Comparative Screening of the Liver Gene Expression Profiles from Type 1 and Type 2 Diabetes Rat Models.

Experimental animal models of diabetes can be useful for identifying novel targets related to disease, for understanding its physiopathology, and for evaluating emerging antidiabetic treatments. This study aimed to characterize two rat diabetes models: HFD + STZ, a high-fat diet (60% fat) combined with streptozotocin administration (STZ, 35 mg/kg BW), and a model with a single STZ dose (65 mg/kg BW) in comparison with healthy rats. HFD + STZ- induced animals demonstrated a stable hyperglycemia range (350-450 mg/dL), whereas in the STZ-induced rats, we found glucose concentration values with a greater dispersion, ranging from 270 to 510 mg/dL. Moreover, in the HFD + STZ group, the AUC value of the insulin tolerance test (ITT) was found to be remarkably augmented by 6.2-fold higher than in healthy animals (33,687.0 ± 1705.7 mg/dL/min vs. 5469.0 ± 267.6, respectively), indicating insulin resistance (IR). In contrast, a more moderate AUC value was observed in the STZ group (19,059.0 ± 3037.4 mg/dL/min) resulting in a value 2.5-fold higher than the average exhibited by the control group. After microarray experiments on liver tissue from all animals, we analyzed genes exhibiting a fold change value in gene expression <-2 or >2 (p-value <0.05). We found 27,686 differentially expressed genes (DEG), identified the top 10 DEGs and detected 849 coding genes that exhibited opposite expression patterns between both diabetes models (491 upregulated genes in the STZ model and 358 upregulated genes in HFD + STZ animals). Finally, we performed an enrichment analysis of the 849 selected genes. Whereas in the STZ model we found cellular pathways related to lipid biosynthesis and metabolism, in the HFD + STZ model we identified pathways related to immunometabolism. Some phenotypic differences observed in the models could be explained by transcriptomic results; however, further studies are needed to corroborate these findings. Our data confirm that the STZ and the HFD + STZ models are reliable experimental models for human T1D and T2D, respectively. These results also provide insight into alterations in the expression of specific liver genes and could be utilized in future studies focusing on diabetes complications associated with impaired liver function.

Quercetin, a Flavonoid with Great Pharmacological Capacity.

Quercetin is a flavonoid with a low molecular weight that belongs to the human diet's phenolic phytochemicals and nonenergy constituents. Quercetin has a potent antioxidant capacity, being able to capture reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive chlorine species (ROC), which act as reducing agents by chelating transition-metal ions. Its structure has five functional hydroxyl groups, which work as electron donors and are responsible for capturing free radicals. In addition to its antioxidant capacity, different pharmacological properties of quercetin have been described, such as carcinostatic properties; antiviral, antihypertensive, and anti-inflammatory properties; the ability to protect low-density lipoprotein (LDL) oxidation, and the ability to inhibit angiogenesis; these are developed in this review.

Automated Computer-Assisted Image Analysis for the Fast Quantification of Kidney Fibrosis.

Chronic kidney disease (CKD) is a common and worldwide health problem and one of the most important causes of morbidity and mortality. Most primary research on this disease requires evaluating the fibrosis index in animal model kidneys, specifically using Masson's trichrome stain. Different programs are used to calculate the percentage of fibrosis; however, the analysis is time-consuming since one image must be performed at a time. CellProfiler™ is a program designed to analyze data obtained from biological samples and can process multiple images through pipelines, and the results can be exported to databases. This article explains how CellProfiler™ can be used to automatically analyze kidney histology photomicrographs from samples stained with Masson's trichrome stain to assess the percentage of fibrosis in an experimental animal model of CKD. A pipeline was created to analyze Masson's trichrome-stained slides in a model of CDK induced by adenine at doses of 50 mg/kg and 100 mg/kg, in addition to samples with the vehicle (75% glycerin). The results were compared with those obtained by ImageJ, and no significant differences were found between both programs. The CellProfiler™ pipeline made here is a reliable, fast, and easy alternative for kidney fibrosis analysis and quantification in experimental animal models.

Escherichia/Shigella, SCFAs, and Metabolic Pathways-The Triad That Orchestrates Intestinal Dysbiosis in Patients with Decompensated Alcoholic Cirrhosis from Western Mexico.

