Saikosaponin-d alleviates hepatic stellate cell activation and liver fibrosis by inhibiting the TGF-β1/Smads signaling pathway and blocking the EMT process

Abstract

BACKGROUND

Liver fibrosis is a compensatory response to chronic liver injuries, such as viral hepatitis, alcohol abuse, and metabolic disorders. Despite this, no United States Food and Drug Administration-approved anti-fibrotic drugs are currently available. Saikosaponin-d (SSd) has demonstrated antifibrotic effects, but its impact on the transforming growth factor-β1 (TGF-β1)/Smads signaling pathway and epithelial-mesenchymal transition (EMT) during fibrosis remains poorly understood.

AIM

To investigate the effect of SSd on the TGF-β1/Smads signaling pathway and EMT during hepatic fibrosis progression.

METHODS

A carbon tetrachloride (CCl₄)-induced liver fibrosis model was established in C57BL/6 mice (CCl₄: olive oil = 1:4, twice weekly for 6 weeks). Mice were assigned to control, CCl₄, and SSd + CCl₄ groups with SSd administered intraperitoneally (1.5 mg/kg) for 6 weeks. The human hepatic stellate cell line LX-2 was activated with TGF-β1, and groups included control, TGF-β1, and TGF-β1 + SSd. The TGF-β1 group was treated with TGF-β1 (5 ng/mL), while the TGF-β1 + SSd group received both TGF-β1 (5 ng/mL) and SSd (5 μmol/L) for 24 hours. Evaluations included serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, liver histology (Masson’s trichrome, Sirius red staining), α-smooth muscle actin immunohistochemistry, cell counting kit-8 cell viability assay, quantitative real-time polymerase chain reaction, and western blotting to assess the effects of SSd on TGF-β1/Smads signaling and EMT.

RESULTS

SSd alleviated liver injury in CCl₄-induced fibrotic mice, significantly reducing serum ALT, AST levels, and collagen fiber deposition (P < 0.05). Importantly, SSd significantly decreased mRNA, α-smooth muscle actin, p-Smad2, TGF-β1, and p-Smad3 levels in fibrotic liver tissue (P < 0.05). Regarding EMT, SSd reduced the protein levels of N-cadherin and vimentin, while increasing E-cadherin expression (P < 0.05). SSd inhibited TGF-β1-induced LX-2 cell proliferation and decreased ALT and AST levels (P < 0.05). Moreover, it significantly suppressed the levels of key molecules in the TGF-β1/Smads pathway and EMT progression in vitro (P < 0.05).

CONCLUSION

SSd mitigates hepatic stellate cell activation and liver fibrosis progression by inhibiting the TGF-β1/Smads signaling pathway and effectively blocking the EMT process. These findings position SSd as a promising therapeutic agent and potential antifibrotic strategy.

Key Words: Saikosaponin-d; Transforming growth factor-β1/Smads; Epithelial-mesenchymal transition; Liver fibrosis; Mechanism

Core Tip: Saikosaponin-d (SSd) is a promising therapeutic agent and potential antifibrotic drug. This study, through in vivo and in vitro models, demonstrates that SSd alleviates hepatic stellate cell activation and liver fibrosis progression by inhibiting the transforming growth factor-β1/Smads signaling pathway and effectively blocking the epithelial-mesenchymal transition process. The study also addresses the reversibility and safety issues of SSd, proposing solutions. These findings enrich the understanding of SSd’s antifibrotic molecular mechanisms and provide a theoretical basis for drug optimization and improvement.

INTRODUCTION

Liver fibrosis is a chronic, unresolved inflammatory condition that induces the activation of collagen-producing hepatic stellate cells (HSCs), leading to their transformation into myofibroblasts[1,2]. This pathology is characterized by an imbalance between the synthesis and degradation of the extracellular matrix (ECM), driven by disrupted fibrogenic and fibrinolytic processes[3,4]. Liver fibrosis typically results from various chronic liver diseases, including alcoholic steatohepatitis, chronic hepatitis B and C, autoimmune biliary diseases, and non-alcoholic steatohepatitis[5,6]. If left unchecked, liver fibrosis progresses to cirrhosis and hepatocellular carcinoma, both of which involve significant hepatic dysfunction and alterations in vascular structure. These pathological conditions are major contributors to disease burden and survival rates, posing substantial global healthcare challenges[7]. To date, no United States Food and Drug Administration-approved anti-fibrotic drugs are available, although several agents are undergoing clinical trials[5,8,9]. Therefore, the development of effective pharmacological treatments to halt fibrosis progression is urgently needed.

HSCs are the central cellular drivers of the fibrotic process. Upon liver injury, quiescent HSCs are activated and transition to a profibrotic state, producing excessive ECM and tipping the balance toward fibrogenesis[6,10]. Activated HSCs undergo significant phenotypic and morphological changes, upregulate fibrogenic genes, and resemble myofibroblasts[11,12]. These activated HSCs secrete transforming growth factor-β1 (TGF-β1), a potent profibrotic mediator that promotes fibrosis in the liver and other organs[13]. TGF-β signaling begins with the formation of a receptor complex consisting of type I and type II serine/threonine kinases. This complex activates Smad2/3, which then associates with Smad4 to form a heterotrimer that translocates to the nucleus. There, it regulates transcription factors such as Snail, Slug, and Twist, as well as key fibrogenic markers including collagens, α-smooth muscle actin (α-SMA), and fibronectin[14]. The TGF-β1/Smad axis further drives the transdifferentiation of HSCs into α-SMA-expressing myofibroblasts, which are the primary contributors to ECM overproduction during fibrosis[15]. Additionally, TGF-β1 induces epithelial-mesenchymal transition (EMT), a process where epithelial cells lose their native characteristics and adopt mesenchymal traits[16]. EMT plays a critical role in fibrotic conditions, and its inhibition has been shown to reduced fibrosis progression[17]. Therefore, targeting these signaling pathways may provide effective therapeutic strategies for protecting the liver from chronic injury.

