As a model for the analysis of the fibrosuppressive role of estradiol, hepatic fibrosis was induced in male and female rats by the administration of a single dose of dimethylnitrosamine (DMN). The fibrotic response of the male liver after DMN treatment was significantly stronger than that of the female liver. In the male DMN model, estradiol reduced hepatic mRNA for type I and III procollagens and the tissue inhibitor of metalloproteinase-1 (TIMP-1), as well as deposition of type I and III collagen protein total hepatic collagen and malondialdehyde (MDA), a product of lipid peroxidation. Concomitant administration of a neutralizing antibody against rat estradiol enhanced fibrogenesis, as judged by the same parameters. Ovariectomy in the female model had a fibrogenic effect, inducing the hepatic expression of both types of procollagen and TIMP-1; in addition, the number of ␣-smooth muscle actin (␣-SMA)-positive cells in the liver increased; estradiol replacement was fibrosuppressive in the castrated-female model. In rat hepatic stellate cells incubated in primary culture with estradiol, cell number, type I collagen production, and ␣-SMA expression were all reduced. These findings suggest that estradiol suppressed the induction of hepatic fibrosis, and may in part underlie the more rapid progression in males of hepatic fibrosis and its complications. (HEPATOLOGY 1999;29:719-727.)Hepatic fibrosis is a consequence of severe liver damage and, in many chronic liver diseases, progresses to cirrhosis. 1,2 It consists of an abnormal accumulation of extracellular matrix proteins, particularly collagens. 3 Hepatocellular carcinoma is strongly associated with cirrhosis. 4 Among patients with cirrhosis and hepatocellular carcinoma, the male:female ratio is in the range of 2.3:1 to 2.6:1. 5,6 Both male and female livers contain high-affinity, low-capacity estrogen receptors 7,8 and respond to estrogens by regulating liver function. 9,10 This suggests that sex hormones may play a role in the progression from hepatic fibrosis to cirrhosis.It is now evident that hepatic stellate cells (HSC) (Ito cells, fat-storing cells, lipocytes) in the space of Disse undergo transformation under inflammatory stimuli into ␣-smooth muscle actin (␣-SMA)-positive myofibroblast-like cells, and are responsible for much of the collagen hypersecretion and nodule formation that occurs during hepatic fibrosis and cirrhosis. 11,12 Our preliminary studies suggested that fibrotic liver-collagen levels initially increase partly as a result of the balance between type I collagen and matrix metalloproteinase-1 (MMP-1) expression rates during the course of hepatic fibrosis in rats, as induced by a single dose of dimethylnitrosamine (DMN), 13 and that estradiol inhibits the myofibroblastic transformation of rat HSC in primary culture. 14 It is also noteworthy that oxygen-derived free radicals and lipid peroxidation have been implicated in hepatic fibrosis. 15-17 HSC may be activated by free radicals, induced by ascorbate/FeSO 4 , as well as by malondialdehyde ...

It has been shown that lipid peroxidation is associated with hepatic fibrosis and stellate cell activation. Shosaiko-to (TJ-9) is an herbal medicine, which is commonly used to treat chronic hepatitis in Japan, although the mechanism by which TJ-9 protects against hepatic fibrosis is not known. As a result, we assayed the preventive and therapeutic effects of TJ-9 on experimental hepatic fibrosis, induced in rats by dimethylnitrosamine (DMN) or pig serum (PS), and on rat stellate cells and hepatocytes in primary culture, and assessed the antioxidative activities and the active components of TJ-9. Male Wistar rats were given a single intraperitoneal injection of 40 mg/kg DMN or 0.5 mL PS twice weekly for 10 weeks. In each model, rats were fed a basal diet throughout, or the same diet, which also contained 1.5% TJ-9, for 2 weeks before treatment or for the last 2 weeks of treatment. TJ-9 suppressed the induction of hepatic fibrosis, increased hepatic retinoids, and reduced the hepatic levels of collagen and malondialdehyde (MDA), a production of lipid peroxidation. Immunohistochemical examination showed that TJ-9 reduced the deposition of type I collagen and the number of ␣-smooth muscle actin (␣-SMA) positive-stellate cells in the liver and inhibited, not only lipid peroxidation in cultured rat hepatocytes that were undergoing oxidative stress, but also the production of type I collagen, ␣-SMA expression, cell proliferation, and oxidative burst in cultured rat stellate cells. In addition, TJ-9 inhibited Fe 2؉ /adenosine 5Ј-diphosphate-induced lipid peroxidation in rat liver mitochondria in a dose-dependent manner and showed radical scavenging activity. Among the active components of TJ-9, baicalin and baicalein were found to be mainly responsible for the antioxidative activity. These findings suggest that Sho-saiko-to (TJ-9) functions as a potent antifibrosuppressant by inhibition of lipid peroxidation in hepatocytes and stellate cells in vivo. (HEPATOLOGY 1999;29:149-160)

