Fifty female Sprague-Dawley rats aged 10 wk (Laboratory Animal Center affiliated to Chinese Academy of Sciences, Shanghai, China) were divided into 5 groups (n = 10). All the rats were initiated with either a bilateral ovariectomy (Ovx) or a sham operation 3 wk before the studies. Control group received subcutaneous injection of olive oil (3 mL/kg every 3 d and first dosage doubled), CCl₄ group was given the same dose of 400 g/L CCl₄, Ovx + CCl₄ group was treated with the same dose of 400 g/L CCl₄ after ovariectomy, β-Est + CCl₄ group along with CCl₄ injection described above was treated with 0.002% β-Est (1 mL/kg per day, Sigma-Aldrich Corporation, St Louis, Missouri, United States), Ovx + β-Est + CCl₄ group was treated the same as β-Est + CCl₄ group with the addition of ovariectomy.

At the end of 6 wk, the number of alive rats in the groups was 10, 7, 8, 10, 9 respectively. Several rats died of infection at the site of injection or hepatic crack by unsuitable handling. After an overnight fast, all the rats were sacrificed. Serum samples obtained were treated with proteinase inhibitor and stored at -20 °C. Liver tissue specimens were rinsed with normal saline containing 0.1 g/L DEPC, some were stored at -80 °C, and the others were fixed in neutral formalin and embedded in paraffin.

Serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed by standard enzymatic methods, hyaluronic acid (HA) and type IV collagen (C IV) concentrations were measured by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Navy Medical Institute, Shanghai, China).

The embedded liver specimens were sliced 5 μm in thickness, mounted on slides, deparaffinised in xylene, and dehydrated in alcohol. For histopathological studies, the liver sections were stained with van Gieson (VG). For alpha-smooth muscle actin (α-SMA) immunohistochemistry, the sections were incubated with 3% (v/v) hydrogen peroxide in methanol for 15 min to block endogenous peroxidase, and then with 1 g/L Triton-X plus 1 g/L BSA in phosphate-buffered saline (PBS) to block non-specific antigens. After absorption of surplus liquid, the samples were incubated with a 1:100 dilution of a polyclonal antibody against α-SMA (Boster Biological Technology Corporation, Wuhan, China) at 37 °C for 2 h. Then the samples were rinsed and incubated with a 1:100 dilution of biotin-conjugated goat anti-rabbit IgG (Boster Biological Technology Corporation, Wuhan, China) at 37 °C for 30 min. Finally, the antigen-antibody complexes were visualized with 3,3’-diaminobenzidine (DAB).

For morphometric analysis, the mean value of collagen content or α-SMA positive cells in six ocular fields per specimen was used as the percentage area at 100x magnification using Zeiss KS400 image analysis system (Kontron Electronics, Eching, Germany).

Serum estradiol concentrations were measured with radioimmunoassay (RIA) using a commercial kit (Diagnostic Products Corporation, Tianjin, China).

HSCs were isolated and purified from the livers of normal female rats weighing about 400 g to 450 g by a combination of pronase-collagenase perfusion and density gradient centrifugation[15-19]. Briefly, the liver was perfused in situ through the portal vein, first with Ca²⁺/Mg²⁺-free Krebs-Ringer (KR) solution at a flow rate of 20 mL/min, followed by pronase and then with collagenase (Sigma-Aldrich Corporation, St Louis, Missouri, USA) in KR solution. The digested liver was excised, minced with scissors, and incubated in KR solution containing pronase and DNase (Sigma-Aldrich Corporation, St Louis, Missouri, United States) for 30 min. The resulting suspension was filtered through nylon mesh (200 μm in diameter) and then washed 3 times by centrifugation in D-Hanks solution at 1700 r/min for 7 min. An HSC-enriched fraction was obtained by centrifugation of the filtrate in an 180 g/L nycodenz (Sigma-Aldrich Corporation, St Louis, Missouri, United States) solution with the volume ratio of 1:2 at 3 200 r/min for 17 min. The cells in the upper layer were washed by centrifugation at 1700 r/min for 7 min and suspended in Dulbecco’s modified Eagle medium (DMEM, GIBCO BRL Life Technologies Incorporation, United States) supplemented with 200 mL/L fetal bovine serum (FBS, GIBCO BRL Life Technologies Incorporation, United States). Both cell viability and purity were over 90%, examined by trypan blue (Sigma-Aldrich Corporation, St Louis, Missouri, United States) exclusion and autofluorescence respectively.

Cells were plated at a density of 1 × 10⁶ cells in 1 mL culture medium on uncoated plastic culture dishes. The culture medium was changed after 24 h and then every 3 d. After cultured for 14 d, the cells were passaged.

The first-passage HSCs were plated at a density of 2 × 10⁵ cells in 1 mL DMEM supplemented with 100 g/L FBS on an uncoated plastic culture dish. Subconfluent HSCs were divided into 10 groups, and different concentrations of β-Est, 2OHE, 2MeOE (Sigma-Aldrich Corporation, St Louis, Missouri, United States) or vehicle were added to the cells. After incubated for 24 h, experiments were done to assess the functional state of HSCs.

HSC proliferation was measured by MTT assay. The cells were continually cultivated with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich Corporation, St Louis, Missouri, United States) in a 96-well plate for 4 h. After the supernatant in each well was aspirated and discarded, the cells were agitated with dimethylsulfoxide (DMSO, Sigma-Aldrich Corporation, St Louis, Missouri, United States) 150 μLfor 10 min. Finally, the optical density of each well was read in an auto reader using a 492 nm filter with a reference at 630 nm.

The levels of HA and C IV in the supernatants of cell were messured as afore-metioned.

