F2-Isoprostanes as markers of oxidative stress and mediators of CCl4-induced liver fibrosis

INTRODUCTION: F2-isoprostanes are considered as the most reliable markers of OS in vivo and mediators of some important biological effects, such as the vasoconstriction of kidney glomerular arterioles. It is known that hepatic fibrosis induced by CCl4 may be linked to oxidative stress (OS). In previous studies, we demonstrated the role of isoprostanes as possible mediators of CCl4-induced hepatic fibrosis. Plasma levels of isoprostanes were found to be elevated in rat chronically treated with CCl4 and correlated with hepatic collagen content. We also demonstrated that in vitro isoprostanes induced a marked increase in DNA and collagen synthesis in cultured hepatic stellate cells (HSC), in the concentration range found in the in vivo studies (1-10 nM). It has been suggested that isoprostanes act through the activation of receptors analogous to those for tromboxane A2 (TxA2r). Thus, the possible occurrence of TxA2r on HSC was investigated. Binding studies with [3H]SQ29548 (a specific antagonist of TxA2r) and competition binding experiments with I-BOP (a specific agonist of TxA2r) demonstrated the existence of TxA2r in HSC and the involvement of TxA2r in Iso-evoked responses. We then examined which signal transduction pathways are set into motion by isoprostanes to exert their fibrogenic effects. METHODS: HSC were isolated from the rat liver and treated with 8-epi-PGE2the most represented isomer). Ins(1,4,5)P3 (IP3) and cAMP levels were determined by commercial kits. Activation of mitogen-activated protein kinases (MAPK) and cyclin 1 was assessed by Western blotting. Cell proliferation and collagen production were assessed by measuring the incorporation of 3H-thymidine and 3H-proline, respectively. RESULTS: 8-epi-PGE2 increased 4 times IP3 and affected cAMP production. The expression of cyclin D1, a molecule involved in cell proliferation, is also increased. Furthermore, 8-epi-PGE2 activated two classes of MAPK: extracellular signal-regulated kinase (ERK) and p38, which have been shown to influence cell proliferation and collagen gene expression. CONCLUSION: On the basis of these data, it is possible to hypothesize that one of the pathways activated by 8-epi-PGF2a is that of Gq/PKC. The binding of isoprostanes to TxA2r could stimulate downstream MAPK activation, via PKC. In particular, p38 is known to increase in HSC collagen production, while ERK is able to increase cyclin D1 expression and then cell proliferation. Thus, fibrogenic effects of isoprostanes in HSC are mediated through TxA2r binding by specific activation of these transduction pathways.