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.

Advances of Nanotechnology in the Diagnosis and Treatment of Hepatocellular Carcinoma.

Nanotechnology has emerged as a promising technology in the field of hepatocellular carcinoma (HCC), specifically in the implementation of diagnosis and treatment strategies. Nanotechnology-based approaches, such as nanoparticle-based contrast agents and nanoscale imaging techniques, have shown great potential for enhancing the sensitivity and specificity of HCC detection. These approaches provide high-resolution imaging and allow for the detection of molecular markers and alterations in cellular morphology associated with HCC. In terms of treatment, nanotechnology has revolutionized HCC therapy by enabling targeted drug delivery, enhancing therapeutic efficacy, and minimizing off-target effects. Nanoparticle-based drug carriers can be functionalized with ligands specific to HCC cells, allowing for selective accumulation of therapeutic agents at the tumor site. Furthermore, nanotechnology can facilitate combination therapy by co-encapsulating multiple drugs within a single nanoparticle, allowing for synergistic effects and overcoming drug resistance. This review aims to provide an overview of recent advances in nanotechnology-based approaches for the diagnosis and treatment of HCC. Further research is needed to optimize the design and functionality of nanoparticles, improve their biocompatibility and stability, and evaluate their long-term safety and efficacy. Nonetheless, the integration of nanotechnology in HCC management holds great promise and may lead to improved patient outcomes in the future.

AdhMMP8 Vector Administration in Muscle: An Alternate Strategy to Regress Hepatic Fibrosis.

The development of several vaccines against the SARS-CoV2 virus and their application in millions of people have shown efficacy and safety in the transfer of genes to muscle turning this tissue into a protein-producing factory. Established advanced liver fibrosis, is characterized by replacement of hepatic parenchyma by tissue scar, mostly collagen type I, with increased profibrogenic and proinflammatory molecules gene expression. Matrix metalloproteinase 8 (MMP-8) is an interstitial collagen-degrading proenzyme acting preferentially on collagen type I when activated. This study was carried out to elucidate the effect of an intramuscularly delivered adenoviral vector containing proMMP-8 gene cDNA (AdhMMP8) in male Wistar rats with experimental advanced liver fibrosis induced by thioacetamide. Therapeutic effects were monitored after 1, 2, or 3 weeks of a single dose (3 × 1011 vp/kg) of AdhMMP8. Circulating and liver concentration of MMP-8 protein remained constant; hepatic fibrosis decreased up to 48%; proinflammatory and profibrogenic genes expression diminished: TNF-α 2.28-fold, IL-1 1.95-fold, Col 1A1 4-fold, TGF-ß1 3-fold and CTGF 2-fold; and antifibrogenic genes expression raised, MMP-9 2.8-fold and MMP-1 10-fold. Our data proposes that the administration of AdhMMP8 in muscle is safe and effective in achieving liver fibrosis regression at a comparable extent as when the adenoviral vector is delivered systemically to reach the liver, using a minimally invasive procedure.

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.

Impaired muscle strength is associated with ultrastructure damage in myositis.

The muscle fiber ultrastructure in Idiopathic Inflammatory Myopathies (IIM) has been scarcely explored, especially in Inclusion Body Myositis. The aim of this study was to implement the Scanning Electron Microscopy (SEM) in a small cohort of IIM patients, together with the characterization of immunological profile for a better understanding of the pathophysiology. For immunological profile characterization, we identified the presence of autoantibodies (Ro-52, OJ, EJ, PL7, PL12, SRP, Jo-1, PMScl75, PMScl100, Ku, SAE1, NXP2, MDA5, TIF1γ, Mi-2α, Mi-2ß) and quantified cytokines (IL-1ß, IFN-α2, IFN-γ, TNF-α, IL-6, IL-10, IL-12p70, IL-17A, IL-18, IL-23, IL-33) and chemokines (CCL2, CXCL8). The histological analysis was made by hematoxylin-eosin staining while the muscle fiber ultrastructure was characterized by SEM. We observed changes in the morphology and structure of the muscle fiber according to muscle strength and muscle enzymes. We were able to find and describe muscle fiber ultrastructure with marked irregularities, porosities, disruption in the linearity and integrity of the fascicle, more evident in patients with increased serum levels of muscle enzymes and diminished muscle strength. Despite the scarce reports about the use of SEM as a tool in all clinical phenotypes of IIM, our work provides an excellent opportunity to discuss and reframe the clinical usefulness of SEM in the diagnostic approach of IIM.

