Integrating Network Pharmacology and Experimental Validation to Elucidate Huaier in Attenuating Liver Fibrosis via the AKT Signaling Pathway

Integrating Network Pharmacology and Experimental Validation to Elucidate Huaier in Attenuating Liver Fibrosis via the AKT Signaling Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Integrating Network Pharmacology and Experimental Validation to Elucidate Huaier in Attenuating Liver Fibrosis via the AKT Signaling Pathway Jiachun Ding, Jiaqiang Ren, Fan Chen, Ye Lu, Yifei Ma, Ting Zhang, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9123161/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Liver fibrosis, a critical pathological precursor to cirrhosis and hepatocellular carcinoma, represents a significant unmet global health challenge due to the lack of effective targeted therapies that can reverse established fibrotic scarring. An integrated strategy was employed. Network pharmacology identified potential targets, followed by molecular docking and dynamics simulations. In vivo validation utilized two established murine models: carbon tetrachloride (CCl₄) and bile duct ligation (BDL)-induced fibrosis. In vitro studies employed human LX-2 hepatic stellate cells to assess anti-fibrotic effects on myofibroblasts. Huaier administration significantly attenuated liver fibrosis, improved liver function and reduced collagen deposition in both animal models. Histopathological analysis confirmed diminished inflammatory infiltration and fibrotic scarring. In vitro, Huaier suppressed LX-2 cell proliferation and migration. Bioinformatics and simulation analyses pinpointed AKT1 as a central target, showing high-affinity binding with Huaier's bioactive steroidal components. Mechanistically, Huaier specifically inhibited the phosphorylation and activation of the AKT signaling pathway in hepatic myofibroblasts. This study provides the first compelling evidence that Huaier granule alleviates liver fibrosis by targeting the AKT pathway to inhibit myofibroblast activation. These findings illuminate its mechanistic basis and reposition Huaier as a promising, multi-targeted therapeutic candidate for combating fibrotic liver disease. Liver fibrosis Huaier network pharmacology AKT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Liver fibrosis, a common pathological outcome of chronic liver injury, is characterized by the excessive accumulation of extracellular matrix (ECM) proteins, which can progress to cirrhosis, liver failure and hepatocellular carcinoma, posing a significant threat to global health[ 1 ]. Despite advances in understanding its molecular mechanisms, primarily involving the activation of hepatic stellate cells (HSCs) and complex cytokine networks, effective anti-fibrotic therapies remain a major unmet medical need[ 2 ]. Current strategies predominantly focus on removing the underlying etiology, such as antiviral therapy for viral hepatitis[ 3 ]. However, this approach often has limited efficacy in reversing established fibrosis and for many patients, the causative agent cannot be eliminated. Consequently, the development of targeted and effective pharmacological agents to halt or reverse the fibrogenic process is an urgent priority and a significant challenge in hepatology[ 4 ]. Huaier (scientific name: Trametes robiniophila Murr.), a traditional medicinal fungus, has been used in China for centuries[ 5 ]. Its extract, the active component of the commercial preparation Huaier granule, is approved by the National Medical Products Administration (NMPA) of China as an adjuvant therapy for hepatocellular carcinoma[ 6 ]. Modern pharmacological studies have revealed that Huaier is rich in various bioactive compounds, including polysaccharides, proteoglycans and amino acids, endowing it with a wide spectrum of biological activities such as immunomodulation, antitumor, anti-inflammatory and antiviral effects[ 7 ]. Notably, in the context of liver diseases, clinical and experimental studies have demonstrated that Huaier granule can improve liver function and inhibit tumor cell proliferation, showcasing its therapeutic potential and safety profile[ 8 ]. Emerging evidence suggests that Huaier holds promise for the treatment of liver fibrosis[ 9 ]. A pioneering multicenter trial explores the potential of Huaier granule, a traditional Chinese medicine, as adjuvant therapy following curative resection of hepatocellular carcinoma (HCC). Conducted across 39 centers with 1,044 patients, the randomized phase IV study demonstrates a significant improvement in recurrence-free survival and overall survival rates in the treatment group compared to controls[ 6 ]. The findings provide compelling evidence that Huaier granule effectively reduces both intrahepatic and extrahepatic tumor recurrence, offering a promising postoperative strategy for HCC management. Its multifaceted actions, particularly its potent anti-inflammatory and immunoregulatory properties, position it as a compelling candidate for targeting the complex pathogenesis of fibrosis. However, the existing body of research is still in its early stages. The precise molecular targets, the key effective components responsible for its anti-fibrotic effects, and the comprehensive mechanisms of action remain largely elusive[ 10 ]. This lack of a systematic understanding hinders its potential application as a targeted anti-fibrotic therapy. Therefore, the present study aims to systematically investigate the therapeutic effects of Huaier on liver fibrosis and to elucidate its underlying mechanisms. To achieve this, we employed an innovative integrated strategy combining network pharmacology with in vivo validation in the mouse model. Network pharmacology was used to predict the potential active compounds[ 11 ], key targets and signaling pathways involved in Huaier's anti-fibrotic action. These predictions were subsequently experimentally validated in a CCl4 and BDL induced mouse model of liver fibrosis through histopathological and molecular biological techniques. This research not only provides a solid scientific basis for the clinical application of Huaier in anti-fibrosis therapy but also offers a novel paradigm for exploring the mechanisms of traditional medicines using a multi-disciplinary approach. Materials and methods Mice and liver fibrosis model Eight-week-old male and female C57BL/6 wild-type mice were obtained from Xi'an Jiaotong University and housed under conventional pathogen-free conditions, with a 12-hour light/dark cycle, ambient temperature maintained at 22 ± 2°C and relative humidity at 50 ± 10%. Food and water were provided ad libitum. To establish chronic liver fibrosis, mice received intraperitoneal injections of carbon tetrachloride (CCl4) at 0.6 mL/kg body weight three times per week for six weeks. Forty-eight hours after the final injection, at 14 weeks of age, mice were humanely euthanized via carbon dioxide inhalation in a euthanasia chamber. Blood and liver tissues were subsequently collected for analysis. In a parallel set of experiments, liver fibrosis was induced via bile duct ligation (BDL). Mice were fasted for 12 hours prior to surgery and sacrificed two weeks post-operatively for tissue and serum collection. All mice were randomly divided into the following two groups (the control group (saline 100 µL/day, gavage) and Huaier group[ 12 ] (2 g/kg/day, gavage). Huaier extract was provided by Gaitianli Medicine Co., Ltd. (Qidong, Jiangsu, China). All experimental procedures were conducted in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" and were approved by the Animal Experiment Administration Committee of Xi’an Jiaotong University to ensure ethical and humane treatment of animals. Histology Harvested liver tissues were gently perfused and fixed in fresh 4% neutral-buffered formalin for 24 hours at 4°C, followed by washing in 1× phosphate-buffered saline (PBS). For histological analysis, samples were dehydrated, cleared and embedded in paraffin. Serial sections of 5 µm thickness were prepared, mounted on glass slides, and stained with Hematoxylin and Eosin (H&E; Cat# ST047, Solarbio), Masson’s Trichrome (Cat# abs9348, Absin) and Sirius Red (Cat# DC0041, Leagene Biotech). Bright-field imaging was performed using an Olympus microscope. Digitized images were analyzed with Image-Pro Plus 6.0 software (Media Cybernetics) to evaluate histological architecture and quantify fibrosis. Immunofluorescence staining For cryosectioning, liver tissues were embedded in Optimal Cutting Temperature (OCT) compound (Cat# 4583, Tissue-Tek) and 5 µm-thick sections were prepared using a cryostat maintained at -20°C. The resulting sections were mounted on adhesive-coated glass slides. Subsequently, the sections were blocked and permeabilized for 1 hour at room temperature with 5% bovine serum albumin (BSA; Cat# V900933, Sigma-Aldrich) and 0.3% Triton X-100 (Cat# T8200, Solarbio). Primary antibodies were applied overnight at the following dilutions: α-SMA-Cy3 (1:100, Cat# C6198, Sigma-Aldrich), Ki-67 (1:100, Cat# ab16667, Abcam), AKT (1:100, Cat# ab8805, Abcam), P-AKT (1:100, Cat# ab192623, Abcam) and Phalloidin-594 (1:100, Cat# 8953S, CST). The following day, slides were incubated for 1 hour at room temperature with appropriate Alexa Fluor-conjugated secondary antibodies (Cat# A11008 and A11012, Invitrogen). Nuclei were visualized by counterstaining with DAPI (1 µg/mL, Thermo Fisher Scientific) for 5 minutes. After three washes with PBS, the sections were coverslipped using an antifade mounting medium (Vector Laboratories). Images were captured with a laser-scanning confocal microscope (Olympus FV3000, Core Facilities Sharing Platform at School of Basic Medical Sciences). Assay for hydroxyproline in liver tissue We use hydroxyproline content assay kit(Cat# BC0250, Solarbio) to measure hydroxyproline content in mouse liver, begin by homogenizing approximately 0.2 g of liver tissue and hydrolyzing it with 2 ml of 6 M HCl at 110°C for 4 hours. After cooling, neutralize the hydrolysate to pH 6–8 using NaOH, then dilute to 4 ml with distilled water. Centrifuge the mixture at 16,000 rpm for 20 minutes and collect the supernatant. Using a multifunction microplate reader (POLARstar OPTIMA; BMG, Offenburg, Germany) set at 560 nm, mix 200 µl of the supernatant with 200 µl of Reagent 1, incubate for 20 minutes, add 200 µL of Reagent 2, and heat at 60°C for another 20 minutes. After cooling, measure the absorbance. A standard curve constructed with hydroxyproline standards enables precise quantification, reflecting collagen metabolism in the liver with accuracy and elegance. Western blot Isolated livers or myofibroblasts were lysed using RIPA buffer (Cat# 9806, Cell Signaling) supplemented with protease and phosphatase inhibitors (Cat# 04906837001, Roche). Following lysis, the homogenates were centrifuged at 11,752 × g for 10 minutes at 4°C. The resulting supernatants were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% skim milk for 1 hour, the membranes were incubated with the following primary antibodies: Collagen I (1:1000, Cat# ab279711, Abcam), α-SMA (1:1000, Cat# AF1032, Affinity), Ki-67 (1:1000, Cat# ab16667, Abcam), AKT (1:100, Cat# ab8805, Abcam), P-AKT (1:100, Cat# ab192623, Abcam), β-Actin (1:1000, Cat# ab6276, Abcam) and α-Tubulin (1:1000, Cat# ab81299, Abcam) were used as loading controls. Protein bands were visualized using the ECL detection system (BioRad). Quantitative RT-PCR analysis Total RNA was extracted from myofibroblasts or liver tissues using Trizol reagent (Cat# 15596018, Invitrogen). cDNA was synthesized from 1 µg of total RNA using a commercial cDNA synthesis kit (Cat# 170–8891, BioRad), following the manufacturer's protocol. Quantitative real-time PCR (qRT-PCR) was performed on a StepOne Plus System (Applied Biosystems) with universal SYBR Green mix (Cat# 172–5122, BioRad). The relative expression of target genes was calculated using the 2 –ΔΔCT method, with GAPDH serving as the internal reference for normalization. Quantitative PCR primer sequences were as follows: Acta2: 5’-gaggcaccactgaaccctaa-3’/5’-tacatggcggggacattgaa-3’; Col1a1, 5’-ttcagggaatgcctggtgaa-3’/5’-acctttgggaccagcatca-3’; Col1a2, 5’- aagggtgctactggactccc-3’/5’-ttgttaccggattctcctttgg-3’; Col2a1, 5’- agcaggtccttggaaacctt-3’/5’-aaggagtttcatctggccct-3’; Ki-67, 5’-atttgcttctggccttcccc-3’/5’-ccaaacaagcaggtgctgag-3’; GAPDH, 5’- gtaacccgttgaaccccatt-3’/5’-ccatccaatcggtagtagcg-3’. Serum ALT and AST Assays Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice were determined using commercial assay kits (ALT:Cat# C009-2-1, Nanjing Jiancheng Bioengineering Institute; AST:Cat# C010-2-1, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions. Serum aliquots were directly subjected to analysis without further dilution. For the assay, samples were incubated with the respective substrate solutions at 37°C for 30 minutes. The enzymatic reactions were then terminated by adding 2,4-dinitrophenylhydrazine. Following a 20-minute incubation at 37°C, the resulting solutions were alkalized and the colored products were allowed to develop at room temperature for 15 minutes. The absorbance was measured at 505 nm for ALT and 510 nm for AST using a multifunction microplate reader (POLARstar OPTIMA; BMG, Offenburg, Germany). The enzyme activities, expressed in Karmen units, were calculated based on standard curves and subsequently converted to international units per liter (U/L). Cell culture and treatment The human hepatic stellate cell line LX-2 (Cat# CL-0560, Procell, Inc.) was cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C under a 5% CO₂ atmosphere. Prior to experimental treatment, cells were serum-starved overnight in serum-free DMEM. Differentiation into myofibroblasts was induced by stimulation with 5 ng/mL TGF-β1 (Cat# HY-P70648, MCE) for 72 hours. Subsequently, the differentiated myofibroblasts were treated with or without Huaier (8 mg/mL) for 48 hours. Cell viability assay Myofibroblasts were seeded in 96-well plates at a density of 3,000 cells per well and allowed to adhere overnight. Following this, the cells were treated with or without Huaier (8 mg/mL) for 48 hours. After treatment, the culture medium was carefully removed, and each well was supplemented with 100 µL of DMEM and 10 µL of Cell Counting Kit-8 (CCK-8) reagent. The plates were then incubated for an additional 3 hours at 37°C under 5% CO₂. Absorbance was measured at 450 nm using a multifunctional microplate reader (POLARstar OPTIMA, BMG, Offenburg, Germany). Cell migration assays Myofibroblasts were seeded in 6-well plates pre-marked with grid lines on the underside. After the cells reached 100% confluence, a straight wound was created in each well using a 200 µl pipette tip. The cells were then treated with or without Huaier (8 mg/mL). Wound closure was monitored at 12 and 24 hours post-treatment by imaging the same predefined locations under an inverted microscope. The extent of wound healing was quantitatively analyzed using Image Pro Plus 6.0 software (Media Cybernetics, USA). Network pharmacology Twenty-six bioactive components of Huaier were identified through a combination of database mining using HERB ( http://herb.ac.cn/ ) and a review of previously published literature. Based on the Swiss Target Prediction platform ( http://www.swisstargetprediction.ch/ ), potential target proteins of the active ingredients from Huaier were predicted. The search parameter was set to “Homo sapiens” and targets were selected under the criterion of “probability > 0”. Potential targets associated with liver cirrhosis were retrieved from multiple public databases, including OMIM ( https://omim.org/ ), DrugBank ( https://go.drugbank.com/ ), TTD ( http://db.idrblab.net/ttd/ ), PharmGkb ( https://www.pharmgkb.org/ ) and GeneCards ( https://www.genecards.org/ ), using the keyword "liver cirrhosis". Common targets between Huaier and liver cirrhosis were identified using the Venny 2.1.0 platform ( https://bioinfogp.cnb.csic.es/tools/venny/index.html ) and their overlap was visualized in a Venn diagram. Based on Venn diagram, compound-target interaction network was constructed using Cytoscape software (v3.10.3). A protein-protein interaction (PPI) network was subsequently constructed with the STRING database ( https://cn.string-db.org ) and visualized using Cytoscape software (v3.10.3). Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, were performed for the key target clusters via the Metascape database ( https://metascape.org ). The results of these enrichment analyses were visualized using an online bioinformatics platform ( https://www.bioinformatics.com.cn/ ). Molecular docking The three-dimensional structures of bioactive compounds derived from Huaier were retrieved from the PubChem database ( https://pubchem.ncbi.nlm.nih.gov/ ). The crystal structure of the target protein was obtained from the Protein Data Bank ( https://www.rcsb.org/ ). Molecular docking simulations were performed using AutoDock software (version 4.2.6) and corresponding binding energies were calculated. For each compound–target pair, five independent docking runs were conducted using different random seeds to ensure reproducibility. The lowest-energy conformation from each run was selected, and binding free energies (ΔG, kcal/mol) are expressed as mean ± standard deviation derived from the five replicates. Molecular dynamics simulation Molecular dynamics simulations were performed using GROMACS 2020.6 to model the ligand–protein interaction over a 100 ns period. The topology of the small molecule ligand was generated with Sobtop 1.0 using the GAFF force field, while the protein topology was constructed with the AMBER99SB-ILDN force field. To neutralize the system, sodium and chloride ions were introduced and the complex was solvated in a truncated octahedral box of TIP3P water molecules with a 1.0 nm buffer distance. Energy minimization was carried out in two stages: first with the steepest descent algorithm for 2500 steps, followed by the conjugate gradient method for another 2500 steps. Subsequently, the system was equilibrated under an NVT ensemble for 100 ps at 298.15 K and then under an NPT ensemble for another 100 ps at the same temperature. Using the PME approach, long-range electrostatic interactions were estimated in an NPT ensemble simulation at 100 nm with the periodic boundary condition. A collision frequency of 2 ps and a non-bond cut-off distance of 1 nm were determined. Trajectories were recorded at 10-ps intervals, the integration step was set to 2 s and the system pressure was kept at 101.325 kPa. Single-cell RNA-seq dataset processing Publicly available single-cell RNA sequencing (scRNA-seq) datasets were downloaded from the Gene Expression Omnibus (GEO) database under accession numbers GSE145086[ 13 ]. Data processing and analysis were performed using the Seurat R package (v4.3.0.1). A Seurat object was created from the expression matrix and accompanying metadata using the Create Seurat Object function. Subsequent dimensionality reduction and visualization were carried out via uniform manifold approximation and projection (UMAP) using the Run UMAP function with 15 principal components (dims = 1:15). Violin plots were generated with ggplot2 (v3.5.1) and feature expression overlays on UMAP projections were rendered using Seurat's visualization utilities. Statistics All statistical analyses were conducted using GraphPad Prism 9. Sample sizes were determined based on preliminary or prior experiments to ensure sufficient statistical power. Samples with inadequate RNA/cDNA quality or suboptimal tissue integrity after processing (below commonly accepted thresholds) were excluded from analysis. Data are expressed as mean ± SEM. Differences between two groups were assessed using an unpaired, two-tailed Student’s t-test and a P-value ≤ 0.05 was considered statistically significant. Results Bioactive composition and molecular structures of Huaier Based on previously published literature and the HERB database[ 7 ], a total of 30 bioactive substances have been identified in processed Huaier (Trametes robiniophila). These active components are broadly classified into four major categories: proteoglycans, bioactive small molecules, steroids and alkaloids. The proteoglycans include Polysaccharide (SP1), water-soluble neutral polysaccharide (W-NTRP), T. robiniophila polysaccharide (TRP) and Proteoglycan (TPG-1). According to existing research, the bioactive small molecules comprise uracil, methyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, cyclo(Leu-Pro), cyclo(Phe-Ala), cyclo(Phe-Gly), methylparaben, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, ethyl 3,4-dihydroxybenzoate, genistein, 4,4'-biphenyldithiol, dehydrofalcarinone, diphenyldisulfide, glyodin, O-acetylserine, pirbuterol, semustine, sulfometuron-methyl, tyrosol 4-sulfate and valinopine. In addition, the steroid constituents include ergosta-7,22-dien-3β-ol, ergosterol, 3β-hydroxystigmast-5,22-dien-7-one and sitogluside. And the alkaloids identified consist of adenosine (Fig. 1 A). The chemical structures of the bioactive molecules identified in Huaier were obtained from the PubChem database (Fig. 1 B). Huaier attenuate the degree of liver fibrosis induced by CCl4 in mice Eight-week-old C57 mice were subjected to intraperitoneal injections of CCl4 at 0.6 mL/kg body weight three times per week for six weeks. Beginning from the second week, control mice received daily oral gavage of normal saline (100 µL/day), while the treatment group was administered Huaier (2 g/kg/day) by oral gavage for five weeks. Forty-eight hours after the final CCl4 injection, 14-week-old mice were euthanized via carbon dioxide inhalation and blood and liver samples were collected (Fig. 2 A). Body weight monitoring during the modeling period revealed that Huaier-treated mice experienced less weight loss compared to the control group, with a statistically significant difference observed by the sixth week (Fig. 2 B). The liver-to-body weight ratio was significantly lower in the Huaier group (Fig. 2 C). Histological analysis showed reduced inflammatory cell infiltration in H&E-stained sections, along with markedly diminished fibrotic scarring in Sirius Red and Masson staining (Fig. 2 D), which was quantitatively confirmed (Fig. 2 E), indicating a significant anti-fibrotic effect of Huaier. Immunofluorescence staining for α-SMA revealed a significant reduction in myofibroblast in the treatment group (Fig. 2 F), as supported by quantification (Fig. 2 G). Further analysis showed a significant decrease in hepatic hydroxyproline content in Huaier-treated mice (Fig. 2 H). Western blot results indicated downregulated protein expression of collagen I and α-SMA in Huaier-treated mice (Fig. 2 I-K) and qPCR showed reduced mRNA levels of fibrogenic genes such as Acta2, Col1a1, Col1a2 and Col2a1 in Huaier-treated mice (Fig. 2 L). Finally, the serum ALT and AST levels were significantly lower in the Huaier group, consistent with improved liver function (Fig. 2 M). Collectively, these findings substantiate the protective role of Huaier against CCl4-induced liver fibrosis in mice. Huaier attenuate the degree of liver fibrosis induced by BDL in mice Eight-week-old C57 mice underwent bile duct ligation (BDL). Starting on the third day after surgery, control mice received daily oral gavage of normal saline (100 µL/day), while the treatment group was administered Huaier (2 g/kg/day) for 12 days. Liver and blood samples were collected two weeks post-modeling (Fig. 3 A). Body weight changes throughout the experimental period revealed that Huaier-treated mice exhibited less pronounced weight loss compared to controls, with a statistically significant difference between the two groups by the second week (Fig. 3 B). The liver-to-body weight ratio was significantly lower in the Huaier-treated group (Fig. 3 C). Histological evaluation demonstrated a marked reduction in inflammatory cell infiltration in H&E-stained liver sections from the treatment group. Additionally, Sirius Red and Masson staining showed substantially attenuated fibrotic scarring, which was quantitatively confirmed, indicating that Huaier significantly alleviates BDL-induced liver fibrosis (Fig. 3 D-E). Immunofluorescence staining for α-SMA revealed a significant decrease in the area positive for myofibroblasts in Huaier-treated mice, as quantified, suggesting suppression of myofibroblast activation (Fig. 3 F-G). Further biochemical analysis indicated that hepatic hydroxyproline content was significantly reduced in the treatment group (Fig. 3 H). Western blot analysis showed downregulated protein expression of collagen I and α-SMA in the livers of Huaier-treated mice (Fig. 3 I-K). Consistent with these findings, there was a reduction in the mRNA expression of fibrosis-related genes, including Acta2, Col1a1, Col1a2 and Col2a1 (Fig. 3 L). Finally, serum levels of ALT and AST were both significantly lower in the Huaier group, reflecting improved liver function (Fig. 3 M). Together, these results substantiate the anti-fibrotic efficacy of Huaier in a BDL-induced liver fibrosis model. Huaier attenuate the degree of liver fibrosis by inhibiting the proliferation of myofibroblasts in vivo and in vitro We employed immunofluorescence to assess the proliferation of myofibroblasts in liver tissues from mice with CCl4-induced liver fibrosis. Myofibroblasts were labeled with α-SMA and proliferating cells were identified using Ki-67. Results showed a marked reduction in the number of proliferating myofibroblasts in the Huaier-treated group compared to the control (Fig. 4 A), which was statistically significant (Fig. 4 B). Similarly, in the BDL-induced liver fibrosis model, Huaier treatment also suppressed the proliferation of myofibroblasts in fibrotic livers (Fig. 4 C-D). In vitro, using an LX-2-derived myofibroblast model, a wound healing assay was performed to evaluate the effect of Huaier on cell migration. We observed that Huaier significantly inhibited the migratory capacity of myofibroblasts (Fig. 4 E-G). Furthermore, a CCK-8 assay revealed that Huaier suppressed the proliferative activity of LX-2 myofibroblasts in culture (Fig. 4 H). Additional immunofluorescence staining for Ki-67 confirmed that Huaier treatment led to a notable decrease in the proportion of proliferating myofibroblasts (Fig. 4 I-J). At the molecular level, Western blot analysis demonstrated that Huaier downregulated Ki-67 protein expression in myofibroblasts (Fig. 4 K-L). Consistent with this, qPCR analysis indicated a reduction in Ki-67 mRNA levels following Huaier treatment (Fig. 4 M). Collectively, these findings illustrate that Huaier effectively curbs both the migration and proliferation of myofibroblasts in vitro, supporting its anti-fibrotic role. Network pharmacology analysis revealing potential mechanisms by which Huaier attenuate the degree of liver fibrosis Twenty-six bioactive components of Huaier were identified through an integrated approach involving database mining via HERB ( http://herb.ac.cn/ ) and a comprehensive review of previously published literature. Using the Swiss Target Prediction platform ( http://www.swisstargetprediction.ch/ ), potential target proteins of these twenty-six bioactive components from Huaier were systematically predicted. A total of 604 protein targets associated with the bioactive components of Huaier were identified. Given the absence of a specific disease category for liver fibrosis in the database, we selected liver cirrhosis-clinically characterized by features of liver fibrosis-as a proxy to identify relevant disease targets. Concurrently, 10,872 therapeutic targets for liver cirrhosis were retrieved from multiple databases, including OMIM ( https://omim.org/ ), DrugBank ( https://go.drugbank.com/ ), TTD ( http://db.idrblab.net/ttd/ ), PharmGkb ( https://www.pharmgkb.org/ ) and GeneCards ( https://www.genecards.org/ ). Among these, 486 overlapping targets were found between the protein targets of Huaier’s bioactive components and the therapeutic targets for liver cirrhosis. A Venn diagram was constructed to illustrate these intersecting therapeutic targets (Fig. 5 A). Based on the intersection results, a compound-target interaction network was established, delineating the relationships between each of the 26 active components of Huaier and the corresponding therapeutic targets for liver cirrhosis (Fig. 5 B). For the 486 liver cirrhosis targets potentially modulated by the active constituents of Huaier, a protein-protein interaction (PPI) network was subsequently constructed using the STRING database ( https://cn.string-db.org ) and visualized with Cytoscape software (v3.10.3). The PPI network was screened via a topological analysis method, with nodes colored in red indicating higher Matthews Correlation Coefficient (MCC) values (MCC ≥ 0.6)[ 14 ]. Among these, AKT1 exhibited the strongest and most prominent expression, followed by GAPDH, IL6, TNF and IL1β (Fig. 5 C–F). GO、KEGG enrichment analysis and molecular docking for identifying the binding beween bioactive composition of Huaier and AKT1 The analysis of GO enrichment for 486 target was conduct with a significance threshold established at P < 0.01. The GO analysis revealed that the potential therapeutic targets of Huaier are involved in 19 biological processes, including Protein kinase activity, Oxidoreductase activity, Kinase binding, Transcription factor binding, Serine hydrolase activity, Protein homodimerization activity, Protein domain specific binding, Hydrolase activity (acting on ester bonds), Histone modifying activity, Protein tyrosine kinase activity, Hormone binding, Nuclear receptor activity, G protein-coupled peptide receptor activity, Protease binding, Exopeptidase activity, G protein–coupled amine receptor activity, Deacetylase activity, Prostanoid receptor activity and Insulin-like growth factor II binding (Fig. 6 A). The experiment conducted Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis (KEGG) on the signaling pathways that may be involved in the alleviation of liver cirrhosis by Huaier. The results showed the top 13 signaling pathways, among which the top 3 enriched pathways