates inflammation, and the up-regulation of Nrf2, which prevents oxidation.

Moreover, quercetin treatment resulted in increased expression of CPT-1a, a key enzyme of fatty acid oxidation. In this study the authors also analyzed the effects of quercetin on the expression of genes related to lipid metabolism in liver, such as FAS involved in de novo lipogenesis , Acetyl-coenzyme A carboxylase α Acaca , apolipoprotein A-IV Apoa4 , ATP-binding cassette, subfamily G, member 5 Abcg5 , Fdft1, farnesyl-diphosphate farnesyltransferase 1 Fdft1 involved in the synthesis of saturated fatty acids and glycerolphosphate acyltransferase mitochondrial GPAM , involved in triacylglycerol synthesis and related to SREBP-1c gen.

All these genes were down-regulated in quercetin-treated mice. Marcolin et al [ 58 ] analyzed the effect of quercetin 0. In this study, a lower degree of steatosis and a reduction in transaminases and oxidative stress were observed in mice treated with quercetin for 4 wk.

Moreover, proinflammatory and profibrotic gene expression was reduced. The number of studies performed with quercetin is lower than that of studies carried out with resveratrol.

The most commonly used animal model in these studies was mice. It is important to point out that a low dose of this polyphenol 0. The only study which analyzed different doses of quercetin[ 54 ] showed a dose response pattern. As far as the potential mechanisms of this polyphenol are concerned, quercetin similarly to resveratrol decreases de novo fatty acid synthesis.

In addition to resveratrol and quercetin, other polyphenols such as antocyanin Cyg, proanthocyanidins, teaflavin a flavanol and ellagic acid a tannin have studied as potential agent for both prevention and treatment of hepatic steatosis Tables 1 and 4.

A reduction in triacylglycerol content was observed due to an inhibition in glycerol-snphosphate acyl transferase 1 GPAT1 , a key enzyme in the synthesis of triacylglycerols. In FAO cells, a rat hepatoma cell line, Baselga-Escudero et al [ 61 ] studied the effect of proanthocyanidins on hepatic lipid metabolism.

miR is a novel class of non-coding RNA that regulates genes involved in fatty acid and triacylglycerol synthesis. Moreover, the protein expression of FAS was also decreased. The study showed that this polyphenol inhibited fat accumulation in a dose dependent manner due to a decrease in SREBP-1c and FAS gene and protein expressions, through the activation of AMPK and SIRT1.

The study showed a reduction in triacylglycerol content and lipid droplets due to an inhibition in translocation of GPAT1 and a reduction in the synthesis of triacylglycerols. These results are in good accordance with their own results obtained in cultured cells.

In the same animal model, but using a high-fat diet supplemented with 0. Moreover, a decrease in macrovesicular steatosis and hepatic triacylglycerol was shown. CPT-1a and PPAR-α mRNA expression was increased but Acaca, SREBP-1c and ACO mRNA expression remained unchanged. Surprisingly, the mRNA expression of FAS was increased.

They observed that theoflavin reduced cell ballooning, micro and macrovesicular steatosis, hepatocyte apoptosis, oxidative stress and inflammatory cells in liver, as well as serum transaminase concentrations.

Baselga-Escudero et al [ 61 ] conducted a study with Wistar rats fed ad libitum with a standard diet. The rats were orally gavaged with lard oil 2. After 3 h a reduction in serum and hepatic triacylglycerol content was observed.

Similar to what these authors observed in cultured cells, increased miR mRNA levels accompanied by decreased FAS mRNA levels were found. It is not possible to compare the above described studies because important differences in terms of type of polyphenol used, dose and animal model exist among them.

Nevertheless, the reported results show that all the polyphenols tested, belonging to different families, show an anti-lipidogenic effect. As in the case of resveratrol, the potential mechanisms that justify this effect are decreased synthesis of fatty acids and triacylglycerols and increased fatty acid oxidation.

In addition to studies conducted with individual polyphenols, several works have used polyphenol extracts with different origins and compositions.

Although using polyphenol extracts makes it quite complicated to assign the beneficial effects observed to a specific molecule, they have two clear advantages.

On the one hand, they better mimic the real situation in our dietary patterns. On the other hand, additive or synergic effects can be observed. In this context, sometimes combinations of molecules present beneficial effects that are not shown when they are administrated separately Tables 1 and 5.

There are few studies analyzing the effect of polyphenols extracts in vitro. An inhibitory effect was observed in triacylglycerols accumulation in a dose-dependent manner. This extract reduced triacylglycerols content due to the up-regulation of CPT-1a, ACO, FAS and Acetyl-CoA carboxylase β Aca-β gene expression and CPT-1a protein expression.