The herb Bupleurum (Chai Hu) is widely used in traditional medicine throughout China and other Asian countries for the treatment of chronic liver inflammation and viral hepatitis. Saikosaponin-d (SSd), a triterpenoid saponin compound derived from Bupleurum, demonstrates broad range pharmacological activities, including anti-inflammatory, antioxidant, anti-apoptotic, antifibrotic, and anticancer properties[18-23]. Our previous studies have shown that SSd exerts significant protective effects against carbon tetrachloride (CCl₄)-induced hepatic fibrosis[24-27]. The underlying mechanisms primarily involve three key pathways: The G protein-coupled estrogen receptor 1/autophagy, reactive oxygen species/NOD-like receptor family pyrin domain containing 3 (NLRP3), and estrogen receptor β (ERβ)/NLRP3 inflammasome pathways[19,24,25]. These findings indicate that SSd ameliorates hepatic fibrosis through a multi-pathway synergistic mechanism, exerting beneficial effects by mitigating inflammatory responses, alleviating oxidative stress, and promoting autophagic homeostasis. Further in vitro experiments revealed that SSd significantly downregulated the mRNA expression levels of TGF-β1 and Smad3 while upregulating the mRNA expression level of Smad7 in rat HSCs (HSC-T6). However, the regulatory effects of SSd on these targets at the protein level remain to be validated, and the precise relationship between SSd and the core pathological mechanisms of hepatic fibrosis remains unclear. To date, no study has comprehensively elucidated the specific molecular mechanisms through which SSd alleviates hepatic fibrosis through the regulation of the TGF-β1/Smad signaling pathway and EMT.

Therefore, this study aimed to investigate the regulatory role of SSd in key pathological processes involved in the progression of hepatic fibrosis. Given that the TGF-β1/Smad signaling pathway serves as the central regulatory axis in fibrogenesis and that EMT plays a crucial role in cellular phenotypic transformation, we used both a mouse model of CCl₄-induced hepatic fibrosis and a TGF-β1-activated human HSC line (LX-2) to investigate the molecular mechanisms through which SSd exerts antifibrotic effects through the TGF-β1/Smad and EMT pathways. The findings not only provide novel insights into the role of SSd in the TGF-β1/Smad-EMT axis in hepatic fibrosis but also offer novel therapeutic targets and a theoretical basis for the treatment of hepatic fibrosis, holding significant scientific value for advancing research in this field.

MATERIALS AND METHODS

Reagents

The cell counting kit-8 (CCK-8) kit (catalog No. 40203ES80), RNA extraction kit (catalog No. 19211ES60), SYBR green mix (catalog No.11201E03), and polyvinylidene fluoride membrane (catalog No. 36126ES03) were purchased from Shanghai Yeasen Biotechnology Co., Ltd., China; RIPA lysis buffer (catalog No. WB0102), protease inhibitor cocktail (catalog No. WB0122), and phosphate-buffered saline (catalog No. WH1165) were obtained from Shanghai Weiao Biotechnology Co., Ltd., China; SSd, (> 97% purity), xylene (catalog No. 1330-20-7), glacial acetic acid (catalog No. 64-19-7), and CCl₄ (catalog No. 5623-5) were supplied by Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; Dulbecco’s modified Eagle medium (catalog No. 8119054) was acquired from Gibco, Thermo Fisher Scientific, MA, United States, and bovine serum albumin (catalog No. SRE0096) was from Sigma-Aldrich, MO, United States. The reverse transcription kit (catalog No. KR118) was sourced from TIANGEN Biotech Co., Ltd., Beijing, China. Masson’s trichrome staining kit (catalog No. G1006), hematoxylin staining solution (catalog No. G1077), and DAB (3,3’-Diaminobenzidine) chromogen kit (catalog No. G1212) for histological analysis were provided by Servicebio Technology Co., Ltd., China. The sirius red staining kit (catalog No. 2610-10-8) was procured from LinkBio Technology Co., Ltd., Shanghai, China. The alanine aminotransferase (ALT, catalog No. C009-2-1) and aspartate aminotransferase (AST, catalog No. C010-2-1) assay kits were supplied by the Nanjing Jiancheng Bioengineering Institute, China. Primary antibodies against α-SMA (catalog No. ab124964) were obtained from Abcam, MA, United States. p-Smad2 (catalog No. 3108), Smad2 (catalog No. 5339), N-cadherin (catalog No. 13116), vimentin (catalog No. 5741S), and E-cadherin (catalog No. 3195) were sourced from Cell Signaling Technology, MA, United States. Vimentin (catalog No. 10366-1-AP), TGF-β1 (catalog No. 81746-2-RR), p-Smad3 (catalog No. 80427-2-RR), and Smad3 (catalog No. 66516-1-Ig) were sourced from Proteintech Biotechnology Co., Ltd., Wuhan, China. β-actin (catalog No. ab27937), horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) (catalog No. GB23301), and HRP-conjugated goat anti-rabbit IgG (catalog No. GB22303) were provided by Servicebio Technology, Wuhan, China.