for the target genes are Neuroactive ligand-receptor interaction, Lipid and atherosclerosis, Hormone signaling (Fig. 6 B). The three-dimensional structures of 26 bioactive compounds derived from Huaier were retrieved from the PubChem database ( https://pubchem.ncbi.nlm.nih.gov/ ). The crystal structure of the AKT1, GAPDH, IL6, TNF and IL1β was obtained from the Protein Data Bank ( https://www.rcsb.org/ ). Molecular docking analyses of the 26 bioactive compounds with the five core protein targets were performed using AutoDock software and the corresponding binding energies were systematically computed. To ensure reproducibility, each compound-target interaction was evaluated through five independent docking runs employing distinct random seed values. From each run, the lowest-energy conformation was selected and the binding free energies (ΔG, kcal/mol) are reported as the mean ± standard deviation based on the five technical replicates. Based on the binding energies calculated from molecular docking between 26 bioactive compounds from Huaier and the target proteins AKT1, GAPDH, TNF, IL1B and IL6, we generated a heatmap to visualize the interaction profiles between Huaier-derived active components and therapeutic targets for liver cirrhosis. The analysis revealed that AKT1 exhibited stronger binding affinities with the 26 compounds compared to the other target proteins. Notably, the highest binding energies were observed between AKT1 and Ergosta-7,22-dien-3β-ol, Ergosterol, 3β-hydroxystigmast-5,22-dien-7-one and Sitogluside, respectively (Fig. 6 C). The molecular docking visualizations revealed distinct binding modes between AKT1 and several key bioactive compounds-namely, Ergosta-7,22-dien-3β-ol, Ergosterol, 3β-hydroxystigmast-5,22-dien-7-one and Sitogluside (Fig. 6 D-G). Collectively, these findings suggest that Huaier may elicit its anti-fibrotic effects in the liver via multi-target and multi-pathway mechanisms, in which the AKT1 signaling pathway appears to play a central role. Molecular dynamics simulation for identifying the binding beween bioactive composition of Huaier and AKT1 To assess the stability of the binding interactions between AKT1 and several key bioactive compounds-namely, Ergosta-7,22-dien-3β-ol, Ergosterol, 3β-hydroxystigmast-5,22-dien-7-one and Sitogluside, molecular dynamics (MD) simulations were performed on the corresponding docked complexes using GROMACS software. The Root Mean Square Deviation (RMSD) was employed as a key metric to evaluate the conformational stability and reliability of each complex. An RMSD fluctuation within 0.2 nm is generally indicative of a structurally stable system[ 15 ]. The RMSD values of the ligands across all systems remained consistently around or below 0.2 nm throughout the simulation trajectory, confirming the overall structural integrity of the complexes (Fig. 7 A-D). Furthermore, the Root Mean Square Fluctuation (RMSF) was utilized to characterize atomic-level flexibility, providing insight into the local mobility of residues within the protein structure[ 16 ]. The RMSF profiles reveal that the peptide residues in all complexes: Ergosta-7,22-dien-3β-ol-AKT1, Ergosterol-AKT1, 3β-hydroxystigmast-5,22-dien-7-one-AKT1 and Sitogluside-AKT1, exhibited fluctuations largely confined within approximately 0.2 nm during the entire simulation (Fig. 7 A-D). These observations suggest that AKT1 maintains considerable structural rigidity upon binding to these bioactive constituents of Huaier, with no marked conformational rearrangements, thereby underscoring the stability of the interactions. Distance between molecules is a crucial observable for characterizing binding affinity and interaction stability. Tracking its fluctuations helps identify stable bound states, transient encounters and dissociation events throughout a simulation. The intermolecular distance profiles demonstrate that throughout the simulation, the binding interfaces in all complexes: Ergosta-7,22-dien-3β-ol-AKT1, Ergosterol-AKT1, 3β-hydroxystigmast-5,22-dien-7-one-AKT1 and Sitogluside-AKT1, exhibited fluctuations predominantly constrained within approximately 1.0 nm (Fig. 7 A-D). As a fundamental metric in molecular simulations, the distance between molecules provides critical insights into interaction stability and the dynamic behavior of complexes. The observed narrow fluctuation range indicates the sustained proximity essential for molecular recognition and effective binding. These results suggest that AKT1, upon binding with these bioactive constituents of Huaier, engages in well-defined molecular interactions characterized by persistent association, with only minor transient variations, reflecting a stable and specific binding mode. The radius of gyration (Rg) was employed to assess the structural compactness of the protein, where a lower value corresponds to a more tightly folded conformation[ 17 ]. Within the 100 ns simulation period, the Rg values of all complexes: Ergosta-7,22-dien-3β-ol-AKT1, Ergosterol-AKT1, 3β-hydroxystigmast-5,22-dien-7-one-AKT1 and Sitogluside-AKT1, rapidly converged to an equilibrium state and stabilized within the range of 2.15–2.20 nm (Fig. 7 A-D). These results indicate that AKT1 is capable of forming compact and stable complexes with the bioactive constituents of Huaier. Such structural consolidation is conducive to the establishment of stable interactions between the active compounds and AKT1, which may underly the pharmacological role of Huaier in mitigating liver fibrosis by modulating myofibroblast activity. AKT1 is highly expressed in myofibroblasts during liver fibrosis in single-cell RNA sequencing By interrogating public repositories for single-cell RNA sequencing data and identifying the dataset GSE145086[ 13 ], which comprises liver samples from normal mouse liver and a mouse model of CCl4-induced liver fibrosis. Subsequent bioinformatic analysis was performed using the Seurat R package, followed by the generation of Uniform Manifold Approximation and Projection (UMAP) visualizations. These plots elucidated the cell clusters across normal and fibrotic liver tissues, revealing distinct cell types including hepatocytes, myofibroblasts, endothelial cells, etc (Fig. 8 A-B). Further investigation into the expression level of AKT1 across these cell clusters demonstrated notably elevated expression of AKT1 in myofibroblasts. This finding suggests a potential biological role for AKT1 within myofibroblasts during the progression of liver fibrosis (Fig. 8 C-D). As is well established, the biological activity of AKT is exerted upon its phosphorylation at key amino acid residues (Thr308 and Ser473)[ 18 ], which converts it into its active form, P-AKT. To investigate this, we measured the protein levels of both AKT and P-AKT in LX-2 cells following Huaier treatment. Our results revealed that while the total protein level of AKT remained largely unchanged, the level of P-AKT was significantly reduced. This indicates that Huaier likely attenuates the biological functions of AKT in liver fibrosis by specifically inhibiting its phosphorylation in myofibroblasts (Fig. 8 E-F). Huaier attenuate the degree of liver fibrosis by inhibiting the proliferation of myofibroblasts via AKT pathway in vivo and in vitro The protein expression of AKT and P-AKT in a mouse model of liver fibrosis was further investigated, using α-SMA as a marker for myofibroblasts. In mice with CCl4-induced liver fibrosis, treatment with Huaier did not significantly alter the expression level of AKT in myofibroblasts, but led to a statistically significant reduction in P-AKT expression (Figure. 9A-B, G). These findings suggest that active components in Huaier indeed suppress the activation of the AKT signaling pathway in myofibroblasts during liver fibrosis. Consistently, we observed the same trend with statistical significance in a BDL-induced liver fibrosis model (Fig. 9 C-D, H). We employed immunofluorescence staining to assess the expression of AKT and P-AKT in cultured LX-2 cells. Consistent with our prior findings, Huaier treatment did not alter the overall protein level of AKT. However, a marked reduction in P-AKT levels was readily detectable, indicating a specific inhibition of AKT phosphorylation (Fig. 9 E-F, I). Discussion The present study elucidates the potent therapeutic effects of Huaier extract on liver injury and fibrosis, positioning this traditional remedy as a promising multi-targeted agent for chronic liver diseases. Our findings demonstrate that Huaier administration significantly attenuates the pathological progression of liver fibrosis by suppressing a single inflammatory pathway. This aligns with the holistic philosophy of its traditional use and provides a compelling molecular basis for its efficacy[ 19 ]. A previous clinical study provides valuable clinical evidence supporting Huaier granule's role in improving the hepatic microenvironment in HBV-related HCC[ 6 ]. The significant histopathological improvements in inflammation and fibrosis suggest that Huaier modifies the "soil" conducive to late recurrence, aligning with the "seed and soil" hypothesis of HCC recurrence[ 9 ]. By demonstrating a tangible reversal of fibrosis—a process often considered irreversible[ 20 ]—the findings position Huaier as a promising adjuvant therapy[ 21 ]. The prolonged recurrence interval, likely attributable to these histological improvements, underscores its potential to alter the natural history of the disease[ 22 ]. However, the retrospective nature of the study necessitates validation through larger, prospective randomized trials. Future research should also focus on elucidating the precise molecular mechanisms by which Huaier achieves these anti-fibrotic and anti-inflammatory effects to optimize its clinical application. A cornerstone of our findings is the significant dampening of the AKT signaling pathway, the principal driver of hepatic stellate cell (HSC) activation and differentiation into myofibroblasts[ 23 ]. By intervening at this critical nexus, Huaier effectively curbs the engine of fibrogenesis. However, the action of Huaier extends beyond this central axis. The AKT signaling pathway emerges as a central regulator of myofibroblast pathogenicity during liver fibrosis[ 24 ], orchestrating a spectrum of cellular responses that collectively drive disease progression. In the injured liver, persistent inflammatory and oxidative stress stimuli converge upon this pathway, leading to the phosphorylation and activation of AKT within myofibroblasts. This activation is not merely an epiphenomenon but a critical molecular switch that sustains the fibrogenic cell population. Furthermore, the pathway acts as a powerful mitogen, driving myofibroblast proliferation through its regulation of the cell cycle, thereby expanding the pool of collagen-producing cells[ 25 ]. Beyond survival and proliferation, AKT activation directly fuels the fibrogenic synthetic program, enhancing the transcription and secretion of major extracellular matrix components, particularly collagen type I, which constitutes the primary scar tissue[ 26 ]. This is often mediated through its interplay with other pro-fibrotic signaling cascades, such as TGF-β. Consequently, the hyperactive AKT pathway in myofibroblasts creates a vicious, self-reinforcing cycle of cell accumulation and ECM deposition[ 27 ]. Our experimental data, demonstrating a significant reduction in the activated P-AKT form following therapeutic intervention without a change in total AKT, underscores that targeted disruption of this pathway's activation state is sufficient to blunt its pro-fibrotic output. This positions the inhibition of AKT phosphorylation not just as an observation, but as a viable therapeutic strategy to disrupt the core engine of fibrogenesis by promoting myofibroblast quiescence, reducing their numbers, and ultimately mitigating scar tissue accumulation. The compelling anti-fibrotic efficacy of Huaier demonstrated in our study necessitates a discussion that bridges its complex pharmacology with the clinical realities of liver fibrosis. The observed downregulation of key profibrogenic pathways, particularly AKT pathway, suggests that Huaier operates not as a mere inhibitor of a single cytokine, but as a multi-target network regulator[ 28 ]. This is of paramount clinical importance. Current therapeutic strategies for liver fibrosis often falter due to the disease's multifaceted pathogenesis and redundant signaling cascades[ 29 ]. A single-target agent may be evaded by the fibrotic microenvironment. Huaier's ability to simultaneously dampen activation of hepatic stellate cells into myofibroblasts, attenuate the proliferation of myofibroblasts, and suppress inflammatory drivers positions it as a promising “pathway-buster.” This poly-pharmacological approach mirrors the complex interplay of the disease itself, offering a potential solution to the limited efficacy seen with many monotherapies. It moves the treatment paradigm from a narrow, linear blockade towards a broader, systems-level restoration of liver homeostasis. Despite the promising results, the journey of Huaier from bench to bedside requires thoughtful navigation of several translational challenges. The very nature of its multi-component composition, while a therapeutic strength, is a regulatory and scientific complexity. Future research must transcend simply confirming efficacy and delve into identifying the key active compounds and their synergistic relationships. This will be crucial for standardizing production to ensure batch-to-batch consistency and predictable clinical outcomes. Moreover, our data hint at variable responses in different preclinical models, underscoring the need for predictive biomarkers. Can we identify patient subpopulations—perhaps based on their dominant fibrogenic pathway (e.g., TGF-β-dominant[ 30 ] vs. PDGF-dominant[ 31 ])—that would derive the maximum benefit from Huaier? The ultimate goal is to evolve from a one-size-fits-all application to a precision medicine approach, where Huaier is deployed in the right patient, at the right disease stage, potentially in rational combination with other agents to create synergistic, regimen-based strategies for halting or reversing liver fibrosis. Declarations Acknowledgements Not applicable. Author information Authors and Affiliations Department of Hepatobiliary Surgery, the First Affiliated Hospital of Xi'an Jiaotong University, Xi’an, Shaanxi, China Jiachun Ding, Jiaqiang Ren, Fan Chen, Ye Lu , Yifei Ma, Zhenchao Gao, Yiqun Song, Jiahui Zeng, Jiaoxing Wu, Zhengyuan Feng, Cancan Zhou, Zheng Wang & Weikun Qian Pancreatic Disease center of Xi’an Jiaotong University Jiachun Ding, Jiaqiang Ren, Fan Chen, Ye Lu , Yifei Ma, Zhenchao Gao, Yiqun Song, Jiahui Zeng, Jiaoxing Wu, Zhengyuan Feng, Cancan Zhou, Zheng Wang & Weikun Qian Department of Cardiac Surgery, the First Affiliated Hospital of Xi’an Jiaotong University, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an, Shaanxi, China Ting Zhang, Zehua Shao, Huijing Tian & Siyu Wang Author Contributions Jiachun Ding designed and conducted the research, performed data curation and formal analysis, and wrote the original draft of the manuscript. Jiaqiang Ren, Fan Chen, Ye Lu, Huijing Tian and Siyu Wang participated in data curation and formal analysis. Yifei Ma, Ting Zhang, Zehua Shao and Zhenchao Gao contributed to the methodology. Yiqun Song, Jiahui Zeng, Jiaoxing Wu and Zhengyuan Feng performed validation experiments. Cancan Zhou supervised the study and was responsible for project administration and conceptualization. Zheng Wang and Weikun Qian reviewed, edited the manuscript, administered the project and acquired funding. Corresponding author Correspondence to Weikun Qian: [email protected] Funding This study was supported by the National Natural Science Foundation of China (No. 82372895); the Key Research and Development Program of Shaanxi (No. 2024SF2-GJHX-03), the Youth Star of Science and Technology Program of Shaanxi (No. 2025ZC-KJXX-126) and the Innovative Team Foundation of Shaanxi Health Commission (No. 2024TD-16). Data availability The dataset generated and/or analyzed during the study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Ethics approval and consent to participate All procedures complied with the NIH Guide for the Care and the animal study protocol was approved by the Ethics Committee of the Animal Ethics Committee of Xi’an Jiaotong University (protocol code 2025-3684). Consent for publication Not applicable. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. References Horn P, Tacke F. Liver Macrophage Diversity in Health and Disease. Results Probl Cell Differ. 2024;74:175–209. Kisseleva T, Brenner D. Molecular and cellular mechanisms of liver fibrosis and its regression. Nat Rev Gastroenterol Hepatol. 2021;18(3):151–66. Lazarus JV, Kopka CJ, Nicolàs A, Karim SA, Bansal MB, Betel M et al. Lessons learned from viral hepatitis testing that inform law and policy responses to steatotic liver disease. 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Supplementary Files fig1.jpg Graphical abstract Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Mar, 2026 Reviews received at journal 23 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviews received at journal 23 Mar, 2026 Reviews received at journal 22 Mar, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviews received at journal 18 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers invited by journal 17 Mar, 2026 Editor assigned by journal 17 Mar, 2026 Submission checks completed at journal 17 Mar, 2026 First submitted to journal 14 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9123161","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608301383,"identity":"676d0c50-370b-4cca-b3cf-2ba0a3b286b7","order_by":0,"name":"Jiachun Ding","email":"","orcid":"","institution":"First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Jiachun","middleName":"","lastName":"Ding","suffix":""},{"id":608301384,"identity":"59fd2bc7-aa22-4e2c-88f9-f8b128664d84","order_by":1,"name":"Jiaqiang Ren","email":"","orcid":"","institution":"First Affiliated Hospital of Xi'an 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14:08:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9123161/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9123161/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105084732,"identity":"20d8438d-ad46-4b12-8403-f514e94fdb70","added_by":"auto","created_at":"2026-03-20 19:10:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":534402,"visible":true,"origin":"","legend":"\u003cp\u003eBioactive composition and molecular structures of Huaier\u003c/p\u003e\n\u003cp\u003e(A) Morphology of harvested Huaier before processing. Proteoglycan include Polysaccharide (SP1), Water-soluble neutral polysaccharide (W-NTRP), T. robiniophila polysaccharide (TRP), Proteoglycan (TPG-1).\u003c/p\u003e\n\u003cp\u003eAdditionally, Huaier comprises the classes of bioactive small molecules, steroids and alkaloids. (B) Molecular structures of A:Uracil, B:Methyl 3,4-dihydroxybenzoate, C:3,4-dihydroxybenzoicacid, D:Cyclo (Leu-Pro), E:Cyclo(Phe-Ala), F:Cyclo (Phe-Gly), G:Methylparaben, H:P-hydroxybenzoic acid, I:P-hydroxybenzaldehyde,\u003c/p\u003e\n\u003cp\u003eJ:Ethyl 3,4-dihydroxybenzoate, K:Genistein, L:4,4'-biphenyldithiol, M:Dehydrofalcarinone, N:Diphenyldisulfide, O:Glyodin,\u003c/p\u003e\n\u003cp\u003eP:O-Acetylserine, Q:Pirbuterol, R:Semustine, S:Sulfometuron-methyl,\u003c/p\u003e\n\u003cp\u003eT:Tyrosol 4-sulfate, U:Valinopine, V:Ergosta-7,22-dien-3β-ol, W:Ergosterol, X:3β-hydroxystigmast-5,22-dien-7-one, Y:Sitogluside, Z:Adenosine.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9123161/v1/8ca484b75d26f7685981feee.jpg"},{"id":105084723,"identity":"4501a1da-6c7f-486e-95a0-eda67be13dd8","added_by":"auto","created_at":"2026-03-20 19:10:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":630871,"visible":true,"origin":"","legend":"\u003cp\u003eHuaier attenuate the degree of liver fibrosis induced by CCl4 in mice.\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of the experimental design. The C57BL/6 mice were challenged to a consecutive intraperitoneal injection of CCl4 to induce liver fibrosis. Then the mice were administered with Huaier or PBS via gavage for 5 consecutive weeks. After 6 weeks, the mice were euthanized for subsequent analysis. (B) The body weight change of control and mice treated with Huaier at 6 weeks. (C) The ratio of liver to body weight in control and mice treated with Huaier. (D) H\u0026amp;E、Sirius red staining and Masson trichrome staining showing the histology of fibrotic livers. Sirius red staining and Masson trichrome staining showing the fibrotic scar in livers. Scale bar:100μm. (E) Quantification of the Sirius red fibrosis and Masson fibrosis area. (F) Immunostaining with α-SMA antibody, a marker for myofibroblasts. Scale bar:250μm. (G) Quantification of the percentage of α-SMA positive area. (H) Hydroxyproline content in the liver of control and mice treated with Huaier. (I-K) Western blot analysis and quantification of the indicated protein levels in liver homogenates of control and mice treated with Huaier. (L) Real-time qPCR analysis of the indicated fibrotic gene mRNA levels in liver of control and mice treated with Huaier. (M) The level of AST and ALT in serum of control and mice treated with Huaier. n=6/group. Shown are mean values ± SEM. Statistical significance was determined by unpaired Student’s t test. ns, no significance;*P \u0026lt; 0.05 was considered significant; **P \u0026lt; 0.01;***P \u0026lt; 0.001. CCl4, carbon tetrachloride; ALT, alanine aminotransferase; AST, aspartate aminotransferase.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9123161/v1/28a181fc95328c7ac063fdd9.jpg"},{"id":105084724,"identity":"6551ac36-aa6c-4e28-b6b1-cd88de536d97","added_by":"auto","created_at":"2026-03-20 19:10:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":599805,"visible":true,"origin":"","legend":"\u003cp\u003eHuaier attenuate the degree of liver fibrosis induced by BDL in mice.\u003c/p\u003e\n\u003cp\u003e(A)Schematic diagram of the experimental design. The C57BL/6 mice were challenged to perform BDL to induce liver fibrosis. Then the mice were administered with Huaier or PBS via gavage for 11 consecutive days. After 2 weeks, the mice were euthanized for subsequent analysis. (B) The body weight change of control and mice treated with Huaier at 2 weeks. (C) The ratio of liver to body weight in control and mice treated with Huaier. (D) H\u0026amp;E、Sirius red staining and Masson trichrome staining showing the histology of fibrotic livers. Sirius red staining and Masson trichrome staining showing the fibrotic scar in livers. Scale bar:100μm. (E) Quantification of the Sirius red fibrosis and Masson fibrosis area. (F) Immunostaining with α-SMA antibody, a marker for myofibroblasts. Scale bar:250μm. (G) Quantification of the percentage of α-SMA positive area. (H) Hydroxyproline content in the liver of control and mice treated with Huaier. (I-K) Western blot analysis and quantification of the indicated protein levels in liver homogenates of control and mice treated with Huaier. (L) Real-time qPCR analysis of the indicated fibrotic gene mRNA levels in liver of control and mice treated with Huaier. (M) The level of AST and ALT in serum of control and mice treated with Huaier. n=6/group. Shown are mean values ± SEM. Statistical significance was determined by unpaired Student’s t test. ns, no significance;*P \u0026lt; 0.05 was considered significant; **P \u0026lt; 0.01;***P \u0026lt; 0.001. BDL, bile duct ligation; ALT, alanine aminotransferase; AST, aspartate aminotransferase.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9123161/v1/fd82725bbf21492096146dbf.jpg"},{"id":105562912,"identity":"85581cab-29f3-4c7b-bae6-2ff5ca5108b8","added_by":"auto","created_at":"2026-03-27 12:45:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":737758,"visible":true,"origin":"","legend":"\u003cp\u003eHuaier attenuate the degree of liver fibrosis by inhibiting the proliferation of myofibroblasts in vivo and in vitro.\u003c/p\u003e\n\u003cp\u003e(A-B) Ki-67 immunostaining showing the proliferating cells in the liver fibrosis induced by CCl4, while α-SMA was co-stained to mark myofibroblasts in control and mice treated with Huaier. The percentage of Ki-67 positive cells among myofibroblasts was quantified. Scale bar: 25 μm, Merge; 100 μm, Magnification. (C-D) Ki-67 immunostaining showing the proliferating cells in the liver fibrosis induced by BDL, while α-SMA was co-stained to mark myofibroblasts in control and mice treated with Huaier. The percentage of Ki-67 positive cells among myofibroblasts was quantified. Scale bar: 25 μm, Merge; 100 μm, Magnification. (E) Effect of Huaier on mobility of LX-2 was assessed by wound healing assays. Scale bar: 0.5 mm. (F-G) Quantification of the relative migration area and rate of LX-2 treated by Huaier. (H) The CCK8 cell viability assay showed the LX-2 cells viability in control group and Huaier group. (I-J) Ki-67 expression in LX-2 under Huaier treatment. Cells were costained with phalloidin. The percentage of Ki-67 positive cells among LX-2 was quantified. Scale bar: 10μm. (K-L) Western blot analysis and quantification of the Ki-67 protein levels of LX-2 under Huaier treatment. β-actin was used as the internal control. (M) Real-time qPCR analysis of the Ki-67 gene mRNA levels of LX-2 under Huaier treatment. n=6/group. Shown are mean values ± SEM. Statistical significance was determined by unpaired Student’s t test. ns, no significance;*P \u0026lt; 0.05 was considered significant; **P \u0026lt; 0.01;***P \u0026lt; 0.001. CCl4, carbon tetrachloride; BDL, bile duct ligation.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9123161/v1/87e5ffe52bfab11c428e4ab8.jpg"},{"id":105563010,"identity":"9cb689b7-0a5e-4228-8cf5-7126920a4571","added_by":"auto","created_at":"2026-03-27 12:45:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":859427,"visible":true,"origin":"","legend":"\u003cp\u003eNetwork pharmacology analysis revealing potential mechanisms by which Huaier attenuate the degree of liver fibrosis.\u003c/p\u003e\n\u003cp\u003e(A) Venn diagram showing the intersecting therapeutic targets of Huaier and liver cirrhosis. (B) Compound-target interaction network constructed based on Venn diagram, illustrating key hub targets in Huaier potentially involved in liver cirrhosis treatment. (C) The original protein-protein interaction (PPI) network constructed based on STRING, illustrating the detailed interactions of the targets against liver cirrhosis. (D-F) The whole screening process for the PPI network through a topological method using Cytoscape 3.10.3, the red color represent higher Matthews Correlation Coefficient (MCC ≥ 0.6) value the AKT1 was noted strong and top expression.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9123161/v1/2cd751acce73b186f1b38aac.jpg"},{"id":105563234,"identity":"0f93e215-788d-4765-bb9c-68029be124a0","added_by":"auto","created_at":"2026-03-27 12:46:27","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":627344,"visible":true,"origin":"","legend":"\u003cp\u003eGO, KEGG enrichment analysis and molecular docking for identifying the binding beween bioactive composition of Huaier and AKT1.\u003c/p\u003e\n\u003cp\u003e(A) Molecular functions of GO enrichment analysis. (B) KEGG pathway enrichment analysis. (C) The heatmap of molecular docking energy between 26 Huaier bioactive components and 5 core targets for the treatment of liver cirrhosis. (D) Molecular docking of AKT1 with Ergosta-7,22-dien-3β-ol、Ergosterol、3β-hydroxystigmast-5,22-dien-7-one and Sitogluside respectively.\u003c/p\u003e","description":"","filename":"fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9123161/v1/bcf88b17b98c068c09ef5af2.jpg"},{"id":105084729,"identity":"afd6c9ed-96e3-4582-b4e4-22a6a31a23e3","added_by":"auto","created_at":"2026-03-20 19:10:16","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":563234,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular dynamics simulation for identifying the binding beween bioactive composition of Huaier and AKT1.\u003c/p\u003e\n\u003cp\u003e(A-D) Root mean square deviation (RMSD), Root mean square fluctuation (RMSF), Distance between molecules and Radius of gyration (Rg) between Ergosta-7,22-dien-3β-ol、Ergosterol、3β-hydroxystigmast-5,22-dien-7-one、Sitogluside and AKT1 respectively.\u003c/p\u003e","description":"","filename":"fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9123161/v1/28d75847f8c16924665c0eaf.jpg"},{"id":105084733,"identity":"444d907d-b2c7-4bce-bb47-ac2204310701","added_by":"auto","created_at":"2026-03-20 19:10:17","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":399951,"visible":true,"origin":"","legend":"\u003cp\u003eAKT1 is highly expressed in myofibroblasts during liver fibrosis in single-cell RNA sequencing.\u003c/p\u003e\n\u003cp\u003e(A)Single cell transcriptomics analysis of published mouse dataset GSE145086. Uniform Manifold Approximation and Projection (UMAP) representation of cell types from CCl4-induced fibrotic mouse livers. (B)\u003c/p\u003e\n\u003cp\u003eUMAP representation of cell types from normal mouse liver, CCl4-induced 2 weeks and 4 weekes fibrotic mouse livers.\u003c/p\u003e\n\u003cp\u003e(C) AKT1 expression in control and fibrotic livers. In the subject plot, each color depicts a distinct subject. (D) Violin plot showed AKT1 expression levels in myofibroblast from control and fibrotic livers. (E-F) Western blot analysis and quantification of the AKT and P-AKT protein levels of LX-2 under Huaier treatment. β-actin was used as the internal control.\u003c/p\u003e","description":"","filename":"fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9123161/v1/fd7596fca82b8c572acdb413.jpg"},{"id":105084728,"identity":"59ed2703-0b63-4a1a-9dd2-3af04fa3b753","added_by":"auto","created_at":"2026-03-20 19:10:16","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":776593,"visible":true,"origin":"","legend":"\u003cp\u003eHuaier attenuate the degree of liver fibrosis by inhibiting the proliferation of myofibroblasts via AKT pathway in vivo and in vitro.