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Fibrose pulmonar (FP) ? o resultado de um processo de les?o cr?nica no qual fibroblastos se tornam ativados e passam a secretar e depositar grandes quantidades de matriz extracelular (ECM). Este fato leva uma dificuldade na degrada??o da ECM, seguido por rigidez tecidual, perda de fun??o tecidual e morte. A frutose-1,6-bisfosfato ? um metab?lito intermedi?rio da rota glicol?tica que vem sendo estudada h? v?rios anos e tem demonstrado a??es protetoras em diferentes tipos celulares e tecidos. H? evid?ncias de que a FBP ? capaz de prevenir o desenvolvimento da FP induzida por bleomicina (BLM) em camundongos, mas o mecanismo pelo qual isso acontece ainda ? desconhecido. Assim, o objetivo deste estudo foi avaliar os efeitos da FBP sobre a preven??o e/ou regula??o do processo fibr?tico em um modelo experimental de FP in vivo e em c?lulas pulmonares in vitro. Para isso, tratamos animais que receberam BLM intratraqueal com FBP por 14 dias. Ainda, utilizamos culturas prim?rias de fibroblastos e miofibroblastos pulmonares extra?dos de camundongos saud?veis e fibr?ticos, respectivamente. Analisamos, a fun??o pulmonar, a express?o g?nica de subtipos de col?genos e outros componentes importantes no processo fibrotico, assim como as taxas de prolifera??o, contra??o e migra??o celular, al?m das prote?nas envolvidas na degrada??o da ECM. No modelo de FP, a FBP foi capaz de melhorar significativamente a fun??o pulmonar, como demonstrado pela redu??o do dep?sito de col?geno e diminui??o da express?o de genes relacionados ? ECM, como col?geno e fibronectina. In vitro, miofibroblastos tratados com FBP por 3 dias demonstraram uma menor taxa de prolifera??o, contra??o e migra??o, o que s?o ind?cios de desativa??o e revers?o fenot?pica. Al?m disso, a FBP tamb?m diminui a express?o de genes e prote?nas relacionados ? ECM, como col?geno, fibronectina e actina de m?sculo liso ? em cultura prim?ria de miofibroblastos. MMP1, uma colagenase respons?vel pela degrada??o da ECM, teve sua express?o aumentada, enquanto a TIMP-1, um inibidor da MMP1, diminuiu. MMP2, que est? associada ao aumento da invas?o do tecido por miofibroblastos, tamb?m teve sua express?o diminu?da. Nossos resultados demonstram que a FBP pode prevenir o desenvolvimento de FP induzida por BLM atrav?s da redu??o da produ??o e dep?sito de col?geno e outros componentes de ECM e isso pode ser mediado atrav?s do aumento da produ??o de MMP1 e diminui??o da TIMP-1, o que promoveria uma degrada??o efetiva da ECM depositada no tecido. Pulmonary fibrosis (PF) is the result of chronic injury where fibroblasts become activated and secrete large amounts of extracellular matrix (ECM), leading to impaired ECM degradation followed by tissue stiffness, loss of lung function and death. Fructose-1,6-bisphosphate is a metabolite from glycolytic pathway that has been studied for many years and showing beneficial actions in different cell lines and tissues. In a recent study from our laboratory, FBP was able to prevent the development of FP bleomycin (BLM)-induced on mice, but the underlying mechanism is still unknown. In this way, the objective of this study was to evaluate the effects of FBP under prevention and regulation of fibrotic process in PF mouse model and lung fibroblasts. In this way, we treated mice that received BLM intratracheally with FBP for 14 days. We also extracted lung fibroblasts and myofibroblasts from healthy and fibrotic animals, respectively. We analyzed lung function, gene expression of collagen subtypes and other important components of ECM, as well proliferation, contraction and migration rates, and proteins involved in ECM degradation. In PF mice model, lung function was improved by FBP as revealed by reduced collagen deposition and downregulation of ECM gene expression such as collagens and fibronectin. Myofibroblasts treated with FBP for 3 days in vitro showed decreased proliferation, contraction, and migration, which are characteristic of myofibroblast to fibroblast phenotype reversal. ECM-related genes and proteins such as collagens, fibronectin and ?-smooth muscle actin, were downregulated in FBP-treated myofibroblasts in vitro. Moreover, MMP1, responsible for ECM degradation, and TIMP-1, a MMP1 inhibitor, were up and down regulated, respectively. MMP2, which is associated with fibroblast invasion on tissue, also was downregulated. These results demonstrate that FBP may prevent bleomycininduced PF development through reduced expression of collagen and other ECM components mediated by a reduced TIMP-1 and increased MMP1 production, which would allow a better ECM degradation. Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES

