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.

Oxidative and Fibrinolytic Mechanisms: Two Important Processes to Consider in Platelet Storage

Objective: Platelet-rich plasma (PRP) is used in multiple coagulation disorders. Its therapeutic effectiveness relies on technical procedures related to PRP procurement and preservation because free radicals induce platelet activation and aging. This work aims to elucidate the oxidative mechanisms involved in activation of platelets obtained from PRP during storage. Materials and Methods: One hundred ten PRP batches were obtained from healthy donors and kept under stirring at a temperature of 20-24 °C. Protein extraction was performed from platelet homogenates and plasma at different times of storage from day 1 to 20. The activities of antioxidant markers such as catalase (CAT), superoxide dismutase, and ceruloplasmin, as well as fibrinolytic protein activity metalloproteases 2 and 3, plasmin, and urokinase plasminogen activator, were analyzed by zymography assays. Oxidized proteins were also determined. Results: Significant activity of antioxidant enzymes and fibrinolytic molecules was observed on day 5 of storage in PRP homogenates, which increased over time and was concomitantly correlated with oxidized protein levels. Reverse enzymatic activity patterns were observed in plasma, except for CAT, which remained unchanged. Conclusion: Storage conditions of platelets from PRP for up to 5 days induced in vitro platelet activation by oxidative damage and proteolysis. This finding confirms the need for proper management of these blood products to preserve their viability and functionality.

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.

The Role of NRF2 in Obesity-Associated Cardiovascular Risk Factors.

The raising prevalence of obesity is associated with an increased risk for cardiovascular diseases (CVDs), particularly coronary artery disease (CAD), and heart failure, including atrial fibrillation, ventricular arrhythmias and sudden death. Obesity contributes directly to incident cardiovascular risk factors, including hyperglycemia or diabetes, dyslipidemia, and hypertension, which are involved in atherosclerosis, including structural and functional cardiac alterations, which lead to cardiac dysfunction. CVDs are the main cause of morbidity and mortality worldwide. In obesity, visceral and epicardial adipose tissue generate inflammatory cytokines and reactive oxygen species (ROS), which induce oxidative stress and contribute to the pathogenesis of CVDs. Nuclear factor erythroid 2-related factor 2 (NRF2; encoded by Nfe2l2 gene) protects against oxidative stress and electrophilic stress. NRF2 participates in the regulation of cell inflammatory responses and lipid metabolism, including the expression of over 1000 genes in the cell under normal and stressed environments. NRF2 is downregulated in diabetes, hypertension, and inflammation. Nfe2l2 knockout mice develop structural and functional cardiac alterations, and NRF2 deficiency in macrophages increases atherosclerosis. Given the endothelial and cardiac protective effects of NRF2 in experimental models, its activation using pharmacological or natural products is a promising therapeutic approach for obesity and CVDs. This review provides a comprehensive summary of the current knowledge on the role of NRF2 in obesity-associated cardiovascular risk factors.

Pirfenidone modifies hepatic miRNAs expression in a model of MAFLD/NASH.

miRNAs are involved in the development of metabolic associated fatty liver disease (MAFLD) and nonalcoholic steatohepatitis (NASH). We aimed to evaluate modifications by prolonged-release pirfenidone (PR-PFD) on key hepatic miRNAs expression in a MAFLD/NASH model. First, male C57BL/6J mice were randomly assigned into groups and fed with conventional diet (CVD) or high fat and carbohydrate diet (HFD) for 16 weeks. At the end of the eighth week, HFD mice were divided in two and only one half was treated with 300 mg/kg/day of PR-PFD mixed with food. Hepatic expression of miRNAs and target genes that participate in inflammation and lipid metabolism was determined by qRT-PCR and transcriptome by microarrays. Increased hepatic expression of miR-21a-5p, miR-34a-5p, miR-122-5p and miR-103-3p in MAFLD/NASH animals was reduced with PR-PFD. Transcriptome analysis showed that 52 genes involved in lipid and collagen biosynthesis and inflammatory response were downregulated in PR-PFD group. The expression of Il1b, Tnfa, Il6, Tgfb1, Col1a1, and Srebf1 were decreased in PR-PFD treated animals. MAFLD/NASH animals compared to CVD group showed modifications in gene metabolic pathways implicated in lipid metabolic process, inflammatory response and insulin resistance; PR-PFD reversed these modifications.

Molecular Mechanisms of Obesity-Linked Cardiac Dysfunction: An Up-Date on Current Knowledge.