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.

Validation of the Spanish-Mexican Version of the Australian Breastfeeding Attitude Questionnaire in Higher Education Health Students.

Positive attitudes towards breastfeeding in health professionals/students have been associated with increasing their confidence to provide support and accompaniment to mothers. In Mexico, there is no valid/reliable tool to assess attitudes towards breastfeeding in this population. The Australian Breastfeeding Attitudes (and Knowledge) Questionnaire (ABAQ) measures attitudes in the Australian population. We aimed to adapt and validate the ABAQ in Mexican health students. We included 264 health students (nursing, nutrition, and medicine) from the University of Guadalajara. Bilingual translators carried out the Spanish adaptation with a reverse translation into English. Experts evaluated the content validity. Reliability was evaluated through an internal consistency analysis (Cronbach's alpha) and construct validity through convergent-divergent validation, item-total correlation, exploratory factor analysis (by principal components), and confirmatory factor analysis. According to the exploratory factor analysis, only one component was identified. Seven items were removed (low correlation between items ≤0.2 and low factor load ≤0.3). The Cronbach's alpha was 0.78. According to the confirmatory factor analysis, the one-factor solution of the ABAQ-13Mx showed a good model fit (X2 = 98.41, G = 62, p = 0.02, CFI = 0.940, and RMSEA = 0.048). The ABAQ-13Mx is a reliable and valid instrument for evaluating attitudes towards breastfeeding in Mexican health degree students.

Gene therapy with urokinase-type plasminogen activator regenerates liver tissue.

BACKGROUND AND OBJECTIVES: Gene therapy using urokinase-type plasminogen activator (uPA) has been shown to induce extracellular matrix degradation, hepatocyte proliferation and liver tissue function restoration in liver cirrhosis models. Physiologically, uPA activates plasminogen conversion to plasmin, which leads, depending on the organ, to thrombolysis or extracellular matrix degradation. The purpose of this study was to compare the regenerative effect of gene therapy with adenoviruses encoding wild-type uPA (huPA), as well as its truncated isoform (ΔhuPA), in healthy and cirrhotic animals. In addition, possible adverse effects on coagulation were assessed. METHODS: 6 x 1011 vp/kg of Ad-huPA or Ad-ΔhuPA were administered via the iliac vein to healthy male Wistar rats or to male Wistar rats with cirrhosis induced by chronic poisoning with carbon tetrachloride (CCl4). The animals were sacrificed at day 2, 4 or 6 post-treatment. Liver fibrosis, proliferating cell nuclear antigen expression, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels and coagulation markers were evaluated. RESULTS: On day 6 post-treatment, a fibrosis reversal of 48.7-41.5% was achieved. AST and ALT levels did not change in cirrhotic animals treated with ΔhuPA, but showed an elevation in healthy animals. Cell proliferation increased in healthy and cirrhotic animals with both transgene isoforms. No coagulation adverse effects were observed in the ΔhuPA group, and by day 6, they had disappeared in the huPA group. CONCLUSIONS: Gene therapy with Ad-huPA and Ad-ΔhuPA favors cell proliferation in cirrhotic animals, without important side effects.

