Detection of cell-free histones in the cerebrospinal fluid of pediatric central nervous system malignancies by imaging flow cytometry.

Introduction: Pediatric brain tumours (PBT) are one of the most common malignancies during childhood, with variable severity according to the location and histological type. Certain types of gliomas, such a glioblastoma and diffuse intrinsic pontine glioma (DIPG), have a much higher mortality than ependymoma and medulloblastoma. Early detection of PBT is essential for diagnosis and therapeutic interventions. Liquid biopsies have been demonstrated using cerebrospinal fluid (CSF), mostly restricted to cell free DNA, which display limitations of quantity and integrity. In this pilot study, we sought to demonstrate the detectability and robustness of cell free histones in the CSF. Methods: We collected CSF samples from a pilot cohort of 8 children with brain tumours including DIPG, medulloblastoma, glioblastoma, ependymoma and others. As controls, we collected CSF samples from nine children with unrelated blood malignancies and without brain tumours. We applied a multichannel flow imaging approach on ImageStream(X) to image indiviual histone or histone complexes on different channels. Results: Single histones (H2A, macroH2A1.1, macroH2A1.2 H2B, H3, H4 and histone H3 bearing the H3K27M mutation), and histone complexes are specifically detectable in the CSF of PBT patients. H2A and its variants macroH2A1.1/macroH2A1/2 displayed the strongest signal and abundance, together with disease associated H3K27M. In contrast, mostly H4 is detectable in the CSF of pediatric patients with blood malignancies. Discussion: In conclusion, free histones and histone complexes are detectable with a strong signal in the CSF of children affected by brain tumours, using ImageStream(X) technology and may provide additive diagnostic and predictive information.

Human centenarian-associated SIRT6 mutants modulate hepatocyte metabolism and collagen deposition in multilineage hepatic 3D spheroids.

Non-alcoholic fatty liver disease (NAFLD), encompassing fatty liver and its progression into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), is one of the rapidly rising health concerns worldwide. SIRT6 is an essential nuclear sirtuin that regulates numerous pathological processes including insulin resistance and inflammation, and recently it has been implicated in the amelioration of NAFLD progression. SIRT6 overexpression protects from formation of fibrotic lesions. However, the underlying molecular mechanisms are not fully delineated. Moreover, new allelic variants of SIRT6 (N308K/A313S) were recently associated with the longevity in Ashkenazi Jews by improving genome maintenance and DNA repair, suppressing transposons and killing cancer cells. Whether these new SIRT6 variants play different or enhanced roles in liver diseases is currently unknown. In this study, we aimed to clarify how these new centenarian-associated SIRT6 genetic variants affect liver metabolism and associated diseases. We present evidence that overexpression of centenarian-associated SIRT6 variants dramatically altered the metabolomic and secretomic profiles of unchallenged immortalized human hepatocytes (IHH). Most amino acids were increased in the SIRT6 N308K/A313S overexpressing IHH when compared to IHH transfected with the SIRT6 wild-type sequence. Several unsaturated fatty acids and glycerophospholipids were increased, and ceramide tended to be decreased upon SIRT6 N308K/A313S overexpression. Furthermore, we found that overexpression of SIRT6 N308K/A313S in a 3D hepatic spheroid model formed by the co-culture of human immortalized hepatocytes (IHH) and hepatic stellate cells (LX2) inhibited collagen deposition and fibrotic gene expression in absence of metabolic or dietary challenges. Hence, our findings suggest that novel longevity associated SIRT6 N308K/A313S variants could favor the prevention of NASH by altering hepatocyte proteome and lipidome.

Gene Editing and Human iPSCs in Cardiovascular and Metabolic Diseases.

The incidence and the burden of cardiovascular disease (CVD), coronary heart disease (CHD), type 2 diabetes mellitus (T2DM), and the metabolic syndrome are greatly increasing in our societies. Together, they account for 31% of all deaths worldwide. This chapter focuses on the role of two revolutionary discoveries that are changing the future of medicine, induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 technology, in the study, and the cure of cardiovascular and metabolic diseases.We summarize the state-of-the-art knowledge about the possibility of editing iPSC genome for therapeutic applications without hampering their pluripotency and differentiation, using CRISPR/Cas technology, in the field of cardiovascular and metabolic diseases.

