Stages of activation of hepatic stellate cells: effects of ellagic acid, an inhibiter of liver fibrosis, on their differentiation in culture

To further explore that hepatic stellate cell (HSC) activation results in physiological protection against environmental insult, the profile of differentiation of HSC has been examined upon treatment with ellagic acid (EA), a plant-derived antioxidant that shows multiple protective effects during liver disease. Sparse rat liver cell cultures were grown in media containing EA (3, 6, 30 and 100 mug/ml) and, as controls, without EA, and inspected until day 7 in culture. The cells were double-labelled with antibodies against glial fibrillary acidic protein (GFAP) and smooth muscle alpha-actin (SMAA), marker proteins of quiescent and activated HSC, respectively. In EA-free culture conditions, the quiescent (SMAA(-)/GFAP(+)) HSC transiently acquired a semi-activated (SMAA(+)/GFAP(+)), phenotype and were further transformed into activated (SMAA(+)/GFAP(-)), pleomorphic HSC. Up to a concentration of 30 mug/ml, EA induced an early synthesis of SMAA in all HSC and inhibited their morphologic differentiation and individual growth throughout the culture period. At a concentration of 6 mug/ml, EA supported the semi-activated (SMAA(+)/GFAP(+)) phenotype of HSC throughout the culture period, whereas treatment with high EA concentrations (30 mug/ml) resulted in an early loss of GFAP expression. In conclusion: (i) the uniform response of HSC to EA by mild activation adds functional significance to cellular features preceding the transformation of HSC to myofibroblasts; (ii) the high sensitivity of HSC to EA treatment suggests their involvement in any mechanisms of protection by this antioxidant; (iii) the maintenance of HSC morphology might be one of the factors playing a role in the prevention or slowing down of liver fibrosis; (iv) because the effects of EA are concentration- and time-dependent, an arbitrary usage of this antioxidant is a matter of potential concern; (v) the various patterns of HSC activation observed might correspond to distinct activities of these cells, which, in turn, might lead to different outcomes of liver fibrosis.