Therapeutic Mechanisms of Ruangan Huayu Formula in Liver Cirrhosis: Network Pharmacology, Molecular Docking and Experimental Validation

Abstract

Background

Liver cirrhosis remains a challenging global health issue for which current treatment options are largely limited to managing acute liver failure, with no definitive curative therapies available. The Ruangan Huayu Formula (RGHY) has shown promising clinical effects in recent years; however, its comprehensive chemical profile and the specific mechanisms underlying its anti-cirrhotic activity remain to be fully elucidated.

Methods

Ultra-high-performance liquid chromatography-Orbitrap-explorative mass spectrometry (UHPLC-OE-MS) was performed to characterize the chemical profile of the RGHY. Network pharmacology and molecular docking were leveraged to identify potential targets and pathways. Antifibrotic efficacy and underlying mechanisms of RGHY were evaluated using a thioacetamide (TAA)-induced liver fibrosis model in male Sprague-Dawley rats, along with transforming growth factor-beta (TGF-β)-stimulated human hepatic stellate cells (LX-2) in vitro.

Results

UHPLC-OE-MS study showed various chemical components of RGHY. Network pharmacology analysis revealed 57 intersecting targets, with the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway standing out. Molecular docking studies returned strong binding scores for AKT1, SRC, PIK3CA, and STAT3 as core targets key to RGHY compounds. RGHY was shown to significantly alleviate TAA-induced liver injury and fibrosis by down-regulating the PI3K/AKT pathway. In vitro, RGHY-containing serum and its key bioactive compounds (7,3’,4’-trihydroxyflavone and sulfuretin) disrupted TGF-β-induced LX-2 cell activation, migration, and fibrogenesis. The PI3K/AKT signaling pathway was confirmed to involve in mediating the antifibrotic effects of RGHY.

Conclusion

RGHY and its bioactive compounds 7,3’,4’-trihydroxyflavone and effectively attenuated liver fibrosis and hepatic stellate cell activation through multicomponent, multitarget actions, primarily by modulating the PI3K/AKT pathway. These findings provide mechanistic evidence supporting its traditional use and its potential as a therapeutic candidate for liver cirrhosis.

Background

As the end stage of chronic liver disease, liver cirrhosis is characterized by growth of diffuse fibrous tissue; it is a leading cause of morbidity and mortality worldwide, and is commonly the sequel of continuing cause, such as viral hepatitis, alcoholism, metabolic dysfunction-associated steatohepatitis or others whose onset is still unknown [1, 2]. Liver injury, inflammation, cell death, activation of hepatic stellate cells (HSCs), and excessive deposition of extracellular matrix proteins all contribute in the cirrhotic process to the non-reversible condition that diminishes regenerative capacity, and despite better understanding of the molecular basis of liver fibrosis, to date few adequate antifibrotic therapies exist [3, 4].

Traditional Chinese Medicine (TCM) is very well positioned to provide alternative holistic and multi-target therapeutic paradigm-delicious, tasty, and supporting different common chronic diseases [5]. Biejia Ruangan tablet, Huangjia Ruangan granules and Shaoyao Ruangan Decoction have shown promising therapeutic effects in treating liver fibrosis and cirrhosis [6–8]. Ruangan granules consist of eight raw materials such as Biejia (Carapax Trionycis), Chishao (Radix Paeoniae Rubra), Huangqi (Radix Astragali Mongolici), Baizhu (Rhizoma Atractylodis Macrocephalae), Danggui (Radix Angelicae Sinensis), Danshen (Radix Salviae Miltiorrhizae), Juemingzi (Semen Cassiae Obtusifoliae), and Xiakucao (Spica Prunellae Vulgaris) [9]. Fuzheng Huayu formula is clinically used for liver fibrosis treatment [10] and has an anticirrhotic effect [11]. Fuzheng Huayu formula comprises Danshen, Dongchongxiacao (Cordyceps), Taoren (Semen Persicae), Jiaogulan (Herba seu Radix Gynostemmatis Pentaphylli), Wuweizi (Fructus Schisandrae Chinensis), and Songhuafen (Pini pollen) [12]. In recent decades, our group found that Ruangan Huayu (RGHY) formula demonstrated good clinical efficacy for the treatment of liver fibrosis by invigorating qi, promoting blood circulation, and softening the hardness of patients. The raw materials of RGHY formula are partly coincident with that of Ruangan granules and Fuzheng Huayu formula, including Biejia, Chishao, Baizhu, and Danshen, which could nourish yin, invigorate the spleen and qi, clear heat, remove blood stasis, and relieve pain [9]. RGHY formula also comprises Taizishen (Radix Pseudostellariae), Fuling (Poria), Mudanpi (Cortex Moutan Radicis), Baihuasheshecao (Herba Hedyotdis), Muli (Concha Ostreae), Foshou (Fructus Citri Sarcodactylis). Among them, Taizishen, Fuling and Baihuasheshecao have anti-inflammatory and antioxidant activities [13]. Mudanpi is widely employed in TCM for its analgesic, antipyretic, antioxidant, anti-inflammatory, and antitumor properties [14]. Muli has sedative and anticonvulsant effects [15], and Foshou has anti-inflammatory effects and blood pressure-lowering activities [16]. However, the potential mechanism of RGHY formula against liver cirrhosis is still unclear.