Profiling of cell-free DNA methylation and histone signatures in pediatric NAFLD: A pilot study.

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in children and adolescents, increasing the risk of its progression toward nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. There is an urgent need for noninvasive early diagnostic and prognostic tools such as epigenetic marks (epimarks), which would replace liver biopsy in the future. We used plasma samples from 67 children with biopsy-proven NAFLD, and as controls we used samples from 20 children negative for steatosis by ultrasound. All patients were genotyped for patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), membrane bound O-acyltransferase domain containing 7 (MBOAT7), and klotho-ß (KLB) gene variants, and data on anthropometric and biochemical parameters were collected. Furthermore, plasma cell-free DNA (cfDNA) methylation was quantified using a commercially available kit, and ImageStream(X) was used for the detection of free circulating histone complexes and variants. We found a significant enrichment of the levels of histone macroH2A1.2 in the plasma of children with NAFLD compared to controls, and a strong correlation between cfDNA methylation levels and NASH. Receiver operating characteristic curve analysis demonstrated that combination of cfDNA methylation, PNPLA3 rs738409 variant, coupled with either high-density lipoprotein cholesterol or alanine aminotransferase levels can strongly predict the progression of pediatric NAFLD to NASH with area under the curve >0.87. Conclusion: Our pilot study combined epimarks and genetic and metabolic markers for a robust risk assessment of NAFLD development and progression in children, offering a promising noninvasive tool for the consistent diagnosis and prognosis of pediatric NAFLD. Further studies are necessary to identify their pathogenic origin and function.

The costs and benefits of senotherapeutics for human health.

Cellular senescence is a major contributor to age-related diseases in humans; however, it also has a beneficial role in physiological and pathological processes, including wound healing, host immunity, and tumour suppression. Reducing the burden of cell senescence in animal models of cardiometabolic disorders, inflammatory conditions, neurodegenerative diseases, and cancer using pharmaceutical approaches that selectively target senescent cells (ie, senolytics) or that suppress senescence-associated secretory phenotype (ie, senomorphics) holds great promise for the management of chronic age-associated conditions. Although studies have provided evidence that senolytics or senomorphics are effective at decreasing the number of senescent cells in humans, the short-term and long-term side-effects of these therapies are largely unknown. In this Review, we systematically discuss the senolytics and senomorphics that have been investigated in clinical trials or have been used off-label, presenting their various adverse effects. Despite the potential of senotherapeutics to transform anti-ageing medicine, a cautionary approach regarding unwanted dose-dependent side-effects should be adopted.

GDF11 inhibits adipogenesis and improves mature adipocytes metabolic function via WNT/ß-catenin and ALK5/SMAD2/3 pathways.

OBJECTIVE: GDF11 is a member of the TGF-ß superfamily that was recently implicated as potential "rejuvenating" factor, which can ameliorate metabolic disorders. The main objective of the presented study was to closely characterize the role of GDF11 signaling in the glucose homeostasis and in the differentiation of white adipose tissue. METHODS: We performed microscopy imaging, biochemical and transcriptomic analyses of adipose tissues of 9 weeks old ob/ob mice and murine and human pre-adipocyte cell lines. RESULTS: Our in vivo experiments employing GDF11 treatment in ob/ob mice showed improved glucose/insulin homeostasis, decreased weight gain and white adipocyte size. Furthermore, GDF11 treatment inhibited adipogenesis in pre-adipocytes by ALK5-SMAD2/3 activation in cooperation with the WNT/ß-catenin pathway, whose inhibition resulted in adipogenic differentiation. Lastly, we observed significantly elevated levels of the adipokine hormone adiponectin and increased glucose uptake by mature adipocytes upon GDF11 exposure. CONCLUSION: We show evidence that link GDF11 to adipogenic differentiation, glucose, and insulin homeostasis, which are pointing towards potential beneficial effects of GDF11-based "anti-obesity" therapy.

Adipocyte-Specific Expression of PGC1α Promotes Adipocyte Browning and Alleviates Obesity-Induced Metabolic Dysfunction in an HO-1-Dependent Fashion.

