Inhibitory Mechanism of Buyang Huanwu Decoction on AGE/RAGE Pathway in Membranous Nephropathy: Integration of Network Pharmacology and Cell Model Validation

Abstract

Purpose

The Buyang Huanwu Decoction (BYHWD) has demonstrated therapeutic potential in renal-related disorders; however, its pharmacological mechanisms are still poorly understood. Therefore, the aim of this study was to elucidate the regulatory mechanisms of BYHWD in membranous nephropathy (MN).

Methods

Network pharmacology was used to identify BYHWD-related target genes for MN. Enrichment analyses were conducted to determine the relevant biological functions and signaling pathways. An integrated “compound-target-pathway” interaction network was established. The binding affinities between the active compounds and target proteins were determined via molecular docking. Two podocyte injury models were established using zymosan-activated serum (ZAS)-induced MPC-5 cells and Angiotensin II (Ang II)-induced AB8/13 cells. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. ELISA was used to quantify the levels of pro-inflammatory cytokines, membrane attack complexes (MAC, C5b-9), and advanced glycation end products (AGE), while Western blotting was performed to determine receptor for advanced glycation end products (RAGE) protein expression.

Results

BYHWD shared 230 genes with the MN-related targets. GO analysis indicated its involvement in regulating cell proliferation, apoptosis, and inflammation. KEGG analysis highlighted the modulation of the AGE-RAGE signaling pathway. IL-1β showed the highest diagnostic value in the machine learning analysis. Molecular docking revealed stable interactions between key compounds (myristic acid, stigmasterol, quercetin, and β-sitosterol) and target proteins. Both ZAS and Ang II inhibited podocyte proliferation and increased the levels of pro-inflammatory cytokines and C5b-9, whereas BYHWD reversed these effects. It also suppressed AGE and RAGE expression, and these effects were counteracted by pathway agonists.

Conclusion

BYHWD may improve podocyte injury by inhibiting AGE/RAGE and suppressing inflammatory responses and complement activation, providing a preliminary basis for its clinical application.

Introduction

Membranous nephropathy (MN) is characterized by autoimmune pathology of the glomeruli.^1,2 The main pathogenic process in MN involves the buildup of immune complexes on the glomerular basement membrane (GBM), leading to podocyte damage, remodeling of the basement membrane, and compromise of the glomerular filtration barrier.³ Clinically, MN is characterized predominantly by nephrotic syndrome and associated complications, such as proteinuria, edema, and dyslipidemia.^2,4 In adults, MN is the most common primary glomerulopathy linked to nephrotic syndrome, accounting for about 25–35% of cases.⁵ Compared with other glomerulopathies (eg, IgA nephropathy, focal segmental glomerulosclerosis), MN exhibits heterogeneous outcomes with approximately 30% of cases progressing to end-stage renal disease.⁶ However, current clinical treatment strategies for MN are often associated with substantial side effects and the risk of developing drug resistance.⁷ Therefore, there is a pressing necessity to develop innovative therapies to improve MN clinical prognosis.

Traditional Chinese medicine (TCM), recognized for its efficacy and favorable safety profile in various conditions, has emerged as a potential source for such novel therapies.^8,9 The Buyang Huanwu Decoction (BYHWD), a classic TCM formula primarily used for stroke rehabilitation, has demonstrated promising renoprotective effects in experimental models of kidney disease.^10–12 Studies report that BYHWD and its modifications can attenuate renal injury by downregulating pro-inflammatory and pro-fibrotic signaling pathways, suppressing oxidative stress, inhibiting specific forms of programmed cell death, and crucially, mitigating podocyte damage and apoptosis.^13–16 Significantly, these documented mechanisms of BYHWD–particularly its ability to protect podocytes, dampen inflammation, counteract oxidative stress, and modulate immune responses–align directly with the core pathological drivers of MN, namely podocyte injury, immune complex-driven glomerular inflammation, and proteinuria. This mechanistic alignment is further supported by studies showing that the key bioactive components of BYHWD herbs exhibit protective effects on podocyte structure and function in vitro and in models of glomerular injury.^17,18 Nevertheless, despite this compelling mechanistic rationale linking the renoprotective properties of BYHWD to MN pathology, the specific therapeutic efficacy and underlying molecular mechanisms of BYHWD in MN remain largely unexplored.

The complex, multi-component nature of traditional Chinese herbal formulas and their multi-pathway actions makes elucidating their mechanisms a major challenge.¹⁹ Network pharmacology has developed into a comprehensive research approach integrating systems biology, polypharmacology, and bioinformatics, centered on multidimensional analyses of the “disease-target-drug” network.^20–24 As such, network pharmacology offers a robust strategy to uncover the mechanisms involved in multi-component herbal formulas and advancing the modernization of TCM research.²⁵ In recent years, numerous research groups have applied network pharmacology to examine the mechanisms by which TCM formulas treat kidney diseases. For example, Zhu et al leveraged network pharmacology to demonstrate that the Yiqi Jianpi Xiaoyu formula could ameliorate acute kidney injury through STING–NCOA4–mediated suppression of ferroptosis, thereby reducing tubular necrosis.¹⁵ In summary, network pharmacology has become an essential methodological approach for elucidating the pharmacological basis of traditional Chinese herbal formulas.