With the advent of systems biology and bioinformatics developed for elucidating the molecular basis of diseases, a combination of network pharmacology, molecular docking, and experimental validation has emerged as a robust approach to investigating the multi-component, multi-target, and multi-pathway mechanisms of TCM formulas. Network pharmacology defines a new paradigm that integrates systems biology, bioinformatics and polypharmacology in a framework to “decipher the complex multi-component, multi-target, multi-pathway mechanisms”, by constructing and analyzing interactive networks via associating drugs, predicted targets as well as related diseases so as to reveal core therapeutic targets and major signaling pathways [17, 18]. Molecular docking, on the other hand predicts the affinity and interaction modes of putative active compounds when binding to target protein structures and provides atomic-level understanding that can validate network-derived hypotheses [19].

In this study, the antifibrotic mechanisms of RGHY are explored, utilizing a “big data” approach to mine the rich information present within the pharmacological entity. In the first step, the drug is profiled by Ultra-high-performance liquid chromatography-Orbitrap-explorative mass spectrometry (UHPLC-OE-MS), and then potential targets and pathways associated with liver cirrhosis are predicted based on this dataset using network pharmacology. several of the important compound-target binding from this predicted spectrum were then verified by docking simulation to corroborate the hypotheses, and finally, the authors test the predicted core mechanism inhibition of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway in vivo (using a thioacetamide [TAA]-induced rat model) and in vitro [transforming growth factor-beta (TGF-β)-stimulated LX-2 cell] models. As with a lot of TCM studies, here there is much more leucine to the understanding of the therapeutic effect, the bridging of the TCM to the more formal pharmacological/mechanistic understanding.

Materials and Methods

Identification of Bioactive Compounds of RGHY

The RGHY was supplied by Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai, China. We employed untargeted metabolomics to identify the main bioactive ingredients of RGHY through the UHPLC-OE-MS method. In brief, RGHY sample solutions were prepared and the supernatant obtained (subjecting to centrifugation at 12,000 rpm for 15 min at 4 °C), with 300 µL of the supernatant then combined with 1000 µL of extraction solution (containing deuterated internal standards), vortexed for 30 s, and ultrasonicated in a 4 °C water bath for 5 min, before incubating at -20 °C for 1 h (to precipitate proteins) and then centrifugating again as above. The supernatant was cautiously poured into new glass vials for analysis. A quality control sample was created by equally mixing all the supernatant. Chromatographic separation was performed using a Thermo Fisher Scientific UHPLC Vanquish system, which included a Phenomenex Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm particle size) and was linked to an Orbitrap Exploris 120 mass spectrometer. The mobile phase comprised (A) 0.01% acetic acid dissolved in water and (B) a mixture of isopropanol and acetonitrile in equal volumes. With the auto-sampler held at 4 °C, a 2 µL injection volume was utilized. Mass spectrometric analysis was carried out on an Orbitrap Exploris 120, operating in information-dependent acquisition mode under Xcalibur software. The electrospray ionization source parameters were established as follows: sheath gas flow at 50 units, auxiliary gas flow at 15 units, and capillary temperature at 320 °C. The full MS resolution was configured at 60,000, with MS/MS resolution at 15,000. The stepped normalized collision energy was adjusted to 20/30/40, and the spray voltage was maintained at + 3.8 kV for positive ion mode or -3.4 kV for negative ion mode. Raw data files were transformed into mzXML format via ProteoWizard software. A custom pipeline developed in R was used for the subsequent data processing tasks of peak detection, feature extraction, alignment, and integration, leveraging the XCMS package for a comprehensive metabolomic analysis.

Acquisition of RGHY and Liver Cirrhosis Targets

A systematic bioinformatics method was used to pinpoint potential therapeutic targets of RGHY for liver cirrhosis. First, the chemical structures of the bioactive compounds in RGHY were obtained from the PubChem database. These structures were subsequently submitted to the SwissTargetPrediction online platform to generate predictions for their probable protein targets. While the authors were collecting potential targets associated with liver cirrhosis. Key in constructing the datasets are the four basic biomedical databases: Online Mendelian Inheritance in Man, DrugBank, the Pharmacogenomics Knowledge Base, and GeneCards. The targets obtained were first merged, and then deduplicated to produce a cirrhosis related gene dataset. In order to find the common targets through the medication, the predicted RGHY targets were then intersected with the list of the cirrhosis targets that had been collected above. The intersected genes were then performed through Venny2.1, finally exposing the common to RGHY and cirrhosis. Finally, the RGHY-target interacted compound(s), and disease (cirrhosis), was merged together and exposed and the Drug-targeting-Disease graph.