The therapeutic potential of Baccharis anomala DC. extracts was evaluated through its cytotoxic and antiproliferative effect and their phenotypic reversion property in activated hepatic stellate cells (HSCs). Baccharis anomala is distributed in Brazil (southeastern and south regions) and used for diuretic effect in folk medicine. Four fractions were obtained from the fractionation of the methanolic extract. Fractions III and IV decreased cell proliferation without increasing cell necrosis markers levels and induced cell cycle arrest in G1 phase. Fraction III induced phenotypic reversion through PPAR-γ activation pathway, while fraction IV did not alter PPAR-α/γ expression levels, suggesting that there is an independent PPAR-α/γ pathway involved. Hydroxybenzoic, chlorogenic and coumaric acids were identified. Fractions III and IV showed antiproliferative effect and ability to induce reversion of activated phenotype of HSCs., EXCLI Journal; 18:Doc91; ISSN 1611-2156

Hemocromatose heredit?ria (HH) ? uma doen?a gen?tica onde o balan?o do ferro est? desregulado e esse metal se acumula no f?gado, causando efeitos t?xicos e, principalmente, fibrose. Fibrose ? uma resposta de cicatriza??o exacerbada com dep?sito de matriz extracelular (ECM). C?lulas estreladas hep?ticas (HSC) quando ativadas s?o as maiores respons?veis pela produ??o de ECM. Frutose-1,6-bisfosfato (FBP) ? um a??car e possui in?meros efeitos ben?ficos, como melhorar o potencial antioxidante da c?lula, proteger o f?gado de les?o e reverter o fen?tipo de HSC ativadas. Por causa disso, nosso objetivo foi testar os efeitos da FBP em uma linhagem imortalizada de HSC (GRX) expostas a ferro livre (Fe), na tentativa de simular o que ocorre em pacientes com HH.O tratamento com Fe (1mg/L) por 8 dias aumentou a prolifera??o celular enquanto o tratamento com Fe + FBP (1mg/L + 0.6mM) a diminuiu para n?veis menores que os do controle. A atividade da LDH, taxa de apoptose e ciclo celular n?o foi alterada em nenhum grupo. A colora??o com OilRed-O (ORO) mostrou uma diminui??o na quantidade de lip?dio intracelular quando as c?lulas foram tratadas com Fe por 8 dias. No grupo Fe + FBP, houve um aumento do conte?do lip?dico e as c?lulas apresentaram caracter?sticas morfol?gicas de c?lulas quiescentes. A express?o de PPAR-γ foi diminu?da no grupo Fe e igual ao controle no grupo Fe + FBP. Ao contr?rio, o Fe aumentou os n?veis de express?o de Col?geno tipo I (Col-1) e o tratamento concomitante com FBP reverteu esse efeito, ficando igual ao controle. A produ??o de TGF-β1 se manteve inalterada no grupo Fe e foi menor que o controle no tratamento com Fe + FBP, mostrando uma atividade antifibr?tica da FBP. O teste de DPPH mostrou que a FBP n?o possui atividade antioxidante em nenhuma dose testada. O teste de Ferrozine mostrou uma diminui??o da absorb?ncia depois de 120 minutos de incuba??o de FBP + Fe em todas as doses testadas, mostrando que a FBP ? um quelante de ferro.Esses dados demonstram que FBP reverte o fen?tipo das c?lulas GRX mesmo quando em presen?a do Fe e que isso pode ser causado pela regula??o da express?o do PPAR-γ e COL-1.Em conclus?o, a FBP diminuiu o crescimentoe reverteu o fen?tipo de c?lulas GRX, mostrando um poss?vel efeito antifibr?tico. Hereditary hemochromatosis (HH) is a genetic disease where iron balance is deregulated and this metal accumulates in the liver, causing toxic effects and fibrosis. Fibrosis is an exacerbated wound-healing response with extracellular matrix (ECM) deposition. Hepatic stellate cells (HSC), when activated, are the main responsible for ECM production. Fructose-1,6-bisphosphate (FBP) is a sugar and possess innumerous beneficial effects, like enhance cell antioxidant potential, protects liver from damage and reverts the phenotype of activated HSC. Because of this, we aimed to test the effects of FBP in immortalized HSC line (GRX) exposed to free iron (Fe) tempting to simulate what occurs in patients with HH.Fe (1mg/L) treatment for 8 days increased cell growth, whereas Fe + FBP (1mg/L + 0.6mM) decreased cell proliferation to levels below of control. LDH activity, apoptosis rate and cell cycle were not altered in any group. Oil Red-O (ORO) staining showed a decrease in lipid content when GRX cells were Fe-treated (1mg/L) for 8 days. In Fe + FBP (1mg/L + 0.6mM), GRX cells showed increased lipid content and characteristics of quiescent HSC. PPAR-γ expression was diminished on Fe group and same as control on Fe + FBP group. On the contrary, Fe treatment rose Col-1 expression and Fe + FBP reversed it to control levels. TGF-β1 was unaltered in Fe group. However, on Fe + FBP group, TGF-β1 levelswas far bellow of control and Fe-treated group, showing an antifibrotic activity. FBP didn t present antioxidant activity by DPPH assay. Ferrozine assay showed a decreased absorbance after 120 min in all FBP-tested doses, demonstrating that FBP is an iron chelator. These data demonstrate that FBP reverse the phenotype of GRX cells even when in Fepresence and that this could be caused by regulation of PPAR-γ and COL-1. In conclusion, FBP diminished the growth rate and reversed the phenotype of GRX cell, showing a possible antifibrotic effect.