Gut microbiota undergoes profound alterations in alcohol cirrhosis. Microbiota-derived products, e.g., short chain fatty acids (SCFA), regulate the homeostasis of the gut-liver axis. The objective was to evaluate the composition and functions of the intestinal microbiota in patients with alcohol-decompensated cirrhosis. Fecal samples of 18 patients and 18 healthy controls (HC) were obtained. Microbial composition was characterized by 16S rRNA amplicon sequencing, SCFA quantification was performed by gas chromatography (GC), and metagenomic predictive profiles were analyzed by PICRUSt2. Gut microbiota in the cirrhosis group revealed a significant increase in the pathogenic/pathobionts genera Escherichia/Shigella and Prevotella, a decrease in beneficial bacteria, such as Blautia, Faecalibacterium, and a decreased α-diversity (p < 0.001) compared to HC. Fecal SCFA concentrations were significantly reduced in the cirrhosis group (p < 0.001). PICRUSt2 analysis indicated a decrease in acetyl-CoA fermentation to butyrate, as well as an increase in pathways related to antibiotics resistance, and aromatic amino acid biosynthesis. These metabolic pathways have been poorly described in the progression of alcohol-related decompensated cirrhosis. The gut microbiota of these patients possesses a pathogenic/inflammatory environment; therefore, future strategies to balance intestinal dysbiosis should be implemented. These findings are described for the first time in the population of western Mexico.

Nanoparticles Formulation Improves the Antifibrogenic Effect of Quercetin on an Adenine-Induced Model of Chronic Kidney Disease.

Renal fibrosis is the final stage of chronic kidney injury characterized by glomerulosclerosis and tubulointerstitial fibrosis with parenchymal destruction. Quercetin belongs to the most studied flavonoids with antioxidant, anti-inflammatory, antifibrogenic, and antitumor activity. It modifies the TGF-ß/Smad signaling pathway, decreasing profibrogenic expression molecules and inducing the expression of antioxidant, anti-inflammatory, and antifibrogenic molecules. However, quercetin exhibits poor water solubility and low absorption and bioavailability. This limitation was solved by developing a nanoparticles formulation that improves the solubility and bioavailability of several bioactive compounds. Therefore, we aimed to investigate the in vivo antifibrogenic effect of a quercetin nanoparticles formulation. Male C57BL/6 mice were induced into chronic renal failure with 50 mg/kg of adenine for four weeks. The animals were randomly grouped and treated with 25, 50, or 100 mg/kg of quercetin, either macroparticles or nanoparticles formulation. We performed biochemical, histological, and molecular analyses to evaluate and compare the effect of macroparticles versus nanoparticles formulation on kidney damage. Here, we demonstrated that smaller doses of nanoparticles exhibited the same beneficial effect as larger doses of macroparticles on preventing kidney damage. This finding translates into less quercetin consumption reaching the desired therapeutic effect.

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.

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.

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.

New gene therapy strategies for hepatic fibrosis.

The liver is the largest internal organ of the body, which may suffer acute or chronic injury induced by many factors, leading to cirrhosis and hepatocarcinoma. Cirrhosis is the irreversible end result of fibrous scarring and hepatocellular regeneration, characterized by diffuse disorganization of the normal hepatic structure, regenerative nodules and fibrotic tissue. Cirrhosis is associated with a high co-morbidity and mortality without effective treatment, and much research has been aimed at developing new therapeutic strategies to guarantee recovery. Liver-based gene therapy has been used to downregulate specific genes, to block the expression of deleterious genes, to delivery therapeutic genes, to prevent allograft rejection and to augment liver regeneration. Viral and non-viral vectors have been used, with viral vectors proving to be more efficient. This review provides an overview of the main strategies used in liver-gene therapy represented by non-viral vectors, viral vectors, novel administration methods like hydrodynamic injection, hybrids of two viral vectors and blocking molecules, with the hope of translating findings from the laboratory to the patient's bed-side.

Nrf2 and Snail-1 in the prevention of experimental liver fibrosis by caffeine.