ANTECEDENTES Y OBJETIVOS: La terapia génica empleando el activador de plasminógeno tipo urocinasa (huPA) ha demostrado que induce la degradación de matriz extracelular, la proliferación de hepatocitos y el restablecimiento de la funcionalidad tisular en modelos de cirrosis hepática. Fisiológicamente, el uPA activa la conversión del plasminógeno en plasmina, lo que conlleva, dependiendo del órgano, una trombólisis o a degradación de la matriz extracelular. El objetivo de este estudio fue comparar el efecto regenerador de la terapia génica con adenovirus codificando la forma silvestre (huPA), así como su isoforma truncada (DhuPA) en animales sanos y cirróticos. Además, se valoraron los posibles efectos adversos sobre la coagulación. MÉTODOS: Se administró 6x1011 pv/kg del Ad-huPA o Ad-ΔhuPA a ratas Wistar macho sanas o con cirrosis por intoxicación crónica con tetracloruro de carbono (CCl4) vía vena ilíaca. El sacrificio fue al día 2, 4 o 6 postratamiento. Se evaluó la fibrosis hepática, la expresión de antígeno nuclear de proliferación celular y niveles séricos de alanina aminotransferasa (ALT) y aspartato aminotransferasa (AST), y marcadores de coagulación. RESULTADOS: Al día 6 postratamiento se alcanzó una reversión de fibrosis del 48.7-41.5%. Los niveles de AST y ALT no cambiaron en animales cirróticos tratados con DhuPA, pero aumentaron en animales sanos comparados con el control sano no tratado. La proliferación aumenta en animales sanos y cirróticos con ambas isoformas del transgén. No se observaron efectos adversos en la coagulación en el grupo DhuPA y para el día 6 habían desaparecido en el grupo huPA. CONCLUSIONES: La terapia génica con Ad-uPA y Ad-ΔhuPA favorece la proliferación celular en animales cirróticos, sin efectos secundarios importantes.

An Update in Epigenetics in Metabolic-Associated Fatty Liver Disease.

Metabolic-associated fatty liver disease (MAFLD) is characterized by hepatic steatosis accompanied by one of three features: overweight or obesity, T2DM, or lean or normal weight with evidence of metabolic dysregulation. It is distinguished by excessive fat accumulation in hepatocytes, and a decrease in the liver's ability to oxidize fats, the accumulation of ectopic fat, and the activation of proinflammatory pathways. Chronic damage will keep this pathophysiologic cycle active causing progression from hepatic steatosis to cirrhosis and eventually, hepatocarcinoma. Epigenetics affecting gene expression without altering DNA sequence allows us to study MAFLD pathophysiology from a different perspective, in which DNA methylation processes, histone modifications, and miRNAs expression have been closely associated with MAFLD progression. However, these considerations also faced us with the circumstance that modifying those epigenetics patterns might lead to MAFLD regression. Currently, epigenetics is an area of great interest because it could provide new insights in therapeutic targets and non-invasive biomarkers. This review comprises an update on the role of epigenetic patterns, as well as innovative therapeutic targets and biomarkers in MAFLD.

Fibrosis regression is induced by AdhMMP8 in a murine model of chronic kidney injury.

Adenoviral vector AdhMMP8 (human Metalloproteinase-8 cDNA) administration has been proven beneficial in various experimental models of liver injury improving liver function and decreasing fibrosis. In this study, we evaluated the potential therapeutic AdhMMP8 effect in a chronic kidney damage experimental model. Chronic injury was induced by orogastric adenine administration (100mg/kg/day) to Wistar rats for 4 weeks. AdhMMP8 (3x1011vp/kg) was administrated in renal vein during an induced-ligation-ischemic period to facilitate kidney transduction causing no-additional kidney injury as determined by histology and serum creatinine. Animals were sacrificed at 7- and 14-days post-Ad injection. Fibrosis, histopathological features, serum creatinine (sCr), BUN, and renal mRNA expression of αSMA, Col-1α, TGF-ß1, CTGF, BMP7, IL-1, TNFα, VEGF and PAX2 were analyzed. Interestingly, AdhMMP8 administration resulted in cognate human MMP8 protein detection in both kidneys, whereas hMMP8 mRNA was detected only in the left kidney. AdhMMP8 significantly reduced kidney tubule-interstitial fibrosis and glomerulosclerosis. Also, tubular atrophy and interstitial inflammation were clearly decreased rendering improved histopathology, and down regulation of profibrogenic genes expression. Functionally, sCr and BUN were positively modified. The results showed that AdhMMP8 decreased renal fibrosis, suggesting that MMP8 could be a possible therapeutic candidate for kidney fibrosis treatment.