Examination of the Protein−Protein Interaction (PPI) Network

The common target genes from the intersection analysis were submitted to the string database, analyzed with the organism ‘Homo sapiens choice and high confidence interaction score (0.9). Nodes that were isolated from the main network were deleted. The interaction data were processed and imported into Cytoscape software (3.9.1) to visualize the network and conduct additional topological analysis. Nodes within the network were colored and sized by ‘degree centrality’ - where centrality is defined as the density of connectors to which the given node is attached (the number or edges), to highlight potential key hub proteins in the pharmacological network of RGHY treating liver cirrhosis.

Analysis of Pathway and Function Enrichment

To understand the biological functions and pathways of the intersecting genes, enrichment analyses were performed using the Bioinformatics Platform. The shared target genes were analyzed for Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and visualized as bubble plots and histograms.

Molecular Docking of Bioactive Compounds and Key Targets

To evaluate the binding affinity between key bioactive compounds and target proteins, molecular docking was conducted. The three-dimensional structures for the bioactive compounds in sdf format were downloaded from the PubChem database. The crystal structure of the relevant target proteins was downloaded simultaneously in pdb format from rcSB protein Data Bank. The procedures were performed using Autodock Tools (1.5.6) and Autodock Vina (1.1.2). Unknowing the potential functional pockets, the crystal structure of target proteins was therefore completely wrapped by Grid Box. The resulting docking poses were inspected with Pymol (2.5.4).

Animal Experiments

Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) supplied male Sprague-Dawley rats weighing between 180 and 200 g. According to the resource equation [20], the maximum sample size for each group was five rats. Therefore, the rats were divided randomly into five experimental groups, each consisting of eight rats. Group 1 (Control) received a daily oral administration of 10% Tween-20 for two months and was injected intraperitoneally with sterile distilled water three times a week. Group 2 (TAA Model) was given daily oral doses of 10% Tween-20 and received intraperitoneal injections of TAA (200 mg/kg) three times a week. Groups 3–5 (TAA+RGHY groups) were given daily oral doses of RGHY granules at low (0.62 g/kg/d, RGHY-L), medium (1.24 g/kg/d, RGHY-M), or high (2.48 g/kg/d, RGHY-H) levels, in addition to 200 mg/kg of TAA administered three times a week. Sterile distilled water was used as the vehicle. The study lasted for two months. At the end of the treatment, after an overnight fast, All rats were put down humanely under general anesthesia, which was induced by an intraperitoneal injection of ketamine (30 mg/kg) and xylazine (3 mg/kg). All animal experimental protocols were approved by the Medical Ethics Committee of the Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine (Approval Number: PZSHUTCM2409290005), and the animal procedures adhered to the ARRIVE guidelines 2.0.

Biochemical Analysis

After euthanasia, blood samples were gathered in gel-activated tubes and centrifuged at 2500 rpm for 15 min. The resulting serum was used to determine liver function markers. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were quantified with kits BC1565 and BC1555 from Beijing Solarbio Science & Technology Co., Ltd. The concentration of serum albumin (ALB) was measured using the assay kit (A028-2-1) from Nanjing Jiancheng Bioengineering Institute.

Histological Analysis

Liver tissues were embedded in paraffin and sectioned, then stained with hematoxylin and eosin (H&E) and Masson’s trichrome and Sirius red, in line with the protocols [21]. Histopathology was evaluated and imaged in a Nikon microscope (Y-ths, Japan).

Immunohistochemistry (IHC) Analysis

Immune-staining for IHC were performed by dewaxing paraffin-embedded sections and rehydrating in descending alcohol gradients, incubating at room temperature for 10 min in 3% hydrogen peroxide to quench endogenous peroxidase activity. The sections were washed three times with PBS, then the antigen retrieval areas were exposed, followed by another wash. They were blocked with 10% non-immune goat serum at room temperature for 20 min and incubated overnight at 4 °C with α-SMA (14395-1-AP, Proteintech). After mixed incubating with PBS three times, the sections treated with secondary antibody for 20 min then treated with streptavidin‐biotin‐peroxidase complex at 37 °C for 10 min. Reactivity was visualized by diaminobenzidine and nuclei counterstained with hematoxylin.

Preparation of RGHY Drug-containing Serum

Sprague-Dawley rats were orally administered RGHY granules at a dose of 2.48 g/kg/d for three consecutive days. Two hours after the final administration, blood was collected from the abdominal aorta under anesthesia. The blood samples were centrifuged at 1000 rpm for 5 min, and the upper serum layer was collected. The serum was then inactivated at 56 °C for 30 min, filtered through a 0.22 μm membrane, aliquoted, and stored at -80 °C until further use.