Recent studies suggest that PGC1-α plays a crucial role in mitochondrial and vascular function, yet the physiological significance of PGC1α and HO expression in adipose tissues in the context of obesity-linked vascular dysfunction remains unclear. We studied three groups of six-week-old C57BL/6J male mice: (1) mice fed a normal chow diet; (2) mice fed a high-fat diet (H.F.D.) for 28 weeks, and (3) mice fed a high-fat diet (H.F.D.) for 28 weeks, treated with adipose-specific overexpression of PGC-1α (transgenic-adipocyte-PGC-1α) at week 20, and continued on H.F.D. for weeks 20-28. R.N.A. arrays examined 88 genes involved in adipocyte proliferation and maturation. Blood pressure, tissue fibrosis, fasting glucose, and oxygen consumption were measured, as well as liver steatosis, and the expression levels of metabolic and mitochondrial markers. Obese mice exhibited a marked reduction of PGC1α and developed adipocyte hypertrophy, fibrosis, hepatic steatosis, and decreased mitochondrial respiration. Mice with adipose-specific overexpression of PGC1-α exhibited improvement in HO-1, mitochondrial biogenesis and respiration, with a decrease in fasting glucose, reduced blood pressure and fibrosis, and increased oxygen consumption. PGC-1α led to the upregulated expression of processes associated with the browning of fat tissue, including UCP1, FGF21, and pAMPK signaling, with a reduction in inflammatory adipokines, NOV/CCN3 expression, and TGFß. These changes required HO-1 expression. The R.N.A. array analysis identified subgroups of genes positively correlated with contributions to the browning of adipose tissue, all dependent on HO-1. Our observations reveal a positive impact of adipose-PGC1-α on distal organ systems, with beneficial effects on HO-1 levels, reversing obesity-linked cardiometabolic disturbances.

Nociceptin/orphanin FQ opioid receptor (NOP) selective ligand MCOPPB links anxiolytic and senolytic effects.

Accumulation of senescent cells may drive age-associated alterations and pathologies. Senolytics are promising therapeutics that can preferentially eliminate senescent cells. Here, we performed a high-throughput automatized screening (HTS) of the commercial LOPAC®Pfizer library on aphidicolin-induced senescent human fibroblasts, to identify novel senolytics. We discovered the nociceptin receptor FQ opioid receptor (NOP) selective ligand 1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole (MCOPPB, a compound previously studied as potential anxiolytic) as the best scoring hit. The ability of MCOPPB to eliminate senescent cells in in vitro models was further tested in mice and in C. elegans. MCOPPB reduced the senescence cell burden in peripheral tissues but not in the central nervous system. Mice and worms exposed to MCOPPB also exhibited locomotion and lipid storage changes. Mechanistically, MCOPPB treatment activated transcriptional networks involved in the immune responses to external stressors, implicating Toll-like receptors (TLRs). Our study uncovers MCOPPB as a NOP ligand that, apart from anxiolytic effects, also shows tissue-specific senolytic effects.

Bioactive Compounds from Lemon (Citrus limon) Extract Overcome TNF-α-Induced Insulin Resistance in Cultured Adipocytes.

The consumption of plant-based food is important for health promotion, especially regarding the prevention and management of chronic diseases such as diabetes. We investigated the effects of a lemon extract (LE), containing ≥20.0% total flavanones and ≥1.0% total hydroxycinnamic acids, on insulin signaling in murine 3T3-L1 adipocytes treated with TNF-α, which was used to mimic in vitro the insulin resistance condition that characterizes diabetes mellitus. Our results showed LE increased PPARγ, GLUT4 and DGAT-1 levels, demonstrating the potential of this lemon extract in the management of insulin resistance conditions associated with TNF-α pathway activation. LE treatment further decreased the release of interleukin 6 (IL-6) and restored triglyceride synthesis, which is the main feature of a healthy adipocyte.