Adenovirus Biodistribution is Modified in Sensitive Animals Compared to Naïve Animals.

Pre-existing immune response against adenovirus could diminish transgene expression efficiency when Ad is employed in humans as gene therapy vector. We previously used Ad-hΔuPA (Recombinant adenovirus expressing human urokinase-type plasminogen activator) as antifibrotic gene therapy in cirrhosis models and demonstrated its effectiveness. As a further clinical approach, transient Cyclosporine A (CsA) immunosuppression was induced in cirrhotic animals to determine whether Ad-hΔuPA administration retained efficacy. Adenovirus sensitization was achieved by systemic administration of non-therapeutic Ad-ßGal (Recombinant adenovirus expressing beta-galactosidase) after 4 weeks of intraperitoneal carbon tetrachloride (CCl4) regimen. Cirrhosis induction continued up to 8 weeks. At the end of CCl4 intoxication, immunosuppression was achieved with three CsA doses (40 mg/kg) as follows: 24 h before administration of Ad-hΔuPA, at the moment of Ad-hΔuPA injection and finally, 24 h after Ad-hΔuPA inoculation. At 2 and 72 h after Ad-hΔuPA injection, animals were sacrificed. Liver, spleen, lung, kidney, heart, brain, and testis were analyzed for Ad-biodistribution and transgene expression. In naïve animals, Ad-hΔuPA genomes prevailed in liver and spleen, while Ad-sensitized rats showed Ad genomes also in their kidney and heart. Cirrhosis and Ad preimmunization status notably diminished transgene liver expression compared to healthy livers. CsA immunosuppression in cirrhotic animals has no effect on Ad-hΔuPA biodistribution, but increments survival.

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.

Effect of Ursolic Acid on Insulin Resistance and Hyperinsulinemia in Rats with Diet-Induced Obesity: Role of Adipokines Expression.

Excess of visceral adipose tissue (VAT) characteristic of obesity leads to a proinflammatory state disrupting the insulin signaling pathway, triggering insulin resistance (IR) and inflammation, the main processes contributing to obesity comorbidities. Ursolic acid (UA), a pentacyclic triterpenoid occurring in a variety of plant foods, exhibits anti-inflammatory properties. The aim of this study was to evaluate UA effects on IR, hyperinsulinemia, and inflammation in experimental diet-induced obesity. Forty male Wistar rats were randomly assigned to eight groups (n = 5). One group was used for time 0. Three groups were labeled as OBE (control): receiving high-fat diet (HFD; fat content 45.24% of energy) during 3, 6, or 9 weeks; three groups UA-PREV: exposed to simultaneous HFD and UA during 3, 6, or 9 weeks to evaluate UA preventive effects; one group UA-REV: receiving HFD for 6 weeks, followed by simultaneous HFD and UA for three additional weeks to analyze UA reversal effects. Measurements were performed after 3, 6, or 9 weeks of treatment. Adiposity was calculated by weighing VAT after sacrifice. Serum markers were quantified through colorimetric and enzyme-linked immunosorbent assay methods. VAT adipokines RNAm expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction. Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests. UA significantly decreased adiposity, IR, hyperinsulinemia, triacylglycerides, and cholesterol levels, and also VAT mRNA expression of MCP-1 (monocyte chemoattractant protein-1), IL (interleukin)-1ß and IL-6, concomitantly increasing adiponectin levels. UA metabolic effects demonstrated in this study support its potential therapeutic utility to improve IR, hyperinsulinemia, and inflammation observed in obesity and diabetes.