Animal grouping and administration

Eighteen male C57BL/6 mice (sun protection factor grade, aged 6-8 weeks, weight 18-22 g) were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd., Shanghai, China, and housed at the Shanghai Municipal Hospital of Traditional Chinese Medicine animal facility. The mice were maintained under controlled conditions (temperature: 25-27 °C) with ad libitum access to sterilized food and water. After a seven-day adaptation period, the experimental procedures began. The mice were randomly divided into three experimental groups (n = 6 per group): (1) Control group (C): Received intraperitoneal (i.p.) injections of olive oil (5 mL/kg body weight); (2) Model group (CCl₄): Received i.p. injections of a 20% (v/v) CCl₄ mixture in olive oil (5 mL/kg body weight)[27,28]; and (3) Treatment group (CCl₄ + SSd): Received co-administration of SSd (1.5 mg/kg body weight, i.p.) and the CCl₄ mixture (5 mL/kg body weight, i.p.)[27]. Treatments were administered twice a week for 6 weeks. Given that our previous studies have shown that administration of SSd alone for 6 weeks in healthy mice has no significant effect on physiological hepatic function and that no pro-fibrotic or anti-fibrotic activity is observed under non-injury conditions, we did not include an SSd-only treatment group (without CCl₄) in this study to optimize the experimental design. In addition, we have previously found that 1.0 mg/kg, 1.5 mg/kg, and 2.0 mg/kg SSd are effective therapeutic doses[26]. Given that the primary objective of this study was to elucidate the mechanisms of action of SSd in alleviating hepatic fibrosis, we selected 1.5 mg/kg as the experimental dose. Studies have demonstrated that the CCl₄-induced liver fibrosis model can complete the typical pathological progression from liver injury to fibrosis within 6 weeks. The initial 2-3 weeks are predominantly characterized by hepatic necrosis (manifested as elevated liver enzymes), followed by the deposition of fibrosis markers (e.g., hydroxyproline) in the subsequent 2-3 weeks. By the 6-week mark, a stable fibrotic structure is established. Extending the duration beyond 8 weeks may lead to cirrhosis progression but significantly increases animal mortality rates[29-31]. The procedures for establishing the liver fibrosis model and administering SSd followed previously documented methodologies[25-27]. During the modeling period, including after each CCl₄ injection, observe the mice’s respiratory rate, heart rate, and activity levels. If abnormalities such as respiratory or cardiac distress, unrelieved pain, signs of fear or suffering, or severe illness are detected, promptly administer an overdose of 1% sodium pentobarbital anesthetic (100-150 mg/kg) via i.p. injection. This will induce rapid unconsciousness followed by euthanasia.

After the experimental period and model establishment, all mice were deeply anesthetized using 1% pentobarbital sodium (50 mg/kg body weight, i.p.). Blood samples were collected via orbital sinus puncture, and the animals were then euthanized by cervical dislocation. Liver tissues were promptly excised. A portion (approximately 1 cm × 1 cm) from the central lobe was immediately fixed in 4% paraformaldehyde for histological examination. The remaining liver segments were placed in pre-cooled cryovials, flash-frozen using liquid nitrogen, and stored at -80 °C for subsequent molecular analyses. All animal experiments were approved by the Animal Ethics Committee of Shanghai Municipal Hospital of Traditional Chinese Medicine (approval No. 2024018).

Cell culture and treatment

The LX-2 was obtained from Shanghai Fankun Biotechnology Co., Ltd, Shanghai, China. Cells were cultured under standard conditions (37 °C, 5% CO₂) in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum. Normal LX-2 cells were then divided into three experimental groups: (1) Control group (Con): Cells cultured in basal medium without treatment for 24 hours; (2) TGF-β1 group: Cells exposed to recombinant human TGF-β1 (5 ng/mL) for 24 hours to induce activation; and (3) TGF-β1 + SSd group: Cells co-treated with TGF-β1 (5 ng/mL) and SSd (5 μmol/L) for 24 hours[25]. The methodology for establishing the activated stellate cell model and the selected concentrations of TGF-β1 and SSd were based on established protocols described in published literature[25].

Measurement of ALT and AST levels

Blood samples collected from mice in each group were allowed to clot at ambient temperature for 4 hours. The serum was then isolated by centrifugation at 3000 × g for 15 minutes. Commercial diagnostic kits were used to measure serum ALT and AST activity levels according to per the supplier’s protocols. ALT and AST levels in cell culture supernatants were determined using the same kit instructions.

Masson’s trichrome and Sirius red: Liver tissue specimens, initially fixed in 4% paraformaldehyde, were kept at ambient temperature for 24 hours. Following fixation, the tissues were processed through dehydration, clearing, paraffin embedding, and sectioning. Tissue sections (typically 4-5 μm thickness) were stained using Masson’s trichrome kit and Sirius red histochemical staining kit according to the manufacturers’ protocols. Stained sections were examined under a light microscope to assess histoarchitectural changes, and digital images were captured. The severity of liver fibrosis and collagen deposition was evaluated based on the stained tissue morphology. Quantitative analysis of Masson and Sirius red-stained images using ImageJ Plus (National Institutes of Health) involves the following steps: First, perform color deconvolution to separate the DAB-stained channel. Next, use the Threshold tool to adjust the positive staining regions (blue or red indicates selected areas), ensuring the “limit to threshold” parameter is enabled. Finally, calculate the collagen volume fraction as the ratio of collagen area to total tissue area (collagen volume fraction = collagen area/total area × 100%).

Immunohistochemical staining for hepatic α-SMA: Deparaffinized liver sections underwent heat-induced antigen retrieval. These specimens were incubated overnight at 4 °C with a primary antibody specific to α-SMA (1:1000 dilution). After extensive buffer washes to remove excess primary antibody, the specimens were incubated with an appropriate HRP-conjugated secondary antibody for 1 hour at ambient temperature. Antibody binding was visualized using a 3,3’-DAB chromogen kit, and counterstaining with hematoxylin was performed to visualize nuclei. Stained sections were assessed under a light microscope at 400× magnification, and representative photomicrographs from randomly selected fields were captured for analysis of α-SMA expression. Quantitative analysis of tissue sections using ImageJ Plus involves the following steps: First, convert the image to an 8-bit grayscale format. Then, perform color deconvolution to separate the DAB-stained channel. Next, use the Threshold tool to adjust the positive staining regions (red indicates selected areas), ensuring the “limit to threshold” parameter is enabled. Finally, measure the area, integrated density, and average optical density to calculate protein expression levels (average optical density = area, integrated density/area).

Assessment of cell viability via CCK-8 assay

The experimental cells were plated in 96-well plates at 4-7 × 10³ cells per well and maintained for 24 hours under standard growth conditions (37 °C, 5% CO₂). Following the specified treatment protocols and incubation durations, cellular viability was measured using the CCK-8 per the supplier’s instructions. Specifically, 10 μL of CCK-8 solution was added to each well, followed by a 3-hour incubation at 37 °C. Optical density values were measured at 450 nm using a microplate reader. Cell viability percentages were calculated by comparing to untreated control wells.

