Pirfenidone Reverts Global DNA Hypomethylation, Promoting DNMT1/UHRF/PCNA Coupling Complex in Experimental Hepatocarcinoma.

Hepatocellular carcinoma (HCC) development is associated with altered modifications in DNA methylation, changing transcriptional regulation. Emerging evidence indicates that DNA methyltransferase 1 (DNMT1) plays a key role in the carcinogenesis process. This study aimed to investigate how pirfenidone (PFD) modifies this pathway and the effect generated by the association between c-Myc expression and DNMT1 activation. Rats F344 were used for HCC development using 50 mg/kg of diethylnitrosamine (DEN) and 25 mg/kg of 2-Acetylaminofluorene (2-AAF). The HCC/PFD group received simultaneous doses of 300 mg/kg of PFD. All treatments lasted 12 weeks. On the other hand, HepG2 cells were used to evaluate the effects of PFD in restoring DNA methylation in the presence of the inhibitor 5-Aza. Histopathological, biochemical, immunohistochemical, and western blot analysis were carried out and our findings showed that PFD treatment reduced the amount and size of tumors along with decreased Glipican-3, ß-catenin, and c-Myc expression in nuclear fractions. Also, this treatment improved lipid metabolism by modulating PPARγ and SREBP1 signaling. Interestingly, PFD augmented DNMT1 and DNMT3a protein expression, which restores global methylation, both in our in vivo and in vitro models. In conclusion, our results suggest that PFD could slow down HCC development by controlling DNA methylation.

Pirfenidone Protects from UVB-Induced Photodamage in Hairless Mice.

BACKGROUND: Ultraviolet radiation (UV) is the main environmental factor that causes histological degenerative changes of the skin giving rise to a chronic process called photodamage. Non-melanoma skin cancer induced by UVB radiation is a result of a cascade of molecular events caused by DNA damage in epidermis cells, including persistent inflammation, oxidative stress, and suppression of T cell-mediated immunity. Retinoids such as tretinoin have been widely used in skin to treat photoaging and photodamage, though its secondary adverse effects have been recognized. Pirfenidone (PFD) has emerged as an antifibrogenic, anti-inflammatory and antioxidant agent, and in this work its efficacy was evaluated in a model of UVB-induced photodamage. METHODS: Epidermal, dermal, and inflammatory changes were measured by histomorphometric parameters. In addition, gene, and protein expression of key molecules in these processes were evaluated. RESULTS: Our results revealed an anti-photodamage effect of topical PFD with absence of inflammatory skin lesions determined by dermoscopy. In addition, PFD reduced elastosis, improved organization, arrangement, and deposition of dermal collagens, downregulated several pro-inflammatory markers such as NF-kB, IL-1, IL-6 and TNFα, and decreased keratinocyte damage. CONCLUSION: Topical pirfenidone represents a promising agent for the treatment of cell photodamage in humans. Clinical trials need to be carried out to explore this premise.

Moringa oleifera Improves MAFLD by Inducing Epigenetic Modifications.

BACKGROUND AND AIMS: Metabolic Associated Fatty Liver Disease (MAFLD) encompasses a spectrum of diseases from simple steatosis to nonalcoholic steatohepatitis (NASH). Here, we investigated the hepatoprotective role of Moringa oleifera aqueous extract on hepatic miRNAs, genes and protein expression, as well as histological and biochemical parameters in an experimental model of NASH. METHODS: Male C57BL/6J mice were fed with a high fat diet (HFD, 60% lipids, 42 gr/L sugar in water) for 16 weeks. Moringa extract was administered via gavage during the final 8 weeks. Insulin Tolerance Test (ITT) and HOMA-IR were calculated. Serum levels of insulin, resistin, leptin and PAI-1 and hepatic expression of miR-21a-5p, miR-103-3p, miR-122-5p, miR-34a-5p and SIRT1, AMPKα and SREBP1c protein were evaluated. Alpha-SMA immunohistochemistry and hematoxylin-eosin, Masson's trichrome and sirius red staining were made. Hepatic transcriptome was analyzed using microarrays. RESULTS: Animals treated with Moringa extract improved ITT and decreased SREBP1c hepatic protein, while SIRT1 increased. Hepatic expression of miR-21a-5p, miR-103-3p and miR-122-5p, miR34a-5p was downregulated. Hepatic histologic analysis showed in Moringa group (HF + MO) a significant decrease in inflammatory nodules, macro steatosis, fibrosis, collagen and αSMA reactivity. Analysis of hepatic transcriptome showed down expression of mRNAs implicated in DNA response to damage, endoplasmic reticulum stress, lipid biosynthesis and insulin resistance. Moringa reduced insulin resistance, de novo lipogenesis, hepatic inflammation and ER stress. CONCLUSIONS: Moringa prevented progression of liver damage in a model of NASH and improved biochemical, histological and hepatic expression of genes and miRNAs implicated in MAFLD/NASH development.