\u003c/p\u003e\n\u003cp\u003e(A-D) In the mice with liver fibrosis induced by CCl4 or BDL, AKT and P-AKT in the cytoplasm of myofibroblasts were detected by immunofluorescence, while α-SMA was co-stained to mark myofibroblasts in control and mice treated with Huaier. Scale bar: 75 μm. (E-F) AKT and P-AKT in the cytoplasm of LX-2 under Huaier treatment. Scale bar: 50 μm. (G-H) Quantification of the AKT and P-AKT positive area in α-SMA positive area in control and mice treated with Huaier. (I) Quantification of the percentage of AKT and P-AKT positive area in LX-2 under Huaier treatment.\u003c/p\u003e","description":"","filename":"fig10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9123161/v1/999f43af5ff7f0e57f67627e.jpg"},{"id":105728013,"identity":"a2a822de-f583-41ae-8b46-2f326ecc75d3","added_by":"auto","created_at":"2026-03-30 11:08:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7046228,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9123161/v1/bf4ba5d4-23f0-4df1-911d-ba3b52729fb7.pdf"},{"id":105084725,"identity":"695b0745-8434-490c-9c2b-a436c7d3339d","added_by":"auto","created_at":"2026-03-20 19:10:16","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":485265,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9123161/v1/eb7a1f6480fab896773d148f.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrating Network Pharmacology and Experimental Validation to Elucidate Huaier in Attenuating Liver Fibrosis via the AKT Signaling Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLiver fibrosis, a common pathological outcome of chronic liver injury, is characterized by the excessive accumulation of extracellular matrix (ECM) proteins, which can progress to cirrhosis, liver failure and hepatocellular carcinoma, posing a significant threat to global health[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite advances in understanding its molecular mechanisms, primarily involving the activation of hepatic stellate cells (HSCs) and complex cytokine networks, effective anti-fibrotic therapies remain a major unmet medical need[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Current strategies predominantly focus on removing the underlying etiology, such as antiviral therapy for viral hepatitis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, this approach often has limited efficacy in reversing established fibrosis and for many patients, the causative agent cannot be eliminated. Consequently, the development of targeted and effective pharmacological agents to halt or reverse the fibrogenic process is an urgent priority and a significant challenge in hepatology[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHuaier (scientific name: Trametes robiniophila Murr.), a traditional medicinal fungus, has been used in China for centuries[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Its extract, the active component of the commercial preparation Huaier granule, is approved by the National Medical Products Administration (NMPA) of China as an adjuvant therapy for hepatocellular carcinoma[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Modern pharmacological studies have revealed that Huaier is rich in various bioactive compounds, including polysaccharides, proteoglycans and amino acids, endowing it with a wide spectrum of biological activities such as immunomodulation, antitumor, anti-inflammatory and antiviral effects[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Notably, in the context of liver diseases, clinical and experimental studies have demonstrated that Huaier granule can improve liver function and inhibit tumor cell proliferation, showcasing its therapeutic potential and safety profile[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEmerging evidence suggests that Huaier holds promise for the treatment of liver fibrosis[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A pioneering multicenter trial explores the potential of Huaier granule, a traditional Chinese medicine, as adjuvant therapy following curative resection of hepatocellular carcinoma (HCC). Conducted across 39 centers with 1,044 patients, the randomized phase IV study demonstrates a significant improvement in recurrence-free survival and overall survival rates in the treatment group compared to controls[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The findings provide compelling evidence that Huaier granule effectively reduces both intrahepatic and extrahepatic tumor recurrence, offering a promising postoperative strategy for HCC management. Its multifaceted actions, particularly its potent anti-inflammatory and immunoregulatory properties, position it as a compelling candidate for targeting the complex pathogenesis of fibrosis. However, the existing body of research is still in its early stages. The precise molecular targets, the key effective components responsible for its anti-fibrotic effects, and the comprehensive mechanisms of action remain largely elusive[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This lack of a systematic understanding hinders its potential application as a targeted anti-fibrotic therapy.\u003c/p\u003e \u003cp\u003eTherefore, the present study aims to systematically investigate the therapeutic effects of Huaier on liver fibrosis and to elucidate its underlying mechanisms. To achieve this, we employed an innovative integrated strategy combining network pharmacology with in vivo validation in the mouse model. Network pharmacology was used to predict the potential active compounds[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], key targets and signaling pathways involved in Huaier's anti-fibrotic action. These predictions were subsequently experimentally validated in a CCl4 and BDL induced mouse model of liver fibrosis through histopathological and molecular biological techniques. This research not only provides a solid scientific basis for the clinical application of Huaier in anti-fibrosis therapy but also offers a novel paradigm for exploring the mechanisms of traditional medicines using a multi-disciplinary approach.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice and liver fibrosis model\u003c/h2\u003e \u003cp\u003eEight-week-old male and female C57BL/6 wild-type mice were obtained from Xi'an Jiaotong University and housed under conventional pathogen-free conditions, with a 12-hour light/dark cycle, ambient temperature maintained at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and relative humidity at 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%. Food and water were provided ad libitum. To establish chronic liver fibrosis, mice received intraperitoneal injections of carbon tetrachloride (CCl4) at 0.6 mL/kg body weight three times per week for six weeks. Forty-eight hours after the final injection, at 14 weeks of age, mice were humanely euthanized via carbon dioxide inhalation in a euthanasia chamber. Blood and liver tissues were subsequently collected for analysis. In a parallel set of experiments, liver fibrosis was induced via bile duct ligation (BDL). Mice were fasted for 12 hours prior to surgery and sacrificed two weeks post-operatively for tissue and serum collection. All mice were randomly divided into the following two groups (the control group (saline 100 \u0026micro;L/day, gavage) and Huaier group[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] (2 g/kg/day, gavage). Huaier extract was provided by Gaitianli Medicine Co., Ltd. (Qidong, Jiangsu, China). All experimental procedures were conducted in accordance with the National Institutes of Health \"Guide for the Care and Use of Laboratory Animals\" and were approved by the Animal Experiment Administration Committee of Xi\u0026rsquo;an Jiaotong University to ensure ethical and humane treatment of animals.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistology\u003c/h3\u003e\n\u003cp\u003eHarvested liver tissues were gently perfused and fixed in fresh 4% neutral-buffered formalin for 24 hours at 4\u0026deg;C, followed by washing in 1\u0026times; phosphate-buffered saline (PBS). For histological analysis, samples were dehydrated, cleared and embedded in paraffin. Serial sections of 5 \u0026micro;m thickness were prepared, mounted on glass slides, and stained with Hematoxylin and Eosin (H\u0026amp;E; Cat# ST047, Solarbio), Masson\u0026rsquo;s Trichrome (Cat# abs9348, Absin) and Sirius Red (Cat# DC0041, Leagene Biotech). Bright-field imaging was performed using an Olympus microscope. Digitized images were analyzed with Image-Pro Plus 6.0 software (Media Cybernetics) to evaluate histological architecture and quantify fibrosis.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eFor cryosectioning, liver tissues were embedded in Optimal Cutting Temperature (OCT) compound (Cat# 4583, Tissue-Tek) and 5 \u0026micro;m-thick sections were prepared using a cryostat maintained at -20\u0026deg;C. The resulting sections were mounted on adhesive-coated glass slides. Subsequently, the sections were blocked and permeabilized for 1 hour at room temperature with 5% bovine serum albumin (BSA; Cat# V900933, Sigma-Aldrich) and 0.3% Triton X-100 (Cat# T8200, Solarbio).\u003c/p\u003e \u003cp\u003ePrimary antibodies were applied overnight at the following dilutions: α-SMA-Cy3 (1:100, Cat# C6198, Sigma-Aldrich), Ki-67 (1:100, Cat# ab16667, Abcam), AKT (1:100, Cat# ab8805, Abcam), P-AKT (1:100, Cat# ab192623, Abcam) and Phalloidin-594 (1:100, Cat# 8953S, CST). The following day, slides were incubated for 1 hour at room temperature with appropriate Alexa Fluor-conjugated secondary antibodies (Cat# A11008 and A11012, Invitrogen). Nuclei were visualized by counterstaining with DAPI (1 \u0026micro;g/mL, Thermo Fisher Scientific) for 5 minutes. After three washes with PBS, the sections were coverslipped using an antifade mounting medium (Vector Laboratories). Images were captured with a laser-scanning confocal microscope (Olympus\u003c/p\u003e \u003cp\u003eFV3000, Core Facilities Sharing Platform at School of Basic Medical Sciences).\u003c/p\u003e\n\u003ch3\u003eAssay for hydroxyproline in liver tissue\u003c/h3\u003e\n\u003cp\u003eWe use hydroxyproline content assay kit(Cat# BC0250, Solarbio) to measure hydroxyproline content in mouse liver, begin by homogenizing approximately 0.2 g of liver tissue and hydrolyzing it with 2 ml of 6 M HCl at 110\u0026deg;C for 4 hours. After cooling, neutralize the hydrolysate to pH 6\u0026ndash;8 using NaOH, then dilute to 4 ml with distilled water. Centrifuge the mixture at 16,000 rpm for 20 minutes and collect the supernatant. Using a multifunction microplate reader (POLARstar OPTIMA; BMG, Offenburg, Germany) set at 560 nm, mix 200 \u0026micro;l of the supernatant with 200 \u0026micro;l of Reagent 1, incubate for 20 minutes, add 200 \u0026micro;L of Reagent 2, and heat at 60\u0026deg;C for another 20 minutes. After cooling, measure the absorbance. A standard curve constructed with hydroxyproline standards enables precise quantification, reflecting collagen metabolism in the liver with accuracy and elegance.\u003c/p\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\u003cp\u003eIsolated livers or myofibroblasts were lysed using RIPA buffer (Cat# 9806, Cell Signaling) supplemented with protease and phosphatase inhibitors (Cat# 04906837001, Roche). Following lysis, the homogenates were centrifuged at 11,752 \u0026times; g for 10 minutes at 4\u0026deg;C. The resulting supernatants were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% skim milk for 1 hour, the membranes were incubated with the following primary antibodies: Collagen I (1:1000, Cat# ab279711, Abcam), α-SMA (1:1000, Cat# AF1032, Affinity), Ki-67 (1:1000, Cat# ab16667, Abcam), AKT (1:100, Cat# ab8805, Abcam), P-AKT (1:100, Cat# ab192623, Abcam), β-Actin (1:1000, Cat# ab6276, Abcam) and α-Tubulin (1:1000, Cat# ab81299, Abcam) were used as loading controls. Protein bands were visualized using the ECL detection system (BioRad).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT-PCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from myofibroblasts or liver tissues using Trizol reagent (Cat# 15596018, Invitrogen). cDNA was synthesized from 1 \u0026micro;g of total RNA using a commercial cDNA synthesis kit (Cat# 170\u0026ndash;8891, BioRad), following the manufacturer's protocol. Quantitative real-time PCR (qRT-PCR) was performed on a StepOne Plus System (Applied Biosystems) with universal SYBR Green mix (Cat# 172\u0026ndash;5122, BioRad). The relative expression of target genes was calculated using the 2\u003csup\u003e\u0026ndash;ΔΔCT\u003c/sup\u003e method, with GAPDH serving as the internal reference for normalization. Quantitative PCR primer sequences were as follows: Acta2: 5\u0026rsquo;-gaggcaccactgaaccctaa-3\u0026rsquo;/5\u0026rsquo;-tacatggcggggacattgaa-3\u0026rsquo;; Col1a1, 5\u0026rsquo;-ttcagggaatgcctggtgaa-3\u0026rsquo;/5\u0026rsquo;-acctttgggaccagcatca-3\u0026rsquo;; Col1a2, 5\u0026rsquo;- aagggtgctactggactccc-3\u0026rsquo;/5\u0026rsquo;-ttgttaccggattctcctttgg-3\u0026rsquo;; Col2a1, 5\u0026rsquo;- agcaggtccttggaaacctt-3\u0026rsquo;/5\u0026rsquo;-aaggagtttcatctggccct-3\u0026rsquo;; Ki-67, 5\u0026rsquo;-atttgcttctggccttcccc-3\u0026rsquo;/5\u0026rsquo;-ccaaacaagcaggtgctgag-3\u0026rsquo;; GAPDH, 5\u0026rsquo;- gtaacccgttgaaccccatt-3\u0026rsquo;/5\u0026rsquo;-ccatccaatcggtagtagcg-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSerum ALT and AST Assays\u003c/h3\u003e\n\u003cp\u003e Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice were determined using commercial assay kits (ALT:Cat# C009-2-1, Nanjing Jiancheng Bioengineering Institute; AST:Cat# C010-2-1, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions. Serum aliquots were directly subjected to analysis without further dilution. For the assay, samples were incubated with the respective substrate solutions at 37\u0026deg;C for 30 minutes. The enzymatic reactions were then terminated by adding 2,4-dinitrophenylhydrazine. Following a 20-minute incubation at 37\u0026deg;C, the resulting solutions were alkalized and the colored products were allowed to develop at room temperature for 15 minutes. The absorbance was measured at 505 nm for ALT and 510 nm for AST using a multifunction microplate reader (POLARstar OPTIMA; BMG, Offenburg, Germany). The enzyme activities, expressed in Karmen units, were calculated based on standard curves and subsequently converted to international units per liter (U/L).\u003c/p\u003e\n\u003ch3\u003eCell culture and treatment\u003c/h3\u003e\n\u003cp\u003eThe human hepatic stellate cell line LX-2 (Cat# CL-0560, Procell, Inc.) was cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37\u0026deg;C under a 5% CO₂ atmosphere. Prior to experimental treatment, cells were serum-starved overnight in serum-free DMEM. Differentiation into myofibroblasts was induced by stimulation with 5 ng/mL TGF-β1 (Cat# HY-P70648, MCE) for 72 hours. Subsequently, the differentiated myofibroblasts were treated with or without Huaier (8 mg/mL) for 48 hours.