Real time quantitative polymerase chain reaction

For real time quantitative polymerase chain reaction analysis, 40-50 mg of liver tissue was collected from each group, and total RNA was extracted following the supplier’s protocol. Reverse transcription was performed using a commercial kit, and the resulting cDNA was diluted tenfold for amplification. Gene expression levels were quantified via SYBR green-based real time quantitative polymerase chain reaction, with β-actin serving as the internal control. Primers targeting α-SMA, TGF-β1, Smad2, Smad3, E-cadherin, N-cadherin, vimentin and β-actin were designed based on GenBank cDNA sequences and synthesized by Shanghai Shanjing Molecular Biotechnology Co., Ltd, Shanghai, China. The process involves retrieving the target gene’s coding sequence from the NCBI database (https://www.ncbi.nlm.nih.gov/gene/), followed by primer design using Primer3 software (https://www.primer3plus.com/) to amplify a fragment of 80-200 bp with optimal melting temperature of 58 °C-62 °C and GC content of 40%-60%. To ensure specificity, the designed primers are then validated using Primer-BLAST (http://blast.ncbi.nlm.nih.gov/) to exclude non-specific binding sites and primer-dimer formation risks. Primer sequences are provided in Supplementary Table 1.

Western blot analysis

The protein concentration of the extracted lysates was determined using a bicinchoninic acid protein assay kit. Equal amounts of protein (20-30 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene fluoride membranes. The membranes were blocked with either 5% non-fat dry milk or 3%-5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 for 1-2 hours at room temperature. After blocking, the membranes were incubated overnight at 4 °C with specific primary antibodies diluted in blocking buffer, with gentle agitation.

Following thorough washing with Tween-20, membranes were incubated with corresponding HRP-conjugated secondary antibodies for 2 hours at room temperature. Signal detection was performed using a chemiluminescence imaging system (Amersham Imager 600, MA, United States), and band intensities were quantified using ImageJ software. The primary antibodies used were α-SMA (1:1000), TGF-β1 (1:1000), p-Smad2 (1:1000), Smad2 (1:1000), p-Smad3 (1:1000), Smad3 (1:2000), N-cadherin (1:1000), vimentin (1:1000), and E-cadherin (1:1000), with goat anti-rabbit or anti-mouse IgG (1:5000) as the secondary antibody.

Virtual molecular docking

The three-dimensional structure of the TGF-β1 protein was retrieved from the Protein Data Bank database (https://www.rcsb.org/), while the molecular structure of silver sulfadiazine (hereafter referred to as the ligand) was obtained from the PubChem Compound database (https://pubchem.ncbi.nlm.nih.gov/). Molecular docking simulations were subsequently performed using AutoDock Vina software (version 1.2.0) to predict the optimal binding conformation of the ligand within the TGF-β1 binding pocket. The docking procedure employed the Lamarckian genetic algorithm with default parameters for energy minimization and conformation sampling. The resulting binding poses were analyzed using PyMOL (Schrödinger, LLC, NY, United States) and visualized through both two-dimensional interaction diagrams and three-dimensional molecular surface representations.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.3.0 (GraphPad Software Inc., San Diego, CA, United States). Unpaired two-tailed t-tests were used for comparisons between two groups, while one-way analysis of variance was applied to evaluate differences among multiple groups. Data are presented as mean ± SEM, with statistical significance defined as P < 0.05.

RESULTS

SSd mitigates liver fibrosis development through suppressing HSC activation

Previous studies have demonstrated SSd’s protective role against hepatic fibrosis[24,27,32,33]. The current experiments further support the therapeutic efficacy of SSd in hepatic fibrosis. After a 6-week experimental period, macroscopic examination of liver specimens revealed notable differences. The livers from the CCl₄ group exhibited pallor (loss of normal deep red color) and a pronounced granular, millet-seed-like texture. In contrast, livers from the SSd-treated group showed significant macroscopic improvement (Figure 1A). Serum analysis revealed a significant increase in both ALT and AST levels in the CCl₄ group compared to the control group (P < 0.001). However, ALT and AST levels were significantly reduced in the SSd-treated group compared to the CCl₄ group (P < 0.01) (Figure 1B and C). Histological examination of liver sections stained with Masson’s trichrome and Sirius red showed severe disruption of hepatic lobular architecture in the CCl₄ group, characterized by extensive pseudolobule formation and collagen fiber deposition, with intense positive staining for collagen. Liver sections from the SSd-treated group exhibited considerable preservation of hepatic architecture, reduced collagen deposition, and absence of fatty infiltration compared to the CCl₄ group (Figure 1D). Immunohistochemistry for α-SMA revealed significantly increased immunoreactivity (brown staining) in the CCl₄ group, particularly around bile ducts and portal areas, compared to the control group. SSd treatment resulted in a notable reduction in both the distribution and intensity of α-SMA-positive staining (Figure 1E). Quantitative polymerase chain reaction analysis confirmed significantly elevated hepatic α-SMA mRNA levels in the CCl₄ group compared to the control group (P < 0.01). SSd substantially reduced α-SMA mRNA expression in the SSd-treated group compared to the CCl₄ group (P < 0.01) (Figure 1F).