Cell Culture and Treatment

Human hepatic stellate cells (LX-2, Procell Life Science & Technology, Wuhan, China) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 50 U/mL streptomycin, and maintained at 37 °C in a humidified 5% CO₂ incubator. For experiments, cells were treated with 10 ng/mL TGF-β in the absence or presence of either 2%, 5%, or 10% RGHY drug-containing serum, or a combination of 0.1 µM 7,3’,4’-trihydroxyflavone (GC46080, GlpBio) and 1 µM sulfuretin (HY-N1193, MedChemExpress), with or without the addition of 10 µM PI3K/AKT pathway activator 740 Y-P (S7865, Selleck). All treatments were administered for a duration of 48 h.

Cell Viability Assay

Using the Cell Counting Kit-8 (CCK-8, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), cell viability was determined in line with the manufacturer’s instructions. A 96-well plate was used to seed LX-2 cells at 5,000 cells per well. Following cell attachment, they were subjected to different doses of 7,3’,4’-trihydroxyflavone (0.1, 0.2, 0.4, 0.8, or 1.6 µM) or sulfuretin (1, 2, 4, 8, or 16 µM). Following the treatment, cells were washed twice with PBS and incubated with a 1:10 dilution of CCK-8 reagent in serum-free medium for 2 h at 37 °C. Absorbance was measured at 450 nm using a Multiskan microplate reader from Thermo Fisher Scientific.

Wound Healing Assay

Cell migration was assessed using a wound healing assay. Following the treatments, LX-2 cells were seeded into 6-well plates at a density of 5 × 10^5 cells per well. After all the cells in each monolayer were uniform and confluent, a full wound was created in each with a pipette tip. After detached cells were removed from washing with PBS, the cells were assigned to a serum-free medium. The closure of the wounds was periodically determined by taking images at same places at 0 h, 24 h and 48 h followed by the assessment of distance migrated.

Measurement of Hydroxyproline (Hyp)

Hyp in the liver samples and cultured LX-2 cells were measured using the commercial Hydroxyproline assay kit (A030-1) made available by Nanjing Jiancheng Bioengineering Institute (Nanjing, China), based on the manufacturer’s instructions.

Western Blot

Proteins were extracted with RIPA lysis buffer, separated on a 10% odium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and transferred to a polyvinylidene fluoride membrane. The membrane was incubated overnight at 4 °C with primary antibodies against α-SMA (14395-1-AP, Proteintech); p-PI3K (#4292) and PI3K (#4228) from Cell Signaling Technology; p-AKT (#2938) from Cell Signaling Technology; AKT (#9018) from Cell Signaling Technology. The protein bands were detected with the help of an enhanced chemiluminescence system from Bio-Rad, USA.

Statistical Analysis

Graphs and analyses were done with GraphPad Prism software (version 8.4.2) and results were displayed and analysed as mean ± standard deviation. One-way analysis of variance and Tukey’s test was used to determined difference between groups, a P-value of < 0.05 was considered statistically significant.

Results

Network Pharmacology Analysis Uncovers Potential Targets and Pathways for RGHY in Managing Liver Cirrhosis

The chemical profiling of the extract of RGHY by UHPLC-OE-MS, together with the full-scan analyses performed in both positive and negative ionization modes to capture the entire profile of the metabolites of the extract is depicted in Fig. 1A and B. The primary bioactive compounds in RGHY were identified through UHPLC-OE-MS (Table 1). A network pharmacology approach was employed to elucidate the potential mechanism of RGHY against liver cirrhosis. A Venn diagram analysis pinpointed 57 targets that intersect between the active components of RGHY and the known therapeutic targets for liver cirrhosis (Fig. 2A). A drug-target-disease network was constructed, visually integrating RGHY, its candidate compounds, the intersecting targets, and liver cirrhosis (Fig. 2B). The PPI network of these 57 intersecting targets was established, revealing core hub proteins such as AKT1, SRC, PIK3CA, EGFR, STAT3, KRAS, MMP9, IL1B, and ESR1 (Fig. 2C). GO enrichment analysis suggested that the main biological processes were responses to bacterial molecules and lipopolysaccharide, while molecular functions were significantly enriched in carboxylic acid binding and serine-type peptidase activity; cellular component analysis highlighted enrichment in vesicle lumen and secretory granule lumen (Fig. 2D). The KEGG analysis showed the top 30 significantly enriched pathways were lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, resistance to EGFR tyrosine kinase inhibitors, and a few cancers (Fig. 2E). Putting what we have seen together strongly indicated that RGHY may affect liver cirrhosis by a multi-target and multi-pathway mechanism.

Fig. 1.

Presents UHPLC-OE-MS Spectra Under Positive Ion Mode (A) And Negative Ion Mode (B)

Table 1.

