PSP (≥98%) was obtained from Xi'an Baoyifeng Biotechnology Co., Ltd. Biejia Ruangan (BJRG) tablets were purchased from Inner Mongolia Furui Medical Science Co., Ltd. and served as a positive control drug. BJRG tablets are used in clinical practice for the treatment of LF and are commonly used as a positive control drug in the study of anti-LF in traditional Chinese medicine. The mechanism of BJRG in anti-LF is associated with the inhibition of collagen fiber synthesis and the TGF-β/Smad signaling pathway; therefore, BJRG tablets were selected as the positive control drug in the present study (21,22). High-fat diet (HFD; consisting of basic feed + 10% lard + 10% egg yolk powder + 1% cholesterol) was used in the present study (Xiaoshuyoutai Biotechnology Co., Ltd.). CCl₄ was acquired from Tianjin Tianli Chemical Co., Ltd. Olive oil was purchased from Shanghai Xinyu Biological Technology Co., Ltd. Aspartate transaminase (AST; cat. no. 04657543190), alanine aminotransferase (ALT; cat. no. 20764957322), alkaline phosphatase (ALP; cat. no. 03333752190), total bilirubin (TBIL; cat. no. 05795648190), total cholesterol (TC; cat no. 04718917190), and triglyceride (TG; cat no. 04657594190) testing kits were sourced from Roche Diagnostics. A Masson's trichrome staining kit (cat. no. MM1007) was obtained from Maokang Biotechnology Co., Ltd. TRIzol^® was obtained from Invitrogen; Thermo Fisher Scientific, Inc. Taq DNA polymerase was purchased from Takara Bio, Inc. ELISA kits for hyaluronic acid (HA; cat. no. MM-0916R1), laminin (LN; cat. no. MM-0843R1), procollagen III N-terminal peptide (PIIINP; cat. no. 0752R1), collagen type IV (Col IV; cat. no. MM-70796R1), hydroxyproline (Hyp; cat no. 43761R1), α-smooth muscle actin (α-SMA; cat no. MM-61545R1), collagen type I (Col I; cat no. MM-0041R1), collagen type III (Col III; cat no. MM-0080R1), IL-1β (cat no. MM-0047R1), TNF-α (cat no. MM-0180R1), IL-10 (cat no. MM-0195R1), superoxide dismutase (SOD; cat no. MM-0386R1), malondialdehyde (MDA; cat no. MM-0576R1) and glutathione peroxidase (GSH-PX; cat no. 0743R1) were purchased from Jiangsu Enzyme Immunoassay Co., Ltd. Goat serum (cat. no. c-0005), MMP2 (cat. no. bs4605R), MMP9 (cat. no. bsm55544m), Col III (cat no. bs-0549R) and Col I (cat no. bs-7158R) antibodies were obtained from Bioss. RIPA lysis buffer (cat. no. c1053) was purchased from Applygen Technologies, Inc. TGF-β1 (cat. no. ab215715), Smad3 (cat. no. ab40854) and Smad7 (cat. no. ab216428) antibodies were acquired from Abcam Co., Ltd. HRP-labeled secondary antibody (cat. no. 111005003) was purchased from Jackson ImmunoResearch Laboratories, Inc. The anti-GAPDH antibody (cat. no. AF1186) and enhanced chemiluminescent reagents (ECL; cat no. P0018FSwere obtained from Beyotime Institute of Biotechnology.

