Juglone was purchased from the Shandong Engineering Research Center for Natural Drugs (Yantai, China), the purity of which was 99.5%, according to high-performance liquid chromatography (acetonitrile: water, 90:10; C₁₈ column). Analytical grade dimethylnitrosamine (DMN) was purchased from Chengdu Kelong Chemical Reagent Factory (Chengdu, China) and silymarin was purchased from Tianshili Pharmaceutical Company (Tianjing, China). A total of 30 male Sprague-Dawley rats, weighing 150±25 g, were obtained from the Experimental Animal Center of Guiyang Medical College (Guiyang, China; Approval no. SCXK 2001–0022). The present study was performed according to instructions approved by the Institutional Ethical Committee of the General Hospital of Chengdu Military Region (Chengdu, China). The rats were randomly divided into the following five groups, each containing six rats: Controlgroup, DMN-induced hepatic fibrosis (model) group, juglone prevention (JP) group, silymarin prevention (SP) group and juglone + silymarin prevention (JP+SP) group. The rats in the model, JP, SP and JP+SP groups received subcutaneous injections of DMN solution at a dose of 0.5 ml/kg twice weekly for 4 weeks, with an initial dose of 0.2 ml/kg. These rats were fed a high-lipid/low-protein diet. The rats in the control group were fed a normal diet. Juglone (200 mg/kg), silymarin (1.0 g/kg) and juglone (200 mg/kg) + silymarin (1.0 g/kg), were administered once a day by gastric gavage to the rats in the In the JP, SP and JP+SP groups, respectively. After 4 weeks, the rats were sacrificed using anesthesia (anaestheticpropofol, 0.1 ml/100 mg) and a midline incision was made to remove the liver; ~2 ml blood was collected from each rat, as well as 0.5–1 mm liver lobe sections. The same section of each liver was removed and fixed in 10% neutral formalin (Sigma-Aldrich, St. Louis, MA, USA). The residual portion of liver was stored at −50°C. Rat serum was prepared by centrifugation at 224 x g for 15 min at room temperature and stored at −50°C.

The serum levels of ALT, AST, HA, LN, PCIII and CIV were measured using commercially available kits as follows: Alanine Aminotransferase BioAssay™ ELISA kit, Aspartate Aminotransferase BioAssay™ ELISA kit, Hyaluronic Acid BioAssay™ ELISA kit, LamininBioAssay™ ELISA kit, Procollagen III BioAssay™ ELISA kit and Collagen Type IV (CIV) BioAssay™ ELISA Kit (Wuhan Boster Biological Engineering Co., Ltd., Wuhan, China) and were performed, according to the manufacturer's instructions.

The tissue samples were homogenized in cold 20 mM HEPES buffer, containing 1 mM EGTA, 150 mMmannitol and 30 mM sucrose (pH 7.1; all Sigma-Aldrich), using a tephlon homogenizer (Polytron RZR 1; Heidolph, Schwabach, Germany). The cell debris was detached by centrifugation at 2,000 × g for 5 min at 4°C. The supernatants were used for the assessment of SOD activity, with assays conducted according to the manufacturer's instructions (Cayman Chemical Company, Ann Arbor, MI, USA). The levels of MDA were also estimated using a thiobarbituric acid method, according to the manufacturer's instructions (Cayman Chemical Company).

Following fixation in 10% formalin for 24 h, the liver samples were embedded in paraffin (Sigma-Aldrich). The samples were then cut into 5-mm tissue sections and mounted onto slides (Sigma-Aldrich). The sections were processed for hematoxylin and eosin (H&E) and Masson's trichrome (both Sigma-Aldrich) staining for assessment of the degree of liver fibrosis. A second set of tissue sections (size, 0.5×0.5 cm) were prepared for immunohistochemical assessment of the tissues and mitotic indexing (MI). The liver tissues were further assessed for histopathological examination by a single observer in a blinded-manner.

The liver sections were incubated in 2% H₂O₂ (Qingdao HiseaChem Co., Ltd., Qingdao, China) for 15 min following deparaffinization in xylene, rehydration in graded ethanol and antigenunmasking by heat treatment using citrate buffer. The sections were subsequently incubated with mouse anti-α-SMA (cat. no. SC-32251; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-Col III (cat. no.SC-80564; Santa Cruz Biotechnology) and rabbit anti-MT (cat. no.SC-11377; Santa Cruz Biotechnology, Inc.)antibodies (all at 1:500 dilution) overnight at 4°C Anti-mouse IgG in rabbit (cat no. M7023; 1:500; Sigma-Aldrich) was used as the secondary antibody. The samples were subsequently processed using an EnVision kit (Dako Denmark A/S, Glostrup, Denmark), according to the manufacturer's instructions. Phosphate-buffered saline (Sigma-Aldrich) served as a negative control. The cells with brown staining in the cytoplasm/nucleus were considered to be positive. A total of five high power microscopic fields (magnification, x500) were randomly selected in each slide and the numbers of positive cells in each field were counted under a microscope (Eclipse E600; Nikon Corporation, Tokyo, Japan (α-SMA and Masson's trichromeimmunopositivity). For the Col III staining, five high power fields were randomly selected in each tissue section and images were captured using a BioMias 2000 image analysis instrument (Image and Figure Research Institute, Sichuan University, China), to quantify the transparency of the immune-positive areas in the tissue section.

Cell proliferation was assessed by counting the number of mitotic cells per high-power field at a magnification of x200. For each slide, 10 randomly selected fields were counted. The MI was defined as the number of mitotic cells per 1,000 hepatocytes in paraffin-embedded liver samples stained with H&E.