The Main Bioactive Compounds of RGHY were Identified by UHPLC-OE-MS

Name	Formula	mzmed	rtmed
Myristic acid	C14H28O2	227.2016	542.7
Undecanoic acid	C11H22O2	185.1546	494.5
Sulfuretin	C15H10O5	269.0454	332.1
Pyrocatechol	C6H6O2	109.0295	226.3
Caffeic acid	C9H8O4	179.0349	265.6
Vanillic acid	C8H8O4	167.0349	259.7
7,3’,4’-Trihydroxyflavone	C15H10O5	269.0454	332.1
Hydroquinone	C6H6O2	109.0295	226.3
(E)-5-(2,3-dimethyl-3-tricyclo[2.2.1.02,6]heptanyl)-2-methyl-pent-2-enoic acid	C15H22O2	233.1546	497.7
1-Naphthol	C10H8O	143.0501	382.5
Royal jelly acid	C10H18O3	185.1181	346.4
2,3-Dihydroxy-4-methoxyacetophenone	C9H10O4	181.0506	249.5
DL-Tryptophan	C11H12N2O2	205.097	206.9
Pyrogallol	C6H6O3	127.0389	115.9
Leucine	C6H13NO2	130.0872	67.9
(R)-5-Oxopyrrolidine-2-carboxylic acid	C5H7NO3	128.0352	64.5
Alanine	C3H7NO2	88.0403	42
β-Alanine	C3H7NO2	88.0403	42
Vanillin	C8H8O3	151.04	277.5
Succinic Acid	C4H6O4	117.0192	81.4
Uric Acid	C5H4N4O3	167.0209	52

Fig. 2.

Network Pharmacology Analysis for RGHY and Liver Cirrhosis. A Venn Diagram of RGHY and Liver Cirrhosis (57 intersecting targets). B Drug-Target-Disease Network Diagram. C PPI Network Is Constructed Based on Intersecting Targets. D GO Three-In-One Histogram (the top 10 entries of each item are selected according to the size of the gene ratio). E The Results of KEGG Pathway Analysis for the Top 30 Entries are Selected According to the Size of the Gene Ratio

Molecular Docking Demonstrates Strong Binding Affinities Between Key RGHY Compounds and Core Target Proteins

To elucidate these possible interactions as foreseen in the network pharmacology results, we further performed molecular docking studies on the core targets and the main bioactive ingredients found in RGHY. Bindings were found amongst the several important pairs. 7,3’,4’-trihydroxyflavone showed high-affinity binding to both AKT1 (binding energy: -10.3 kcal/mol) and SRC (binding energy: -8.3 kcal/mol) (Fig. 3A and B). Sulfuretin also showed strong binding to PIK3CA (binding energy: -8.8 kcal/mol) (Fig. 3C). Similarly, caffeic acid maintained stable binding with PIK3CA (-6.5 kcal/mol) and STAT3 (-5.8 kcal/mol) (Fig. 3D and F). Lastly, 2,3-Dihydroxy-4-methoxyacetophenone show good docking with PI3KCA (binding energy: -6.3 kcal/mol) (Fig. 3E). Overall, these molecular docking explored the possibilities of direct interactions among the major bioactive ingredients of RGHY while also illuminating the major target proteins purportedly implicated in the anti-cirrhotic mechanism.

Fig. 3.

Molecular Docking Results of Bioactive Compounds of RGHY with Target Proteins. 2D and 3D structure diagrams of 7,3’,4’-trihydroxyflavone with (A) AKT1 or (B) SRC protein, (C) sulfuretin, (D) vaffeic acid or (E) 2,3-Dihydroxy-4-methoxyacetophenone with PK3CA protein, and (F) caffeic acid with STAT3 protein

RGHY Treatment Alleviates TAA-Induced Liver Fibrosis and Regulates PI3K/AKT Pathway in Rats

A TAA-induced male rat model of liver fibrosis was used to evaluate the therapeutic efficacy of RGHY in vivo. Histopathological examination revealed that TAA administration caused severe hepatic injury, characterized by marked inflammatory infiltration, collagen deposition, and strong α-SMA (alpha-smooth muscle actin) expression (Fig. 4A). Treatment with RGHY, at low, medium and high doses, significantly ameliorated these pathological changes, reducing necrosis, inflammation, collagen accumulation, and α-SMA expression in a dose-dependent manner. Biochemical tests indicated that medium and high doses of RGHY treatment significantly decreased by 34.91% and 54.04% for serum AST (P < 0.001; Fig. 4B) and 35.31% and 54.51% for serum ALT (P < 0.001; Fig. 4C) in TAA-induced rats, respectively. Conversely, low, medium, and high doses of RGHY treatment significantly induced 1.45-, 1.77- and 1.97-fold increases in serum ALB in TAA-induced rats, respectively (P < 0.001; Fig. 4D). Furthermore, the Hyp content, a direct indicator of collagen deposition, was increased in the TAA group and significantly decreased by 16.77%, 23.56%, and 38.47% in low, medium, and high doses of RGHY groups, respectively (P < 0.001; Fig. 4E). Western blot analysis revealed that low, medium, and high doses of RGHY decreased α-SMA protein levels by 27.97%, 52.58%, and 58.83% in fibrotic liver tissues, respectively (P < 0.001; Fig. 4F, G). Low, medium, and high doses of RGHY also decreased p-PI3K levels by 14.76%, 36.09%, and 62.62% (P < 0.05; Fig. 4F, H) and p-AKT levels by 15.38%, 44.15%, and 54.20% (P < 0.05; Fig. 4F, I) in fibrotic liver tissues, respectively. Collectively, these results indicate that RGHY effectively protects against TAA-induced liver fibrosis, improves liver function, and its mechanism is associated with the blocking of the PI3K/AKT signaling pathway.