Mild exacerbation of obesity- and age-dependent liver disease progression by senolytic cocktail dasatinib + quercetin.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is increasingly prevalent and represents a growing challenge in terms of prevention and treatment. A minority of affected patients develops inflammation, subsequently fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCC is a leading cause of cancer-related death. An increased number of senescent cells correlate with age-related tissue degeneration during NAFLD-induced HCC. Senolytics are promising agents that target selectively senescent cells. Previous studies showed that whereas a combination of the senolytic drugs dasatinib and quercetin (D + Q) reduced NAFLD in mice, D + Q lacked efficacy in removing doxorubicin-induced ß-gal-positive senescent cells in human HCC xenografted mice. Whether D + Q has an effect on the age-associated spectrum of NAFLD-inflammation-HCC remains unknown. METHODS: Here, we utilized an established model of age- and obesity-associated HCC, the low dose diethylnitrosamine (DEN)/high fat diet (HFD), a regimen promoting liver inflammation and tumorigenesis over a long period of 9 months. Four groups of mice each were created: group 1 included control untreated mice; group 2 included mice treated with D + Q; group 3 included mice undergoing the DEN/HFD protocol; group 4 included mice undergoing the DEN/HFD protocol with the administration of D + Q. At the end of the chemical/dietary regimen, we analyzed liver damage and cell senescence by histopathology, qPCR and immunoblotting approaches. RESULTS: Unexpectedly, D + Q worsened liver disease progression in the DEN/HFD mouse model, slightly increasing histological damage and tumorigenesis, while having no effect on senescent cells removal. CONCLUSIONS: In summary, using an animal model that fully recapitulates NAFLD, we demonstrate that these compounds are ineffective against age-associated NAFLD-induced HCC. Video Abstract.

In vitro effects of bioflavonoids rich lemon extract on pre-adipocyte differentiation.

Lemon fruit is a source of bioactive compounds, which has many beneficial effects on health. Obesity is characterized by over-accumulation of adipose tissue as a result of increased adipocyte size and number. Adipogenesis is mediated and assisted by various transcription factors that induce lipid-metabolizing enzymes followed by an increase of perilipin expression and lipid droplets generation. Here, we evaluate the effect of lemon extract (LE) as radical scavenger and the consequent regulation of adipocyte differentiation and lipid accumulation. 3T3-L1 murine pre-adipocytes were differentiated and treated with different LE concentrations. The high percentages of flavonoid contained in LE led to a significant inhibition of DPPH radical and reactive oxygen species, demonstrating a strong radical scavenger activity. Treatment of 3T3-cells with LE showed a significant decrease of perilipin expression, subsequently confirmed by the reduction of lipid droplet accumulation, resulting from Oil Red O Staining and by the downregulation of PPARγ and DGAT-1mRNA.

Redox and Epigenetics in Human Pluripotent Stem Cells Differentiation.

Significance: Since their discovery, induced pluripotent stem cells (iPSCs) had generated considerable interest in the scientific community for their great potential in regenerative medicine, disease modeling, and cell-based therapeutic approach, due to their unique characteristics of self-renewal and pluripotency. Recent Advances: Technological advances in iPSC genome-wide epigenetic profiling led to the elucidation of the epigenetic control of cellular identity during nuclear reprogramming. Moreover, iPSC physiology and metabolism are tightly regulated by oxidation-reduction events that mainly occur during the respiratory chain. In theory, iPSC-derived differentiated cells would be ideal for stem cell transplantation as autologous cells from donors, as the risks of rejection are minimal. Critical Issues: However, iPSCs experience high oxidative stress that, in turn, confers a high risk of increased genomic instability, which is most often linked to DNA repair deficiencies. Genomic instability has to be assessed before iPSCs can be used in therapeutic designs. Future Directions: This review will particularly focus on the links between redox balance and epigenetic modifications-in particular based on the histone variant macroH2A1-that determine DNA damage response in iPSCs and derived differentiated cells, and that might be exploited to decrease the teratogenic potential on iPSC transplantation. Antioxid. Redox Signal. 34, 335-349.

Milk thistle seed cold press oil attenuates markers of the metabolic syndrome in a mouse model of dietary-induced obesity.

