Adriana Salazar-Montes

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.

Pirfenidone Prevents Capsular Contracture After Mammary Implantation

BackgroundPirfenidone (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.MethodsWistar 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.ResultsTypical 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-β (TGF-β) decreased 85% and collagen 1 gene expression 60%, compared with the control group.ConclusionThe 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.

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

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.

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 β1 (TGF-β1), 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-β1 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.

Differential gene expression of pro-inflammatory and anti-inflammatory cytokines in acute and chronic liver injury

Background: acute carbon tetrachloride (CCl4) intoxication in rats is strongly characterized by the expression of numerous cytokines playing an important role in the pathophysiology of liver diseases. We investigated the chronology of appearance of pro-inflammatory and anti-inflammatory cytokines in the liver of rats treated either acutely or chronically with CCl4. Methods: by using a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) methodology, we investigated pro-inflammatory and anti-inflammatory cytokines gene expression in liver of rats intoxicated acutely and chronically with CCl4. Results: we found high levels of mRNA transcripts of all pro-inflammatory cytokines [TNF-a, TGF-b, IL-1b, IL-6 and magrophage inflammatory protein-2 (MIP-2)] studied at 24‐48 h after acute administration with CCl4, decreasing thereafter and returning to basal levels within 1 week. Specifically, MIP-2 gene expression was detected as early as 6 h after intoxication disappearing at 48 h after treatment. TGF-b was increased at 24 h and peaking at 48 h. IL-10 mRNA was increased at 24 h post-CCl4 and maintained high levels 1 week after intoxication, in spite of the fact, that the inflammatory reaction was already resolved. On the contrary, IL-4 mRNA was not detected at any time analyzed. In chronic damage, pro-inflammatory cytokines gene expression was strong and sustained throughout the study. IL-4 transcripts were not detected. Conclusions: the counterbalance present between the activity of pro- and anti-inflammatory cytokines in acute damage could be responsible of the control and:or resolution of the inflammatory process.

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-1β) 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-1β 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-1β, but not IL-6. Conclusions. In cirrhotic CLD patients elevated plasma IL-1β 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.

Chemically induced liver regeneration is characterized by specific IL-6 gene expression

Abstract IL-6 and TNFα have been shown as important cytokines in liver regeneration induced in partially hepatectomized rats. Nonetheless, IL-6 gene expression in the course of chemically induced hepatic regeneration, resembling what occurs in humans infected by hepatitis virus in acute manner, remains obscure. To gain an insight on this matter we determined IL-6 and TNFα gene expression in Wistar rats after acute and chronic liver intoxication with CCl4 and/or turpentine. We used a semiquantitative method of RT-PCR to analyze cytokine gene expression and, quite interestingly, IL-6 was detected only at 24 h after acute damage as opposed to TNFα expression that was increased from 6 to 48 h after acute injury. A single intradermally-injected dose of turpentine did not suffice to induce IL-6 gene expression at any studied times, but, as expected, turpentine-treated animals showed decreased albumin mRNA transcripts as compared with the control rats. Furthermore, livers from cirrhotic animals did not show detectable IL-6 gene transcripts, but, IL-6 expression re-appeared in cirrhotic rats treated with an additional acute CCl4 dose, indicating that IL-6 is a critical component of the brisk regenerative response induced by CCl4.

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 patients bed-side.

Correction: 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-β1, TGF-β3, HIF-1α, and HIF-1β. 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.