AIM: To determine the molecular mechanisms involved in experimental hepatic fibrosis prevention by caffeine (CFA). METHODS: Liver fibrosis was induced in Wistar rats by intraperitoneal thioacetamide or bile duct ligation and they were concomitantly treated with CFA (15 mg/kg per day). Fibrosis and inflammatory cell infiltrate were evaluated and classified by Knodell index. Inflammatory infiltrate was quantified by immunohistochemistry (anti-CD11b). Gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction for collagen I (Col-1), connective tissue growth factor (CTGF), transforming growth factor ß1 (TGF-ß1), tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, superoxide dismutase (SOD) and catalase (CAT). Activation of Nrf2 and Snail-1 was analyzed by Western-blot. TNF-α expression was proved by enzyme-linked immunosorbant assay, CAT activity was performed by zymography. RESULTS: CFA treatment diminished fibrosis index in treated animals. The Knodell index showed both lower fibrosis and necroinflammation. Expression of profibrogenic genes CTGF, Col-1 and TGF-ß1 and proinflammatory genes TNF-α, IL-6 and IL-1 was substantially diminished with CFA treatment with less CD11b positive areas. Significantly lower values of transcriptional factor Snail-1 were detected in CFA treated rats compared with cirrhotic rats without treatment; in contrast Nrf2 was increased in the presence of CFA. Expression of SOD and CAT was greater in animals treated with CFA showing a strong correlation between mRNA expression and enzyme activity. CONCLUSION: Our results suggest that CFA inhibits the transcriptional factor Snail-1, down-regulating profibrogenic genes, and activates Nrf2 inducing antioxidant enzymes system, preventing inflammation and fibrosis.

Quercetin improves hepatic fibrosis reducing hepatic stellate cells and regulating pro-fibrogenic/anti-fibrogenic molecules balance.

BACKGROUND AND AIM: Development of hepatic cirrhosis involves oxidative stress, inflammation, hepatic stellate cells (HSC)s activation and fibrosis. On the other hand, quercetin, a natural flavonoid is a potent antioxidant and activator of superoxide dismutase and catalase. The aim was to determinate the effect of quercetin on HSCs and development of hepatic fibrosis. METHODS: Wistar male rats were chronically intoxicated with CCl(4) for 8 weeks and concomitantly treated with 100 mg/kg per day of quercetin. Oxidative state, inflammation and fibrosis were evaluated. Effect of quercetin on apoptosis of HSC was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling reaction. RESULTS: Sixty percent of reduction in fibrosis index was observed with quercetin treatment compared with control animals. Considerable reduction on hepatic enzymes was detected in the quercetin group. Expression of pro-fibrotic genes (transforming growth factor-ß [TGF-ß], Collagen 1α [Col-1α] and connective tissue growth factor [CTGF]) were decreased by quercetin. Quercetin increased gene expression and functional activity of antioxidant enzymes superoxide dismutase and catalase. Inflammatory index was highly reduced as determined by H-E staining and pro-inflammatory cytokines expression and nuclear factor-κB activation were also inhibited. A significant reduction of 65% on activated HSC number was detected when rats were treated with quercetin. Quercetin also induced activation of matrix metalloproteinases MMP2 and MMP9 contributing to decreased index of fibrosis. CONCLUSIONS: Treatment with quercetin reduces oxidation and inflammation and also prevents liver fibrosis, through induction of HSC apoptosis and activation of MMPs.

Potent antioxidant role of pirfenidone in experimental cirrhosis.

Three important features must be considered when proposing therapeutic strategies in liver cirrhosis: inflammation, oxidative stress and fibrogenesis. Pirfenidone is a synthetic molecule which oxidative action has not been tested in cirrhosis. Cirrhosis was induced in rats by ligation of the common bile duct or carbon tetrachloride (CCl(4)) chronic intoxication and treated with pirfenidone or diphenyleneiodonium (a potent known antioxidant) for the last two weeks for bile duct ligation model or for the last three weeks for CCl(4) chronic intoxication. A 60% reduction in fibrosis index for bile duct ligation model and 42% for CCl(4) along with reduced inflammation was observed. Considerable reduction on hepatic enzymes and total and direct bilirubins were detected with pirfenidone in both models. Pirfenidone antioxidant capacity rendered a 28% and 30% reduction in nitrites and malonyldealdehide concentration in bile duct ligation and 52% and 38% in CCl(4). With respect to gene expression, fibrotic genes like transforming growth factor-beta (TGF-beta) and collagen Ialpha (Col-1alpha) were down-regulated by pirfenidone and increased expression of regenerative genes like hepatocyte growth factor (HGF) and c-met . Superoxide dismutase (SOD), catalase (CAT) and inducible nitric oxide synthase (iNOS) gene expression were importantly down-regulated where nuclear factor kappa B (NF-kappaB) binding activity also decreased with pirfenidone treatment. Also, SOD and CAT functional activity decreased after pirfenidone action. On the other hand, diphenyleneiodonium induced a drop in oxidative stress similar in extent to pirfenidone, but it was not as effective as pirfenidone in reducing fibrosis. In this work, we showed antioxidant properties of pirfenidone beyond its well-known antifibrotic effect. These features make pirfenidone an attractive drug for trying fibrotic diseases accompanied by oxidative stress processes.

