Preparation of animals

Male Wistar rats weighing 200 g were used for the DMN model and 120 g rats were used for the PS model. The rats were housed in air conditioned animal quarters with lighting from 0800 to 2000, with unrestricted access to a basal diet (CE-2; Nihon Clea, Tokyo, Japan) and water. As the experimental course for the DMN and PS models required two and 10 weeks, respectively, the lower weight animals were used for the latter model. Twenty five animals were used for the DMN model and 15 for the PS model (eight groups of five each) (fig 1).

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Figure 1

Experimental schedule in dimethylnitrosamine (DMN) and pig serum (PS) treated rats. Twenty five male Wistar rats were used for the DMN model and 15 for the PS model of hepatic fibrosis (eight groups of five each).

The DMN animals used in groups 2–5 were administered a single intraperitoneal injection of DMN (Sigma, St Louis, Missouri, USA) diluted with saline at a dose of 40 mg/kg body weight.18 ,24 Control animals for the DMN model in group 1 received a single injection of 0.5 ml of saline without DMN. The PS animals in groups 7 and 8 were administered intraperitoneal injections of PS (0.5 ml) twice per week for 10 weeks.19 The control animals for the PS model in group 6 received biweekly injections of saline (0.5 ml) for 10 weeks.

For the DMN model, the rats in groups 3, 4, and 5 received intraperitoneal injections of oestradiol valerate (Mochida Pharmaceutical Co., Tokyo, Japan) in olive oil at a dose of 0.033, 0.33, and 3.3 mg/kg/day, respectively, for two weeks after the single injection of DMN. For the PS model, the rats in group 8 received intraperitoneal oestradiol valerate in olive oil at a dose of 3.3 mg/kg/day for two weeks followed by a dose of 10 mg/kg twice per week for two weeks during the last four weeks of PS treatment. Animals in groups 1, 2, 6, and 7 received injections of olive oil without oestradiol.

All animals were anaesthetised using sodium pentobarbital (40 mg/kg, intraperitoneally) and killed after terminal exsanguination two weeks after DMN treatment in groups 1–5, and 10 weeks after PS treatment for groups 6–8. Blood samples were drawn from the inferior vena cava and used for radioimmunoassay of oestradiol. Liver tissue specimens were used for light microscopy and immunohistochemistry using anti-α-SMA and antidesmin antibodies. The remaining liver tissue was promptly removed and cut into small pieces; some were used for the direct determination of hepatic contents of collagen and RP, a main retinyl ester,11 while others were frozen in liquid nitrogen and stored at −80°C for RNA extraction. All animals used were treated humanely according to the Japanese National Guidelines.

Light microscopy and immunohistochemistry

For light microscopic examination, liver specimens were fixed overnight in phosphate buffered formaldehyde, embedded in paraffin wax, and stained with haematoxylin and eosin and Mallory azan. For immunohistochemical studies, tissue block sections were mounted on slides, deparaffinised in xylene, and rehydrated in alcohol. Endogenous peroxidase was blocked with a 1% hydrogen peroxide solution in methanol. Incubation with a monoclonal antibody directed against α-SMA (clone 1A4; Dako, Glostrup, Denmark; diluted 1/50) or desmin (Dako; diluted 1/25) for one hour at room temperature was preceded by digestion in a 0.1% trypsin solution in phosphate buffered saline (PBS) at 37°C for 30 minutes. The secondary antibody was a biotin conjugated rabbit antimouse IgG F(ab′)2 fragment (Dako; diluted 1/200) followed by incubation with an avidin-biotin complex (Vectastain ABC reagent, Vector Laboratories, Burlingame, California, USA). Reaction products were visualised using an ABC-peroxidase-diaminobenzidine method. A control for immunostaining specificity in which the primary antibody was substituted with non-immune mouse serum, was negative. Liver tissue sections were photographed using a light microscope (Carl Zeiss, Heidenheim, Germany) or a differential interference contrast (DIC) microscope (Axioskop, Carl Zeiss).

