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)

As of this writing, the most common cause of hepatic fibrosis is chronic hepatitis C virus infection (HCV), the characteristic feature of which is hepatic steatosis. Hepatic steatosis leads to an increase in lipid peroxidation in hepatocytes, which in turn activates hepatic stellate cells (HSCs). HSCs are also thought to be the primary target cells for inflammatory stimuli, and produce extracellular matrix components. Based on available clinical information, chronic hepatitis C appears to progress more rapidly in men than in women, and cirrhosis is predominately a disease of men and postmenopausal women. It should be noted that estradiol (E2) is a potent endogenous antioxidant. A recent study has shown that hepatic steatosis became evident in an aromatase-deficient mouse and was diminished in animals, after treatment with E2. Our studies showed that E2 suppressed hepatic fibrosis in hepatic fibrosis models, inhibited the activation of activator protein 1 and nuclear factor-kappa B in cultured hepatocytes undergoing oxidative stress, and attenuated HSC activation in primary culture. Recently, variant oestrogen receptors (ERs) were found to be expressed to a greater extent in male patients with chronic liver disease than in female subjects. We also demonstrated decreased levels of ERs in postmenopausal women and cirrhotic patients of both genders. The actions of E2 are mediated through ER alpha and beta. HSCs have also been found to possess functional ER beta but not ER alpha. A better understanding the basic mechanisms underlying the gender-associated differences observed in the development of hepatic fibrosis would provide valuable information relative to the search for effective antifibrogenic therapies.

Chronic hepatitis B virus (HBV) infection is the most common cause of hepatic fibrosis and hepatocellular carcinoma (HCC), mainly as a result of chronic necroinflammatory liver disease. A characteristic feature of chronic hepatitis B infection, alcoholic liver disease and nonalcoholic fatty liver disease (NAFLD) is hepatic steatosis. Hepatic steatosis leads to an increase in lipid peroxidation in hepatocytes, which, in turn, activates hepatic stellate cells (HSCs). HSCs are the primary target cells for inflammatory and oxidative stimuli, and these cells produce extracellular matrix components. Chronic hepatitis B appears to progress more rapidly in males than in females, and NAFLD, cirrhosis and HCC are predominately diseases that tend to occur in men and postmenopausal women. Premenopausal women have lower hepatic iron stores and a decreased production of proinflammatory cytokines. Hepatic steatosis has been observed in aromatase-deficient mice, and has been shown to decrease in animals after estradiol treatment. Estradiol is a potent endogenous antioxidant which suppresses hepatic fibrosis in animal models, and attenuates induction of redox sensitive transcription factors, hepatocyte apoptosis and HSC activation by inhibiting a generation of reactive oxygen species in primary cultures. Variant estrogen receptors are expressed to a greater extent in male patients with chronic liver disease than in females. These lines of evidence suggest that the greater progression of hepatic fibrosis and HCC in men and postmenopausal women may be due, at least in part, to lower production of estradiol and a reduced response to the action of estradiol. A better understanding of the basic mechanisms underlying the sex-associated differences in hepatic fibrogenesis and carciogenesis may open up new avenues for the prevention and treatment of chronic liver disease.

The existence and distribution of glucagon-like peptide-1 (GLP-1) and its receptor in rat brain in relation to that of glucagon were examined. The concentration of GLP-1 immunoreactivity (GLP-1-IR), measured by a specific and sensitive RIA established in this study with anti GLP-1 serum (LMT-01), was found to be highest in the thalamus-hypothalamus, followed by the medulla oblongata. The distribution of glucagon-like immunoreactivity was similar to that of GLP-1-IR. However, appreciable glucagon immunoreactivity was detected only in the thalamus-hypothalamus. Gel filtration analysis showed the presence of GLP-1-IR of various molecular weights in the extract of thalamus-hypothalamus including that eluted at the same position as synthetic GLP-1 (1-37); moreover, HPLC analysis also confirmed the presence of GLP-1-IR, eluted at the exact position as synthetic GLP-1 (1-37). The distribution of receptors for GLP-1 corresponded with that of GLP-1-IR in the rat brain, except in the pituitary gland. The distribution of these receptors was also similar to that of glucagon receptors. The thalamus-hypothalamus, pituitary gland, and medulla oblongata were rich in GLP-1 and glucagon-binding sites. The binding affinities of GLP-1 and glucagon were in the nanomolar range [disocciation constant Kd approximately equal to 4 nM]. The presence of specific, high affinity receptors for GLP-1 was confirmed by demonstrating that GLP-1 stimulated cAMP formation in the thalamus-hypothalamus and the pituitary gland. The concentration of GLP-1 required for half-maximal stimulation of cAMP formation in these regions was about 1 nM. These results suggest that GLP-1 may be synthesized in certain parts of the brain and play a role as a neurosignal transmitter.