Figure 1 Saikosaponin-d attenuates the progression of hepatic fibrosis by inhibiting hepatic stellate cells activation. A: Fresh liver images of mice in the control (C), carbon tetrachloride (CCl₄), and CCl₄ + saikosaponin-d (SSd) groups; B: Serum alanine aminotransferase levels in mice from the C, CCl₄, and CCl₄ + SSd groups, n = 6; C: Serum aspartate aminotransferase levels in mice from the C, CCl₄, and CCl₄ + SSd groups, n = 6; D: Masson’s trichrome and Sirius red staining of liver tissue from mice in the C, CCl4, and CCl₄ + SSd groups, magnification × 200; E: Immunohistochemical staining for α-smooth muscle actin (α-SMA) in liver tissue from mice in the C, CCl₄, and CCl₄ + SSd groups, magnification × 100; F: MRNA expression levels of α-SMA in liver tissue from mice in the C, CCl₄, and CCl₄ + SSd groups, n = 6; G: The changes in cell viability of LX-2 cells after 24 hours of intervention with different concentrations of SSd, n = 6; H: Cell viability in the control (Con), transforming growth factor-β1 (TGF-β1), and TGF-β1 + SSd groups, n = 4; I: Aspartate aminotransferase levels in LX-2 cells from the Con, TGF-β1, and TGF-β1 + SSd groups, n = 4; J: Alanine aminotransferase levels in LX-2 cells from the Con, TGF-β1, and TGF-β1 + SSd groups, n = 4; K: MRNA expression levels of α-SMA in LX-2 cells from the Con, TGF-β1, and TGF-β1 + SSd groups, n = 4; L: Protein expression levels of α-SMA in LX-2 cells from the Con, TGF-β1, and TGF-β1 + SSd groups, n = 4. C: Control; CCl₄: Carbon tetrachloride; SSd: Saikosaponin-d; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; α-SMA: Α-smooth muscle actin; TGF-β1: Transforming growth factor-β1.

To validate the findings, the anti-fibrotic mechanism of SSd was investigated using the LX-2 cell line. CCK-8 assay revealed that SSd treatment reduced the viability of LX-2 cells in a concentration-dependent manner. At an SSd concentration of 5 μmol/L, the cell survival rate was approximately 100%, and no cytotoxicity was observed. However, when the SSd concentration was > 5 μmol/L, cell viability decreased below 80% (Figure 1G). These findings are consistent with those of our previous studies, validating that 5 μmol/L is the optimal concentration of SSd for LX-2 cells[19,25]. Therefore, in subsequent experiments, LX-2 cells were treated with 5 μmol/L SSd to investigate its mechanism of action in ameliorating hepatic fibrosis. After 24 hours of TGF-β1 stimulation, LX-2 cells exhibited a significant increase in cell viability and ALT and AST levels (P < 0.001). SSd treatment effectively counteracted these changes, significantly reducing viability and ALT and AST levels in LX-2 cells (P < 0.01) (Figure 1H-J). In addition, TGF-β1 stimulation notably increased the mRNA and protein expression levels of α-SMA in LX-2 cells, whereas SSd treatment resulted in a marked pronounced of α-SMA mRNA and protein levels compared to the TGF-β1-induced group (P < 0.001) (Figure 1K and L).

SSd attenuates hepatic fibrosis progression by inhibiting the TGF-β1/Smad signaling cascade

Molecular docking results revealed that SSd interacted with TGF-β1, forming a stable binding conformation (Figure 2A and B). The docking score of -5.3 kcal/mol indicates significant binding affinity between SSd and TGF-β1. Binding site analysis revealed that key residues such as Glu B:313, AsnA:82, and GluA:75286 contributed to the stability of the SSd-TGF-β1 complex through hydrogen bonding and hydrophobic interactions (Figure 2C). These findings suggest that SSd directly binds to TGF-β1, thereby inhibiting its signaling activity through steric hindrance or allosteric modulation. SSd downregulates the mRNA expression of TGF-β1 and Smad3 while enhancing Smad7 expression in the rat HSC line HSC-T6[32]. These results suggest that SSd may regulate the TGF-β1/Smad signaling pathway, which could contribute to its antifibrotic effect[32]. The present study further supports these findings. In a CCl₄-induced murine model of hepatic fibrosis, mRNA levels of TGF-β1, Smad2, and Smad3 were significantly elevated, accompanied by increased protein expression of TGF-β1, p-Smad2, and p-Smad3 (P < 0.01) (Figure 2D-G). In the CCl₄ + SSd group, however, mRNA expression of TGF-β1, Smad2, and Smad3, as well as protein levels of TGF-β1, p-Smad2, and p-Smad3, were markedly reduced compared to the CCl₄ group (P < 0.05 (Figure 2D-G). Similar inhibitory effects were observed in vitro in TGF-β1-stimulated human HSCs (LX-2) (P < 0.01) (Figure 2H-K). These results confirm that SSd effectively suppresses the TGF-β1/Smad signaling pathway. However, the specific downstream molecular events responsible for this inhibition remain to be fully elucidated.

Figure 2 Saikosaponin-d attenuates hepatic fibrosis progression by inhibiting the transforming growth factor-β1/Smad signaling pathway. A and B: The overall three-dimensional structure of the interaction between silver sulfadiazine molecules and transforming growth factor-β1 (TGF-β1) protein; C: The overall 2D structure of the interaction between silver sulfadiazine molecules and TGF-β1 protein; D: MRNA expression levels of TGF-β1 in mice from the control (C), carbon tetrachloride (CCl₄), and CCl₄ + saikosaponin-d (SSd) groups, n = 6; E: MRNA expression levels of Smad2 in mice from the C, CCl₄, and CCl₄ + SSd groups, n = 6; F: MRNA expression levels of Smad3 in mice from the C, CCl₄, and CCl₄ + SSd groups, n = 6; G: Protein expression levels of TGF-β1, p-Smad2, and p-Smad3 in mice from the C, CCl₄, and CCl₄ + SSd groups, n = 4; H: MRNA expression levels of TGF-β1 in LX-2 cells from the control (Con), TGF-β1, and TGF-β1 + SSd groups, n = 4; I: MRNA expression levels of Smad2 in LX-2 cells from the Con, TGF-β1, and TGF-β1 + SSd groups, n = 4; J: MRNA expression levels of Smad3 in LX-2 cells from the Con, TGF-β1, and TGF-β1 + SSd groups, n = 4; K: Protein expression levels of TGF-β1, p-Smad2, and p-Smad3 in LX-2 cells from the Con, TGF-β1, and TGF-β1 + SSd groups, n = 4. C: Control; CCl₄: Carbon tetrachloride; SSd: Saikosaponin-d; TGF-β1: Transforming growth factor-β1.

