All animal experiments were performed in accordance with the National Institutes of Health Guidelines for the Handling of Laboratory Animals (15) and were approved by the Ethics Committee of The Eighth Affiliated Hospital of Xinjiang Medical University (Urumqi, China; approval no. 2020-035). A total of 40 male Kunming (KM) mice (weight, 20±2 g; 4 weeks-old; Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimated for 1 week in an environment of 25˚C with a 12-h light/dark cycle, relative humidity of 55%, and free access to food and water. Subsequently, 1 ml CCl₄ (Sinopharm Chemical Reagent Co., Ltd.) was dissolved in 100 ml olive oil and mixed well to prepare a 1% CCl₄ solution. RSG was purchased from Chengdu Hengrui Pharmaceutical Co., Ltd.

KM mice were divided into the following four groups: i) Control; ii) RSG; iii) CCl₄; and iv) RSG + CCl₄ groups. Mice in the control and CCl₄ group were separately treated with an intraperitoneal injection of a single dose of saline or 1% CCl₄ (10 ml/kg), respectively. Prior to CCl₄ injection, mice in the RSG + CCl₄ group were treated with 10 mg/kg RSG by oral gavage for 5 consecutive days. Mice in the RSG group were treated as outlined in the RSG + CCl₄ group, except with an additional (10 ml/kg) saline injection in place of CCl₄. All mice were fasted and had only free access to water for 24 h after modeling. Subsequently, mice were anesthetized using sodium pentobarbital (100 mg/kg; intraperitoneal) and the abdominal cavity was opened through an incision along the midline of the abdomen to expose the abdominal aorta. Following blood collection (0.5 ml) with a syringe, the mice were sacrificed by cervical dislocation. The liver tissues were immediately obtained, washed with PBS and were then stored at -80˚C.

The liver tissue samples were fixed in 4% paraformaldehyde at 4˚C for 6 h, dehydrated with gradient alcohol and embedded in paraffin. Subsequently, the tissue samples were cut into 5-µm slices. Sections were immersed in xylene, rehydrated with gradient alcohol and water, and stained with hematoxylin for 3 min and eosin for 3 min (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature. Following dehydration with gradient alcohol, the tissues were treated with xylene for 3 min. Physiological changes in the tissue samples were observed under a light microscope (magnification, x400; Olympus Corporation).

The blood was allowed to stand at 4˚C for 30 min and the serum was collected following centrifugation at 1,000 x g for 10 min at 4˚C. The serum levels of ALT and AST were measured using ALT (cat. no. C009-1-1) and AST assay kits (cat. no. C010-1-1; both from Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's instructions. Briefly, the serum sample was added to a tube, mixed with ALT or AST matrix solution for 30 min in a 37˚C water bath followed by the addition of a color developing agent. The reaction was terminated following incubation of the samples with stop solution for 20 min in a water bath at 37˚C. After incubation for 5 min at room temperature, a microplate reader (Thermo Fisher Scientific, Inc.) was used to measure the optical density (OD) value of each well at a wavelength of 505 nm.

The mass of tissue samples used to measure indices of hepatic function was calculated according to the formula: Liver mass (g): volume (ml)=1:9. Subsequently, the tissue samples were added into pre-cooled physiological saline and homogenized on ice. The homogenate was then centrifuged at 1,500 x g at 4˚C for 10 min and the supernatant was collected. SOD (cat. no. A001-1), catalase (CAT; cat. no. A007-1-1), GSH (cat. no. A005-1), NO (cat. no. A012-1-2) and malondialdehyde (MDA; cat. no. A003-1-1) assay kits (all from Nanjing Jiancheng Bioengineering Institute) were used to assess the content of the corresponding indices, according to the manufacturer's instructions. The OD values were measured using a microplate reader (Thermo Fisher Scientific, Inc.) at a wavelength of 550 (for SOD and NO), 405 (for CAT), 412 (for GSH) and 532 nm (for MDA).

The determination of ROS was performed using a ROS assay kit (cat. no. HR7814; Beijing BioRab Technology Co. Ltd.). The tissue sections were soaked in the cleaning solution for 15 sec and then the residual solution on the surfaces were wiped off. The sections were incubated in the staining working solution for 30 min at 37˚C in the dark. After washing twice with PBS, the results were observed under a fluorescence microscope (magnification, x100; Olympus Corporation).

Serum samples were collected as aforementioned. The secretion levels of TNF-α (cat. no. 88-7324), IL-6 (cat. no. 88-7064) and IL-1β (cat. no. 88-7013; all from Thermo Fisher Scientific, Inc.) in the serum were determined using the corresponding ELISA kits according to the manufacturer's instructions. Finally, a microplate reader (Thermo Fisher Scientific, Inc.) was used to measure the OD values in each well at a wavelength of 450 nm.

Total RNA was extracted from tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The extracted RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. The mRNA expression levels were quantified using the QuantiTect SYBR Green PCR kit (Qiagen, Inc.) on the ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR amplification conditions were as follows: 95˚C for 10 min, followed by 40 cycles at 95˚C for 30 sec, 64˚C for 34 sec and 72˚C for 30 sec. The relative gene expression levels were normalized to that of the housekeeping gene, GAPDH. The 2^-ΔΔCq method was utilized (16). The primer sequences used for qPCR are listed in Table I.

