Carbon tetrachloride (CCl 4 )-induced hepatic fibrosis has been considered to be linked to oxidative stress and mediated by aldehydic lipid peroxidation products. In the present study, we investigated whether collagen synthesis is induced by F 2 -isoprostanes, the most proximal products of lipid peroxidation and known mediators of important biological effects. By contrast with aldehydes, F 2 -isoprostanes act through receptors able to elicit definite signal transduction pathways. In a rat model of CCl 4 -induced hepatic fibrosis, plasma F 2 -isoprostanes were markedly elevated for the entire experimental period; hepatic collagen content also increased. When hepatic stellate cells (HSCs) from normal liver were cultured with F 2 -isoprostanes in the concentration range found in the in vivo studies (10 À9 -10 À8 M), a striking increase in DNA synthesis (reversed by the thromboxane A 2 antagonist SQ 29 548), in cell proliferation and in collagen synthesis was observed. Total collagen content was similarly increased. Moreover, F 2 -isoprostanes markedly increased the production of transforming growth factor-b1 by U937 cells, considered a model of liver macrophages. The data provide evidence for the possibility that F 2 -isoprostanes generated by lipid peroxidation in hepatocytes mediate HSC proliferation and collagen production seen in hepatic fibrosis.
F 2 -isoprostanes are considered as the most reliable markers of oxidative stress and can be used to evaluate the oxidative status in a number of human pathologies. Besides being markers of oxidative stress, F 2 -isoprostanes proved to be mediators of important biological effects and would act through the activation of receptors analogous to those for thromboxane A 2 . In a previous work, we provided evidence that F 2 -isoprostanes, generated during carbon tetrachlorideinduced hepatic fibrosis, mediate hepatic stellate cell (HSC) proliferation and collagen hyperproduction. To investigate whether TxA 2 receptor (TxA 2 r or TPr) is involved in the effects of F 2 -isoprostanes on HSC, experiments on DNA synthesis were carried out in the presence of 8-epi-prostaglandin F 2a (8-epi-PGF 2a ) or the TxA 2 r-specific agonist I-Both agonists significantly stimulated DNA synthesis, which was almost completely inhibited by the TxA 2 r-specific, suggesting that much of the effect of 8-epi-PGF 2a is mediated by the TxA 2 r. Further studies showed that increasing concentrations of SQ29548 progressively inhibit DNA synthesis, suggesting a possible competitive antagonism between the two molecules. In addition, we demonstrated that the stimulatory effect of 8-epi-PGF 2a on collagen synthesis could be mediated by TxA 2 r. The occurrence of TxA 2 r on HSC was also investigated using western blotting analysis and immunocytochemistry, which reveals that TP is distributed both on plasma membranes and within the cells. Moreover, binding studies indicated the presence of a specific binding site for 3 H-SQ29548 on HSC. Competition binding studies indicated that 8-epi-PGF 2a and I-BOP were both able to displace 3 H-SQ29548 binding with a very different affinity (K i ¼ 4.14±1.9 Â 10 À6 M and K i ¼ 1.15±0.3 Â 10 À9 M, respectively), suggesting the involvement of a modified form of isoprostane receptor, homologous to the classic thromboxane A 2 -binding site in F 2 -isoprostanes-evoked responses on HSC.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.