Lee et al [ 67 ] studied the effect of an extract of Hibiscus sabdariffa L. including 8. Cells were treated with different doses of this polyphenol extract 0. The results showed that Hibiscus sabdariffa L. reduced triacylglycerol content.

Moreover, it was able to eliminate the release of intermembrane proteins and to reduce cell death. Mice were treated with 3 doses of extract of Hibicus sabdariffa L. The study showed that this polyphenol extract reduced transaminases in a dose-dependent manner, and oxidative stress.

Moreover, decreased liver damage and steatosis were shown by histopathological analysis. Beltrán-Debón et al [ 68 ] administered an aqueous extract of Aspalathus linearis L.

This extract reduced serum triacylglycerols and free fatty acids. Moreover, histopathological analysis showed that steatosis degree was also lower in mice supplemented with rooibos extract.

Animals were supplemented with an extract of Nelumbo nucifera L. lotus root; proanthocyanidins, carechin, gallocatechin at a dose of 0. In addition, the activity of lipogenic enzymes FAS and ME were decreased and CPT-1a, an enzyme related to fatty acid oxidation, remained unchanged. Green tea catechins reduced serum triacylglycerols and ALT.

In liver, this polyphenol extract reduced weight and the amount of triacylglycerols. All these results were observed during both treatment periods. In order to analyze the potential mechanisms of action, gene expression of SREBP-1c, PPAR-γ and ACC was assessed, but only in the group treated for 22 wk.

All these genes showed down-regulation induced by green tea. The effects of an extract of grape skin were tested by Park et al [ 71 ]. They showed decreased serum free fatty acids and leptin and increased adiponectin concentrations. With regard to liver, a reduction in triacylglycerols was observed.

In order to determine the potential mechanisms of action, gene expression and activity of enzymes related to hepatic triacylglycerol metabolism were analyzed. Both the expression and the activity of enzymes involved in de novo lipogenesis, such as FAS, glucosephosphate dehydrogenase G6PDH and ME and triacylglycerol synthesis, such as phosphatidate phosphohydrolase PAP , were decreased.

Accordingly, gene expression of PPAR-γ was also reduced. CPT-1a and PPAR-α mRNA levels, as well as β-oxidation were increased. Surprisingly, no changes were observed in CPT-1a activity. Feillet-Coudray et al [ 72 ] carried out a study using Wistar rats fed a high-fat diet supplemented 0.

The histological analysis revealed a decrease in macroesteatosis and fat droplets in treated animals. A reduction in hepatic lipid peroxidation was also observed. The potential mechanisms of action underlying these effects were further reported by this research group[ 73 ].

Provinol ® increased protein expression of SIRT1, without changing SCD1, SREBP-1c, FAS, hepatocyte nuclear factor 4 α HNF-4α , PGC-1α, CPT-1a and phosphorylated AMPK. Moreover, a decrease in phosphorylated ACC was observed.

Therefore, the authors suggested that the reduction in liver triacylglycerol accumulation was, at least in part, regulated by the inhibition of ACC, the limiting enzyme in de novo lipogenesis, probably through the activation of SIRT1 deacetylase.

hop pomace; flavonoids, procyanidins for 70 d. Liver weight and hepatic triacylglycerol content showed a tendency towards reduced values.

A reduction in de novo lipogenesis was observed, without changes in ACO and CPT-1a activity. All the polyphenol extracts analyzed were able to reduce liver fat accumulation. As expected, the mechanisms underlying this effect were those reported in studies carried out with isolated polyphenols.

As a general conclusion, it can be stated that polyphenols are biomolecules which present hepatoprotective effects because they reduce liver fat accumulation and decrease oxidative stress and inflammation, the two main factors responsible for liver damage.

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Academic Content and Language Evaluation of This Article. Answering Reviewers PDF Peer-Review Report PDF. Citation of this article. Aguirre L, Portillo MP, Hijona E, Bujanda L. Effects of resveratrol and other polyphenols in hepatic steatosis.

World J Gastroenterol ; 20 23 : [PMID: DOI: Corresponding Author of This Article. portillo ehu. Publishing Process of This Article.

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Copyright © Baishideng Publishing Group Inc. All rights reserved. World J Gastroenterol. Jun 21, ; 20 23 : Published online Jun 21, doi: Leixuri Aguirre , Maria Puy Portillo , Elizabeth Hijona , Luis Bujanda.