Hepatocarcinogenesis Prevention by Pirfenidone Is PPARγ Mediated and Involves Modification of Nuclear NF-kB p65/p50 Ratio.

Targeted therapies for regulating processes such as inflammation, apoptosis, and fibrogenesis might modulate human HCC development. Pirfenidone (PFD) has shown anti-fibrotic and anti-inflammatory functions in both clinical and experimental studies. The aim of this study was to evaluate PPARγ expression and localization in samples of primary human tumors and assess PFD-effect in early phases of hepatocarcinogenic process. Human HCC tissue samples were obtained by surgical resection. Experimental hepatocarcinogenesis was induced in male Fischer-344 rats. TGF-ß1 and α-SMA expression was evaluated as fibrosis markers. NF-kB cascade, TNFα, IL-6, and COX-2 expression and localization were evaluated as inflammation indicators. Caspase-3, p53, and PARP-1 were used as apoptosis markers, PCNA for proliferation. Finally, PPARα and PPARγ expression were evaluated to understand the effect of PFD on the activation of such pathways. PPARγ expression was predominantly localized in cytoplasm in human HCC tissue. PFD was effective to prevent histopathological damage and TGF-ß1 and α-SMA overexpression in the experimental model. Anti-inflammatory effects of PFD correlate with diminished IKK and decrease in both IkB-phosphorylation/NF-kB p65 expression and p65-translocation into the nucleus. Pro-apoptotic PFD-induced effects are related with p53 expression, Caspase-3 p17 activation, and PARP-1-cleavage. In conclusion, PFD acts as a tumor suppressor by preventing fibrosis, reducing inflammation, and promoting apoptosis in MRHM.

PPARs as Metabolic Sensors and Therapeutic Targets in Liver Diseases.

Carbohydrates and lipids are two components of the diet that provide the necessary energy to carry out various physiological processes to help maintain homeostasis in the body. However, when the metabolism of both biomolecules is altered, development of various liver diseases takes place; such as metabolic-associated fatty liver diseases (MAFLD), hepatitis B and C virus infections, alcoholic liver disease (ALD), and in more severe cases, hepatocelular carcinoma (HCC). On the other hand, PPARs are a family of ligand-dependent transcription factors with an important role in the regulation of metabolic processes to hepatic level as well as in other organs. After interaction with specific ligands, PPARs are translocated to the nucleus, undergoing structural changes to regulate gene transcription involved in lipid metabolism, adipogenesis, inflammation and metabolic homeostasis. This review aims to provide updated data about PPARs' critical role in liver metabolic regulation, and their involvement triggering the genesis of several liver diseases. Information is provided about their molecular characteristics, cell signal pathways, and the main pharmacological therapies that modulate their function, currently engaged in the clinic scenario, or in pharmacological development.

Liver Cancer: Therapeutic Challenges and the Importance of Experimental Models.

Liver cancer is one of the main causes of death related to cancer worldwide; its etiology is related with infections by C or B hepatitis virus, alcohol consumption, smoking, obesity, nonalcoholic fatty liver disease, diabetes, and iron overload, among other causes. Several kinds of primary liver cancer occur, but we will focus on hepatocellular carcinoma (HCC). Numerous cellular signaling pathways are implicated in hepatocarcinogenesis, including YAP-HIPPO, Wnt-ß-catenin, and nuclear factor-κB (NF-κB); these in turn are considered novel therapeutic targets. In this review, the role of lipid metabolism regulated by peroxisome proliferator-activated receptor gamma (PPARγ) in the development of HCC will also be discussed. Moreover, recent evidence has been obtained regarding the participation of epigenetic changes such as acetylation and methylation of histones and DNA methylation in the development of HCC. In this review, we provide detailed and current information about these topics. Experimental models represent useful tools for studying the different stages of liver cancer and help to develop new pharmacologic treatments. Each model in vivo and in vitro has several characteristics and advantages to offer for the study of this disease. Finally, the main therapies approved for the treatment of HCC patients, first- and second-line therapies, are described in this review. We also describe a novel option, pirfenidone, which due to its pharmacological properties could be considered in the future as a therapeutic option for HCC treatment.