For the morphometric analysis of activated stellate cells in fibrotic livers, the mean value of α-SMA or desmin positive cells in five ocular fields per specimen was used as the percentage area at 40× magnification using an NIH image analysis system. As α-SMA was strongly stained in vascular smooth muscle cells and desmin was detected in perisinusoidal cells and vascular smooth muscle cells,15 the mean value in five control liver specimens from rats of groups 1 or 6 was subtracted from each experimental specimen and α-SMA or desmin positive cells were expressed as a percentage of the total area of the specimen.

Collagen content determination

Collagen was determined as previously reported.18 A portion of each liver was homogenised in 33 volumes (ml/g) of 0.5 M acetic acid at 4°C using a Polytron PCU-2 homogeniser (Kinematica, Switzerland), and the homogenate was disrupted by freeze thawing and sonication for two minutes. The fraction of insoluble collagen after the acid extraction, which was comprised of cross linked collagen, was then heated at 80°C for 60 minutes followed by conversion into soluble gelatin. The gelatin contents of the acid extract were assayed using the Sircol collagen assay kit (Biocolor, Belfast, Northern Ireland) according to the manufacturer’s directions, and the results were expressed as micrograms of collagen per 100 mg of dry weight liver.

Retinoid analysis

Retinoid analysis was performed as previously reported.21 Another portion of each liver was homogenised in 40 volumes (ml/g) of distilled water at 4°C using a Polytron PCU-2 homogeniser. For extraction, RP and retinyl acetate were added as the internal standard and the homogenate was mixed with two volumes of ethanol and n-hexane (containing 0.02% butylated hydroxytoluene). After shaking for five minutes, the mixture was centrifuged at 200 g to separate then-hexane layer. Then-hexane layer was removed and dried under a stream of N₂. The residue was resuspended in a small volume of n-hexane and injected directly into a reverse phase high performance liquid chromatography (HPLC) column, TSKgel ODS-80Tm (Tosho, Tokyo, Japan) using an eluent composed of ethanol and distilled water (95:5) at a flow rate of 15 ml/h. Retinoids were detected by fluorescence spectorophotometry with an excitation wave length of 325 nm and an emission wave length of 470 nm. Hepatic RP content was quantified by comparing the peak areas of the extract samples with the peak area of authentic RP as an internal standard (peak b in fig 2), and corrected for differences in molar absorption coefficients as determined in standard solutions. For a series of 20 HPLC determinations, the efficiency of RP extraction was 78 (8)% with an intra-assay variability of 8 (4)%. The lowest detectable concentration of RP was 8.7 IU/injection (7 IU/g liver), evaluated as the concentration resulting in a response of 2 SD from the zero dose response.

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Figure 2

Representative HPLC chromatograms of (A) retinyl acetate and retinyl palmitate (RP) standards; and (B) a rat liver extract. Peaks: a, retinyl acetate (retention time, 1.9 minutes); b, RP, internal standard (retention time, 6.7 minutes).

RNA extraction and northern blot analysis

For total RNA extraction from liver tissue, 100 mg of the frozen liver tissue was homogenised in 2 ml of RNAzol B (Biotex Laboratories, Friendswood, Texas, USA) at 4°C using a Polytron PCU-2 homogeniser. Chloroform (200 μl) was added to the homogenate and the aqueous phase was collected after centrifugation at 12 000g at 4°C for 15 minutes. The RNA was precipitated with an equal volume of isopropanol and washed with 75% ethanol. RNA pellets were resuspended in diethylpyrocarbonate treated distilled water, and the amount of RNA was determined by spectrophotometric absorption at 260 nm. Samples of 10 μg of total RNA were electrophoresed under denaturing conditions, transferred to Hybond N⁺ filters (Amersham, UK), and fixed by ultraviolet cross linking. The filters were prehybrised and then hybridised with cDNA probes that were labelled with ³²P-deoxcytidine triphosphate using a Random Prime DNA labelling kit (Boehringer Mannheim, Indianapolis, Indiana, USA).