Leixuri Aguirre, Maria Puy Portillo, Centro de Investigación Biomédica en Fisiopatología de la Obesidad y Nutrición CIBERobn , Instituto de Salud Carlos III, Vitoria, Spain. Elizabeth Hijona, Luis Bujanda, Centro de Investigación Biomédica en Enfermedades Hepáticas CIBERehd , Instituto de Salud Carlos III, San Sebastian, Spain.

Author contributions : Hijona E and Bujanda L wrote the paragraphs concerning the introduction and the physiopathology of liver steatosis; Aguirre L and Portillo MP wrote the paragraphs concerning the effects of polypehnols on liver steatosis; and Portillo MP was responsible for the general and final revision of the manuscript.

Correspondence to : Dr. Received: October 24, Revised: December 4, Accepted: January 19, Published online: June 21, Key Words: Polyphenols , Resveratrol , Quercetin liver , Steatosis , Non-alcoholic fatty liver disease.

Citation: Aguirre L, Portillo MP, Hijona E, Bujanda L. Table 1 In vitro studies performed with polyphenols. RSV: Resveratrol; ACC: Acetyl CoA carboxylase; FAS: Fatty acid synthase; AMPK: AMP-activated kinase; SREBP1c: Sterol regulatory element binding protein 1c; SIRT1: Sirtuin 1; Q: Quercetin; GPAT1: Glycerol-snphosphate acyltransferase 1; mtGPAT1: Mitochondrial glycerol-snphosphate acyltransferase 1; LXRα: Liver X receptor α; pJNK: Activated c-Jun N-terminal kinase; AIF: Apoptosis inducing factor; GSH: Glutathione; CPT-1a: Carnitine palmitoyltransferase 1a; ACO: Acyl-coenzyme A oxidase 1; Acacβ: Acetyl-coenzyme A carboxylase β.

Table 2 In vivo studies carried out with resveratrol. EFFECTS OF QUERCETIN ON STEATOSIS. Table 3 In vivo studies carried out with quercetin. STZ: Streptozotocin; PPAR-γ: Peroxisome proliferator activated receptor γ; SREBP-1c: Sterol regulatory element binding protein 1c; FAS: Fatty acid synthase; CD Cluster of differentiation 36; PPAR-α: Peroxisome proliferator activated receptor α; BW: Body weight; CPT-1a: Carnitine palmitoyltransferase 1a; Acaca: Acetyl-coenzyme A carboxylase α; Apoa4: Apolipoprotein A-IV; Abcg: ATP-binding cassette, subfamily G, member 5; Fdft1: Farnesyl-diphosphate farnesyltransferase 1; Gpam: Glycerolphosphate acyltransferase, mitochondrial.

Table 4 In vivo studies carried out with other polyphenols. GPAT1: Glycerol-snphosphate acyltransferase 1; BW: Body weight; FAS: Fatty acid synthase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; Acaca: Acetyl-CoA carboxylase α; SREBP-1c: Sterol regulatory element binding protein 1c; ACO: Acyl-coenzyme A oxidase; CPT-1a: Carnitine palmitoyltransferase 1a; PPAR-α: Peroxisome proliferator activated receptor α.

Table 5 In vivo studies carried out with polyphenol extracts. SIRT: Sirtuin 1; SCD1: Stearoyl-CoA desaturase; AMPK: AMP-activated kinase; pAMPK: Phosphorylated AMP-activated kinase; SREBP-1c: Sterol regulatory element binding protein 1c; FAS: Fatty acid synthase; HNF-4α: Hepatocyte nuclear factor 4 α; PGC-1α: Peroxisome proliferator-activated receptor-c coactivator 1α; CPT-1a: Carnitine palmitoyltransferase 1a; ME: Malic enzyme; ALT: Alanine aminotransferase; PPAR-γ: Peroxisome proliferator activated receptor γ; ACC: Acetyl CoA carboxylase; pACC: Phosphorylated acetyl CoA carboxylase; AST: Aspartate aminotransferase; p-JNK: Activated c-Jun N-terminal kinase; AIF: Apoptosis inducing factor; Acaa1: acetyl-coenzyme A acyltransferase 1; Slc25a Solute carrier family 25, member 20; Hadh: Hydroxyacyl-coenzyme A dehydrogenase; ACO: Acyl-coenzyme A oxidase; PPAR-α: Peroxisome proliferator activated receptor α; RXR-α: Retinoid X receptor α; PAP: Phosphatidate phosphohydrolase.

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Chang et al. and Ekhlasi et al. showed a significant improvement of anthropometric parameters after treatment with polyphenols. observed a reduction in weight, body mass index, waist circumference, liver enzymes, and steatosis degree, while resveratrol was associated with lifestyle changes and particularly with physical activity.

in association with a significant improvement of insulin resistance assessed by homeostasis model assessment insulin resistance index.

described a reduction in NF-ϰB activity in the peripheral blood mononuclear cells. reported the reduction of high liver enzymes and improvement of total antioxidant capacity in the NAFLD patients treated with pomegranate juice.