Roles of Nrf2 in Liver Diseases: Molecular, Pharmacological, and Epigenetic Aspects.

Liver diseases represent a critical health problem with 2 million deaths worldwide per year, mainly due to cirrhosis and its complications. Oxidative stress plays an important role in the development of liver diseases. In order to maintain an adequate homeostasis, there must be a balance between free radicals and antioxidant mediators. Nuclear factor erythroid 2-related factor (Nrf2) and its negative regulator Kelch-like ECH-associated protein 1 (Keap1) comprise a defense mechanism against oxidative stress damage, and growing evidence considers this signaling pathway as a key pharmacological target for the treatment of liver diseases. In this review, we provide detailed and updated evidence regarding Nrf2 and its involvement in the development of the main liver diseases such as alcoholic liver damage, viral hepatitis, steatosis, steatohepatitis, cholestatic damage, and liver cancer. The molecular and cellular mechanisms of Nrf2 cellular signaling are elaborated, along with key and relevant antioxidant drugs, and mechanisms on how Keap1/Nrf2 modulation can positively affect the therapeutic response are described. Finally, exciting recent findings about epigenetic modifications and their link with regulation of Keap1/Nrf2 signaling are outlined.

Pirfenidone Is an Agonistic Ligand for PPARα and Improves NASH by Activation of SIRT1/LKB1/pAMPK.

Nonalcoholic steatohepatitis (NASH) is recognized by hepatic lipid accumulation, inflammation, and fibrosis. No studies have evaluated the prolonged-release pirfenidone (PR-PFD) properties on NASH features. The aim of this study is to evaluate how PR-PFD performs on metabolic functions, and provide insight on a mouse model of human NASH. Male C57BL/6J mice were fed with either normo diet or high-fat/carbohydrate diet for 16 weeks and a subgroup also fed with PR-PFD (300 mg/kg/day). An insulin tolerance test was performed at the end of treatment. Histological analysis, determination of serum hormones, adipocytokines measurement, and evaluation of proteins by western blot was performed. Molecular docking, in silico site-directed mutagenesis, and in vitro experiments using HepG2 cultured cells were performed to validate PR-PFD binding to peroxisome proliferator-activated receptor alpha (PPAR-α), activation of PPAR-α promoter, and sirtuin 1 (SIRT1) protein expression. Compared with the high-fat group, the PR-PFD-treated mice displayed less weight gain, cholesterol, very low density lipoprotein and triglycerides, and showed a significant reduction of hepatic macrosteatosis, inflammation, hepatocyte ballooning, fibrosis, epididymal fat, and total adiposity. PR-PFD restored levels of insulin, glucagon, adiponectin, and resistin along with improved insulin resistance. Noteworthy, SIRT1-liver kinase B1-phospho-5' adenosine monophosphate-activated protein kinase signaling and the PPAR-α/carnitine O-palmitoyltransferase 1/acyl-CoA oxidase 1 pathway were clearly induced in high fat + PR-PFD mice. In HepG2 cells incubated with palmitate, PR-PFD induced activation and nuclear translocation of both PPARα and SIRT1, which correlated with increased SIRT1 phosphorylated in serine 47, suggesting a positive feedback loop between the two proteins. These results were confirmed with both synthetic PPAR-α and SIRT1 activators and inhibitors. Finally, we found that PR-PFD is a true agonist/ligand for PPAR-α. Conclusions: PR-PFD provided an anti-steatogenic effect and protection for inflammation and fibrosis.

Participation of the anti-inflammatory and antioxidative activity of docosahexaenoic acid on indomethacin-induced gastric injury model.