A total of 70 male Sprague Dawley (SD) rats (weight, 180–220 g; age, 6 weeks) were obtained from Liaoning Changsheng Biotechnology Co., Ltd. [animal production license no. SCXK (Liao) 2020-002]. The rats were housed in a specific pathogen-free environment with a 12-h light/dark cycle, a relative humidity of 50±5% and a room temperature of 22±2°C. The control group consisted of 10 rats, while the other groups had 8 rats each. A total of 10 male SD rats were randomly assigned as the control group receiving standard feed and purified water ad libitum, whereas the remaining rats (n=60 SD rats) underwent LF induction through a 7-week protocol comprising: i) HFD administered twice daily (150–200 g/feeding) and ad libitum access to purified water; ii) daily 20% ethanol gavage (10 ml/kg; prepared from 56% alcohol by volume) and iii) subcutaneous CCl₄ injections (initial 5 ml/kg; followed by 3 ml/kg 40% CCl₄ in olive oil every 3 days for 14 total injections) (23). Control group rats received equivalent olive oil injections. Rats were randomly divided into the model group, BJRG group (administered 1.70 g/kg BJRG), PSP_L group (administered 0.80 g/kg PSP) or PSP_H group (administered 1.60 g/kg PSP). All groups consisted of 10 rats each. The control and model groups received a daily oral gavage of purified water (10 ml/kg), whereas treatment groups were intragastrically administered either PSP aforementioned doses or BJRG tablets (positive control drug) at the same volume and frequency for 4 weeks. The Chinese Pharmacopoeia stipulates a daily dosage range of 9–15 g P. sibiricum for adult human use (24) For the experimental design of the present study, a maximum recommended human dose of 15 g/day was used as the reference point. Through interspecies dose conversion based on body surface area normalization, the equivalent dose in a rat was calculated to be 1.35 g/kg/day. Considering a crude polysaccharide extraction efficiency of ~60% from the raw herb, the daily administration dose of PSP for rats was calculated as 0.8 g/kg/day, which was designated as the medium dose (MD) in the protocol of the present study. The high dose group received 1.6 g/kg/day (2X the MD), while the low dose group was administered 0.4 g/kg/day (0.5X the MD). Preliminary investigations demonstrated that the 0.4 g/kg/day dose failed to elicit significant therapeutic effects in the LF rat model. Consequently, formal investigation focused on the two higher doses, ultimately selecting 0.8 and 1.6 g/kg/day for comprehensive evaluation in the present study. On the final day of the present study, the rats were anesthetized with 3% sodium pentobarbital (40 mg/kg; intraperitoneal injection). After confirming surgical anesthesia (loss of righting/pedal reflexes), blood was collected via abdominal aortic puncture (7–10 ml from each rat) and the liver samples were surgically resected. After tissue collection, the rats were euthanized by cervical dislocation. Death was confirmed by cardiac arrest, respiratory cessation (≥5 min) and rigor mortis. A portion of the liver tissue was fixed in 4% paraformaldehyde for 24 h at room temperature for histological analysis, while the remaining tissue was stored at −80°C. All animal procedures were approved by the Animal Ethics Committee of Heilongjiang University of Chinese Medicine (approval no. 2021101101; Harbin, China).

The livers were washed with saline and swabbed dry, weighed on an analytical balance, liver weights were recorded and images were captured. Liver index (%) was calculated using the formula=liver weight/body weight ×100.

Following anesthesia, blood was drawn from the abdominal aorta and allowed to stand for 2.5 h. The supernatant (serum) was carefully collected after centrifugation at 3,010 × g for 15 min at 4°C. ALT, AST, ALP, TBIL, TC and TG test kits were used according to the manufacturers' instructions. The serum samples were placed in the designated wells, and an automatic biochemical analyzer (Roche Diagnostics) was used to measure the levels of ALT, AST, ALP, TC and TG.

Liver tissues were fixed in 4% paraformaldehyde for 24 h at room temperature, dehydrated through a graded ethanol series [50% (45 min), 70% (45 min), 80% (45 min), 90% (45 min), 95% (45 min), 100% ethanol I (30 min) and 100% ethanol II (20 min)], cleared in xylene (twice, 30 min each) and embedded in paraffin wax at 65°C. Sections (4 µm) were cut for deparaffinization. The slides were sequentially immersed in xylene I (10 min), xylene II (10 min), 100% ethanol I (2 min), 100% ethanol II (2 min), 95% ethanol (2 min), 90% ethanol (2 min) and 80% ethanol (2 min), and then rinsed thoroughly in distilled water (4 min). Subsequently, the tissues were subjected to H&E or Masson's trichrome staining for ~2 h at room temperature. The stained sections were examined under an optical microscope (Motic Incorporation, Ltd.). Relative statistical analysis of Masson-stained collagen fiber deposition was carried out using ImageJ 1.54f software (National Institutes of Health). Meta-analysis of Histological Data in Viral Hepatitis (METAVIR) scores were used to evaluate LF (25).

Paraffin-embedded sections (4 µm) were dewaxed twice using xylene, followed by gradient ethanol hydration and antigen retrieval using sodium citrate solution at 100°C. To inhibit endogenous peroxidase activity, the sections were treated with 3% hydrogen peroxide for 20 min at room temperature. Subsequently, the sections were blocked with goat serum for 20 min at room temperature and incubated overnight at 4°C with primary antibodies targeting MMP2 (1:150), MMP9 (1:200), Col III (1:200) and Col I (1:500). On the following day, the sections were incubated with appropriate HRP-labelled secondary antibodies (1:1,000) for 30 min at room temperature. Protein expression was visualized using 3′-diaminobenzidine staining, after which the sections were counterstained with hematoxylin for 50 sec at room temperature and examined under an optical microscope (Motic Incorporation, Ltd.). Using the ImageJ 1.54f software, the integrated optical density values of positively expressed cells in each group were calculated.