Obesity is defined as excessive body fat accumulation, and worldwide obesity has nearly tripled since 1975. Excess of free fatty acids (FFAs) and triglycerides in obese individuals promote ectopic lipid accumulation in the liver, skeletal muscle tissue, and heart, among others, inducing insulin resistance, hypertension, metabolic syndrome, type 2 diabetes (T2D), atherosclerosis, and cardiovascular disease (CVD). These diseases are promoted by visceral white adipocyte tissue (WAT) dysfunction through an increase in pro-inflammatory adipokines, oxidative stress, activation of the renin-angiotensin-aldosterone system (RAAS), and adverse changes in the gut microbiome. In the heart, obesity and T2D induce changes in substrate utilization, tissue metabolism, oxidative stress, and inflammation, leading to myocardial fibrosis and ultimately cardiac dysfunction. Peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of carbohydrate and lipid metabolism, also improve insulin sensitivity, triglyceride levels, inflammation, and oxidative stress. The purpose of this review is to provide an update on the molecular mechanisms involved in obesity-linked CVD pathophysiology, considering pro-inflammatory cytokines, adipokines, and hormones, as well as the role of oxidative stress, inflammation, and PPARs. In addition, cell lines and animal models, biomarkers, gut microbiota dysbiosis, epigenetic modifications, and current therapeutic treatments in CVD associated with obesity are outlined in this paper.

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.

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.

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.

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.

Novel Triamcinolone Acetonide-Loaded Liposomes Topical Formulation for the Treatment of Cystoid Macular Edema After Cataract Surgery: A Pilot Study.

PURPOSE: To report tolerability, safety, and efficacy of a topical triamcinolone acetonide-loaded liposomes formulation (TA-LF) in targeting the macular area in patients with refractory pseudophakic cystoid macular edema (PCME). METHODS: For tolerability, safety and efficacy evaluation, 12 eyes of 12 patients with refractory PCME were exposed to one drop of TA-LF (TA at 0.2%) every 2 h for 90 days or until best-corrected visual acuity (BCVA) was achieved. Intraocular pressure (IOP), slit lamp examination, and central foveal thickness (CFT) were analyzed at every visit. RESULTS: Patients with refractory PCME under TA-LF therapy showed a significant improvement in BVCA and CFT without significant IOP modification (P = 0.94). On average CFT decreased to 206.75 ± 135.72 µm and BCVA improved to 20.08 ± 10.35 letters (P < 0.0005). BCVA was achieved at 10.58 ± 6.70 weeks (range 2-18). TA-LF was well tolerated in all cases. Neither ocular surface abnormalities nor adverse events were recorded. CONCLUSION: TA-LF was well tolerated and improved BCVA and CFT on patients with refractory PCME.

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

[This corrects the article DOI: 10.1371/journal.pone.0187907.].

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.

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.

IRS-1 pY612 and Akt-1/PKB pT308 Phosphorylation and Antiinflammatory Effect of Diindolylmethane in Adipocytes Cocultured with Macrophages.

BACKGROUND: 3,3'-Diindolylmethane (DIM) is a condensation product of indole-3-carbinol, a glucosinolate naturally occurring in Brassica genus vegetables. The antiinflammatory properties of DIM through the inhibition of NF-κB, as well as its ameliorating effects on glucose tolerance and hyperglicemic states, have been described. A subclinical proinflammatory profile resultant from the interaction of adipocytes and macrophages has been reported in obesity, affecting the insulin signaling pathway, contributing to insulin resistance. OBJECTIVE: The aim of this study was to evaluate the effect of DIM on proinflammatory cytokines and phosphorylation of IRS-1 pY612 and Akt-1/PKB pT308 in an obesity-induced inflammation model. METHODS: Differentiated 3T3-L1 adipocytes were co-cultured with RAW 264.7 macrophages and exposed to 20 µM, 40 µM and 60 µM DIM for 24 h followed by 100 nM insulin for 20 min. MCP-1, IL-6 and TNFα were quantified in the supernatant through individual ELISAs. Adipocyte lysates were used to determine the relative expression of the proinflammatory mediators by qPCR, and the phosphorylation of IRS-1 pY612 and Akt-1/PKB pT308 proteins by western blot analysis. RESULTS: DIM significantly (p<0.05) reduced the production and mRNA expression of MCP-1, IL-6, and TNFα in a DIM concentration dependent manner, concomitantly increasing the abundance of IRS-1 pY612 and Akt-1/PKB pT308. CONCLUSION: Our results suggest that DIM influences the insulin transduction pathway by exerting an antiinflammatory effect. The potential therapeutic benefits of DIM in the treatment of glucose metabolic disorders deserve further studies.

Pirfenidone Accelerates Wound Healing in Chronic Diabetic Foot Ulcers: A Randomized, Double-Blind Controlled Trial.

BACKGROUND: Diabetic foot ulcers are one disabling complication of diabetes mellitus. Pirfenidone (PFD) is a potent modulator of extracellular matrix. Modified diallyl disulfide oxide (M-DDO) is an antimicrobial and antiseptic agent. AIM: To evaluate efficacy of topical PFD + M-DDO in a randomized, double-blind trial versus ketanserin in the treatment of noninfected chronic DFU. METHODS: Patients received PFD + M-DDO or ketanserin for 6 months. Relative ulcer volume (RUV) was measured every month; biopsies were taken at baseline and months 1 and 2 for histopathology and gene expression analysis for COL-1α, COL-4, KGF, VEGF, ACTA2 (α-SMA), elastin, fibronectin, TGF-ß1, TGF-ß3, HIF-1α, and HIF-1ß. RESULTS: Reduction of median RUV in the PFD + M-DDO group was 62%, 89.8%, and 99.7% at months 1-3 and 100% from months 4 to 6. Ketanserin reduced RUV in 38.4%, 56%, 60.8%, 94%, 94.8%, and 100% from the first to the sixth month, respectively. Healing score improved 4.5 points with PFD + M-DDO and 1.5 points with ketanserin compared to basal value. Histology analysis revealed few inflammatory cells and organized/ordered collagen fiber bundles in PFD + M-DDO. Expression of most genes was increased with PFD + M-DDO; 43.8% of ulcers were resolved using PFD + M-DDO and 23.5% with ketanserin. CONCLUSION: PFD + M-DDO was more effective than ketanserin in RUV reduction.