Adverse gastrointestinal (GI) effects caused by nonsteroidal anti-inflammatory drugs (NSAIDs), including indomethacin, are recognized as the major limitation to their clinical use. NSAID-induced gastric damage is generated by cyclooxygenase inhibition, activation of inflammatory processes, and oxidative stress. Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, has shown gastroprotective effects; however, the molecular mechanisms underlying these effects have not been fully explained. As a result, the aim of this study was to examine DHA's anti-inflammatory and antioxidative actions in a mouse model of indomethacin-induced gastric injury. Oral administration of DHA (3, 10, 30, and 100mg/kg) caused a reduction in indomethacin-induced gastric hemorrhagic lesions. We found that the gastroprotective effects of DHA treatment (100mg/kg) were accompanied by decreases in several parameters: in leukocyte recruitment; gastric levels of myeloperoxidase; leukotriene B4; intercellular adhesion molecule-1; tumor necrosis factor alpha; and nuclear translocation of nuclear factor-кB. Concurrently, we observed an improvement in antioxidant defenses produced by the increase in superoxide dismutase and glutathione activities but not catalase; in addition, a decrease in some oxidative damage markers such as malondialdehyde and carbonyl proteins in lipids and proteins was observed. Furthermore, resolvin D1 production and expression of free fatty acid receptor 4 were stimulated by DHA. Therefore, this study identified the antioxidant and anti-inflammatory actions of DHA as the main mechanisms involved in DHA's gastroprotective effects against indomethacin-induced gastric damage.

Behavior of Oxidative Stress Markers in Alcoholic Liver Cirrhosis Patients.

Alcohol is the most socially accepted addictive substance worldwide, and its metabolism is related with oxidative stress generation. The aim of this work was to evaluate the role of oxidative stress in alcoholic liver cirrhosis (ALC). This study included 187 patients divided into two groups: ALC, classified according to Child-Pugh score, and a control group. We determined the levels of reduced and oxidized glutathione (GSH and GSSG) and the GSH/GSSG ratio by an enzymatic method in blood. Also, protein carbonyl and malondialdehyde (MDA) content were estimated in serum. MDA levels increased in proportion to the severity of damage, whereas the GSH and GSSG levels decreased and increased, respectively, at different stages of cirrhosis. There were no differences in the GSH/GSSG ratio and carbonylated protein content between groups. We also evaluated whether the active consumption of or abstinence from alcoholic beverages affected the behavior of these oxidative markers and only found differences in the MDA, GSH, and GSSG determination and the GSH/GSSG ratio. Our results suggest that alcoholic cirrhotic subjects have an increase in oxidative stress in the early stages of disease severity and that abstinence from alcohol consumption favors the major antioxidant endogen: GSH in patients with advanced disease severity.

Trolox mitigates fibrosis in a bile duct ligation model.

Several studies suggest that free radicals may play a role in cholestatic liver injury. The aim of this work was to evaluate the role of trolox in chronic bile duct ligation (BDL). Liver injury was induced by 28-day BDL to male Wistar rats. Animals were divided in four groups of six rats. Trolox was administered daily (50 mg/kg, p.o.). Alanine aminotransferase (ALT) was quantified in serum. Fibrosis was assessed measuring liver hydroxyproline content. Reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, catalase (CAT), and glutathione peroxidase (GPx) activities were measured in liver. Transforming growth factor-ß (TGF-ß), interleukin-6 (IL-6), and interleukin-10 (IL-10) were determined by western blot and quantified densitometrically. Our results show that trolox treatment in BDL rats prevented the increase in ALT. Collagen was increased by chronic BDL, but trolox administration preserved the normal collagen concentration. BDL produced high levels of the cytokine TGF-ß1, IL-6, and IL-10 levels. Trolox administration was effective to partially prevent the increase of TGF-ß1 and IL-6, and it was able to further augment the levels of IL-10. Oxidative stress (assessed by lipid peroxidation and liver glutathione content) was increased by BDL; this process was normalized by trolox. The activities of CAT and GPx were altered by BDL, and trolox prevented these events. We found that there is a close relationship between cholestatic liver damage and oxidative stress generation, and this was effectively prevented by trolox. Our study shows that the beneficial effects of trolox are because of its important antioxidant and immunomodulatory properties.

Antifibrotic and antioxidant effects of N-acetylcysteine in an experimental cholestatic model.

OBJECTIVES: Several studies have suggested that oxidative stress may play an important role in the pathogenesis of hepatic injury during cholestasis in rats and humans. The aim of this study was to evaluate the ability of N-acetylcysteine (NAC) to prevent the damage induced by bile duct ligation (BDL) for 28 days in male Wistar rats. METHODS: NAC was administered daily (300 mg/kg, orally) for 28 days. Alanine aminotransferase was quantified in the serum; lipid peroxidation, glutathione, and catalase activity were measured in the liver. Fibrosis was assessed by measuring the liver hydroxyproline content; transforming growth factor-ß (TGF-ß), interleukin (IL)-6, and IL-10 were determined in the liver by a western blot and quantified densitometrically. RESULTS: The induction of cholestatic damage by BDL was associated with an increase in alanine aminotransferase. Oxidative stress was also evaluated; lipid peroxidation increased, whereas the liver glutathione content and catalase activity decreased by BDL. NAC treatment prevented these alterations. Hydroxyproline was increased by chronic BDL, but NAC preserved the normal hydroxyproline levels. Cytokines TGF-ß, IL-6, and IL-10 increased after 28 days of BDL. NAC was effectively significant in preventing TGF-ß and IL-6 expression and further augmented the IL-10 expression. CONCLUSION: Our data indicate that in the development to cholestatic liver damage, oxidative stress plays an important role and this in turn leads to fibrosis. This study shows that the beneficial effects of NAC are because of its antioxidant and immunomodulatory properties.