Fig. 4.

RGHY Alleviates TAA-Induced Liver Injury in Rats. A Hematoxylin and Eosin (H&E), Masson Trichrome, Sirius Red and α-SMA Immunohistochemical Staining in Histopathological Examination of Liver Tissue Section (scale bar, 100 μm; magnification, 200⋅). Serum Levels Of B AST, C ALT and D ALB were measured by biochemical analysis. E Hyp content and F-I expression of α-SMA, p-PI3K, PI3K, p-AKT, and AKT in liver tissues were measured by biochemical analysis and Western blot, respectively. ***P < 0.001 vs. control; ^###P < 0.001 vs. TAA

RGHY-Containing Serum Inhibited TGF-β-Induced LX-2 Cell Activation, Migration, and Fibrogenesis in Vitro

Wound healing assay showed that the TGF-β-induced increase in the LX-2 cell migration was decreased by RGHY-containing serum treatment (P < 0.001; Fig. 5A, B). Biochemical analysis showed that the 2%, 5%, and 10% RGHY-containing serum treatment decreased Hyp content by 9.90%, 30.05%, and 44.35% in TGF-β-induced LX-2 cells, respectively (P < 0.001; Fig. 5C). Furthermore, Western blot analysis revealed that TGF-β markedly upregulated the expression of α-SMA, while 2%, 5%, and 10% RGHY-containing serum suppressed this upregulation by 20.64%, 56.31%, and 66.60%, respectively (P < 0.001; Fig. 5D, E). Further in vitro studies on study indicate that indeed act by directly also inhibiting the profibrotic TGF-β responses of hepatic stellate cells, including blocking their migratory capacity and replication, which also suggests its mechanisms in ameliorating liver fibrosis at cellular-level mechanisms.

Fig. 5.

RGHY Inhibits TGF-β-induced Migration and α-SMA Expression in LX-2 cells. LX-2 Cells were Treated with TGF-β in the Absence or Presence of RGHY Drug-Containing Serum. (A, B) Cell Migration was Measured by Wound Healing (scale bar, 200 μm; magnification, 100⋅). (C) Hyp Content and (D, E) α-SMA Expression were Measured by Biochemical Analysis and Western Blot, Respectively. ***P < 0.001 vs. Control; ^###P < 0.001 vs. TGF-β

The Bioactive Compounds 7,3’,4’-Trihydroxyflavone and Sulfuretin Synergistically Inhibited TGF-β-Induced LX-2 Cell Activation, Migration, and the PI3K/AKT Pathway

To elucidate the contribution of specific components within RGHY, the effects of its two key bioactive compounds, 7,3’,4’-trihydroxyflavone and sulfuretin, were investigated individually and in combination (TS) on TGF-β-stimulated LX-2 cells. Cell viability assays confirmed that both compounds were non-toxic to LX-2 cells at the 0.1 µM and 1 µM concentrations (Fig. 6A, B). When used in combination, TS treatment significantly and dose-dependently inhibited the TGF-β-induced migration of LX-2 cells (P < 0.001; Fig. 6C, D). Furthermore, TS treatment markedly reduced the TGF-β-induced increase in Hyp content by 42.72% (P < 0.001; Fig. 6E). Western blot analysis showed that TS treatment inhibited the expression of α-SMA, p-PI3K, and p-AKT by 20.26%, 21.58%, and 33.66%, respectively (P < 0.05; Fig. 6F-I). In summary, the combined treatment with 7,3’,4’-trihydroxyflavone and sulfuretin recapitulates the core antifibrotic effects of RGHY extract by hindering the activation, migration, and collagen production of hepatic stellate cells via the suppression of the PI3K/AKT signaling pathway.

Fig. 6.

The Bioactive Compounds of RGHY Inhibit TGF-β-Induced Migration, α-SMA Expression and PI3K/AKT Pathway in LX-2 Cells. Cell Viability of LX-2 Cells Treated with Different Concentrations of (A) 7,3’,4’-Trihydroxyflavone or (B) dulfuretin. LX-2 Cells were Treated with TGF-β in the Absence or Presence of 7,3’,4’-Trihydroxyflavone Plus Sulfuretin (TS). (C, D) Cell Migration was Measured by Wound Healing (scale bar, 200 μm; magnification, 100⋅). (E) Hyp content and (F-I) Expression of α-SMA, p-PI3K, PI3K, p-AKT, and AKT were Measured by Biochemical Analysis and Western Blot, Respectively. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; ^#P < 0.05, ^#P < 0.01, ^###P < 0.001 vs. TGF-β