The following cDNA probes were used for northern blots: a 3.55 kb EcoR1/EcoR1 fragment of clone NJ3 3.55 coding for pro-α₂(I) collagen25; a 1.3 kb EcoR1/HindIII fragment of clone Hf934 coding for pro-α₁(III) collagen26; and a 1.2 kb EcoR1/HindIII fragment of clone pHcGAP coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).27 The probes were purchased from the American Type Culture Collection (Rockville, Maryland, USA). Hybridisation was performed overnight at 42°C in 50% formamide, 0.05 M sodium phosphate, 0.8 M NaCl, 1 mM EDTA, 0.2% sodium dodecyl sulphate (SDS), 2.5× Denhardt’s solution, and 250 μg/ml denatured salmon sperm DNA. Posthybridisation washes were performed in 2× standard saline citrate (1× SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.4) containing 0.1% SDS three times for 10 minutes at room temperature and also in 0.1× SSC containing 0.1% SDS three times for 20 minutes at 50°C. The filters were then exposed to Kodak XAR film (Eastman Kodak, Rochester, New York, USA) at −70°C. The filters were boiled in 0.1× SSC containing 0.1% SDS for 30 minutes to strip off the radioactive probes and rehybridised with another ³²P-labelled cDNA probe in a similar manner. Densitomery tracings were recorded using a laser densitometer UltraScan XL (Pharmacia LKB, Uppsala, Sweden).

Radioimmunoassay of oestradiol

Serum oestradiol concentrations were measured radioimmunologically using a commercial kit (CIS Diagnostic, Tokyo, Japan). The lower limit of detection for the oestradiol assay system was 1.0 pg/ml.

Isolation and cultivation of stellate cells

Stellate cells were isolated from the livers of male Wistar rats (500–600 g) as described by Friedman et al.28 Briefly, the liver was perfused in situ through the portal vein with a 16 gauge cannula, first with Ca²⁺/Mg²⁺ free Krebs-Ringer (KR) solution at 37°C for 10 minutes at a flow rate of 10 ml/min, followed by 0.1% pronase E (Merck, Darmstadt, Germany) in KR solution for 10 minutes, and then with 0.3% collagenase (Wako, Osaka, Japan) in KR solution for 30 minutes. The digested liver was excised, minced with scissors, and incubated in KR solution containing 0.05% pronase E and 20 μg/ml DNAse (Boehringer Mannheim) for 30 minutes at pH 7.2. The resulting suspension was filtered through nylon mesh (150 μm in diameter). A stellate cell enriched fraction was obtained by centrifugation of the filtrate in an 8.2% Nycodenz (Nycomed, Oslo, Norway) solution at 1400g at 4°C for 20 minutes. The cells in the upper layer were washed by centrifugation at 450g at 4°C for 10 minutes and suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Flow, McLean, Virginia, USA), 100 U/ml penicillin, 100 μg/ml streptomycin, and 1% l-glutamine. The purity of the isolated stellate cells was assessed by direct cell counting under a phase contrast microscope, by intrinsic vitamin A autofluorescence, and by immunocytochemistry using a monoclonal antibody against desmin (Dako; diluted 1/25); cell viability was examined by Trypan blue exclusion. Both cell purity and viability were in excess of 90%.

Cells were plated at a density of 5×10⁵ cells per well in 1 ml of culture medium on uncoated plastic culture dishes (Nunc, Naperville, Illinois, USA). Culture medium was changed two days after plating to a culture medium containing β oestradiol (Wako) and was changed every other day thereafter. β Oestradiol was first prepared as an ethanolic stock solution (10⁻² M) and then diluted in culture medium to the appropriate working solution (for example, 10× of the final concentration). Cells were maintained at 37°C in a 5% CO₂ atmosphere and 100% humidity, and maintained in culture for 10 days.