Delphinidin Ameliorates Hepatic Triglyceride Accumulation in Human HepG2 Cells, but Not in Diet-Induced Obese Mice.

Anthocyanin consumption is linked to benefits in obesity-related metabolic alterations and non-alcoholic fatty liver disease (NAFLD), though the functional role of delphinidin (Dp) is yet to be established. Therefore, this study examined the effects of Dp on metabolic alterations associated with NAFLD, and molecular mechanisms in HepG2 cells and diet-induced obese mice. Cells incubated with palmitate to induce lipid accumulation, concomitantly treated with Dp, reduced triglyceride accumulation by ~53%, and downregulated gene expression of CPT1A, SREBF1, and FASN without modifying AMP-activated protein kinase (AMPK) levels. C57BL/6Nhsd mice were fed a standard diet (control) or a high-fat/high-carbohydrate diet (HFHC) for 16 weeks. Mice in the HFHC group were subdivided and treated with Dp (HFHC-Dp, 15 mg/kg body weight/day) or a vehicle for four weeks. Dp did not affect body weight, energy intake, hyperglycemia, insulin resistance, or histological abnormalities elicited by the HFHC diet. Furthermore, the messenger RNA (mRNA) expressions of Acaca, and Fasn in hepatic or epididymal adipose tissue, and the hepatic sirtuin 1 (SIRT1)/liver kinase B1 (LKB1)/AMPK and proliferator-activated receptor alpha (PPARα) signaling axis did not significantly change due to the HFHC diet or Dp. In summary, Dp effectively reduced triglyceride accumulation in vitro through the modulation of lipid metabolic gene expression. However, a dose of Dp administrated in mice simulating the total daily anthocyanin intake in humans had no effect on either metabolic alterations or histological abnormalities associated with HFHC diets.

Interferon-α Silencing by Small Interference RNA Increases Adenovirus Transduction and Transgene Expression in Huh7 Cells.

Adenoviruses are the most common vectors used in clinical trials of gene therapy. In 2017, 21.2% of clinical trials used rAds as vectors. Systemic administration of rAds results in high tropism in the liver. Interferon types α and ß are the major antiviral cytokines which orchestrate the host's immune response against rAd, limiting therapeutic gene expression and preventing subsequent vector administration. siRNA is small double-strand RNAs that temporally inhibit the expression of a specific gene. The aim is to evaluate the effect of IFN-α blocking by a specific siRNA on Ad-GFP transduction and on transgene expression in Huh7 cells in culture. Huh7 cells were cultured in DMEM and transfected with 70 nM of siRNA-IFN-α. Six hours later, the cells were exposed to 1 × 109 vp/ml of rAd-GFP for 24 h. Expression of IFN-α, TNF-α and the PKR gene was determined by RT-qPCR. Percentage of transduction was analyzed by flow cytometry and by qPCR. GFP expression was determined by western blot. 70 nM of siRNA-IFN-α inhibited 96% of IFN-α and 65% of TNF-α gene expression compared to an irrelevant siRNA. Percentage of transduction and transgene expression increased in these cells compared to an irrelevant siRNA. Inhibition of IFN-α expression by siRNA-IFN-α enabled a higher level of transduction and transgene expression GFP, highlighting the role of IFN-α in the elimination of adenovirus in transduced cells and thus suggesting that its inhibition could be an important strategy for gene therapy in clinical trials using adenovirus as a vector directed to liver diseases.

Protective Effects of Moringa oleifera on HBV Genotypes C and H Transiently Transfected Huh7 Cells.