SSd inhibits EMT progression by suppressing the TGF-β1/Smad signaling pathway

Accordingly, the expression of E-cadherin, N-cadherin, and vimentin in hepatic tissues and cells was examined. Compared to the group (C), the CCl₄ group showed significantly decreased mRNA and protein levels of E-cadherin (P < 0.05) (Figure 3), along with markedly elevated expression of N-cadherin and vimentin mRNA and protein (P < 0.05) (Figure 3B-D) in murine liver tissues. Treatment with CCl₄ + SSd significantly increased E-cadherin mRNA and protein levels (P < 0.05) (Figure 3A and D) and markedly reduced N-cadherin and vimentin expression (P < 0.05) (Figure 3B-D) relative to the CCl₄ group. Similar results were observed in TGF-β1-stimulated human hepatic stellate LX-2 cells, where the TGF-β1 group exhibited significantly downregulated E-cadherin mRNA and protein levels (P < 0.001) (Figure 3E and H) but upregulated N-cadherin and vimentin (P < 0.01) (Figure 3F-H) compared to the control group. TGF-β1 + SSd treatment conversely elevated E-cadherin expression while suppressing N-cadherin and vimentin levels relative to the TGF-β1 group (P < 0.05) (Figure 3E-H). These results collectively suggest that SSd attenuates fibrotic progression by inhibiting the TGF-β1/Smads pathway-mediated EMT.

Figure 3 Saikosaponin-d inhibits epithelial-mesenchymal transition progression by suppressing the transforming growth factor-β1/Smad signaling pathway. A: MRNA expression levels of E-cadherin in mice from the control (C), carbon tetrachloride (CCl₄), and CCl₄ + saikosaponin-d (SSd) groups, n = 6; B: MRNA expression levels of N-cadherin in mice from the C, CCl₄, and CCl₄ + SSd groups, n = 6; C: MRNA expression levels of vimentin in mice from the C, CCl₄, and CCl₄ + SSd groups, n = 6; D: Protein expression levels of E-cadherin, N-cadherin, and vimentin in mice from the C, CCl₄, and CCl₄ + SSd groups, n = 4; E: MRNA expression levels of E-cadherin in LX-2 cells from the control (Con), transforming growth factor-β1 (TGF-β1), and TGF-β1 + SSd groups, n = 4; F: MRNA expression levels of N-cadherin in LX-2 cells from the Con, TGF-β1, and TGF-β1 + SSd groups, n = 4; G: MRNA expression levels of vimentin in LX-2 cells from the Con, TGF-β1, and TGF-β1 + SSd groups, n = 4; H: Protein expression levels of E-cadherin, N-cadherin, and vimentin in LX-2 cells from the Con, TGF-β1, and TGF-β1 + SSd groups, n = 4. C: Control; CCl₄: Carbon tetrachloride; SSd: Saikosaponin-d; TGF-β1: Transforming growth factor-β1.

DISCUSSION

The TGF-β1/Smad signaling cascade is a key mediator in the development of liver fibrosis[34,35]. The TGF-β1/Smad pathway promotes EMT through the increased expression of Smad proteins. TGF-β activation stimulates kinases such as TGF-β-activated kinase 1 and activates downstream signaling cascades, including c-Jun N-terminal kinase, extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and p38 mitogen-activated protein kinase. Moreover, TGF-β induces the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase mitogen-activated protein kinase signaling cascade, leading to elevated expression of type I collagen and vimentin. These actions amplify the profibrotic and pro-EMT effects of TGF-β1[36-38]. The TGF-β1-induced EMT process may initially be reversible. However, EMT becomes irreversible once stable genetic and epigenetic changes profoundly alter the mesenchymal phenotype[39-41]. The reversibility and plasticity of EMT are critically dependent on TGF-β1 expression levels. Sustained high levels of TGF-β promote irreversible EMT, contributing to hepatic fibrosis and potentially facilitating malignant transformation[42,43]. Collectively, these studies suggest that targeting the TGF-β1/Smad and EMT pathways offers a promising therapeutic strategy for managing liver fibrosis.

SSd provides protection against CCl₄-induced liver fibrosis[19,44]. Recent research has offered clearer mechanistic insights into its action. An in vitro study demonstrated that SSd induces apoptosis in HSC-T6 and LX-2 cells, potentially through both caspase-3-dependent and -independent pathways. SSd triggers the translocation of Bcl-2-associated X protein and Bcl-2 homologous antagonist/killer from the cytosol to the mitochondria, leading to mitochondrial dysfunction and disruption of membrane potential[45]. Ultimately, SSd promotes the release of pro-apoptotic factors. Using both TGF-β1-stimulated in vitro models and CCl₄-induced in vivo hepatic fibrosis models, our research team observed that SSd significantly attenuates key fibrotic markers, specifically collagen type 1 and α-SMA. Furthermore, SSd mitigates the progression of hepatic fibrosis in primary HSCs and TGF-β1-treated LX-2 cells by modulating the G protein-coupled estrogen receptor 1/autophagy pathway[19]. SSd inhibits oxidative stress-induced activation of HSC-T6 cells, thereby attenuating fibrosis progression, a process dependent on the regulation of ERβ[33]. SSd may thus function as a novel phytoestrogen with estrogen-like effects. Subsequent findings suggest that SSd exerts its antifibrotic effects, at least in part, by modulating the ERβ/NLRP3 inflammasome pathway, which involves key components such as NLRP3, interleukin-18, and interleukin-1β[24]. Further research reinforced these findings, demonstrating that SSd mitigates hepatic fibrosis through ERβ pathway activation, which subsequently downregulates the reactive oxygen species/NLRP3 inflammasome axis[25]. Our most recent study revealed that SSd ameliorates hepatic fibrosis and restores circadian rhythms via the Esr1-Per2 axis[27]. Collectively, these studies highlight the substantial therapeutic potential of SSd in treating hepatic fibrosis. Preliminary studies have indicated that SSd ameliorates hepatic fibrosis by modulating inflammatory responses, apoptosis, oxidative stress, and autophagy. However, its impact on the core pathological processes of hepatic fibrosis, such as excessive ECM deposition and EMT, remains unclear. To date, no study has elucidated the specific molecular mechanisms through which SSd alleviates hepatic fibrosis through the regulation of the TGFβ1/Smad signaling pathway and EMT.