The Antifibrotic Effect of RGHY’s Bioactive Compounds on LX-2 Cells was Mediated Through the PI3K/AKT Pathway

To confirm that the inhibition of the PI3K/AKT pathway is a central mechanism underlying the effects of RGHY’s bioactive compounds, a rescue experiment was conducted using the specific PI3K/AKT pathway activator 740 Y-P. LX-2 cells were treated with TGF-β in the presence or absence of the compound combination TS (7,3’,4’-trihydroxyflavone plus sulfuretin) and the activator 740 Y-P. As previously observed, TS treatment significantly inhibited TGF-β-induced cell migration (P < 0.001; Fig. 7A, B), Hyp production (P < 0.001; Fig. 7C) and α-SMA expression (P < 0.001; Fig. 7D, E). The PI3K/AKT activator 740 Y-P, administered in combination with TS, effectively rescued the inhibitory effects of TS, which was particularly prominent in TS-induced cellular models of hepatic fibrosis. 740 Y-P treatment partlly restored the migratory capacity of LX-2 cells (P < 0.01; Fig. 7A, B) and abrogated TS-induced downregulation in Hyp content (P < 0.001; Fig. 7C) and α-SMA protein expression (P < 0.001; Fig. 7D, E). Collectively, these tads tell of heights and counteraction of TS antifibrotic actions by pharmacologically activating PI3K/AKT pathway.

Fig. 7.

The Bioactive Compounds of RGHY Inhibit TGF-β-Induced Migration and α-SMA Expression In LX-2 Cells via the PI3K/AKT Pathway. LX-2 Cells were Treated with TGF-β in the Absence or Presence of 7,3’,4’-Trihydroxyflavone Plus Sulfuretin (TS) or PI3K/AKT Pathway Activator 740 Y-P. (A, B) Cell Migration was Measured by Wound Healing (scale bar, 200 μm; magnification, 100⋅). (C) Hyp content and (D, E) α-SMA Expression were Measured by Biochemical Analysis and Western Blot, Respectively. ***P < 0.001 vs. control; ^###P < 0.001 vs. TGF-β; ^ΔΔP < 0.01, ^ΔΔΔP < 0.001 vs. TGF-β + TS

Discussion

Liver cirrhosis is a chronic condition seen following preceding hepatitis/chronic inflammatory process to fibrosis. As a major global health burden, it has limited therapeutic options [22, 23]. This study investigates the mechanism by which RGHY ameliorates the pathophysiology of liver cirrhosis.

Identifying bioactive may deepen pharmacodynamic knowledge of a TCM formula. Using UHPLC-OE-MS, we identified 21 major bioactive compounds in RGHY, including sulfuretin, 7,3’,4’-trihydroxyflavone, caffeic acid, vanillic acid etc. Accumulating literature suggests these compounds are anti-inflammatory, antioxidant and antifibrotic [24–26]. The presence of these bioactive compounds in RGHY implies that it might achieve therapeutic effects via a synergistic effect of multiple phytochemicals. We found 57 targets at the intersection of RGHY and liver cirrhosis in Network pharmacology analysis, suggesting it may act therapeutically via a multi-targeted strategy. The drug-target-disease network diagram shows complex interactions between the bioactive compounds of RGHY and the key targets of liver cirrhosis, reflecting the whole-based regulatory characteristics of TCM. Analysis of the PPI network revealed a key role for AKT1, SRC, PIK3CA, and STAT3, all of which are crucial for the regulation of fibrosis and cirrhosis [27, 28].

Through KEGG pathway analysis, we found an enrichment in pathways to liver fibrosis and inflammation such as PI3K/AKT signaling. The PI3K/AKT signaling pathway also play an important role in the antifibrotic and anticirrhotic effects of Ruangan granules, Fuzheng Huayu formula, Rougan Keli, and Gan-tang-yi decoction [9, 29–31]. The core targets such as AKT1 and PIK3CA are notably enriched in the PI3K/AKT signaling pathway. The active components of Ruangan granules such as quercetin, genistein, and kaempferol exhibited stable binding affinities for phosphatase and tensin homolog (PTEN) and AKT1, therefore regulating PI3K/AKT pathway to attenuate liver fibrosis [32].Dihydroartemisinin, artesunate and caffeic acid as active ingredients of Qinghao Biejia Decoction targeted PIK3CA to affect PI3K/AKT pathway and inflammatory transformation of chronic liver disease [33]. Our molecular docking results indicated that 7,3’,4’-trihydroxyflavone has strong binding affinity with AKT1 (-10.3 kcal/mol) and SRC (-8.3 kcal/mol), whereas sulfuretin showed strong binding affinity with PIK3CA (-8.8 kcal/mol). This supports that the bioactive compounds of RGHY may interact directly with core targets involved in liver fibrosis and suggest a potential molecular basis for the therapeutic efficacy of RGHY.