Impaired counter-regulation of interleukin-1 by the soluble IL-1 receptor type II in patients with chronic liver disease.

OBJECTIVE: To assess the production of the endogenous IL-1 modulators IL-1 receptor antagonist (IL-1Ra), type I and II soluble IL-1 receptors (IL-1sRI and II) in patients with chronic liver disease (CLD). MATERIAL AND METHODS: Plasma levels of IL-1beta (IL-1beta) and IL-1 modulators were assessed in 126 CLD patients and 39 healthy controls. IL-1sRII was also measured in the supernatants of primary hepatocyte cultures. RESULTS: Plasma IL-1sRI and IL-1Ra levels were significantly higher in cirrhotic CLD patients than in non-cirrhotic CLD patients and in controls. Levels did not depend on the etiology of CLD. Likewise, plasma IL-1beta levels were elevated in CLD patients compared with those in controls. In contrast, IL-1sRII levels did not differ between CLD patients and controls. Cultures of human primary hepatocytes showed that IL-1sRII is induced by IL-1beta, but not IL-6. CONCLUSIONS: In cirrhotic CLD patients elevated plasma IL-1beta is not counteracted by endogenous levels of IL-1sRII, whereas high IL-1sRI is expected to neutralize the naturally occurring antagonist IL-1Ra, resulting in a dysregulation of the IL-1 system that might enhance pro-inflammatory activity of IL-1.

Pirfenidone prevents capsular contracture after mammary implantation.

BACKGROUND: Pirfenidone (PFD), a new antifibrotic and antiinflammatory agent, prevents and resolves fibrous tissue. This study evaluated the effect of PFD on adverse events in mammary implants using an animal model. Mammary implantation, the most frequent aesthetic surgery, may present several complications after surgery such as swelling, capsule contracture, hardness, and pain. METHODS: Wistar rats underwent submammary implantation with either smooth or textured silicone gel implants and were administrated 200 mg/kg of PFD daily. The control group received saline. The animals were killed at 8 weeks. The capsular tissue of both implants was removed for histologic and molecular analyses. RESULTS: Typical postaugmentation periimplant capsules with opacity on adjacent tissues developed 8 weeks after silicone implantation. No significant differences were observed between the textured and smooth implants in any analyzed parameter. Clearly, PFD reduced capsule thickness around submmamary tissue, fibroblast-like cell proliferation, and recruitment of inflammatory cells. The total cell numbers per field were reduced as well. In contrast, the control group presented abundant mononuclear cell infiltration and fibroblast-like cell proliferation. The total content of collagen in the PFD group was 50% less than in the control group. Fibroblast cells displayed 45% less activated phenotype in the PFD group than in the control group, as determined by immunohistochemistry techniques. In the PFD animals, transforming growth factor-beta (TGF-beta) decreased 85% and collagen 1 gene expression 60%, compared with the control group. CONCLUSION: The findings show a positive effect of PFD on mammary contracture in 10 rats. Despite the small number of animals, the differences found in 10 control rats encourage the authors to propose a larger study later and to suggest PFD as a potential preventive strategy in human mammary implantation surgery.

Increased DNA binding activity of NF-kappaB, STAT-3, SMAD3 and AP-1 in acutely damaged liver.

AIM: To investigate the role of genes and kinetics of specific transcription factors in liver regeneration, and to analyze the gene expression and the activity of some molecules crucially involved in hepatic regeneration. METHODS: USING gel-shift assay and RT-PCR, transcription factors, such as NF-kappaB, STAT-3, SMAD3 and AP-1, and gene expression of inducible nitric oxide synthase (iNOS), hepatocyte growth factor (HGF) and c-met were analyzed in an animal model of chemically induced hepatectomy. RESULTS: Gene expression of HGF and its receptor c-met peaked at 3 h and 24 h after acute CCl(4) intoxi-cation. iNOS expression was only observed from 6 to 48 h. Transcriptional factor NF-kappaB had an early activation at 30 min after acute liver damage. STAT-3 peaked 3 h post-intoxication, while AP-1 displayed a peak of activation at 48 h. SMAD3 showed a high activity at all analyzed times. CONCLUSION: TNF-alpha and IL-6 play a central role in hepatic regeneration. These two molecules are responsible for triggering the cascade of events and switch-on of genes involved in cell proliferation, such as growth factors, kinases and cyclins which are direct participants of cell proliferation.