Total proteins were extracted from liver tissue samples using RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was measured using a BCA protein assay kit (cat. no. A045-3-2; Nanjing Jiancheng Bioengineering Institute). Total protein extracts (20 µg/lane) were then separated by SDS-PAGE on 10% gels and were transferred onto PVDF membranes. After blocking with 5% skimmed milk at room temperature for 2 h, the membranes were incubated with primary antibodies against cleaved caspase-8 (cat. no. 9748; dilution, 1:1,000), caspase-8 (cat. no. 4927; dilution, 1:1,000; both from Cell Signaling Technology, Inc.), cleaved caspase-3 (cat. no. ab32042; dilution, 1:1,000), caspase-3 (cat. no. ab184787; dilution, 1:2,000), cleaved poly(ADP-ribose) polymerase (PARP; cat. no. ab32064; dilution, 1:1,000), PARP (cat. no. ab191217; dilution, 1:1,000), PPARγ (cat. no. ab178860; dilution, 1:1,000), Nrf2 (cat. no. ab62352; dilution, 1:1,000), NAD(P)H quinone oxidoreductase 1 (NQO1; cat. no. ab213239; dilution, 1:500), HO-1 (cat. no. ab52947; dilution, 1:2,000), NOD-like receptor protein 3 (NLRP3; cat. no. ab263899; dilution, 1:1,000), caspase-1 (cat. no. ab138483; dilution, 1:1,000), IL-1β (cat. no. ab234437; dilution, 1:1,000), apoptosis-associated speck-like protein (ASC; cat. no. ab283684; dilution, 1:1,000) and the loading control, β-actin (cat. no. ab8226; dilution, 1:1,000; all from Abcam) at 4˚C overnight. Following incubation with the primary antibodies, the membranes were incubated with HRP-conjugated anti-rabbit (cat. no. ab97051; dilution, 1:10,000) or anti-mouse (cat. no. ab6728; dilution, 1:2,000; both from Abcam) antibodies at 37˚C for 2 h. The bands were visualized with an ECL reagent (MilliporeSigma) at room temperature for 2 min. The density of each band was semi-quantified using ImageJ software (v1.8; National Institutes of Health).

All experiments were repeated three times. Data are expressed as the mean ± SD. One-way ANOVA followed by Tukey's post hoc test was carried out to compare the differences among multiple groups. Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

The changes in liver histology among the four groups of mice were evaluated by H&E staining. The results showed that hepatocytes in the control and RSG groups were a normal shape and were tightly arranged. In the CCl₄ group, the cell arrangement was disordered, and inflammatory cell infiltration and cell necrosis were observed. However, pretreatment of CCl₄-treated mice with RSG markedly improved inflammatory infiltration. In addition, the cell arrangement was more compact in the RSG + CCl₄ group compared with that in the CCl₄ group (Fig. 1A). Furthermore, no differences were observed in the serum levels of ALT and AST between the control and RSG groups. However, in the CCl₄ group, the levels of both transaminases were significantly increased, whereas the levels were reduced following pretreatment with RSG (Fig. 1B). Subsequently, the levels of the hepatic biochemical indicators SOD, CAT, GSH, MDA, NO and ROS were determined in each group of mice using the corresponding kits. No statistically significant differences in the activity of the aforementioned indicators were observed between the control and RSG groups. However, the levels of SOD, CAT and GSH were reduced, whereas those of MDA and NO were increased in the CCl₄ group. This effect was abrogated following pretreatment with RSG (Fig. 1C). In addition, the ROS fluorescence of the CCl₄ group was significantly enhanced compared with that in the control group and RSG pretreatment reduced the ROS fluorescence intensity (Fig. 1D). These findings suggested that pretreatment of mice with RSG could reduce the severity of CCl₄-induced hepatic injury.

Since hepatic injury is closely associated with inflammation, the present study aimed to determine the expression levels of inflammatory factors in serum samples and liver tissues isolated from mice in different groups. Therefore, the secretion levels of TNF-α, IL-6 and IL-1β in the serum were measured using ELISA. The secretion levels of the aforementioned inflammatory factors were all enhanced in the CCl₄ group compared with those in the control group. However, the levels of TNF-α, IL-6 and IL-1β were reduced in the RSG + CCl₄ group compared with the CCl₄ group (Fig. 2A). The mRNA expression levels of TNF-α, IL-6 and IL-1β in the liver tissues were detected using RT-qPCR. No statistically significant differences were observed in the expression levels of the inflammatory factors between the control and RSG groups, whereas the increase in the expression of these factors was less prominent in the RSG + CCl₄ group compared with the CCl₄ group (Fig. 2B). Additionally, the expression levels of apoptosis-related proteins were evaluated by western blot analysis. Cleaved capase-3, cleaved caspase-8 and cleaved PARP were all upregulated in the CCl₄ group compared with in the control group. In addition, the expression levels of the apoptosis-related proteins were reduced in the RSG + CCl₄ group compared with those in the CCl₄ group, thus suggesting the RSG may protect hepatocytes from apoptosis to a certain degree (Fig. 2C).

The aforementioned results indicated that RSG could alleviate hepatic injury; therefore, subsequent experiments focused on uncovering the possible mechanism underlying the effects of RSG on hepatic injury. The expression levels of PPARγ and of proteins associated with the Nrf2 signaling pathway were determined using western blot analysis. The results showed that PPARγ, Nrf2, NQO1 and HO-1 were downregulated in the CCl₄ group compared with in the control group. However, pretreatment with RSG reversed the CCl₄-mediated decreased expression of these factors (Fig. 3A). The mRNA expression levels of Nrf2, NQO1 and HO-1 were measured by RT-qPCR. Consistently, the expression levels of Nrf2, NQO1 and HO-1 were downregulated in the CCl₄ group and were restored following pretreatment with RSG (Fig. 3B). Additionally, the expression levels of the NLRP3 signaling pathway-related proteins were detected using western blot analysis. NLRP3, caspase-1, IL-1β and ASC were significantly upregulated in the CCl₄ group compared with those in the control group. However, the expression these factors was significantly decreased in the RSG + CCl₄ group compared with in the CCl₄ group (Fig. 4).