Immunohistochemistry

Stellate cells which were initially cultured for two days in DMEM supplemented with 10% FBS were then maintained either in the presence or absence of oestradiol for an additional four days. Cells were then fixed with 0.5% paraformaldehyde for one hour at 4°C, washed twice with ice cold PBS, and incubated with 0.25% bovine testicular hyaluronidase for 30 minutes at 4°C. Cells were again washed twice with PBS at room temperature and incubated with a monoclonal antibody against α-SMA (Dako; diluted 1/50) for one hour at 37°C in a humid chamber. Cells were then washed twice with PBS and incubated with the secondary antibody, a biotin conjugated rabbit antimouse IgG F(ab′)2 fragment (Dako; diluted 1/200), and finally by the avidin-biotin complex (Vectastain ABC reagent, Vector Laboratories). Reaction products were visualised by an ABC-peroxidase-diaminobenzidine method.

ELISA analysis

Stellate cells cultured for two days were further incubated in the presence or absence of oestradiol for the indicated time period. The culture media were stored at −20°C, and the concentration of soluble type I collagen in these media was determined by a sensitive antigen enzyme linked immunosorbent assay (ELISA) method. Briefly, a murine monoclonal antibody to human type I collagen (Calbiochem, Cambridge, Massachusetts, USA) was pipetted at a 1/500 dilution in 100 μl of carbonate-bicarbonate buffer (pH 9.5) into Nunc-Immuno Module MaxiSorp 96 well plates. This solid phase was then allowed to absorb overnight at room temperature. Each plate was then aspirated and treated with PBS containing 0.5% non-fat milk (330 μl/well) overnight at 4°C. Following aspiration, the samples were diluted 1/1 with assay buffer (PBS with 0.05% Tween 20 and 0.2% non-fat milk) and subjected to a primary incubation for two hours at room temperature. The plates were washed five times with PBS containing 0.05% Tween 20 and then incubated with the second antibody, rabbit antihuman type I collagen (Chemicon, Temecula, California, USA) conjugated with horseradish peroxidase (HRP) using Pierce’s EZ-Link Plus activated peroxidase (Rockford, Illinois, USA) at a dilution of 1/500 in the assay buffer with 5% normal rabbit serum for two hours at room temperature. The plates were washed five times as described above and treated with 100 μl of 0.05 M citrate-phosphate buffer (pH 5.0) containing 1.0 mg/ml o-phenylene-diamine per well for 20 minutes at room temperature. The reaction was stopped by the addition of 1 N sulphuric acid. The plates were read in an Auto Reader (MTP-120, Corona Electric, Katsuta, Japan) using a 492 nm filter with a reference at 630 nm, after blanking the machine with a reagent blank plate. Sample concentrations were determined from a plot of optical density versus concentration of type I collagen standard over a range of 0–100 μg/ml.

Cell proliferation

Using the Biotrak cell proliferation ELISA system (Amersham, Little Chalfont, UK), DNA synthesis was measured by incorporation of the pyrimidine analogue bromodeoxyuridine (BrdU) into the DNA of proliferating cells.29 Stellate cells cultured in 96 well plates for four days were further incubated in the presence or absence of oestradiol for 24 hours and were labelled with 10 μM BrdU for another 24 hours. After removing the culture medium, the cells were fixed and the incorporated BrdU was detected by the subsequent substrate reaction according to the manufacturer’s recommended protocol.

Western blot analysis of α-SMA

Stellate cells were cultured in 1 ml of culture medium with or without oestradiol for the indicated time period, and were then washed twice with ice cold PBS and lysed directly in SDS loading buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol). The samples were boiled for 10 minutes and applied to a standard 12% SDS-polyacrylamide protein gel. After electrophoresis, protein transfer was performed onto Hybond-ECL (Amersham, Arlingtone Heights, Illinois, USA) using a semi-dry blotting apparatus. The membrane was treated first with 10% non-fat milk in PBS and next with the monoclonal anti-α-SMA antibody (Dako; diluted 1/500) for two hours at room temperature. After vigorous washing, the membrane was then incubated with HRP conjugated goat antimouse IgG (Amersham; diluted 1/1000) for one hour at room temperature. Immunoreactive bands were visualised using the ECL (enhanced chemiluminescence) western blotting detection system kit (Amersham) according to the manufacturer’s recommended protocol.