To address this knowledge gap, this study investigated the regulatory effects of SSd on the TGF-β1/Smad signaling pathway and systematically elucidated its action mechanisms in controlling the activation of HSCs and the expression of EMT-related markers (e.g., E-cadherin, α-SMA, and vimentin). Results revealed that SSd treatment inhibited the expression of TGF-β1/Smad pathway components (TGF-β1, p-Smad2, and p-Smad3) and EMT markers (N-cadherin and vimentin) and increased E-cadherin protein levels, thereby alleviating hepatic fibrosis (Figure 4). These findings provide specific molecular targets for antifibrotic therapy and establish a theoretical basis for the developing of novel small-molecule drugs to address the current lack of United States Food and Drug Administration-approved antifibrotic agents. The findings indicate that SSd reduces collagen deposition and restores E-cadherin expression, suggesting its potential in reversing early-stage fibrosis rather than only delaying its progression. These findings have significant clinical implications for treatment at pre-cirrhotic stages.

Figure 4 The mechanism diagram of saikosaponin-d suppressing epithelial-mesenchymal transition progression and alleviating liver fibrosis by inhibiting the transforming growth factor-β1/Smad signaling pathway. Upon hepatic injury or stimulation by endotoxins and inflammatory cytokines, hepatic stellate cells (HSCs), Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells secrete substantial amounts of transforming growth factor-β1 (TGF-β), contributing to hepatic immune regulation and inflammation. HSCs are key cellular players in the fibrogenic process. In response to liver injury, quiescent HSCs undergo activation and transition into activated HSCs, which secrete excessive extracellular matrix, driving fibrosis progression. Activated HSCs also secrete TGF-β, a major mediator of liver fibrosis. TGF-β signals through a heteromeric complex of two serine/threonine kinase receptors, type I and type II, which phosphorylate Smad2/Smad3 proteins. This leads to the activation of fibrogenic mediators, including collagen, α-smooth muscle actin, and fibronectin. Additionally, TGF-β1 is a potent inducer of the epithelial-mesenchymal transition, a process where epithelial cells lose their characteristic phenotype and acquire mesenchymal features, contributing to fibrosis. Saikosaponin-d attenuates HSC activation and mitigates liver fibrosis progression, partly by inhibiting the TGF-β1/Smad signaling pathway and the downstream epithelial-mesenchymal transition progression. ROS: Reactive oxygen species; TGF-β1: Transforming growth factor-beta 1; SSd: Saikosaponin-d; LSEC: Liver sinusoidal endothelial cell; TGF-βR: Transforming growth factor-beta receptor; EMT: Epithelial-mesenchymal transition; TNF-α: Tumor necrosis factor-alpha; IL-1β: Interleukin-1 beta; HSC: Hepatic stellate cell.

To investigate the molecular mechanisms through which SSd inhibits the TGF-β1/Smad signaling pathway and EMT, we used molecular docking to analyze the interaction between SSd and TGF-β1. Results revealed a stable binding conformation between the two molecules, with a docking score of -5.3 kcal/mol indicating significant binding affinity. Subsequently, binding site analysis identified key residues (e.g., Glu B:313, AsnA:82, and GluA:75286) that contributed to the stability of the SSd-TGF-β1 complex through hydrogen bonding and hydrophobic interactions. These findings suggest that SSd directly binds to TGF-β1, thereby inhibiting its signaling activity through steric hindrance or allosteric modulation. EMT is a crucial downstream effector pathway of the TGF-β1/Smad signaling pathway[46-48]. Upon binding to its receptor, TGF-β1 activates Smad2/3 through phosphorylation. The resulting Smad2/3-Smad4 complex translocates to the nucleus, where it directly regulates the expression of transcription factors such as Snail, Twist, and zinc-finger E-box-binding homeobox 1. These transcription factors drive EMT, which plays a key role in diseases such as hepatic fibrosis, by suppressing the expression of epithelial markers (e.g., E-cadherin) and inducing the production of mesenchymal markers (e.g., N-cadherin and vimentin)[42,47,49]. Based on these findings, we speculate that SSd exerts antifibrotic effects by directly binding to TGF-β1, thereby inhibiting its signaling activity, blocking the activation of the TGF-β1/Smad pathway, and eventually suppressing the activation of EMT. However, this speculation warrants further experimental validation.

However, this study has several limitations. First, the experimental models used have inherent constraints, as this research primarily relies on a CCl₄-induced murine liver fibrosis model and a TGF-β1-activated LX-2 model. Given the etiological complexity of hepatic fibrosis in humans, these models may not fully represent the comprehensive antifibrotic effects of SSd in vivo. Second, estrogen secretion may influence liver fibrosis progression. Early studies have shown that chronic liver diseases such as fibrosis or cirrhosis are more prevalent in male populations compared to females[50,51]. Experimental research suggests that estrogen in female mice may reduce sensitivity to inducers like CCl₄, significantly lowering their modeling rates, potentially through mechanisms that inhibit HSC activation and reduce oxidative stress[52-55]. Recent one study has reported that the SRY gene on the Y chromosome is highly expressed in male mice, exacerbating hepatic fibrosis progression, whereas female mice, lacking this gene, exhibit relatively milder fibrosis[56]. ER are expressed at higher levels in the livers of female mice. Our previous study also demonstrated that saikosaponin d can upregulate ERα and ERβ protein expression in HSC-T6 cells, indicating its role as an ER modulator[33]. Therefore, to avoid the confounding effects of endogenous estrogen in female mice on CCl₄-induced hepatic fibrosis, male mice were selected for this experiment. The use of single-sex experimental designs may compromise the generalizability of research findings, particularly in drug development where it could lead to an underestimation of medication risks in women. Therefore, it is imperative to investigate whether gender differences exist in the efficacy and safety of SSd for treating liver fibrosis models. Third, as a naturally derived small-molecule compound, the impact of SSd on liver fibrosis primarily stems from its role as a biological signaling molecule regulating the TGF-β1/Smad and EMT pathways. This effect is directly mediated by the drug’s pharmacological action without altering DNA genetic information, implying that no long-term irreversible effects would persist after drug metabolism. However, further validation is still required. Finally, despite experimental evidence supporting SSd’s antifibrotic potential, its clinical translatability requires further validation through pharmacokinetic profiling, bioavailability assessments, and rigorous evaluation of safety and efficacy in human subjects.