Milk thistle cold press oil (MTO) is an herbal remedy derived from Silybum marianum which contains a low level of silymarin and mixture of polyphenols and flavonoids. The effect of MTO on the cardiovascular and metabolic complications of obesity was studied in mice that were fed a high-fat diet (HFD) for 20 weeks and treated with MTO for the final 8 weeks of the diet. MTO treatment attenuated HFD-induced obesity, fasting hyperglycemia, hypertension, and induced markers of mitochondrial fusion and browning of white adipose. Markers of inflammation were also attenuated in both adipose and the liver of MTO-treated mice. In addition, MTO resulted in the improvement of liver fibrosis. These results demonstrate that MTO has beneficial actions to attenuate dietary obesity-induced weight gain, hyperglycemia, hypertension, inflammation, and suggest that MTO supplementation may prove beneficial to patients exhibiting symptoms of metabolic syndrome. PRACTICAL APPLICATIONS: Natural supplements are increasingly being considered as potential therapies for many chronic cardiovascular and metabolic diseases. Milk thistle cold press oil (MTO) is derived from Silybum marianum which is used as a dietary supplement in different parts of the world. The results of the present study demonstrate that MTO supplementation normalizes several metabolic and cardiovascular complications arising from dietary-induced obesity. MTO supplementation also had anti-inflammatory actions in the adipose as well as the liver. These results suggest that supplementation of MTO into the diet of obese individuals may afford protection against the worsening of cardiovascular and metabolic disease and improve inflammation and liver fibrosis.

Cold Press Pomegranate Seed Oil Attenuates Dietary-Obesity Induced Hepatic Steatosis and Fibrosis through Antioxidant and Mitochondrial Pathways in Obese Mice.

AIM: Obesity is associated with metabolic syndrome, hypertension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), and type 2 diabetes. In this study, we investigated whether the dietary supplementation of pomegranate seed oil (PSO) exerted a protective effect on liver lipid uptake, fibrosis, and mitochondrial function in a mouse model of obesity and insulin resistance. METHOD: In this in vivo study, eight-week-old C57BL/6J male mice were fed with a high fat diet (HFD) for 24 weeks and then were divided into three groups as follows: group (1) Lean; group (n = 6) (2) HF diet; group (n = 6) (3) HF diet treated with PSO (40 mL/kg food) (n = 6) for eight additional weeks starting at 24 weeks. Physiological parameters, lipid droplet accumulation, inflammatory biomarkers, antioxidant biomarkers, mitochondrial biogenesis, insulin sensitivity, and hepatic fibrosis were determined to examine whether PSO intervention prevents obesity-associated metabolic syndrome. RESULTS: The PSO group displayed an increase in oxygen consumption, as well as a decrease in fasting glucose and blood pressure (p < 0.05) when compared to the HFD-fed mice group. PSO increased both the activity and expression of hepatic HO-1, downregulated inflammatory adipokines, and decreased hepatic fibrosis. PSO increased the levels of thermogenic genes, mitochondrial signaling, and lipid metabolism through increases in Mfn2, OPA-1, PRDM 16, and PGC1α. Furthermore, PSO upregulated obesity-mediated hepatic insulin receptor phosphorylation Tyr-972, p-IRB tyr1146, and pAMPK, thereby decreasing insulin resistance. CONCLUSIONS: These results indicated that PSO decreased obesity-mediated insulin resistance and the progression of hepatic fibrosis through an improved liver signaling, as manifested by increased insulin receptor phosphorylation and thermogenic genes. Furthermore, our findings indicate a potential therapeutic role for PSO in the prevention of obesity-associated NAFLD, NASH, and other metabolic disorders.

Senescence-like phenotype in post-mitotic cells of mice entering middle age.

Staining mice tissues for ß-galactosidase activity is a fundamental tool to detect age- or disease-associated cellular senescence. However, reported analyses of positivity for senescence-associated ß-galactosidase activity or for other markers of senescence in post-mitotic cells of healthy murine tissues have been fragmentary or inconclusive. Here, we attempted to independently deepen this knowledge using multiple senescence markers within the same cells of wild type mice entering middle age (9 months of age). A histochemistry protocol for the pH-dependent detection of ß-galactosidase activity in several tissues was used. At pH 6, routinely utilized to detect senescence-associated ß-galactosidase activity, only specific cellular populations in the mouse body (including Purkinje cells and choroid plexus in the central nervous system) were detected as strongly positive for ß-galactosidase activity. These post-mitotic cells were also positive for other established markers of senescence (p16, p21 and DPP4), detected by immunofluorescence, confirming a potential senescent phenotype. These data might contribute to understanding the functional relation between the senescence-associated ß-galactosidase activity and senescence markers in post-mitotic cells in absence of disease or advanced aging.

Non-competitive heme oxygenase-1 activity inhibitor reduces non-small cell lung cancer glutathione content and regulates cell proliferation.