Following anesthesia, blood was drawn from the abdominal aorta and allowed to stand for 2.5 h. The supernatant was carefully collected after centrifugation at 805 × g for 15 min at 4°C. The serum levels of HA, LN, PIIINP Col IV, IL-1β, TNF-α, IL-10, SOD, MDA and GSH-PX were determined by ELISA according to the manufacturer's instructions.

In addition, to measure hepatic Hyp, Col I, Col III and α-SMA contents, 0.1 g liver tissues were homogenized with 900 µl ice-cold PBS using a glass homogenizer on crushed ice until complete tissue disruption was achieved. The homogenate was centrifuged at 3,010 × g for 15 min in a refrigerated centrifuge (4°C) to remove cellular debris. The supernatant was then collected and hepatic Hyp, Col I, Col III and α-SMA contents were measured using ELISA kits according to the manufacturer's protocols.

Total RNA was isolated from liver tissues using TRIzol, according to the manufacturer's instructions. cDNA synthesis was carried out using a two-step method with a prime script RT reagent kit (Takara Bio, Inc.) at 37°C for 15 min and 85°C for 5 sec. qPCR was performed using the TB green premix kit (Takara Bio, Inc.) under the following conditions: i) Initial denaturation: 95°C for 30 sec (1 cycle); ii) Amplification: 40 cycles of 95°C for 5 sec and 60°C for 30 sec; iii) Melt curve analysis: 65°C to 95°C with 0.5°C increments (5 sec/step). Cycle thresholds were normalized to GAPDH levels in the same sample. The 2^−ΔΔCq method was employed to analyze the mRNA data (26), and the expression levels are presented as fold changes relative to GAPDH. The sequences of the primers used are provided in Table I.

Proteins were extracted from liver tissue using RIPA buffer. Protein concentrations were determined using the BCA assay (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. A total of 10 µg protein/lane was separated by SDS-PAGE on 10% gels, followed by transfer to a PVDF membrane (MilliporeSigma). The membranes were blocked with 5% non-fat dried milk for 2 h at room temperature and washed three times with 1X TBS-0.1%Tween, then incubated overnight at 4°C with primary antibodies against TGF-β1 (1:500), Smad3 (1:1,000), Smad7 (1:1,000), and GAPDH (1:1,000). The following day, the membranes were incubated with HRP-labelled secondary antibodies (1:10,000) for 1 h at room temperature. After applying the ECL working solution evenly onto the protein bands, the bots were incubated in the dark for 5 min at room temperature, and were detected using a gel imaging system (Tanon Science and Technology Co., Ltd.). The gray values were normalized to those of GAPDH and were semi-quantified using ImageJ 1.54f software.

The data were analyzed using GraphPad Prism 9.0 software (Dotmatics) and are presented as the mean ± standard deviation. The METAVIR score was analyzed using Kruskal-Wallis test and Dunn's post hoc test. One-way ANOVA was used for comparisons between groups when the data followed a normal distribution, with Dunnett's method applied for post hoc analyses when the variance was homogeneous, and Tamhane T2 method was used when the variance was heterogeneous. P<0.05 was considered to indicate a statistically significant difference.

To initially evaluate the anti-LF effects of PSP, LF model rats were fed a HFD combined with alcohol and CCl₄. After modeling, the rats were given PSP for 4 weeks (Fig. 1A). The overall liver condition and function were subsequently assessed. The liver in the model group appeared enlarged, yellowish and dull in color, with a greasy texture, rough surface, visible particles of varying sizes, a slightly firm texture and blunted margins (Fig. 1B). The livers of rats treated with PSP or BJRG revealed decreased liver volume, a healthy red color, non-greasy texture, smooth surface, reduced number of particles and a soft texture. Additional testing revealed that the liver weight, liver index and levels of biochemical markers (ALT, AST, ALP, TBIL, TC and TG) in the model group were significantly higher compared with those in the control group (Fig. 1C-K). However, following treatment with PSP or BJRG, these parameters were significantly decreased compared with those in the model group, suggesting that PSP may exert anti-LF effects.

Collagen deposition and structural disorganization of the liver lobules are typical pathological features of LF (27). To further evaluate the effect of PSP on LF, liver tissue histology was examined using H&E and Masson's trichrome staining. H&E staining revealed disruption of the liver lobule structure in the model group, with hepatocytes arranged irregularly, marked focal necrosis, nuclear atrophy, extensive steatosis, inflammatory cell infiltration and fibrous hyperplasia (Fig. 2A). For samples treated with PSP or BJRG, the liver lobules presented a more organized structure, and hepatocytes had a more regular morphology and size, with only mild disorganization in the arrangement, no steatosis or focal necrosis, reduced inflammatory cell infiltration and almost no fibrous hyperplasia.