Background-Hepatic stellate cells play a key role in the pathogenesis of hepatic fibrosis. Aims-To examine the inhibitory eVect of oestradiol on stellate cell activation. Methods-In vivo, hepatic fibrosis was induced in rats by dimethylnitrosamine or pig serum. In vitro, rat stellate cells were activated by contact with plastic dishes resulting in their transformation into myofibroblast-like cells. Results-In the dimethylnitrosamine and pig serum models, treatment with oestradiol at gestation related doses resulted in a dose dependent suppression of hepatic fibrosis with restored content of hepatic retinyl palmitate, reduced collagen content, lower areas of stellate cells which express smooth muscle actin ( -SMA) and desmin, and lower procollagen type I and III mRNA levels in the liver. In cultured stellate cells, oestradiol inhibited type I collagen production, -SMA expression, and cell proliferation. These findings suggest that oestradiol is a potent inhibitor of stellate cell transformation. Conclusion-The antifibrogenic role of oestradiol in the liver may contribute to the sex associated diVerences in the progression from hepatic fibrosis to cirrhosis. (Gut 1999;44:127-136)

Aims-To examine the eVects of oestradiol and testosterone on the early carcinogenic changes expressed in rat liver from the diethylnitrosamine (DEN), 2-acetylaminofluorene (AAF), partial hepatectomy (PH) model of hepatocarcinogenesis. Methods-Preneoplastic liver lesions were evaluated using immunohistochemical analysis of glutathione-S-transferase placental form (GST-P) expression; oestrogen and androgen receptor levels were measured by radioimmunoassay. Results-Oestradiol administration to non-castrated DEN-AAF-PH treated males resulted in a decrease in the area of GST-P positive foci, while testosterone increased the serum oestradiol level and reduced the area. In males, castration alone and castration with oestradiol replacement significantly reduced the GST-P positive area, and increased the hepatic oestrogen receptor level. In DEN-AAF-PH treated females, castration with testosterone replacement was associated with a significant increase in the GST-P positive area and the hepatic androgen receptor level. Conclusion-These findings suggest that exogenous and endogenous oestradiol can suppress chemical hepatocarcinogenesis. It appears that oestrogen receptors may be involved in the inhibition of malignant transformation of preneoplastic liver cells, while androgens and androgen receptors are involved in hepatocarcinogenesis.

— Aims/Background: We wished to clarify the mechanisms that account for the increase in hepatic collagen accumulation during hepatic fibrosis. Methods: The gene expression of type I and type III procollagens and matrix metalloproteinase‐1 (MMP‐1) was measured by Northern blot analysis; immunolocalization of both types of collagen was estimated by indirect immunohistochemical assay; and the hepatic content of collagen and malondialdehyde (MDA), a product of lipid peroxidation, were assayed in hepatic fibrosis induced in rats with a single dose of dimethylnitrosamine (DMN). Results: During the experimental period, more type I procollagen mRNA was found than type III procollagen mRNA. The immunoreactive intensity of type I collagen was greater in necrotic areas near central veins 3 days after DMN treatment than it was on day 9, whereas the type III collagen immunodeposition for the latter period of the hepatic fibrosis was stronger than it was on day 3. As compared with controls, hepatic collagen content increased significantly after 3 days and continued, increasing gradually, as did type I and III procollagen mRNA levels. On day 14, fibrosis was greatest and both types of procollagen gene expression were at their highest, and type I and III procollagen mRNA levels and hepatic collagen content increased as the dosage of DMN was raised. MMP‐1 mRNA levels increased early in hepatic fibrogenesis, and increased on day 14 when DMN dosages were low. Hepatic MDA levels increased rapidly for 3 days after DMN treatment, remaining significantly higher than control values and showing a significant increase even in response to low DMN doses on day 14. Conclusions: Our results suggested that fibrotic liver collagen content may make its first notable increase due in part to the balance between type I collagen and MMP‐1 expression rates. Also, lipid peroxidation may be important in the mechanism of hepatofibrogenesis.