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eMyofibroblasts were seeded in 96-well plates at a density of 3,000 cells per well and allowed to adhere overnight. Following this, the cells were treated with or without Huaier (8 mg/mL) for 48 hours. After treatment, the culture medium was carefully removed, and each well was supplemented with 100 \u0026micro;L of DMEM and 10 \u0026micro;L of Cell Counting Kit-8 (CCK-8) reagent. The plates were then incubated for an additional 3 hours at 37\u0026deg;C under 5% CO₂. Absorbance was measured at 450 nm using a multifunctional microplate reader (POLARstar OPTIMA, BMG, Offenburg, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell migration assays\u003c/h2\u003e \u003cp\u003eMyofibroblasts were seeded in 6-well plates pre-marked with grid lines on the underside. After the cells reached 100% confluence, a straight wound was created in each well using a 200 \u0026micro;l pipette tip. The cells were then treated with or without Huaier (8 mg/mL). Wound closure was monitored at 12 and 24 hours post-treatment by imaging the same predefined locations under an inverted microscope. The extent of wound healing was quantitatively analyzed using Image Pro Plus 6.0 software (Media Cybernetics, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNetwork pharmacology\u003c/h2\u003e \u003cp\u003eTwenty-six bioactive components of Huaier were identified through a combination of database mining using HERB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://herb.ac.cn/\u003c/span\u003e\u003cspan address=\"http://herb.ac.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and a review of previously published literature. Based on the Swiss Target Prediction platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), potential target proteins of the active ingredients from Huaier were predicted. The search parameter was set to \u0026ldquo;Homo sapiens\u0026rdquo; and targets were selected under the criterion of \u0026ldquo;probability\u0026thinsp;\u0026gt;\u0026thinsp;0\u0026rdquo;. Potential targets associated with liver cirrhosis were retrieved from multiple public databases, including OMIM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/\u003c/span\u003e\u003cspan address=\"https://omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), DrugBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://go.drugbank.com/\u003c/span\u003e\u003cspan address=\"https://go.drugbank.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), TTD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://db.idrblab.net/ttd/\u003c/span\u003e\u003cspan address=\"http://db.idrblab.net/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), PharmGkb (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pharmgkb.org/\u003c/span\u003e\u003cspan address=\"https://www.pharmgkb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and GeneCards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), using the keyword \"liver cirrhosis\". Common targets between Huaier and liver cirrhosis were identified using the Venny 2.1.0 platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfogp.cnb.csic.es/tools/venny/index.html\u003c/span\u003e\u003cspan address=\"https://bioinfogp.cnb.csic.es/tools/venny/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and their overlap was visualized in a Venn diagram. Based on Venn diagram, compound-target interaction network was constructed using Cytoscape software (v3.10.3). A protein-protein interaction (PPI) network was subsequently constructed with the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and visualized using Cytoscape software (v3.10.3). Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, were performed for the key target clusters via the Metascape database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org\u003c/span\u003e\u003cspan address=\"https://metascape.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The results of these enrichment analyses were visualized using an online bioinformatics platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eThe three-dimensional structures of bioactive compounds derived from Huaier were retrieved from the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The crystal structure of the target protein was obtained from the Protein Data Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Molecular docking simulations were performed using AutoDock software (version 4.2.6) and corresponding binding energies were calculated. For each compound\u0026ndash;target pair, five independent docking runs were conducted using different random seeds to ensure reproducibility. The lowest-energy conformation from each run was selected, and binding free energies (ΔG, kcal/mol) are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation derived from the five replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dynamics simulation\u003c/h2\u003e \u003cp\u003eMolecular dynamics simulations were performed using GROMACS 2020.6 to model the ligand\u0026ndash;protein interaction over a 100 ns period. The topology of the small molecule ligand was generated with Sobtop 1.0 using the GAFF force field, while the protein topology was constructed with the AMBER99SB-ILDN force field. To neutralize the system, sodium and chloride ions were introduced and the complex was solvated in a truncated octahedral box of TIP3P water molecules with a 1.0 nm buffer distance. Energy minimization was carried out in two stages: first with the steepest descent algorithm for 2500 steps, followed by the conjugate gradient method for another 2500 steps. Subsequently, the system was equilibrated under an NVT ensemble for 100 ps at 298.15 K and then under an NPT ensemble for another 100 ps at the same temperature. Using the PME approach, long-range electrostatic interactions were estimated in an NPT ensemble simulation at 100 nm with the periodic boundary condition. A collision frequency of 2 ps and a non-bond cut-off distance of 1 nm were determined. Trajectories were recorded at 10-ps intervals, the integration step was set to 2 s and the system pressure was kept at 101.325 kPa.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSingle-cell RNA-seq dataset processing\u003c/h2\u003e \u003cp\u003ePublicly available single-cell RNA sequencing (scRNA-seq) datasets were downloaded from the Gene Expression Omnibus (GEO) database under accession numbers GSE145086[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Data processing and analysis were performed using the Seurat R package (v4.3.0.1). A Seurat object was created from the expression matrix and accompanying metadata using the Create Seurat Object function. Subsequent dimensionality reduction and visualization were carried out via uniform manifold approximation and projection (UMAP) using the Run UMAP function with 15 principal components (dims\u0026thinsp;=\u0026thinsp;1:15). Violin plots were generated with ggplot2 (v3.5.1) and feature expression overlays on UMAP projections were rendered using Seurat's visualization utilities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll statistical analyses were conducted using GraphPad Prism 9. Sample sizes were determined based on preliminary or prior experiments to ensure sufficient statistical power. Samples with inadequate RNA/cDNA quality or suboptimal tissue integrity after processing (below commonly accepted thresholds) were excluded from analysis. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Differences between two groups were assessed using an unpaired, two-tailed Student\u0026rsquo;s t-test and a P-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBioactive composition and molecular structures of Huaier\u003c/h2\u003e \u003cp\u003eBased on previously published literature and the HERB database[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], a total of 30 bioactive substances have been identified in processed Huaier (Trametes robiniophila). These active components are broadly classified into four major categories: proteoglycans, bioactive small molecules, steroids and alkaloids.\u003c/p\u003e \u003cp\u003eThe proteoglycans include Polysaccharide (SP1), water-soluble neutral polysaccharide (W-NTRP), T. robiniophila polysaccharide (TRP) and Proteoglycan (TPG-1). According to existing research, the bioactive small molecules comprise uracil, methyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, cyclo(Leu-Pro), cyclo(Phe-Ala), cyclo(Phe-Gly), methylparaben, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, ethyl 3,4-dihydroxybenzoate, genistein, 4,4'-biphenyldithiol, dehydrofalcarinone, diphenyldisulfide, glyodin, O-acetylserine, pirbuterol, semustine, sulfometuron-methyl, tyrosol 4-sulfate and valinopine.\u003c/p\u003e \u003cp\u003eIn addition, the steroid constituents include ergosta-7,22-dien-3β-ol, ergosterol, 3β-hydroxystigmast-5,22-dien-7-one and sitogluside. And the alkaloids identified consist of adenosine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The chemical structures of the bioactive molecules identified in Huaier were obtained from the PubChem database (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHuaier attenuate the degree of liver fibrosis induced by CCl4 in mice\u003c/h2\u003e \u003cp\u003eEight-week-old C57 mice were subjected to intraperitoneal injections of CCl4 at 0.6 mL/kg body weight three times per week for six weeks. Beginning from the second week, control mice received daily oral gavage of normal saline (100 \u0026micro;L/day), while the treatment group was administered Huaier (2 g/kg/day) by oral gavage for five weeks. Forty-eight hours after the final CCl4 injection, 14-week-old mice were euthanized via carbon dioxide inhalation and blood and liver samples were collected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Body weight monitoring during the modeling period revealed that Huaier-treated mice experienced less weight loss compared to the control group, with a statistically significant difference observed by the sixth week (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The liver-to-body weight ratio was significantly lower in the Huaier group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Histological analysis showed reduced inflammatory cell infiltration in H\u0026amp;E-stained sections, along with markedly diminished fibrotic scarring in Sirius Red and Masson staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), which was quantitatively confirmed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), indicating a significant anti-fibrotic effect of Huaier. Immunofluorescence staining for α-SMA revealed a significant reduction in myofibroblast in the treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), as supported by quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Further analysis showed a significant decrease in hepatic hydroxyproline content in Huaier-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Western blot results indicated downregulated protein expression of collagen I and α-SMA in Huaier-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-K) and qPCR showed reduced mRNA levels of fibrogenic genes such as Acta2, Col1a1, Col1a2 and Col2a1 in Huaier-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). Finally, the serum ALT and AST levels were significantly lower in the Huaier group, consistent with improved liver function (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM). Collectively, these findings substantiate the protective role of Huaier against CCl4-induced liver fibrosis in mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eHuaier attenuate the degree of liver fibrosis induced by BDL in mice\u003c/h2\u003e \u003cp\u003eEight-week-old C57 mice underwent bile duct ligation (BDL). Starting on the third day after surgery, control mice received daily oral gavage of normal saline (100 \u0026micro;L/day), while the treatment group was administered Huaier (2 g/kg/day) for 12 days. Liver and blood samples were collected two weeks post-modeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Body weight changes throughout the experimental period revealed that Huaier-treated mice exhibited less pronounced weight loss compared to controls, with a statistically significant difference between the two groups by the second week (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The liver-to-body weight ratio was significantly lower in the Huaier-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Histological evaluation demonstrated a marked reduction in inflammatory cell infiltration in H\u0026amp;E-stained liver sections from the treatment group. Additionally, Sirius Red and Masson staining showed substantially attenuated fibrotic scarring, which was quantitatively confirmed, indicating that Huaier significantly alleviates BDL-induced liver fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). Immunofluorescence staining for α-SMA revealed a significant decrease in the area positive for myofibroblasts in Huaier-treated mice, as quantified, suggesting suppression of myofibroblast activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G). Further biochemical analysis indicated that hepatic hydroxyproline content was significantly reduced in the treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Western blot analysis showed downregulated protein expression of collagen I and α-SMA in the livers of Huaier-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-K). Consistent with these findings, there was a reduction in the mRNA expression of fibrosis-related genes, including Acta2, Col1a1, Col1a2 and Col2a1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Finally, serum levels of ALT and AST were both significantly lower in the Huaier group, reflecting improved liver function (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). Together, these results substantiate the anti-fibrotic efficacy of Huaier in a BDL-induced liver fibrosis model.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHuaier attenuate the degree of liver fibrosis by inhibiting the proliferation of myofibroblasts in vivo and in vitro\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe employed immunofluorescence to assess the proliferation of myofibroblasts in liver tissues from mice with CCl4-induced liver fibrosis. Myofibroblasts were labeled with α-SMA and proliferating cells were identified using Ki-67. Results showed a marked reduction in the number of proliferating myofibroblasts in the Huaier-treated group compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), which was statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Similarly, in the BDL-induced liver fibrosis model, Huaier treatment also suppressed the proliferation of myofibroblasts in fibrotic livers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). In vitro, using an LX-2-derived myofibroblast model, a wound healing assay was performed to evaluate the effect of Huaier on cell migration. We observed that Huaier significantly inhibited the migratory capacity of myofibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G). Furthermore, a CCK-8 assay revealed that Huaier suppressed the proliferative activity of LX-2 myofibroblasts in culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Additional immunofluorescence staining for Ki-67 confirmed that Huaier treatment led to a notable decrease in the proportion of proliferating myofibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-J). At the molecular level, Western blot analysis demonstrated that Huaier downregulated Ki-67 protein expression in myofibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK-L). Consistent with this, qPCR analysis indicated a reduction in Ki-67 mRNA levels following Huaier treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM). Collectively, these findings illustrate that Huaier effectively curbs both the migration and proliferation of myofibroblasts in vitro, supporting its anti-fibrotic role.