To reveal its in vivo therapeutic effects, we develop a rat model of liver cirrhosis using TAA. TAA is a well-established hepatotoxin that induces severe liver injury, inflammation, and fibrosis, thereby mimicking the pathological processes occurring during human liver cirrhosis [34]. In the present study, treatment with RGHY significantly ameliorated liver injury, reduced collagen deposition and α-SMA expression, and inhibited activation of the PI3K/AKT pathway. These in vivo results together confirmed that RGHY exerted protective effects against TAA-induced liver cirrhosis, and confirmed our network pharmacology and molecular docking results. HSCs are the main effector cells of liver fibrosis, and their activation is an important step in the progression of liver cirrhosis [29, 35]. As an important pro-fibrotic cytokine, TGF-β can rapidly stimulate the activation of the HSCs, promoting the production of α-SMA and collagens [36, 37]. To further investigate the in vitro action of RGHY, we used TGF-β on LX-2 cells in the presence or absence of RGHY-containing serum. The RGHY-containing serum dramatically reduced the TGF-β-induced migration of LX-2 cells, Hyp content and α-SMA expression, implying its role in mediating the activation and migration of HSCs under fibrogenic stimulation.

In order to identify the key bioactive compounds responsible for the antifibrotic effects of RGHY, we selected two flavonoids 7,3’,4’-trihydroxyflavone and sulfuretin according to the docking results and treated LX-2 cells with these two compounds. The combined administration of these two compounds was found to significantly inhibit TGF-β-induced migration of LX-2 cells, cause a reduction in Hyp content, downregulate expression of α-SMA, and inhibit the activation of PI3K/AKT signaling. Sulfuretin was found to inhibit HSC activation and collagen deposition in liver fibrosis models [26, 38]. Moreover, sulfuretin protects SH-SY5Y cell apoptosis by inhibiting PI3K/AKT signaling [39]. 4’,5,7-trihydroxyflavone has anti-fibrotic effects by inhibiting oxidative stress, inflammation response, and HSC activation via the PI3K/AKT pathway [40]. 7,3’,4’-trihydroxyflavone has antioxidant and anti-inflammatory activities [41]. To further validate the PI3K/AKT pathway mediates the antifibrotic effects of RGHY, we treated cells with 740 Y-P, a specific activator of the pathway. Our research shown that the 740 Y-P counteracted the inhibiting effect of TS on the TGF-β induced LX-2 cell migration, Hyp content and α-SMA expression. It demonstrates that inhibiting PI3K/AKT pathway is a key part of the antifibrotic mechanism of RGHY.

There are some limitations of this study. This research primarily relies on animal models and in vitro experiments, and the efficacy and safety of RGHY need to be validated in clinical settings. Additionally, the quantification data for the main bioactive compounds of RGHY requires further investigation. Future research needs to further explore and analyze the possible targets, signaling pathway, and molecular mechanisms by which the specific active components of RGHY inhibit liver cirrhosis. Second, the feasibility and effectiveness of combining RGHY with existing treatment methods should be investigated. These studies will help facilitate the transition of RGHY from laboratory research to clinical application, providing new treatment options for chronic liver disease.

Conclusion

Overall, our study sheds light on the therapeutic mechanism of RGHY in liver cirrhosis using network pharmacology, molecular docking, and experimental approaches, and may provide foundational insights that will enable future researchers to address existing gaps and further elucidate RGHY’s potential in liver cirrhosis.

Abbreviations

RGHY

Ruangan Huayu Formula

UHPLC-OE-MS

Ultra-High-Performance Liquid Chromatography-Orbitrap-Explorative Mass Spectrometry

TAA

Thioacetamide

TCM

Traditional Chinese Medicine

PPI

Protein−Protein Interaction

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

AST

Aspartate aminotransferase

ALT

Alanine Aminotransferase

ALB

Albumin

IHC

Immunohistochemistry

TGF-β

Transforming Growth Factor-Beta

CCK-8

Cell Counting Kit-8

Hyp

Hydroxyproline

Authors' Contributions

Haina Xie: Writing – original draft, Visualization, Project administration. Yujie Wang: Investigation, Methodology, Data curation. Mengqian Zhu: Methodology, Formal analysis. Luming Hou: Methodology, Resources. Xin Sun: Validation, Visualization. Xiaodi Gao: Formal analysis, Validation. Sicheng Gao: Software, Supervision, Writing-review and editing. Junfeng Zhu: Conceptualization, Writing-review and editing, Funding acquisition.

Funding

This study was supported by the Specialized Department Construction for Traditional Chinese Medicine Strengths of Yueyang Hospital [YW(2025–2026)-01-04] and Zhu Junfeng Traditional Chinese Medicine Inheritance and Innovation Studio of Shanghai (2025CXGZS-16).

Data Availability

Data will be made available on request.

Declarations

Ethics Approval and Consent to Participate

All animal experimental protocols were approved by the Medical Ethics Committee of the Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine (Approval Number: PZSHUTCM2409290005) and adhered to the ARRIVE guidelines 2.0.

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.