For immunohistochemical examination of α-SMA, estrogen receptor (ER)α and ERβ, the cells were mounted on slides and fixed with 40 g/L paraformaldehyde for 5 min at 4 °C. Following incubation with 30 mL/L H₂O₂ for 15 min, the cells were blocked by 1 g/L Triton-X plus 1 g/L BSA in PBS for 15 min at room temperature. They were incubated for 2 h at 37 °C with a 1:100 dilution of polyclonal antibody against α-SMA, ERα or ERβ (Santa Cruz Biotechnology Incorporation, California, United States) in a humid chamber. After the samples were rinsed, they were incubated with a 1:200 dilution of biotin-conjugated goat anti-rabbit IgG, and then visualized with DAB. Integral light density (ILD) analysis was performed on 20 cells in up, down, left, right quadrant of each section, data were calculated from average of 4 ILDs.

Data are presented as mean ± SE unless otherwise indicated. Parametric data were compared using one-way analysis of variance, followed by multiple pair-wise comparisons according to the least-significant difference t-test. Pearson’s analysis was performed to compare the correlation between serum estradiol and fibrotic area percentage. A P value less than 0.05 was accepted as statistically significant.

Table 1 shows the liver enzymes and fibrosis markers in CCl₄-treated female rats. Treatment with CCl₄ caused a significant increase in the activities of serum ALT and AST compared with the control animals. The enzyme levels in ovariectomized model rats were higher than those in CCl₄ model group, whereas CCl₄ plus β-Est group showed a significant decrease in enzyme levels compared with CCl₄ group. There was no significant difference between CCl₄ group and Ovx + β-Est + CCl₄ group.

The changes of fibrosis markers, HA and C IV, were in parallel with those of the enzyme levels in all groups.

The control livers showed a distinct lobular architecture with collagens only surrounding the central and portal veins (Figure 1A). Administration of CCl₄ induced inflammation, necrosis and collagen deposition in the livers. Ovariectomy significantly increased hepatic fibrosis induced by CCl₄ (Figure 1B), whereas β-Est had the opposite effect. The fibrotic area percentage of Ovx + CCl₄ + β-Est group was similar to that of CCl₄ group (Figure 1C).

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Figure 1 Histopathological findings of representative liver sections from female rats treated with CCl_4. VG staining, original magnification ×100. A: Control group; B: Ovx+CCl₄ group; C: Ovx + CCl₄ + β-Est group.

Immunohistochemistry of α-SMA, an activation marker of rat HSC[20-22], showed strong staining around vascular wall, but not the sinusoids in control female rat livers (Figure 2A). After administration of CCl₄, fibrotic change was evident with significant increases of α-SMA positive cells in centrilobular and periportal fibrotic bands. Ovariectomy significantly increased the percentage area of staining (Figure 2B), whereas β-Est had the opposite effect. There was no significant difference in the fibrotic area percentage of staining between CCl₄ and Ovx + CCl₄ + β-Est groups (Figure 2C).

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Figure 2 Immunohistochemistry of α-SMA in representative liver sections from female rats treated with CCl₄, DAB staining. A: control group, original magnification ×200; B: Ovx + CCl₄ group, original magnification × 400; C: Ovx + CCl₄ + β-Est group, original magnification ×400.

Figure 3 summarizes the histopathological and immunohist-ochemical changes in CCl₄-treated female rats.

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Figure 3 Histopathological and immunohistochemical changes in CCl₄-treated female rats (fibrotic area percentage of total field), ^aP<0. 05 vs CCl₄; ^bP<0.01 vs control.

Figure 4 shows the serum estradiol levels in the rats. The levels did not change in animals treated with CCl₄ alone. Ovariectomy produced significantly lower serum estradiol levels. In rats model treated with exogenous β-Est, the levels increased significantly as compared with those of control or CCl₄ model groups. Twenty μg/kg per day β-Est could rectify the low serum estradiol levels in ovariectomized rats.

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Figure 4 Serum estradiol levels in CCl₄–treated female rats, ^bP<0. 01 vs control, ^dP<0.01 vs CCl₄.

In 34 CCl₄-treated rats, there was a negative correlation between the fibrotic area percentage and serum estradiol level, the calculated correlation coefficient was -0.57 (P < 0.01, Figure 5).

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Figure 5 Negative correlation between fibrotic area percentage and serum estradiol level (r = 0. 57, P < 0.01).

β-Est, 2OHE and 2MeOE (10^-9-10^-7 mol/L) inhibited HSC proliferation (Figure 6A) and ECM secretion (Figure 6B and C) in a dose-dependent manner. Only the high concentration (≥ 10^-7 mol/L) of β-Est exerted inhibitory effects, while a concentration of 2MeOE as low as 10^-9 mol/L had a similar effect. The order of potency was 2MeOE > 2OHE > β-Est. At a concentration of 10^-7 mol/L, β-Est, 2OHE and 2MeOE all could inhibit HSCs in expressing α-SMA, and the potency of 2MeOE was significantly stronger than that of β-Est (Figure 6D)

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Figure 6 Effects of estradiol metabolites on function of HSCs proliferation (A), HA (B) and C IV (C) secretion in HSCs, effects of 10^-7 mol/L estradiol metabolites on α-SMA expression (D) of HSCs. Values for each point represent mean ± SD from 5 separate experiments, ^aP < 0.05 vs control, ^dP < 0.05 vs 10^-7 mol/L β-Est.

A representative immunohistochemistry showed the expression of ER-β (Figure 7B) but not ER-α (Figure 7A) on rat HSCs.

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Figure 7 Representative immunohistochemistry showing expression of ER on HSCs used for studies, original magnification ×200. No expression of ER-α(A) but ER-β(B) was observed.