Chitosan (98% purity, by high-performance liquid chromatography analysis) was purchased from Shandong Jinan Haidebei Marine Bioengineering Co., Ltd. (Shandong, China) and the chemical structure is presented in Fig. 1. CCl₄, xylene, alcohol, chloral hydrate and formaldehyde were purchased from Beijing Chemical Factory (Beijing, China). ALT (cat. no. A7526), ALP (cat. no. A7516) and AST (cat. no. A7561) Elisa kits were purchased from Pointe Scientific, Inc. (Canton, MI, USA). Anti-AST antibody (cat. no. AV43932), anti-α-SMA antibody (cat. no. A2547), secondary antibodies were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Anti-β-actin was purchased from Abcam (cat. no. ab8227, Cambridge, UK). Poly-L-lysine and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from DGCS-Biology Technology, Inc. (cat. no. 9806; Nanjing, China). The enhanced chemiluminescent western blotting kit was purchased from GE Healthcare (Chicago, IL, USA).

The present study was approved by the medical ethics committee of The First Hospital of Jilin University (Changchun, China). Male Sprague-Dawley (SD) rats aged 8 weeks, weighing 190±10 g, were purchased from the Laboratory Research Center of Jilin University (certificate no. 2000-042; Changchun, China). All animals were housed under standard laboratory conditions. Rats were fed and housed in groups in plastic-bottomed cages containing sawdust. Animals were bred in a temperature-controlled environment (25±2°C) with lighting between 6:00 a.m. and 6:00 p.m. From a total of 72 SD male rats, 16 were randomly selected as the control group and the remaining 56 were used as the CCl₄ treatment groups. Rats in the control group received intraperitoneal injections of olive oil (1.75 ml/kg) 3 times a week and for 7 weeks. The CCl₄ group rats received intraperitoneal injections of CCl₄ and an olive oil mixture (1.75 ml/kg) 3 times a week and for 7 weeks (CCl₄:olive oil=4:6) to induce liver fibrosis. After 7 weeks, 6 rats in the control group and 6 rats in the CCl₄ treatment group were sacrificed randomly for morphometric analysis of liver tissue; to measure the serum levels of ALT, AST, and ALP; to quantify the expression levels of the COL3 gene and α-SMA protein in the liver tissue; and to assess the establishment of the HF model. The remaining 50 rats in the CCl₄ group were randomly divided into five groups (n=10): The HF group; glycyrrhizinate group; low-dose chitosan group (2.5 mg/kg); middle-dose chitosan group (5.0 mg/kg); and high-dose chitosan group (10.0 mg/kg). Subsequently, the six groups were unified by a continuous administration of daily tail vein injections for 28 days. Control groups and HF groups were administered normal saline; glycyrrhizinate and chitosan groups were administered the corresponding regents. Blood was collected from the inner canthus vein on days 7, 14, 21 and 28 to detect the ALT, AST and ALP levels. On day 28, rats were anesthetized via an intraperitoneal injection of 10% chloral hydrate (300 mg/kg) and sacrificed by heart puncture for blood extraction. Subsequently, the abdominal cavity was opened using a U-shaped incision; the rat liver was harvested, cut into two uniform blocks, washed with saline and dried with filter paper. A piece of liver tissue was fixed in 10% formalin at 4°C for 24 hours for morphometric analysis, whilst the other piece was wrapped in foil and stored in liquid nitrogen for western blot analysis. Following 7 days of fixation, the fixed liver tissues were embedded in paraffin for sectioning.

Serum levels of AST, ALT and ALP were measured using commercial assay kits, according to the manufacturer's protocols.

Livers were fixed in 10% neutral buffered formalin. The fixed tissues were dehydrated through an ascending alcohol series (70, 80, 90 and 100% v/v) and paraffinized in benzene, prior to being embedded in paraffin. Paraffin sections (5 µm) were cut using a microtome for hematoxylin and eosin (H&E) staining to evaluate liver injury. In brief, the paraffin sections were stained with hematoxylin for 5 mins at room temperature, then washed in running water for 5 mins. Following immersion in 1% hydrochloric acid for 30 sec, the sections were stained with 0.5% eosin. The extent of HF in each sample was assessed based on the scoring criteria presented in Table I. This grading system was proposed by Brunt et al in 1999 for scoring fibrosis (21). A modified non-alcoholic fatty liver disease fibrosis score (22) was used for scoring inflammatory lesions.

Liver tissue lysates were prepared by homogenizing liver samples in RIPA buffer. The supernatant was transferred into a 1.5-ml Eppendorf tube, kept at 4°C in the refrigerator for 40 min and centrifuged at room temperature for 10 min at 12,000 × g. Protein concentration in the supernatants were measured using a Bradford protein assay according to the manufacturer's protocol. A total of 50 µg protein per sample was separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked for 2 h in PBS containing 10% non-fat dried milk and 0.1% Tween 20. Subsequently, membranes were incubated at 37°C for 1 h with anti-α-SMA (1:200) or anti-β-actin (1:40,000) antibodies in PBS containing 5% milk, and then incubated with the goat anti-rat secondary antibody (1:2,000) at room temperature for 2 h. Following washing, the membranes were visualized using the enhanced chemiluminescent western blot kit and densitometric analysis was performed using Image J version 1.44 (National Institutes of Health, Bethesda, MD, USA).