Chronic hepatitis B infection treatment implicates a long-lasting treatment. M. oleifera extracts contain compounds with antiviral, antioxidant, and antifibrotic properties. In this study, the effect of M. oleifera was evaluated in Huh7 cells expressing either HBV genotypes C or H for the antiviral, antifibrotic, anti-inflammatory, and antioxidative responses. Huh7 cells were treated with an aqueous extract of M. oleifera (leaves) at doses of 0, 30, 45, or 60 µg/mL. The replicative virus and TGF-ß1, CTGF, CAT, IFN-ß1, and pgRNA expressions were measured by real time. HBsAg and IL-6 titers were determined by ELISA. CTGF, TGF-ß1, IFN-ß1, and pgRNA expressions decreased with M. oleifera treatment irrespective of the HBV genotype. HBsAg secretion in the supernatant of transfected Huh7 cells with both HBV genotypes was decreased regardless of the dose of M. oleifera. Similar effect was observed in proinflammatory cytokine IL-6, which had a tendency to decrease at 24 hours of treatment. Transfection with both HBV genotypes strongly decreased CAT expression, which is retrieved with M. oleifera treatment. M. oleifera treatment reduced fibrosis markers, IL-6, and HBsAg secretion in HBV genotypes C and H. However, at the level of replication, only HBV-DNA genotype C was slightly reduced with this treatment.

Human adipose derived stem cells regress fibrosis in a chronic renal fibrotic model induced by adenine.

BACKGROUND AND AIMS: ADSCs transplantation had been shown in some experimental models of kidney damage that it improves kidney function and reduces fibrosis. In this study we evaluated the effect of human adipose tissue-derived stem cell (hADSC) therapy in a chronic kidney damage experimental model. METHODS: A chronic kidney injury was induced by daily orogastric administration of adenine (100mg/kg) to male Wistar rats for 28 days. hADSCs were isolated, expanded and characterized before transplantation. hADSC administration was performed in a tail vein at a dose of 2 x106 cells/animal. Animals were sacrificed at 7 days post-treatment. The percentage of fibrotic tissue, serum and urine levels of urea, creatinine, total protein and renal mRNA of COL1A1, TGFB1, CTGF, ACTA2, IL6, IL10, TNF were analyzed. RESULTS: hADSCs treatment significantly reduces kidney fibrosis, improves urea and creatinine serum and urine levels, and diminishes COL1A1, TGFB1, CTGF, ACTA2 mRNA kidney levels. CONCLUSIONS: These results showed that cell therapy using hADSCs improves renal function and reduces fibrosis.

Simultaneous Administration of ADSCs-Based Therapy and Gene Therapy Using Ad-huPA Reduces Experimental Liver Fibrosis.

BACKGROUND AND AIMS: hADSCs transplantation in cirrhosis models improves liver function and reduces fibrosis. In addition, Ad-huPA gene therapy diminished fibrosis and increased hepatocyte regeneration. In this study, we evaluate the combination of these therapies in an advanced liver fibrosis experimental model. METHODS: hADSCs were expanded and characterized before transplantation. Ad-huPA was simultaneously administrated via the ileac vein. Animals were immunosuppressed by CsA 24 h before treatment and until sacrifice at 10 days post-treatment. huPA liver expression and hADSCs biodistribution were evaluated, as well as the percentage of fibrotic tissue, hepatic mRNA levels of Col-αI, TGF-ß1, CTGF, α-SMA, PAI-I, MMP2 and serum levels of ALT, AST and albumin. RESULTS: hADSCs homed mainly in liver, whereas huPA expression was similar in Ad-huPA and hADSCs/Ad-huPA groups. hADSCs, Ad-huPA and hADSCs/Ad-huPA treatment improves albumin levels, reduces liver fibrosis and diminishes Collagen α1, CTGF and α-SMA mRNA liver levels. ALT and AST serum levels showed a significant decrease exclusively in the hADSCs group. CONCLUSIONS: These results showed that combinatorial effect of cell and gene-therapy does not improve the antifibrogenic effects of individual treatments, whereas hADSCs transplantation seems to reduce liver fibrosis in a greater proportion.