However, the information obtained by non-invasive tools like the liver ultrasound reported a significant improvement of liver damage and liver steatosis degree when polyphenol supplementation is associated with lifestyle changes.

NAFLD is the liver board of over nutrition and an upcoming challenge for hepatologists and health systems worldwide. Patients with liver steatosis have an increased prevalence of chronic diseases.

Therefore, the treatment of NAFLD patients should be focused on reducing predisposing factors, such as insulin resistance, oxidative stress, and dyslipidemia. In this way, nutraceuticals may have an important role in the treatment of NAFLD.

Supported by literature data, it can be concluded that adherence to the traditional Mediterranean diet characterized by the consumption of antioxidant-rich foods in general and of polyphenols in particular, can be considered as a potential new approach in the treatment of NAFLD, and even a valuable instrument of prevention of this disorder.

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We thank Dr. Ján Morovič for his support in image elaboration. This study was supported by Hepatic Sciences Inc. Nutramed S. Complesso Ninì Barbieri, Roccelletta di Borgia, Catanzaro, Italy.

Sanyal biotechnology, E Leigh St, Richmond, VA, , USA. Macquarie University Medical School, Sydney, Australia. Institute of Cellular Medicine, University of Newcastle, Newcastle, UK. Department of Pharmacology and Physiology, Saint Louis University School of Medicine, South Grand Blvd, St.

Louis, MO, , USA. You can also search for this author in PubMed Google Scholar. and A. conceptualized and designed the study. Vincenzo Musolino, M.

and M. performed the experiments. and J. performed the G. and I. performed liver staining. reviewed all the histology. analyzed the data. and R. interpreted the data. and Vincenzo Musolino wrote the manuscript. Correspondence to Vincenzo Musolino or Arun J. AJS is President of Sanyal Bio.

He has stock options in Genfit, Galmed, Exhalenz, Durect, Tiziana and Indalo. He has served as a consultant to Intercept, Gilead, Bristol Myers Squibb, Novartis, Pfizer, Lilly, Novo Nordisk, Astra Zeneca, Medimmune, Merck, Allergan, Albireo, Affimmune, Birdrock, Boehringer Ingelhiem, Artham, Celgene, Conatus, Genentech, Tern, Sundise Pharmaceuticals, Takeda, Hemoshear, Immuron, Surrozen, Poxel, Prosciento, Second Genome, Liposcience, Cymabay, Salix, Ferring and Teva.

His institution has received grants from Inercept, Gilead, Novartis, Merck, Salix and Bristol Myers Squibb. Rebecca Caffrey is the Co-founder, part-owner and employee of Sanyal Bio.

Jonathan Marionaux is employed by Sanyal Bio. All other authors have nothing to disclose. Open Access This article is licensed under a Creative Commons Attribution 4. Reprints and permissions. Bergamot Polyphenols Improve Dyslipidemia and Pathophysiological Features in a Mouse Model of Non-Alcoholic Fatty Liver Disease.

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Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma. Skip to main content Thank you for visiting nature. nature scientific reports articles article. Download PDF. Subjects Metabolic syndrome Mouse Non-alcoholic fatty liver disease Non-alcoholic steatohepatitis Target identification.

Abstract There is a need for continued drug development for nonalcoholic steatohepatitis NASH. Introduction Nonalcoholic fatty liver disease NAFLD is a major cause of liver-related morbidity and mortality for which there are no approved therapies 1. Study design Mice of 8—12 weeks of age and weight of Figure 1.

Full size image. Results A total of 25 mice was randomly divided to receive chow diet, Western diet with ad libitum sugar at 8 weeks of age from the same birth cohort of mice. Effect of BPF99 on body weight Body weight of mice fed a WD SW diet and treated with vehicle, increased significantly, compared to normal chow-fed controls Fig.

BPF99 improved glucose tolerance and insulin resistance Following glucose administration during a GTT, mice on WD SW had significantly elevated circulating glucose levels with both higher peak glucose and overall glucose area under the curve AUC Fig. Figure 2. Table 1 Serum biochemical parameters.

Full size table. Figure 3. Table 2 Liver histological findings at the end of the study. Figure 4. Figure 5. Figure 6. Figure 7. Discussion NAFLD is the leading cause of liver-related morbidity and mortality. References Estes, C. Article CAS PubMed Google Scholar Kleiner, D. Article PubMed Google Scholar Kleiner, D.

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