A total of 10 normal male Sprague-Dawley rats (6–8 weeks old, >400 g) were obtained from The Shanghai Laboratory Animal Center, Chinese Academy of Sciences Shanghai, China. Rats were housed in a temperature controlled environment (23±2°C), at a relative humidity of 60±10%, with a 12-h light/dark cycle and free access to food and water. Primary HSCs were purified from Sprague-Dawley rats by perfusion with pronase and collagenase, followed by Nycodenz density-gradient centrifugation at 1,450 g for 22 min at 25°C (18). The isolated cells were cultured with Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). On the third day, the primary HSCs were used for subsequent experiments. The protocol was approved by the Committee on the Ethics of Animal Experiments of Shanghai University of Traditional Chinese Medicine, China. All animals received humane care during the study.

HSCs were plated into 96-well plates and incubated at 37°C for 24 h. Next, the cells were treated with 0.1 µM Ang II (Sigma-Aldrich; Merck KGaA) with or without Lev A (2.4, 6 and 15 µM; Shanghai Winherb Medical Technology Co., Ltd.,) or losartan (1 µM; Sigma-Aldrich; Merck KGaA) for another 24 h at 37°C. Cell proliferation was detected using the Cell-Light 5-ethynyl-2′-deoxyuridine (EdU) Cell Proliferation assay kit (cat. no. 10310; Guangzhou RiboBio Co., Ltd.) according to the manufacturer's protocols. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich; Merck KGaA) at a concentration of 1 µg/ml for 10 min at room temperature. EdU-positive and EdU-negative cells were determined by fluorescent imaging. Images were taken using Cellomics ArrayScan VTI HCS Reader and analyzed using Cellomics Cell Health Profiling BioApplication software (version 3.1; Thermo Fisher Scientific, Inc.).

HSCs were seeded into 96-well plates. After 24 h, the cells were incubated with Ang II (0.1 µM) with or without Lev A (2.4, 6 and 15 µM) for another 24 h at 37°C. The cells were then washed twice with cold phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 10 min at 4°C. F-actin and α-SMA were determined by immunofluorescence staining as previously described (19). Images were captured using Cellomics ArrayScan VTI HCS Reader and analyzed using Cellomics Cell Health Profiling BioApplication software (Thermo Fisher Scientific, Inc.).

A total of 24 male Wistar rats (6–8 weeks old; 240–260 g) were obtained from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences Shanghai, China. The animals were housed with a 12-h light/dark cycle under temperature control at 23±2°C, with a relative humidity of 60±10% and free access to food and water. The rats were randomly assigned into 3 groups: Control group, model group and Lev A group. The animals in model and Lev A groups given intraperitoneal injections of CCL₄ (5 ml/kg) for three consecutive days per week over a period of 4 weeks (20). From the third week of CCL₄ injections, rats in the Lev A group were administered with Lev A at a dose of 3 mg/kg/day. Rats in the control and model groups were treated with an equal amount of vehicle. After 2 weeks of administration, rats were anaesthetized with 2% pentobarbital sodium and the blood samples were obtained from the inferior vena cava. A portion of each liver was fixed in 10% phosphate-buffered formalin for 24 h at room temperature for histological studies following paraffin embedding. The remainder was snap-frozen in liquid nitrogen and stored at −80°C for western blot analysis. The protocol was approved by the Committee on the Ethics of Animal Experiments of Shanghai University of Traditional Chinese Medicine, China. All animals received humane care during the study.

The hematoxylin-eosin and sirius red staining were performed as previously described (21). Briefly, paraffin embedded-liver samples were cut into 4 µm-thick sections and then stained with H&E and sirius red. A random selection six microscopic fields were observed at a magnification of ×200 with an inverted microscope (Mode: IX70, Olympus Corporation).

Kits were used to measure levels of serum alanine aminotransferase (ALT; cat. no. C009-2-1), aspartate aminotransferase (AST cat. no. C010-2-1), albumin (Alb; cat. no. A028-2-1) and total bilirubin (TBil; cat. no. C019-1-1), according to the manufacturer's protocols (Nanjing Jiancheng Bioengineering Institute), as previously described (21).

Total protein was isolated from HSCs and liver tissue of rats using radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM PMSF, 1X Roche complete mini protease inhibitor cocktail, Roche PhosSTOP phosphatase inhibitor cocktail; Beyotime Institute of Biotechnology). The protein concentration was determined by the bicinchoninic acid method (Thermo Fisher Scientific, Inc.). Equal amounts of protein (20 µg) were separated via SDS-PAGE on a 10% gel under denaturing and non-reducing conditions and then transferred to a nitrocellulose membrane. The membrane was blocked with odyssey blocking buffer (LI-COR Biosciences) at room temperature for 1 h and then incubated with primary antibodies against AT1R (1:200; cat. no. sc-1173; Santa Cruz Biotechnology, Inc.), phosphorylated (p)-ERK1/2 (1:200; cat. no. sc-7383; Santa Cruz Biotechnology, Inc.), total ERK2 (1:200; cat. no. sc-1647; Santa Cruz Biotechnology, Inc.), GAPDH (1:5,000; cat. no. sc-166574; Santa Cruz Biotechnology, Inc.), α-SMA (1:1,000; cat. no. ab5649; Abcam), c-Jun (1:1,000; cat. no. 9165; Cell Signaling Technology, Inc.) and p-c-Jun (1:1,000; cat. no. 9261; Cell Signaling Technology, Inc.) at 4°C overnight. Following washing in PBS with 0.1% Tween-20, the blots were incubated for 1 h at room temperature with a IRdye^® 680CW anti-rabbit (1:20,000; cat. no. 926-32223) or IRDye 800CW anti-mouse secondary antibody (1:20,000; cat. no. 926-32210), both purchased from LI-COR Biosciences. The signals were visualized and analyzed using Li-Cor Odyssey Imaging software (version 2.1; Li-Cor Biosciences).