N-acetylcysteine prevents carbon tetrachloride-induced liver cirrhosis: role of liver transforming growth factor-beta and oxidative stress.

OBJECTIVES: N-acetylcysteine (NAC) is an antioxidant, a precursor of reduced glutathione, and an inhibitor of the profibrotic cytokine liver transforming growth factor-beta (TGF-beta). Carbon tetrachloride (CCl4) cirrhosis is characterized by oxidative stress and fibrosis. Therefore, the aim of this work was to study the effect of NAC on experimental cirrhosis. METHODS: CCl4 was chronically administered for 8 weeks along with 300 mg/kg of NAC orally once a day. Alkaline phosphatase, alanine aminotransferase, and gamma-glutamyltranspeptidase were measured in plasma. Hydroxyproline, glycogen, lipid peroxidation, glutathione were determined in liver samples by colorimetric methods. TGF-beta was evaluated by western blotting, and a histopathological analysis was performed. RESULTS: Serum markers of liver damage increased by CCl4 intoxication (P<0.05), whereas cotreatment with NAC prevented these increases (P<0.05); glycogen was depleted in the cirrhotic group (P<0.05), but preserved by NAC (P<0.05). Lipid peroxidation increased and glutathione decreased by the administration of CCl4 (P<0.05), again NAC prevented both effects (P<0.05). Importantly, collagen increased by about seven-fold in the CCl4 group (P<0.05); administration of NAC preserved the normal levels of collagen (P<0.05). Biochemical determinations were corroborated by hematoxylin and eosin, and trichromic stains. Western blots revealed a four-fold increase in TGF-beta in the group receiving CCl4, NAC cotreatment abolished TGF-beta signal (P<0.05). CONCLUSION: Our results strongly suggest that NAC prevents experimental cirrhosis by two mechanisms: by preventing oxidative stress and by downregulating the profibrogenic cytokine TGF-beta. As NAC is currently used in humans intoxicated with paracetamol, it can be tested in fibrotic or cirrhotic patients under controlled trials.

Trolox down-regulates transforming growth factor-beta and prevents experimental cirrhosis.

Cirrhosis is a very common disease and its treatment is limited due to lack of effective drugs. Some studies indicate that this disease is associated with oxidative stress. Therefore, we decided to study the effect of trolox, an effective antioxidant, on experimental cirrhosis. Cirrhosis was induced by CCl4 administration (0.4 g/kg, intraperitoneally, three times per week, for 8 weeks) to Wistar male rats. Trolox was administered daily (50 mg/kg, orally). Fibrosis was assessed histologically and by measuring liver hydroxyproline content. Glutathione, lipid peroxidation and glycogen were measured in liver; serum markers of liver damage were also quantified. Transforming growth factor-beta (TGF-beta) was determined by Western blot and quantified densitometrically. Alkaline phosphatase, gamma-glutamyl transpeptidase and alanine aminotransferase increased in the group receiving CCl4; trolox completely or partially prevented these alterations. Glycogen was almost depleted by CCl4 but was partially preserved by trolox. Lipid peroxidation increased while glutathione decreased by CCl4 administration; trolox corrected both effects. Histology showed thick bands of collagen, necrosis and distortion of the hepatic parenchyma in the CCl4 group, such effects were prevented by trolox. Hydroxyproline content increased 5-fold by CCl4, while the group receiving both CCl4 and trolox showed no significant difference compared to the control group. CCl4 increased 3-fold TGF-beta, while trolox completely prevented this increase. We found that trolox effectively prevented cirrhosis induced with CCl4 in the rat. Our results suggest that the beneficial effects of trolox may be associated to its antioxidant properties and to its ability to reduce the profibrogenic cytokine TGF-beta expression.