A total of 30 Sprague-Dawley rats (male; 6 week old; weight, 180–200 g) obtained from the Liaoning Chang Sheng Biotechnology Co., Ltd. (Benxi, China) were used in the experiment. After adapting for 1 week under standard animal laboratory conditions (25±2°C; air humidity 50±10%; 12-h light/dark cycle) with free access to food and water, the rats were randomly divided into five groups (6/group): Control group, 100 mg/kg ALA group, CCl₄-treated group, CCl₄+50 mg/kg ALA group and CCl₄+100 mg/kg ALA group. The animal raising and handling procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (16) and approved by the Animal Experimental Ethics Committee of the Henan University of Chinese Medicine (Zhengzhou, China).

A CCl₄-induced liver cirrhosis model was established as previously described (17,18). Rats were intraperitoneally injected with 50% CCl₄ (purchased from Shanghai Aladdin Bio-Chem Technology Co. Ltd., Shanghai, China; 1:1 diluted with olive oil) three times a week for 8 consecutive weeks. In addition to treatment with CCl₄, rats in the CCl₄+50 mg/kg ALA or CCl₄+100 mg/kg ALA groups received 50 or 100 mg/kg ALA (Sangon Biotech Co. Ltd., Shanghai, China) orally every day for 8 weeks. Rats in the control group were intraperitoneally injected with an equal volume of olive oil instead of CCl₄ 3 times a week and received 0.5% sodium carboxymethylcellulose (the solvent of ALA) 0.5 ml orally every day. Rats in the 100 mg/kg ALA group were intraperitoneally injected with an equal volume of olive oil instead of CCl₄ three times a week and received 100 mg/kg ALA orally every day for 8 weeks. At the end of treatment, rats were anesthetized with isoflurane (2.5–3%), subsequently, blood was collected from the orbital sinus as Parasuraman et al (19) described previously. The rats were sacrificed with 200 mg/kg pentobarbital sodium by intraperitoneal injection and the liver tissues were harvested.

Liver tissues were fixed in 4% paraformaldehyde for 48 h at 4°C, and were subsequently embedded in paraffin and cut into 5-µm thick sections. Following washing with xylene and hydrating in graded ethanol, the sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome, according to standard procedures (20). For immunohistochemical analysis, the deparaffinized liver sections were incubated with rabbit anti-α-smooth muscle actin (α-SMA) antibody or rabbit anti-transforming growth factor (TGF)-β antibody (both 1:50; α-SMA, cat. no. 55135-1-AP; TGF-β, cat. no. 21898-1-AP; Wuhan Sanying Biotechnology, Wuhan, China) at 4°C overnight. Subsequently, the specific proteins were detected with biotinylated goat anti-rabbit immunoglobulin G antibody (1:200; cat. no. A0208; Beyotime Institute of Biotechnology, Haimen, China) at 37°C for 30 min. The reactions were finally analyzed with horseradish peroxidase-conjugated streptavidin (Beyotime Institute of Biotechnology). Following visualization using a diaminobenzidine substrate kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), the sections were imaged under a light microscope (BX53; Olympus Corporation, Tokyo, Japan; magnification, ×100-200).

Serum was isolated from blood samples by centrifugation at 1,100 × g for 10 min at 4°C. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) in the serum were detected using commercial kits (ALT, cat. no. C009-2; AST, cat. no. C010-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's protocol. The hydroxyproline level in liver tissues was measured using a hydroxyproline assay kit (cat. no. A030-2; Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocol.

Total RNA from liver tissues was extracted using the China TRIpure Total RNA Extraction kit (BioTeke Corporation, Beijing, China). cDNA was synthesized using Super M-MLV Reverse Transcriptase (BioTeke Corporation) according to the manufacturer's protocol and the temperature protocol was: 25°C for 10 min, 42°C for 50 min and 80°C for 10 min. The primer sequences used are presented in Table I. RT-qPCR reactions were performed on an Exicycler^™ 96 (Bioneer Corporation, Daejeon, Korea) with the 2X Power Taq PCR Master Mix (BioTeke Corporation) and SYBR Green (Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturer's protocol. The thermocycling conditions were 94°C for 5 min, 94°C for 10 sec, 60°C for 20 sec, 72°C for 30 sec, 40 cycles; 72°C for 2.5 min; 40°C for 1.5 min; melting at 60–94°C, every 1°C per second; incubation at 25°C for 2 min. The relative expression levels were calculated using the 2^−ΔΔCq method (21).