Masson's trichrome staining revealed that the model group exhibited extensive deposition of interconnected collagen fibers, primarily localized in portal confluence areas and perivenular regions, with progressive extension into hepatic lobules forming irregularly thickened fibrous septa, accompanied by severe steatosis and hepatocellular necrosis (Fig. 2B). By contrast, treatment with either PSP or BJRG attenuated these pathological changes, demonstrating a reduction of collagen deposition in portal confluence zones, marked suppression of fibrous septum formation and concurrent alleviation of steatosis and necrotic lesions. Meanwhile, hepatic collagen fiber area and Metavir scores were significantly increased in the model group compared with those in the control group (Fig. 2C and D). PSP and BJRG treatment significantly reduced both collagen deposition and Metavir scores. These results indicated that PSP exerts potent anti-fibrotic effects possibly through targeted modulation of collagen remodeling and hepatoprotective mechanisms.

HA, LN, PIIINP and Col IV are key serum markers used in diagnosing LF, and their levels markedly rise during LF (28). The present results demonstrated that the serum levels of HA, LN, PIIINP and Col IV were significantly elevated in the model group compared with those in the control group; however, these serum levels were markedly reduced in groups treated with PSP or BJRG (Fig. 3A-D). To further assess collagen synthesis in liver tissue, the levels of Hyp were measured. Analysis revealed significantly increased Hyp levels in the liver tissue of the model group compared with those in the control group; however, treatment with PSP or BJRG led to a significant reduction in Hyp content compared with that in the model group (Fig. 3J). Moreover, the levels of Col I and Col III in the liver, the primary collagen types in the liver, were significantly increased in the model group compared with those in the control group (Fig. 3E-I). By contrast, treatment with PSP and BJRG resulted in decreased expression of Col I and Col III. Notably, PSP inhibited the expression of collagen synthesis-related factors and proteins. Overall, PSP was revealed to inhibit hepatic collagen production in LF rats.

MMP2 and MMP9 are key proteins involved in the degradation of Col IV. In LF, the integrity of the vascular basement membrane serves a crucial role and Col IV is the main collagen in the vascular basement membrane (29). Our previous studies revealed significantly elevated levels of Col IV in the serum of LF, but the underlying mechanism remains unclear. To explore the effect of PSP on the vascular basement membrane in LF model rats, the expression of MMP2 and MMP9 was assessed in liver tissue. The results revealed that the protein expression levels of MMP2 and MMP9 were significantly increased in the model group compared with those in the control group; however, treatment with PSP or BJRG led to a significant decrease in the expression of MMP2 and MMP9 (Fig. 4A-C). These findings suggested that PSP might protect the integrity of the basement membrane by reducing MMPs.

LF is often accompanied by inflammation and oxidative stress, where the levels of proinflammatory factors (IL-1β and TNF-α) and peroxidation markers (MDA) are elevated, whereas the levels of anti-inflammatory (IL-10) and antioxidant factors (SOD and GSH-PX) are reduced (30). Pathological analysis of the liver revealed a notable inflammatory response in the model group compared with in the control group. The findings indicated that the serum levels of IL-1β, TNF-α and MDA were significantly elevated in the model group compared with those in the control group (Fig. 5A, B and E). By contrast, the levels of IL-10, SOD and GSH-PX were significantly reduced compared with those in the control group (Fig. 5C, D and F). However, treatment with PSP or BJRG markedly reduced the levels of IL-1β, TNF-α and MDA, while substantially increasing the levels of IL-10, SOD and GSH-PX. These results suggested that PSP may effectively alleviate the inflammatory response and oxidative stress in rats with LF.

In the development of LF, the TGF-β/Smad signaling pathway serves a key role, since the activation of HSCs promotes the release of TGF-β1. To investigate whether PSP affects the TGF-β/Smad pathway and contributes to the reduction in LF, levels of α-SMA and other components of the TGF-β/Smad pathway were assessed. PSP and BJRG treatment significantly reduced α-SMA expression levels in liver tissue, suggesting potential inhibition of HSC activation (Fig. 6A). Furthermore, the protein and mRNA levels of TGF-β1 and Smad3 were significantly elevated in the model group compared with those in the control group, whereas Smad7 levels were notably decreased (Fig. 6B-H). By contrast, following treatment with PSP, the expression of TGF-β1 and Smad3 was reduced, whereas Smad7 expression levels were significantly increased.