The liver tissues were treated with TRIzol protein extraction reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions, and the protein concentrations were determined using Lowry's method (23). The protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sigma-Aldrich) and electroblotted onto polyvinylidenedifluoride membranes (Millipore Corporation, Billerica, MA, USA). Following blocking with 1.5% bovine serum albumin (Sigma-Aldrich), the membranes were incubated overnight at 4°C with primary antibodies against peroxisome proliferator-activated receptor (PPAR)-γ (mouse anti-PPAR-γ; cat. no. SC-7273), α-SMA (mouse anti-α-SMA; cat. no.SC-32251), TGF-β1 (rabbit anti-TGF-β1; cat. no.SC-146), TIMP-1 (rabbit anti-TIMP-1; cat. no.SC-5538) and MMP-2 (rabbit anti-MMP-2; cat. no. SC-10736) obtained from Santa Cruz Biotechnology, Inc. The membranes were further incubated with secondary antibody for 1 h at room temperature. The protein bands were detected using an enhanced chemiluminescence detection system (Pierce Biotechnology, Inc., Rockford, IL, USA) and the protein expression levels were determined using Quantity One software version 4.5 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Statistical analyses were performed using one-way analysis of variance followed by Bonferroni's post-hoc test, using SPSS 20.0 software (SPSS, Inc., Chicago, IL USA). P<0.05 was considered to indicate a statistically significant difference.

The effect of juglone treatment on functional liver enzymes is shown in Fig. 1. Biochemical analysis revealed that the DMN-treated rats exhibited a marked increase in the serum levels of ALT and AST, which were significantly higher, compared with those in the normal rats (P<0.01; n=10). However, the increased levels of these liver enzymes were efficiently reduced by treatments with silymarin and juglone (P<0.01; n=10). Juglone significantly restored the biochemical parameters to levels comparable with those following treatment with the silymarin, used as a standard drug in the present study.

The results of the effect of juglone on collagenic accumulation in the livers of rats are shown in Fig. 2. The results demonstrated that, in rats treated with DMN (the liver fibrosis model) significantly increased serum levels of HA, LN, PCIII and CIV were observed, compared with the control (P<0.01; n=10). However, following treatment with silymarin and juglone, a gradual decrease in the activities of HA, LN, PCIII and CIV were observed in the fibrotic tissues (P<0.01, vs. model group; n=10). These results suggested that treatment with juglone inhibited DMN-induced collagenic changes in these animals. The effect of juglone effect was more marked, compared with that of silymarin, which was used as the standard.

The antioxidant activity of the DMN group indicated that SOD was significantly reduced compared with the normal group. The JP group or SP group significantly restored the SOD level (Fig. 3). MDA level of liver homogenate was significantly high in the model group (DMN-treated group), however, administration of juglone or silymarin significantly reduced the level of MDA. The results of JP group were comparable with the SP group (Fig. 4). The combination of juglone and silymarin demonstrated a significant effect on the decrease of MDA in rat liver homogenate.

The appearance of the liver tissues in each group are shown in Fig. 5. It was evident that, with the exception of the DMN-treated group, which exhibited considerable nodules and irregularities, the liver tissues in all the groups generally exhibited smooth and even surfaces with no signs of nodules. Furthermore, the normal group exhibited normal growth, whereas the rats in the DMN-treated model group exhibited lower body weights. The protective effects of juglone on liver injury were comparable to those of silymarin. These two groups exhibited similar smoothness of the surface of the liver.

Masson's trichrome staining was performed to estimate the extent of fibrosis following treatment with DMN. As shown in Fig. 6A, histological analysis of the rat liver sections revealed that those from the normal group exhibited no collagen deposition. The DMN-treated model groupexhibited proliferation of the bile duct with thick fibrous septa and increased deposition of collagen fibers around the clogged central vein, indicative of significant fibrosis (Fig. 6B). Theliver sections from the rats in the JP group exhibited fewer fibrous septa and uneven regenerating nodules (Fig. 6C). The protective effect of silymarin on liver fibrosis was observedin the SP group (Fig. 6D), treated with silymarin as a standard drug for liver fibrosis. The collagen deposition patterns appeared comparable between the JP group, SP group and JP+SP (Fig. 6E) group. These observations demonstrated the hepatoprotective effect of juglone on hepatic fibrosis in rats.

The effect of treatment with juglone on the expression ofα-SMA is shown in Fig. 7A–E. Since the expression levels of α-SMA and CoI III are markers for liver fibrosis, the present study investigated the effect of juglone on the expression levels of α-SMA and CoI III. The results of the immunohistochemical staining demonstrated that, in the normal group, α-SMA-positive staining was limited to the vascular walls in the central veins and portal areas, whereas significantly marked immune staining was observed in the central veins, portal areas and surrounding the bile ductules in the DMN-treated model group. In the JP, SP and JP+SP groups, the expression levels of α-SMA were considerably decreased, compared with those in the model group (P<0.05; Fig. 7).

In terms of the expression levels of Col III, an increase in the numbers of Col III positive fibers were observed in the hepatic sinusoids and periportal areas in the model group, whereas a lower expression level of Col III was identified in the normal control group. In the JP, SP and JP+SP treatment groups, the expression levels of Col III were also significantly decreased, compared with the levels in the model group (P<0.05; Fig. 8A–E).

As shown in Fig. 9, treatment with DMNsignificantly suppressed the mRNA expression of PPAR-γ and increased the mRNA expression of α-SMA in the liver tissues (P<0.01). Compared with the model group, silymarin promoted the effect of DMN on the mRNA expression levels of PPAR-γ and α-SMA. By contrast, juglone increased the mRNA expression of PPAR-γ and reduced the mRNA expression of α-SMA, compared with the model and silymarin groups (P<0.01; Fig. 9).