Total protein was isolated from liver tissues using radioimmunoprecipitation assay solution (Beyotime Institute of Biotechnology). Protein quantitation was conducted using the enhanced Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology). Subsequently, 40 µg protein was separated by SDS-PAGE on 10–15% gels and electrotransferred onto polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA). Subsequent to blocking with 5% nonfat milk at room temperature for 1 h, the membranes were incubated with specific primary antibodies against α-SMA (1:1,000; cat. no. 55135-1-AP; Wuhan Sanying Biotechnology), TGF-β (1:500; cat. no. 21898-1-AP; Wuhan Sanying Biotechnology), phosphorylated (p)-Smad3 (1:1,000; cat. no. 9520; Cell Signaling Technology, Inc., Danvers, MA, USA), Smad3 (1:1,000; cat. no. 9523; Cell Signaling Technology, Inc.,), Beclin-1 (1:1,500; cat. no. 11306-1-AP; Wuhan Sanying Biotechnology), light chain (LC)3II/LC3I (1:1,000; cat. no. 2775; Cell Signaling Technology, Inc.), p62 (1:1,000; cat. no. 39749; Cell Signaling Technology, Inc.), protein kinase B (AKT; 1:2,000; cat. no. 9272; Cell Signaling Technology, Inc.), p-AKT (1:1,000; cat. no. 4060; Cell Signaling Technology, Inc.), mammalian target of rapamycin (mTOR; 1:1,000; cat. no. 2972; Cell Signaling Technology, Inc.), p-mTOR (1:1,000; cat. no. 2971; Cell Signaling Technology, Inc.) or β-actin (1:500; cat. no. bsm-33139M; BIOSS, Beijing, China) at 4°C overnight. The membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (1:5,000; HRP-conjugated goat anti-rabbit antibody, cat. no. A0208; HRP-conjugated goat anti-mouse antibody, cat. no. A0216; Beyotime Institute of Biotechnology) at 37°C for 45 min. Finally, the specific proteins were detected using the Beyo Enhanced Chemiluminescent kit (Beyotime Institute of Biotechnology). The relative protein expression level was detected by densitometry using Gel-Pro Analyzer Version 3.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Data are presented as the mean ± standard deviation of six independent experiments. Differences between groups were analyzed using one-way analysis of variance, followed by the Bonferroni post hoc test using Image-pro plus 6.0 software (Media Cybernetics, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Histopathological alterations in the liver following treatment with CCl₄ and different doses of ALA were examined by H&E staining (Fig. 1). The livers of healthy and 100 mg/kg ALA-treated rats exhibited normal liver architecture. Treatment with CCl₄ resulted in visible lesions with the formation of fibrotic septa, the congestion of cytoplasmic vacuolation and hepatocyte necrosis. However, these histopathological alterations induced by CCl₄ were alleviated in the liver of 50- or 100 mg/ml-ALA treated rats. To further examine liver function, the activity of two key enzymes, ALT and AST, in the serum were determined. As presented in Fig. 1B and C, the activity of ALT and AST in CCl₄-treated rats were significantly increased 2.58- and 3.83-fold, respectively, compared with the control rats (P<0.01). By contrast, the activity of ALT and AST was reduced in a dose-dependent manner following treatment with ALA. These results suggested that ALA attenuated CCl₄-induced liver injury.

The collagen deposition of livers was determined using Masson's trichrome staining (Fig. 2A). Liver tissues from normal rats and 100 mg/kg ALA-treated rats demonstrated little collagen deposition, whereas those from CCl₄-treated rats demonstrated dense bundles of collagen fibers around lobules with disordered liver structure. The liver of CCl₄-exposed rats treated with 50 or 100 mg/ml demonstrated decreased collagen deposition. Furthermore, the marker of collagen deposition, hydroxyproline, was additionally significantly increased in the liver of CCl₄-treated rats and decreased in ALA-treated liver-cirrhosis-rats (P<0.01; Fig. 2B). Consistent with these alterations, the mRNA expression level of collagen α-1(I) chain (COL1A1) was significantly upregulated in the liver of rats exposed to CCl₄ and significantly reduced following treatment with 50 or 100 mg/ml ALA compared with the CCl₄ group (P<0.01; Fig. 2C). These data indicated that CCl₄-induced severe fibrosis was improved by treatment with ALA.

The marker of activated HSCs, α-SMA, was detected for the purpose of evaluating the effect of ALA on HSCs. Immunohistochemical staining results demonstrated that the expression level of α-SMA was significantly increased in the liver of CCl₄-treated rats and was significantly reduced following treatment with ALA (P<0.01; Fig. 3A and B). These alterations in the α-SMA level were additionally confirmed by western blot analysis (Fig. 3C). Therefore, the administration of ALA was demonstrated to result in a decrease in the degree of HSC activation in the liver of CCl₄-treated rats.

To investigate the mechanism behind the hepatoprotective effect of ALA, the activation of the TGF-β/Smad3 pathway, an important mediator of liver fibrosis, was detected (22). As presented in Fig. 4A, immunohistochemical analysis demonstrated an increased TGF-β level in the liver tissue of CCl₄-treated rats, when compared with that in the liver tissue of the control rats, while the elevation of TGF-β was decreased by ALA. These alterations in the TGF-β levels were additionally confirmed by RT-qPCR and western blot analysis (Fig. 4B and C). Consistent with the results of TGF-β, the protein level of p-Smad3 in the liver was significantly elevated by CCl₄ (P<0.01) and decreased by ALA. However, the level of Smad3 remained unaltered (Fig. 4D). These results demonstrated that ALA inhibited the activation of the TGF-β/Smad3 pathway in the liver of CCl₄-treated rats.

Autophagy has been implicated in CCl₄-induced liver cirrhosis (18) and thus, the expression of autophagy-associated factors was examined in the present study. RT-qPCR and western blot analysis demonstrated that the mammalian autophagy protein, Beclin-1, was significantly increased in the liver of CCl₄-treated rats (P<0.01), and was decreased by ALA in a dose-dependent manner (Fig. 5A). In addition, the marker of autophagosome formation, LC3II, which was significantly upregulated following treatment with CCl₄, was additionally significantly reduced following treatment with ALA (P<0.01; Fig. 5B). Furthermore, the expression of the autophagy substrate p62 was significantly decreased following CCl₄ treatment (P<0.01) and increased following the administration of ALA, in a dose-dependent manner (Fig. 5C). In combination, these results suggested that treatment with ALA reversed CCl₄-induced hepatocyte autophagy.

The upstream signaling pathway of autophagy, the AKT/mTOR pathway, was further examined (23). As expected, the p-AKT and p-mTOR expression levels were significantly reduced in the liver of CCl₄-treated rats (P<0.01), and alterations were prominently restored by the administration of ALA (Fig. 6). These results indicated that ALA was able to activate the AKT/mTOR pathway in the liver, which had previously been repressed by treatment with CCl₄.