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.

Efecto del ácido alfa lipoico y la pirfenidona en la modulación antioxidante celular contra el daño oxidativo / Effects of alpha lipoic acid and pirfenidone on liver cells antioxidant modulation against oxidative damage

Background: Liver fibrogenic processes are related to cellular redox state. Glutathione (GSH) is the major cellular antioxidant. GSH induced activation could be related to antifibrogenic effects. Aim: To explore the association between the antifibrogenic effect and pro-antioxidant mechanisms of alpha-lipoic acid (ALA) and pirfenidone (PFD). Material and Methods: HepG2 cells and primary HSC cultures were exposed to menadione 0.1 μM (MEN) as oxidative stress inducer and treated to ALA (5 mM) or PFD (10 μM, 100 μM y 1000 μM). Results: In HSC, PFD decreased cell proliferation and the expression of COL1A1, TGF-β1, TIMP1, IL6, TNFα and MCP1 induced by MEN. Furthermore it was confirmed that ALA and PFD activate diverse antioxidants mediators, however MEN decreases this response. Then, MEN, ALA and PFD induce an antioxidant response, the first one as a response to injury and the latter two as pro-antioxidant inducers. Therefore, when cells are exposed to oxidative stress, endogenous systems activate a battery of mediators that increase the antioxidant potential. When these cells are treated with ALA and PFD, de novo formation of protective genes decreases since previous elicited protection induced in response to injury, enhance ALA and PFD effects. Conclusion: Regardless of the route of action, ALA and PFD induce the biosynthesis of antioxidants mediators which is associated with modulation of fibrogenic processes.

[Effects of alpha lipoic acid and pirfenidone on liver cells antioxidant modulation against oxidative damage]. / Efecto del ácido alfa lipoico y la pirfenidona en la modulación antioxidante celular contra el daño oxidativo.

BACKGROUND: Liver fibrogenic processes are related to cellular redox state. Glutathione (GSH) is the major cellular antioxidant. GSH induced activation could be related to antifibrogenic effects. AIM: To explore the association between the antifibrogenic effect and pro-antioxidant mechanisms of alpha-lipoic acid (ALA) and pirfenidone (PFD). MATERIAL AND METHODS: HepG2 cells and primary HSC cultures were exposed to menadione 0.1 µM (MEN) as oxidative stress inducer and treated to ALA (5 mM) or PFD (10 µM, 100 µM y 1000 µM). RESULTS: In HSC, PFD decreased cell proliferation and the expression of COL1A1, TGF-ß1, TIMP1, IL6, TNFα and MCP1 induced by MEN. Furthermore it was confirmed that ALA and PFD activate diverse antioxidants mediators, however MEN decreases this response. Then, MEN, ALA and PFD induce an antioxidant response, the first one as a response to injury and the latter two as pro-antioxidant inducers. Therefore, when cells are exposed to oxidative stress, endogenous systems activate a battery of mediators that increase the antioxidant potential. When these cells are treated with ALA and PFD, de novo formation of protective genes decreases since previous elicited protection induced in response to injury, enhance ALA and PFD effects. CONCLUSION: Regardless of the route of action, ALA and PFD induce the biosynthesis of antioxidants mediators which is associated with modulation of fibrogenic processes.

Genotoxic evaluation of pirfenidone using erythrocyte rodent micronucleus assay.

Pirfenidone is a non-steroidal antifibrotic compound that has been proposed in clinical protocols and experimental studies as a pharmacological treatment for fibroproliferative diseases. The objective of this study was to determine the genotoxicity or cytotoxicity of three doses of pirfenidone using the micronuclei test in peripheral blood erythrocytes of rodent models. Pirfenidone was administered orally to Balb-C mice for 3 days, and also was administered topically to hairless Sprague Dawley rats during the final stage of gestation. Mice were sampled every 24 h over the course of 6 days; pregnant rats were sampled every 24 h during the last 6 days of gestation, and pups were sampled at birth. Blood smears were analyzed and the frequencies of micronucleated erythrocytes (MNEs), micronucleated polychromatic erythrocytes (MNPCEs), and the proportion of polychromatic erythrocytes (PCEs), were recorded in samples from mice, pregnant rats and rat neonates. Increases in MN frequencies (p<0.03) were noted only in the positive control groups. No genotoxic effects or decreased PCE values were observed neither in newborn rats transplacentally exposed to pirfenidone, or in two adult rodent models when pirfenidone was administered orally or topically.