Total RNA was extracted from liver tissues using the TRIzol^® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. A total of 2 µg RNA was reverse transcribed to complementary (c)DNA at 37°C for 15 min and at 85°C for 5 seconds using random hexamers and the SuperScript™ III First-Strand Synthesis system (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was PCR-amplified in tandem using specific primers for COL3 and GAPDH, and Taq polymerase (cat. no. EP0402; Thermo Fisher Scientific, Inc.). The primer sequences were as follows: COL3 (169 bp product), forward 5′-AACCCTGCTCGGAATTGCAG-3′, reverse 5′-TCTGTCCACCAGTGCTTCCG-3′; GAPDH (687 bp product), forward 5′-GGGTGATGCTGGTGCTGAGTATGT-3′, reverse 5′-AAGAATGGGAGTTGCTGTTGAAGT-3′. Amplification was performed for 30 cycles with a denaturation temperature of 94°C, annealing temperature of 58°C and extension temperature of 74°C for COL3 and GAPDH in a thermal cycler (Veriti; Thermo Fisher Scientific, Inc.). PCR products were verified on 1.2% agarose gels and visualized under UV following ethidium bromide staining. Band intensities were analyzed using the Image Master Total Lab image analysis software version 2.01 (Nonlinear Dynamics, Ltd., Newcastle upon Tyne, UK). GAPDH was used as an internal control to normalize target gene expression.

All data were analyzed and assessed for significance using the Pearson omnibus normality test. All data are presented as the mean ± standard error of the mean. The significance between experimental groups and control group was determined by Dunnett-t test using SPSS version≈20.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Rat livers were visually assessed to evaluate morphological alterations following treatment with CCl₄. As presented in Fig. 2A, the livers from rats treated with CCl₄ (right) became pale, hard, rough, shrunken and thick, indicating serious liver injury, compared with the control group (left). The serum levels of ALT, AST and ALP in the CCl₄-treated group were three- to four-fold higher compared with in the control group (P<0.05), demonstrating that the rat model of liver fibrosis was successfully established (Fig. 2B). In addition, the expression levels of α-SMA and COL3 in liver tissues were evaluated using western blotting and RT-sqPCR, respectively (Fig. 2C). The expression levels of the α-SMA protein and COL3 gene in the CCl₄-treated group were approximately two-fold higher compared with the control group (P<0.05; Fig. 2D), indicating HSC activation and liver fibrosis.

The physical status of rats in the different treatment groups was recorded. Rats in the control group were healthy and active, and had glossy, smooth coats with bright eyes. Conversely, HF rats exhibited dull eyes, swollen paws, slow movement, decreased food intake, unkempt back hair, trichomadesis and hard abdominal swelling. Rats treated with chitosan or glycyrrhizinate increased their food intake, and edema and trichomadesis were reduced. Following 7 days of treatment with chitosan, there was a significant reduction in ALT, AST and ALP enzymes compared with the HF group (Fig. 3). The recovery of rats in the middle- and high-dose chitosan treatment groups was also better compared with the low-dose chitosan treatment group. In addition, the effect of treatment with chitosan was dependent on the duration of administration, with improved recovery occurring after 21 days of treatment.

The liver tissue from rats in the HF group exhibited serious liver injury, as identified by substantial macrosteatosis, hepatocellular ballooning, transparent cytoplasm and nuclei being pushed to the side, suggesting cellular necrosis. In addition, pseudolobuli were visible, which are characteristic structures of HF. In the glycyrrhizinate group, inflammation was markedly reduced and the pseudolobule was not visible; however, ballooning degeneration and fatty degeneration remained. The low-dose chitosan group exhibited obvious pseudolobule formation and alveolar inflammatory cell infiltration. Treatment with middle-dose chitosan produced similar results to the glycyrrhizinate group. The high-dose chitosan group had the best outcome, with liver tissues exhibiting no signs of pseudolobules, ballooning degeneration or fatty degeneration. The functional status of the high-dose group improved rapidly. The other groups exhibited certain improvements, although their overall recovery was worse compared with the high-dose chitosan group (Fig. 4 and Table II). These results indicated that chitosan reduced hepatocellular damage and improved liver function in HF rats, with the highest dose having the best treatment effect.

The expression levels of α-SMA were significantly decreased in all treatment groups compared with the HF group (Fig. 5A). α-SMA expression levels were lowest in the liver tissues of rats in the high-dose chitosan group and in the glycyrrhizinate group, The α-SMA expression levels were decreased to a lesser extent in the low-dose chitosan group. Notably, alterations in α-SMA expression levels in each experimental group were in line with the corresponding H&E staining results. The expression levels of COL3 in the liver tissues of the treatment groups were lower compared with the HF group (Fig. 5B). The high-dose chitosan group displayed a significant reduction in COL3 expression compared with the HF group (P<0.01). The middle-dose and glycyrrhizinate groups displayed a less significant reduction after 21 days of treatment (P<0.05).