To address these limitations, future investigations should incorporate a broader range of hepatic fibrosis models (e.g., cholestatic fibrosis, alcohol-induced fibrosis) with larger sample sizes to improve representativeness and reliability. Additionally, employing advanced molecular biology and proteomics techniques will help clarify the specific mechanisms by which SSd modulates the TGF-β1/Smads pathway and EMT processes, providing a solid theoretical foundation for the development of novel antifibrotic drugs. To better understand the gender-specific therapeutic effects of SSd on liver fibrosis, murine models stratified by sex should be utilized to explore its regulatory effects on the TGF-β1/Smads pathway and EMT in hepatic tissues.

Although current studies have not directly reported the reversibility of the effects of SSd, future research could be conducted to further validate this reversibility. First, SSd could be withdrawn at different time points to observe the dynamic recovery of cellular functions. Second, a deeper investigation into the impact of SSd on key signaling pathways and the recovery mechanisms after drug withdrawal is warranted. Third, the reversibility of SSd’s effects could be validated in animal models, providing more direct evidence for clinical applications. Through these studies, a more comprehensive understanding of the potential therapeutic value of SSd in liver fibrosis treatment and the reversibility of its effects can be achieved. For clinical translation, advanced formulations of SSd, such as liposome- or polymer nanoparticle-encapsulated SSd, should be developed to enhance hepatic targeting and bioavailability while minimizing systemic toxicity. Comprehensive safety assessments, including pharmacokinetic profiling and maximum tolerated dose determination in healthy populations, are crucial. To enhance antifibrotic efficacy, targeted combination therapies should be explored: (1) SSd combined with silymarin to synergistically mitigate oxidative stress-induced hepatocyte damage through antioxidant potentiation; and (2) SSd co-administered with entecavir to simultaneously suppress hepatitis B virus replication and TGF-β1/Smad-mediated fibrogenesis in viral hepatitis. As a promising antifibrotic agent, SSd’s unique mechanism of inhibiting HSC activation via TGF-β1/Smad blockade and its potential to reverse early-stage fibrosis (as evidenced by E-cadherin restoration and collagen reduction) position it as a disease-modifying therapeutic. Further mechanistic investigations will help optimize its therapeutic index, and its applications may extend to extrahepatic fibrotic disorders, underscoring the need for increased research investment to improve outcomes in fibrotic diseases.

Although SSd as a natural compound exhibits significant antitumor and antifibrotic activities, its clinical application must be strictly guided by comprehensive toxicological profiling to ensure therapeutic safety. The hepatotoxicity of SSd is primarily associated with its saponin structure, and long-term or excessive use may lead to elevated transaminase levels, which are related to the potential hepatotoxicity of SSd[57,58]. Our previous studies have shown that treatment with SSd at doses of 1.0, 1.5, and 2.0 mg/kg significantly alleviates CCl₄-induced liver injury and necrosis, thereby improving liver function and exerting hepatoprotective effects[26]. However, some studies have suggested that SSd can promote liver damage. For instance, Chen et al[57] reported that SSd induced hepatotoxicity by blocking the PDGF-βR/p38 pathway in LO2 hepatocytes, thereby stimulating mitochondrial apoptosis. In addition, Zhang et al[58] found that SSd administered at a dose of 300 mg/kg for one week induced apoptosis in mouse hepatocytes. On the contrary, SSd (0.2-0.4 mg/mL) has been shown to inhibit tumor growth, improve liver function (as indicated by reduced glutamic pyruvic transaminase levels), and modulate immune function (as evidenced by an increased CD4+/CD8+ ratio) in a dose-dependent manner in a rat model of hepatocellular carcinoma. In this study, the SSd concentration used (1.5 mg/kg) did not induce any hepatotoxic effects. These contradictory findings highlight the narrow therapeutic window of SSd. Specifically, low doses can exert therapeutic effects through immune regulation and liver function improvement, whereas high doses can induce hepatotoxicity, thereby negating therapeutic benefits. This narrow therapeutic window presents significant challenges for clinical translation. The first issue is dose selection. It is necessary to precisely define the safe and effective dosage range to minimize the risk of hepatotoxicity while achieving desired therapeutic efficacy. The second issue is individualized treatment. Before administration, a patient’s baseline liver function must be assessed, and those with hepatic insufficiency should not be recommended for SSd therapy. In addition, regular monitoring of liver function is required during treatment. The third issue is the potential risk of drug interactions. SSd may enhance the effects of certain medications (e.g., anticoagulants and hypoglycemic agents) when used in combination, further narrowing the safe dosage range. Therefore, the clinical translation of SSd therapy is highly dependent on a precise understanding of its therapeutic window. Future studies should focus on elucidating the action mechanisms of SSd to clarify the molecular targets that differentiate its hepatotoxic and antitumor effects. Furthermore, dose titration should begin at subtherapeutic levels, and strict avoidance of prolonged treatment is necessary to prevent hepatotoxicity. Moreover, developing detoxification strategies, such as structural modification or formulation optimization (e.g., nanodispersions), can help enhance the safety of SSd. Finally, establishing a comprehensive monitoring system for pre-treatment screening of hepatic, renal, and cardiovascular functions, along with continuous monitoring of liver enzymes (ALT/AST), hemoglobin profiles, and electrocardiogram parameters, can provide real-time monitoring schemes for clinical drug use. In conclusion, the “double-edged sword” nature of SSd necessitates that safety be prioritized in clinical settings. Through further investigation, the therapeutic benefits of SSd can be maximized while minimizing its potential risks.

CONCLUSION

In conclusion, SSd inhibits EMT progression by blocking the TGF-β1/Smad signaling cascade, thereby reducing HSC activation and mitigating the development of hepatic fibrosis.