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eNetwork pharmacology analysis revealing potential mechanisms by which Huaier attenuate the degree of liver fibrosis\u003c/h2\u003e \u003cp\u003eTwenty-six bioactive components of Huaier were identified through an integrated approach involving database mining via HERB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://herb.ac.cn/\u003c/span\u003e\u003cspan address=\"http://herb.ac.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and a comprehensive review of previously published literature. Using the Swiss Target Prediction platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), potential target proteins of these twenty-six bioactive components from Huaier were systematically predicted. A total of 604 protein targets associated with the bioactive components of Huaier were identified. Given the absence of a specific disease category for liver fibrosis in the database, we selected liver cirrhosis-clinically characterized by features of liver fibrosis-as a proxy to identify relevant disease targets. Concurrently, 10,872 therapeutic targets for liver cirrhosis were retrieved from multiple databases, including OMIM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/\u003c/span\u003e\u003cspan address=\"https://omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), DrugBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://go.drugbank.com/\u003c/span\u003e\u003cspan address=\"https://go.drugbank.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), TTD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://db.idrblab.net/ttd/\u003c/span\u003e\u003cspan address=\"http://db.idrblab.net/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), PharmGkb (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pharmgkb.org/\u003c/span\u003e\u003cspan address=\"https://www.pharmgkb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and GeneCards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Among these, 486 overlapping targets were found between the protein targets of Huaier\u0026rsquo;s bioactive components and the therapeutic targets for liver cirrhosis. A Venn diagram was constructed to illustrate these intersecting therapeutic targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Based on the intersection results, a compound-target interaction network was established, delineating the relationships between each of the 26 active components of Huaier and the corresponding therapeutic targets for liver cirrhosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFor the 486 liver cirrhosis targets potentially modulated by the active constituents of Huaier, a protein-protein interaction (PPI) network was subsequently constructed using the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and visualized with Cytoscape software (v3.10.3). The PPI network was screened via a topological analysis method, with nodes colored in red indicating higher Matthews Correlation Coefficient (MCC) values (MCC\u0026thinsp;\u0026ge;\u0026thinsp;0.6)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Among these, AKT1 exhibited the strongest and most prominent expression, followed by GAPDH, IL6, TNF and IL1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;F).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGO、KEGG enrichment analysis and molecular docking for identifying the binding beween bioactive composition of Huaier and AKT1\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe analysis of GO enrichment for 486 target was conduct with a significance threshold established at P\u0026thinsp;\u0026lt;\u0026thinsp;0.01. The GO analysis revealed that the potential therapeutic targets of Huaier are involved in 19 biological processes, including Protein kinase activity, Oxidoreductase activity, Kinase binding, Transcription factor binding, Serine hydrolase activity, Protein homodimerization activity, Protein domain specific binding, Hydrolase activity (acting on ester bonds), Histone modifying activity, Protein tyrosine kinase activity, Hormone binding, Nuclear receptor activity, G protein-coupled peptide receptor activity, Protease binding, Exopeptidase activity, G protein\u0026ndash;coupled amine receptor activity, Deacetylase activity, Prostanoid receptor activity and Insulin-like growth factor II binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The experiment conducted Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis (KEGG) on the signaling pathways that may be involved in the alleviation of liver cirrhosis by Huaier. The results showed the top 13 signaling pathways, among which the top 3 enriched pathways for the target genes are Neuroactive ligand-receptor interaction, Lipid and atherosclerosis, Hormone signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The three-dimensional structures of 26 bioactive compounds derived from Huaier were retrieved from the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The crystal structure of the AKT1, GAPDH, IL6, TNF and IL1β was obtained from the Protein Data Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Molecular docking analyses of the 26 bioactive compounds with the five core protein targets were performed using AutoDock software and the corresponding binding energies were systematically computed. To ensure reproducibility, each compound-target interaction was evaluated through five independent docking runs employing distinct random seed values. From each run, the lowest-energy conformation was selected and the binding free energies (ΔG, kcal/mol) are reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation based on the five technical replicates. Based on the binding energies calculated from molecular docking between 26 bioactive compounds from Huaier and the target proteins AKT1, GAPDH, TNF, IL1B and IL6, we generated a heatmap to visualize the interaction profiles between Huaier-derived active components and therapeutic targets for liver cirrhosis. The analysis revealed that AKT1 exhibited stronger binding affinities with the 26 compounds compared to the other target proteins. Notably, the highest binding energies were observed between AKT1 and Ergosta-7,22-dien-3β-ol, Ergosterol, 3β-hydroxystigmast-5,22-dien-7-one and Sitogluside, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The molecular docking visualizations revealed distinct binding modes between AKT1 and several key bioactive compounds-namely, Ergosta-7,22-dien-3β-ol, Ergosterol, 3β-hydroxystigmast-5,22-dien-7-one and Sitogluside (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-G). Collectively, these findings suggest that Huaier may elicit its anti-fibrotic effects in the liver via multi-target and multi-pathway mechanisms, in which the AKT1 signaling pathway appears to play a central role.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMolecular dynamics simulation for identifying the binding beween bioactive composition of Huaier and AKT1\u003c/h2\u003e \u003cp\u003eTo assess the stability of the binding interactions between AKT1 and several key bioactive compounds-namely, Ergosta-7,22-dien-3β-ol, Ergosterol, 3β-hydroxystigmast-5,22-dien-7-one and Sitogluside, molecular dynamics (MD) simulations were performed on the corresponding docked complexes using GROMACS software. The Root Mean Square Deviation (RMSD) was employed as a key metric to evaluate the conformational stability and reliability of each complex. An RMSD fluctuation within 0.2 nm is generally indicative of a structurally stable system[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The RMSD values of the ligands across all systems remained consistently around or below 0.2 nm throughout the simulation trajectory, confirming the overall structural integrity of the complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D).\u003c/p\u003e \u003cp\u003eFurthermore, the Root Mean Square Fluctuation (RMSF) was utilized to characterize atomic-level flexibility, providing insight into the local mobility of residues within the protein structure[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The RMSF profiles reveal that the peptide residues in all complexes: Ergosta-7,22-dien-3β-ol-AKT1, Ergosterol-AKT1, 3β-hydroxystigmast-5,22-dien-7-one-AKT1 and Sitogluside-AKT1, exhibited fluctuations largely confined within approximately 0.2 nm during the entire simulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). These observations suggest that AKT1 maintains considerable structural rigidity upon binding to these bioactive constituents of Huaier, with no marked conformational rearrangements, thereby underscoring the stability of the interactions.\u003c/p\u003e \u003cp\u003eDistance between molecules is a crucial observable for characterizing binding affinity and interaction stability. Tracking its fluctuations helps identify stable bound states, transient encounters and dissociation events throughout a simulation. The intermolecular distance profiles demonstrate that throughout the simulation, the binding interfaces in all complexes: Ergosta-7,22-dien-3β-ol-AKT1, Ergosterol-AKT1, 3β-hydroxystigmast-5,22-dien-7-one-AKT1 and Sitogluside-AKT1, exhibited fluctuations predominantly constrained within approximately 1.0 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). As a fundamental metric in molecular simulations, the distance between molecules provides critical insights into interaction stability and the dynamic behavior of complexes. The observed narrow fluctuation range indicates the sustained proximity essential for molecular recognition and effective binding. These results suggest that AKT1, upon binding with these bioactive constituents of Huaier, engages in well-defined molecular interactions characterized by persistent association, with only minor transient variations, reflecting a stable and specific binding mode.\u003c/p\u003e \u003cp\u003eThe radius of gyration (Rg) was employed to assess the structural compactness of the protein, where a lower value corresponds to a more tightly folded conformation[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Within the 100 ns simulation period, the Rg values of all complexes: Ergosta-7,22-dien-3β-ol-AKT1, Ergosterol-AKT1, 3β-hydroxystigmast-5,22-dien-7-one-AKT1 and Sitogluside-AKT1, rapidly converged to an equilibrium state and stabilized within the range of 2.15\u0026ndash;2.20 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). These results indicate that AKT1 is capable of forming compact and stable complexes with the bioactive constituents of Huaier. Such structural consolidation is conducive to the establishment of stable interactions between the active compounds and AKT1, which may underly the pharmacological role of Huaier in mitigating liver fibrosis by modulating myofibroblast activity.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAKT1 is highly expressed in myofibroblasts during liver fibrosis in single-cell RNA sequencing\u003c/h2\u003e \u003cp\u003eBy interrogating public repositories for single-cell RNA sequencing data and identifying the dataset GSE145086[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which comprises liver samples from normal mouse liver and a mouse model of CCl4-induced liver fibrosis. Subsequent bioinformatic analysis was performed using the Seurat R package, followed by the generation of Uniform Manifold Approximation and Projection (UMAP) visualizations. These plots elucidated the cell clusters across normal and fibrotic liver tissues, revealing distinct cell types including hepatocytes, myofibroblasts, endothelial cells, etc (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B). Further investigation into the expression level of AKT1 across these cell clusters demonstrated notably elevated expression of AKT1 in myofibroblasts. This finding suggests a potential biological role for AKT1 within myofibroblasts during the progression of liver fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-D). As is well established, the biological activity of AKT is exerted upon its phosphorylation at key amino acid residues (Thr308 and Ser473)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], which converts it into its active form, P-AKT. To investigate this, we measured the protein levels of both AKT and P-AKT in LX-2 cells following Huaier treatment. Our results revealed that while the total protein level of AKT remained largely unchanged, the level of P-AKT was significantly reduced. This indicates that Huaier likely attenuates the biological functions of AKT in liver fibrosis by specifically inhibiting its phosphorylation in myofibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHuaier attenuate the degree of liver fibrosis by inhibiting the proliferation of myofibroblasts via AKT pathway in vivo and in vitro\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe protein expression of AKT and P-AKT in a mouse model of liver fibrosis was further investigated, using α-SMA as a marker for myofibroblasts. In mice with CCl4-induced liver fibrosis, treatment with Huaier did not significantly alter the expression level of AKT in myofibroblasts, but led to a statistically significant reduction in P-AKT expression (Figure. 9A-B, G). These findings suggest that active components in Huaier indeed suppress the activation of the AKT signaling pathway in myofibroblasts during liver fibrosis. Consistently, we observed the same trend with statistical significance in a BDL-induced liver fibrosis model (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC-D, H). We employed immunofluorescence staining to assess the expression of AKT and P-AKT in cultured LX-2 cells. Consistent with our prior findings, Huaier treatment did not alter the overall protein level of AKT. However, a marked reduction in P-AKT levels was readily detectable, indicating a specific inhibition of AKT phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE-F, I).