Quantificação das células estreladas ativadas / miofibroblastos e análise da apoptose das células do fígado durante a terapia celular na fibrose hepática em ratos

Abstract

Hepatic fibrosis is the result of a scarring response due to continued injury to the liver, and is featured by excessive accumulation of extracellular matrix (MEC) proteins in hepatic parenchyma. These proteins include collagen, fibronectin, elastin, laminin and proteoglicans, along with the participation of different cell populations within the liver. The main cells responsible for the synthesis of MEC proteins are activated hepatic stellate cells and myofibroblasts, which appear after inflammatory stimuli and are characterized by the expression of alpha-smooth muscle actin (α-SMA). It is known that hepatic fibrosis progression is accompanied by hepatocyte death and its substitution by α-SMA+ fibrogenic cells. Therefore, apoptosis of these fibrogenic cells is of main relevance to fibrosis regression and hepatic regeneration. In the later years, bone marrow stem cell therapy has been used to stimulate hepatic regeneration in different experimental models and clinical protocols. The adult bone marrow mononuclear fraction contains two stem cell populations particularly important in the treatment of diverse hepatic diseases: hematopoietic stem cells and mesenchymal stem cells. The aim of this study was to analyze α-SMA expression and the apoptotic process in hepatic cells during hepatic fibrosis induced by bile duct ligation (BDL) and after bone marrow mononuclear cell (BMMC) transplantation. Livers were collect from rats of the following groups: normal, 14 days of BDL, 21 days of BDL and rats that received BMMC 14 days after BDL and were analyzed after 7 days (total of 21 days of BDL). To quantify α-SMA expression by fibrogenic cells in the experimental groups, immunoperoxidase to α-SMA followed by morphometry in the Image Pro Plus software was performed. To analyze apoptosis in hepatic cells, immunoperoxidase and western blotting (WB) against caspase-3 (apoptotic protein) were used, along with double immunofluorescence against caspase-3 and α-SMA to confocal microscopy analysis. Results of α-SMA quantification by morphometry showed that α-SMA expression increased significantly 14 and 21 days after BDL. However, this expression was significantly decreased in the BMMC treated group, which presented a more preserved hepatic parenchyma in relation to the group with 21 days of BDL. Immunoperoxidase, WB and confocal microscopy results showed that caspase-3 is decreased in fibrotic livers with 14 and 21 days of BDL in comparison to normal group, and was significantly augmented in the BMMC treated group. Confocal microscopy analysis showed that were cells coexpressing α-SMA and caspase-3 in rats treated with BMMC, suggesting fibrogenic cells death and hepatic remodeling.