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related death mainly due to its high metastatic rate. Impairment of redox homeostasis mechanisms has been previously described in NSCLC and is associated with the disease itself as well as with comorbidities such as smoking. The aim of the present in vitro study was to evaluate the effect of selective and non-competitive inhibition of heme oxygenase-1 (HO-1) on cancer redox homeostasis with particular regards to glutathione (GSH) metabolism related enzymes. NSCLC cell line (A549) was treated with the HO-1 activity inhibitor VP13/47 (10 µM) and we further evaluated cell viability, apoptosis, mitochondrial dysfunction and oxidative stress. Our results showed that VP13/47 significantly reduced HO-1 expression and total HO activity thus, resulting in a significant reduction of cell viability, proliferation and increased apoptosis, mitochondrial dysfunction and oxidative stress. Consistently with increased oxidative stress, we also showed that reduced GSH was significantly decreased and such effect was also accompanied by a significant downregulation of the enzymes involved in its biosynthesis. Taken all together our results show that selective HO-1 inhibition significantly impairs NSCLC progression and may represent a possible pharmacological strategy for new chemotherapy agents.

Ginseng and heme oxygenase-1: The link between an old herb and a new protective system.

Ginseng is an ancient herb, belonging to Asian traditional medicine, that has been considered as a restorative to enhance vitality for centuries. It has been demonstrated that the antioxidant action of ginseng may be mediated through activation of different cellular signaling pathways involving the heme oxygenase (HO) system. Several compounds derived from ginseng have been studied for their potential role in brain, heart and liver protection, and the Nrf2 pathway seems to be the most affected by these natural molecules to exert this effect. Ginseng is also popularly used in cancer patients therapy for the demonstrated capability to defend tissues from chemotherapy-induced damage. Reported results suggest that the effect of ginseng is primarily associated with ROS scavenging, mainly exerted through the activation of Nrf2 pathway, and the consequent induction of HO-1 levels. This review aims to discuss the connection between the antioxidant properties of ginseng and the activation of the HO system, as well as to outline novel therapeutic applications of this medicinal plant to human health.

Inhibition of Heme Oxygenase Antioxidant Activity Exacerbates Hepatic Steatosis and Fibrosis In Vitro.

The progression of non-alcoholic fatty liver disease (NAFLD) and the development of hepatic fibrosis is caused by changes in redox balance, leading to an increase of reactive oxygen species (ROS) levels. NAFLD patients are at risk of progressing to non-alcoholic steatohepatitis (NASH), associated to cardiovascular diseases (CVD), coronary heart disease and stroke. Heme Oxygenase-1 (HO-1) is a potent endogenous antioxidant gene that plays a key role in decreasing oxidative stress. The present work was directed to determine whether use of an inhibitor of HO-1 activity affects lipid metabolism and fibrosis process in hepatic cells. Oil Red assay and mRNA analysis were used to evaluate the triglycerides content and the lipid metabolism pathway in HepG2 cells. ROS measurement, RT-PCR and Soluble collagen assay were used to assess the intracellular oxidant, the fibrosis pathway and the soluble collagen in LX2 cells. The activity of HO-1 was inhibited using Tin Mesoporphyrin IX (SnMP). Our study demonstrates that a non-functional HO system results in an increased lipid storage and collagen release in hepatocytes. Consequently, an increase of HO-1 levels may provide a therapeutic approach to address the metabolic alterations associated with NAFLD and its progression to NASH.

Synthesis, in vitro and in silico studies of HO-1 inducers and lung antifibrotic agents.

Aim: Dimethyl fumarate (DMF) analogs were synthesized to obtain inducers of HO-1 and antifibrotic agents. Methods: HO-1 expression levels were measured on lung fibroblasts (MRC5). NMR and docking studies were performed. Heme oxygenase activity, gene levels and protein expression have been measured for the most active compound 1a. Collagen production by fibroblast after exposure to TGF-ß was measured. Results: Compound 1a showed to be a strong HO-1 inducer. Its activity seems to be mediated by activation of nuclear factor erythroid 2 related factor 2 (Nrf2). TGF-ß-induced collagen production was significantly decreased on MRC5, pretreated with DMF or 1a. DMF and 1a have a high potential for treatment of lung fibrotic injuries.