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study elucidates the potent therapeutic effects of Huaier extract on liver injury and fibrosis, positioning this traditional remedy as a promising multi-targeted agent for chronic liver diseases. Our findings demonstrate that Huaier administration significantly attenuates the pathological progression of liver fibrosis by suppressing a single inflammatory pathway. This aligns with the holistic philosophy of its traditional use and provides a compelling molecular basis for its efficacy[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA previous clinical study provides valuable clinical evidence supporting Huaier granule's role in improving the hepatic microenvironment in HBV-related HCC[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The significant histopathological improvements in inflammation and fibrosis suggest that Huaier modifies the \"soil\" conducive to late recurrence, aligning with the \"seed and soil\" hypothesis of HCC recurrence[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. By demonstrating a tangible reversal of fibrosis\u0026mdash;a process often considered irreversible[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u0026mdash;the findings position Huaier as a promising adjuvant therapy[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The prolonged recurrence interval, likely attributable to these histological improvements, underscores its potential to alter the natural history of the disease[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the retrospective nature of the study necessitates validation through larger, prospective randomized trials. Future research should also focus on elucidating the precise molecular mechanisms by which Huaier achieves these anti-fibrotic and anti-inflammatory effects to optimize its clinical application.\u003c/p\u003e \u003cp\u003eA cornerstone of our findings is the significant dampening of the AKT signaling pathway, the principal driver of hepatic stellate cell (HSC) activation and differentiation into myofibroblasts[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. By intervening at this critical nexus, Huaier effectively curbs the engine of fibrogenesis. However, the action of Huaier extends beyond this central axis.\u003c/p\u003e \u003cp\u003eThe AKT signaling pathway emerges as a central regulator of myofibroblast pathogenicity during liver fibrosis[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], orchestrating a spectrum of cellular responses that collectively drive disease progression. In the injured liver, persistent inflammatory and oxidative stress stimuli converge upon this pathway, leading to the phosphorylation and activation of AKT within myofibroblasts. This activation is not merely an epiphenomenon but a critical molecular switch that sustains the fibrogenic cell population. Furthermore, the pathway acts as a powerful mitogen, driving myofibroblast proliferation through its regulation of the cell cycle, thereby expanding the pool of collagen-producing cells[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Beyond survival and proliferation, AKT activation directly fuels the fibrogenic synthetic program, enhancing the transcription and secretion of major extracellular matrix components, particularly collagen type I, which constitutes the primary scar tissue[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This is often mediated through its interplay with other pro-fibrotic signaling cascades, such as TGF-β. Consequently, the hyperactive AKT pathway in myofibroblasts creates a vicious, self-reinforcing cycle of cell accumulation and ECM deposition[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our experimental data, demonstrating a significant reduction in the activated P-AKT form following therapeutic intervention without a change in total AKT, underscores that targeted disruption of this pathway's activation state is sufficient to blunt its pro-fibrotic output. This positions the inhibition of AKT phosphorylation not just as an observation, but as a viable therapeutic strategy to disrupt the core engine of fibrogenesis by promoting myofibroblast quiescence, reducing their numbers, and ultimately mitigating scar tissue accumulation.\u003c/p\u003e \u003cp\u003eThe compelling anti-fibrotic efficacy of Huaier demonstrated in our study necessitates a discussion that bridges its complex pharmacology with the clinical realities of liver fibrosis. The observed downregulation of key profibrogenic pathways, particularly AKT pathway, suggests that Huaier operates not as a mere inhibitor of a single cytokine, but as a multi-target network regulator[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This is of paramount clinical importance. Current therapeutic strategies for liver fibrosis often falter due to the disease's multifaceted pathogenesis and redundant signaling cascades[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A single-target agent may be evaded by the fibrotic microenvironment. Huaier's ability to simultaneously dampen activation of hepatic stellate cells into myofibroblasts, attenuate the proliferation of myofibroblasts, and suppress inflammatory drivers positions it as a promising \u0026ldquo;pathway-buster.\u0026rdquo; This poly-pharmacological approach mirrors the complex interplay of the disease itself, offering a potential solution to the limited efficacy seen with many monotherapies. It moves the treatment paradigm from a narrow, linear blockade towards a broader, systems-level restoration of liver homeostasis.\u003c/p\u003e \u003cp\u003eDespite the promising results, the journey of Huaier from bench to bedside requires thoughtful navigation of several translational challenges. The very nature of its multi-component composition, while a therapeutic strength, is a regulatory and scientific complexity. Future research must transcend simply confirming efficacy and delve into identifying the key active compounds and their synergistic relationships. This will be crucial for standardizing production to ensure batch-to-batch consistency and predictable clinical outcomes. Moreover, our data hint at variable responses in different preclinical models, underscoring the need for predictive biomarkers. Can we identify patient subpopulations\u0026mdash;perhaps based on their dominant fibrogenic pathway (e.g., TGF-β-dominant[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] vs. PDGF-dominant[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e])\u0026mdash;that would derive the maximum benefit from Huaier? The ultimate goal is to evolve from a one-size-fits-all application to a precision medicine approach, where Huaier is deployed in the right patient, at the right disease stage, potentially in rational combination with other agents to create synergistic, regimen-based strategies for halting or reversing liver fibrosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Hepatobiliary Surgery, the First Affiliated Hospital of Xi\u0026apos;an Jiaotong University, Xi\u0026rsquo;an, Shaanxi, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJiachun Ding, Jiaqiang Ren, Fan Chen, Ye Lu , Yifei Ma, Zhenchao Gao, Yiqun Song, Jiahui Zeng, Jiaoxing Wu, Zhengyuan Feng, Cancan Zhou, Zheng Wang \u0026amp; Weikun Qian\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePancreatic Disease center of Xi\u0026rsquo;an Jiaotong University\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJiachun Ding, Jiaqiang Ren, Fan Chen, Ye Lu , Yifei Ma, Zhenchao Gao, Yiqun Song, Jiahui Zeng, Jiaoxing Wu, Zhengyuan Feng, Cancan Zhou, Zheng Wang \u0026amp; Weikun Qian\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Cardiac Surgery, the First Affiliated Hospital of Xi\u0026rsquo;an Jiaotong University, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi\u0026rsquo;an, Shaanxi, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTing Zhang, Zehua Shao, Huijing Tian\u0026nbsp;\u0026amp;\u0026nbsp;Siyu Wang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003eJiachun Ding designed and conducted the research, performed data curation and formal analysis, and wrote the original draft of the manuscript. Jiaqiang Ren, Fan Chen, Ye Lu, Huijing Tian and Siyu Wang participated in data curation and formal analysis. Yifei Ma, Ting Zhang, Zehua Shao and Zhenchao Gao contributed to the methodology. Yiqun Song, Jiahui Zeng, Jiaoxing Wu and Zhengyuan Feng performed validation experiments. Cancan Zhou supervised the study and was responsible for project administration and conceptualization. Zheng Wang and Weikun Qian reviewed, \u0026nbsp;edited the manuscript, administered the project and acquired funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Weikun Qian:[email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was supported by the National Natural Science Foundation of China (No. 82372895); the Key Research and Development Program of Shaanxi (No. 2024SF2-GJHX-03), the Youth Star of Science and Technology Program of Shaanxi (No. 2025ZC-KJXX-126) and the Innovative Team Foundation of Shaanxi Health Commission (No. 2024TD-16).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset generated and/or analyzed during the study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003eAll procedures complied with the NIH Guide for the Care and the animal study protocol was approved by the Ethics Committee of the Animal Ethics Committee of Xi\u0026rsquo;an Jiaotong University (protocol code 2025-3684).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u0026nbsp;\u003c/strong\u003eThis article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eother third party material in this article are included in the article\u0026rsquo;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026rsquo;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHorn P, Tacke F. Liver Macrophage Diversity in Health and Disease. Results Probl Cell Differ. 2024;74:175\u0026ndash;209.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKisseleva T, Brenner D. Molecular and cellular mechanisms of liver fibrosis and its regression. 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J Transl Med. 2025;23(1):1036.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang A, Dong H, Cui Y, Zhu W, Ye J, Jin L et al. USP13 promotes hepatic stellate cells activation and aggravates liver fibrosis through deubiquitinating SMAD3. Hepatol Int. 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Liu H, Wang Y, Wang P, Yi Y, Lin Y, et al. Novel protein C6ORF120 promotes liver fibrosis by activating hepatic stellate cells through the PI3K/Akt/mTOR pathway. J Gastroenterol Hepatol. 2024;39(7):1422\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Maadawy WH, Amer M, Mostafa A, Hassany AA, Shafie NS, Lethy AW, et al. Ticagrelor attenuates cholestasis-induced liver fibrosis by inhibiting S1PR2-dependent Akt/ERK signaling, NLRP3 inflammasome activation. Int Immunopharmacol. 2026;168(Pt 1):115834.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFakher HE, Mehanna ET, Zidan AA, Mesbah NM, Abo-Elmatty DM, El-Afify SR, et al. Integrating biochemical and computational approaches to identify targeted therapeutic strategies for liver fibrosis: Effects of Telaglenastat (CB-839) on the glutaminase pathway. Biochem Biophys Res Commun. 2026;795:153101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao L, Peng F, Qi P, Zhang H, Chi H, Deng L et al. TNAP-induced CD47 membrane expression enhances TGF-β1 conversion in liver fibrosis. Hepatol Commun. 2025;9(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Luo X, Li T, Wang X, Yang J, Hu J. Cichoriin suppresses hepatic lipid accumulation and fibrosis in mice metabolic dysfunction associated steatohepatitis via the AMPK pathway. Biochem Pharmacol. 2026;245:117630.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Liver fibrosis, Huaier, network pharmacology, AKT","lastPublishedDoi":"10.21203/rs.3.rs-9123161/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9123161/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLiver fibrosis, a critical pathological precursor to cirrhosis and hepatocellular carcinoma, represents a significant unmet global health challenge due to the lack of effective targeted therapies that can reverse established fibrotic scarring. An integrated strategy was employed. Network pharmacology identified potential targets, followed by molecular docking and dynamics simulations. In vivo validation utilized two established murine models: carbon tetrachloride (CCl₄) and bile duct ligation (BDL)-induced fibrosis. In vitro studies employed human LX-2 hepatic stellate cells to assess anti-fibrotic effects on myofibroblasts. Huaier administration significantly attenuated liver fibrosis, improved liver function and reduced collagen deposition in both animal models. Histopathological analysis confirmed diminished inflammatory infiltration and fibrotic scarring. In vitro, Huaier suppressed LX-2 cell proliferation and migration. Bioinformatics and simulation analyses pinpointed AKT1 as a central target, showing high-affinity binding with Huaier's bioactive steroidal components. Mechanistically, Huaier specifically inhibited the phosphorylation and activation of the AKT signaling pathway in hepatic myofibroblasts. This study provides the first compelling evidence that Huaier granule alleviates liver fibrosis by targeting the AKT pathway to inhibit myofibroblast activation. These findings illuminate its mechanistic basis and reposition Huaier as a promising, multi-targeted therapeutic candidate for combating fibrotic liver disease.\u003c/p\u003e","manuscriptTitle":"Integrating Network Pharmacology and Experimental Validation to Elucidate Huaier in Attenuating Liver Fibrosis via the AKT Signaling Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-20 19:10:11","doi":"10.21203/rs.3.rs-9123161/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-23T19:10:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T13:32:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167015816181338439434521965431402471025","date":"2026-03-23T08:01:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T04:18:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-22T21:41:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"236633965702375321849781388059020514169","date":"2026-03-20T01:23:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T12:45:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32445979626889827391274037762449488271","date":"2026-03-18T12:03:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215344452724226506766253825559193663367","date":"2026-03-18T01:27:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240530964380550431865111633307579369717","date":"2026-03-18T00:53:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-17T19:48:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-17T06:04:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-17T06:03:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical and Experimental Medicine","date":"2026-03-14T13:57:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"08c5ef93-2923-472c-a7be-1427c4c38cfd","owner":[],"postedDate":"March 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-19T06:53:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-20 19:10:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9123161","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9123161","identity":"rs-9123161","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}