Data are expressed as means ± standard deviation, and were analyzed using one-way analysis of variance, followed by a post hoc least significant difference test. Statistical analysis was conducted using SPSS version 18.0 software (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

After 24 h incubation, Ang II significantly increased the cell number of HSCs compared with the control, as determined by EdU assay (P<0.01; Fig. 1A and B). Co-treatment with Lev A significantly decreased the number of HSCs proliferating at concentrations of 6 and 15 µM (P<0.01). However, 2.4 µM of Lev A exhibited little effect on the proliferation Ang II-induced HSCs. The blocker of AT1R, losartan, also significantly suppressed the Ang II-induced proliferation of HSCs (P<0.01; Fig. 1A and B).

To investigate the effect of Lev A on the activation of Ang II-induced HSCs, the expression of α-SMA and F-actin was determined. Ang II treatment significantly enhanced the expression of α-SMA (P<0.01), whereas co-treatment with losartan or Lev A significantly suppressed Ang II-induced α-SMA upregulation (P<0.05; Fig. 2A and B). Western blot analysis also demonstrated that Lev A inhibited Ang II-induced α-SMA upregulation (P<0.01; Fig. 2C and D). The present study also determined the expression of F-actin after Ang II treatment using immunofluorescence staining. Ang II induced a significant upregulation of F-actin (P<0.01; Fig. 2E and F) and Lev A partly reversed the effect of Ang II on F-actin (P<0.05).

Our previous study (21) revealed that Ang II is able to increase the level of its receptor AT1R in HSCs and so the effect of Lev A on AT1R expression was examined. Ang II treatment significantly upregulated the expression of AT1R (P<0.01; Fig. 3A and B). Losartan and Lev A treatment both repressed Ang II-induced AT1R upregulation (P<0.01). Activation of ERK and c-Jun is involved in Ang II-induced HSCs proliferation and activation (12,21). Therefore, the effect of Lev A on the activation of ERK and c-Jun was also studied. Ang II treatment induced significant increases of p-ERK1/2 and p-c-Jun (P<0.01; Fig. 3A, C and D). Pretreatment with Lev A significantly inhibited the phosphorylation of ERK1/2 and c-Jun induced by Ang II (P<0.05).

To evaluate the in vivo effect of Lev A, a CCL₄-induced rat hepatic fibrosis model was used. CCL₄ significantly decreased the body weight, and increased the liver/body weight ratio (P<0.01; Fig. 4A and B). Treatment with Lev A significantly improved the liver/body weight ratio (P<0.01) but it did not alter the body weight. In the model group, CCL₄ treatment significantly decreased the albumin level and increased the ALT, AST, γ-GT, IBIL and TBil levels (P<0.01; Fig. 4C-H). Lev A treatment significantly improved the liver functions of experimental rats with hepatic fibrosis. H&E staining demonstrated that CCL₄ induced evident centrilobular hepatic necrosis and infiltrating lymphocytes, and Lev A treatment partly prevented the histopathological changes (Fig. 4I).

CCL₄ treatment led to a notable deposition of collagen in rat livers as determined by sirius red staining (Fig. 5A). Treatment with Lev A for 2 weeks resulted in an alleviation of collagen deposition (Fig. 5A). As determined by western blotting, α-SMA and collagen I expression was significantly increased in CCL₄-treated rats and was suppressed by Lev A (P<0.01; Fig. 5B-D). In CCL₄-treated rats, the hydroxyproline (Hyp) content was also significantly upregulated compared with the control group (P<0.01; Fig. 5E). Treatment with Lev A significantly decreased the Hyp content (P<0.01).

To further evaluate the role of Lev A on RAS in vivo, the molecules of ACE-Ang II-AT1R axis in rats were next determined. In the model group, the expression of AT1R was significantly upregulated compared with the control group (P<0.01; Fig. 6A and B). p-ERK, the downstream molecule of Ang II-AT1R, was also significantly increased in the model group compared with the control (P<0.05; Fig. 6A and C). Lev A treatment significantly decreased the CCL₄-induced increase of AT1R, Ang II and p-ERK (P<0.01). Compared with the control group, the plasma Ang II level was also significantly increased in the CCL₄ group (P<0.01; Fig. 6D).