This study adopted a network pharmacology-based strategy to investigate the mechanistic pathways through which BYHWD treats MN. Based on these findings, an in vitro MN model was established using MPC-5 cells, followed by pharmacological intervention. In summary, this study integrated a network pharmacology framework centered on the interactions among bioactive compounds, molecular targets, and disease pathways using molecular biology techniques to clarify the therapeutic effects of BYHWD on MN, thereby offering novel insights for its clinical application.

Materials and Methods

Construction of the BYHWD Bioactive Compound-Gene Interaction Network

All active compounds and corresponding target information of BYHWD were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP, https://old.tcmspe.com/tcmsp.php), a widely used platform that compiles detailed information on traditional Chinese medicine chemicals, their targets, and the interactions linking bioactive compounds to genes. Oral bioavailability (OB ≥ 30%) reflects the proportion of an orally administered drug that enters systemic circulation and is one of the key pharmacokinetic parameters used to characterize traditional Chinese medicines. Additionally, drug-likeness (DL ≥ 1.8) scores are used to assess the potential of active ingredients to be developed as therapeutic agents based on their structural similarity to known drugs. The target genes related to MN were characterized using the GeneCards database (https://www.genecards.org). BYHWD-related therapeutic targets for MN were graphically represented using the VennDiagram package in R Studio (4.2.1), and the intersecting genes were illustrated the Venny 2.1.0 online tool (https://bioinfogp.cnb.csic.es/tools/venny). Furthermore, Cytoscape (version 3.9.1) was used to generate and display the BYHWD ingredient-gene network.

Gene Ontology (GO) Analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis

GO and KEGG enrichment analyses of BYHWD-associated genes linked to MN were conducted using R software (version 4.2.0) in combination with several packages, including “org.Hs.eg.db,” “ggplot2,” “clusterProfiler” and “enrichplot.” GO analysis covered three domains: Biological Process (BP), Molecular Function (MF), and Cellular Component (CC). From the resulting biological processes, the five most significant terms with adjusted P < 0.05 were selected for interpretation. For KEGG enrichment analysis, the ten pathways with the highest significance were identified for in-depth examination.

Construction of the Bioactive Compound-Target-Pathway Network of BYHWD

Using Cytoscape software (version 3.9.1), a bioactive compound–target–pathway network of BYHWD was generated by integrating 41 active constituents identified from the preceding analysis with their 29 corresponding target genes and 20 related pathways. Network representation was constructed according to the betweenness centrality (BC) values calculated using the CytoNCA plugin.

Evaluation of the Diagnostic Value of Core Target Genes of BYHWD

The clinical diagnostic performance of the potential core targets of BYHWD in MN was evaluated using receiver operating characteristic (ROC) curves. The GEO database (https://www.ncbi.nlm.nih.gov/gds/?term=) provided expression data for the core targets (AKT1, IL-1β, IL6, INS, MMP9, PTGS2, and TNF) via the dataset GSE104948. ROC curves were performed utilizing the “pROC” package in R software (4.2.1). The diagnostic value of each target gene for MN was evaluated by calculating the area under the ROC curve (AUC), with an AUC exceeding 0.80 considered indicative of good diagnostic performance.

Molecular Docking

The two-dimensional structures of the active compounds in BYHWD were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) and were used as ligand files for molecular docking. The protein structure files of ALB, IL-6, TNF, and AKT1 were downloaded from the Protein Data Bank (PDB) online repository (https://www.rcsb.org). Molecular docking was performed using AutoDock Vina software, and docking results between BYHWD active compounds and target proteins were visualized using Discovery Studio software.

Preparation of BYHWD

BYHWD primarily consists of the following medicinal herbs: Astragali Radix (Huangqi, 60 g), Radix Paeoniae Rubra (Chishao, 18 g), Rhizoma Ligustici Wallichii (9 g), Angelica sinensis (18 g), Pheretima aspergillum (9 g), Amygdalus persica (9 g), and Carthamus tinctorius (9 g).²⁶ All herbs were procured from Jiaxing Hospital of Traditional Chinese Medicine. The constituent herbs were weighed according to the prescribed dosage, mixed, and soaked in distilled water for 30 minutes. Subsequently, distilled water was added to eight times the original volume and the mixture was boiled for 1 h to extract the active compounds. The extraction procedure was repeated once. The combined extracts were centrifuged at 6000 × g for 20 min, and the supernatant was concentrated under reduced pressure using a rotary evaporator (water bath concentration) to a final concentration of 1 g/mL, expressed as the total dry weight of crude herbs per milliliter of decoction. The prepared BYHWD decoction was stored at −20°C until further use.

Cell Culture and Treatment

The immortalized mouse podocyte cell line, MPC-5, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MPC-5 cells were cultured in RPMI-1640 medium (Gibco, New, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Waltham, USA), 100 U/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco). Cells were maintained at 33°C with 10 U/mL interferon-γ (IFN-γ; Peprotech, Rocky Hill, USA) to preserve the undifferentiated state. For differentiation, cells were incubated at 37°C and cultured without IFN-γ for 10–14 days. Differentiated podocytes were used in subsequent experiments.

To establish an in vitro model of C5b-9-mediated podocyte injury, zymosan-activated serum (ZAS)-induced injury model in MPC-5 podocytes was established as follows: 0.15 mol/L sodium chloride was mixed with 1% zymosan and boiled in double-distilled water for 1 hour. The mixture was cooled to 25 °C and centrifuged at 4000 rpm for 30 minutes, after which the supernatant was discarded, and the precipitate retained for further use. According to previously reported methods, MPC-5 cells were incubated at 37 °C for 1 hour in culture medium containing 10% ZAS.^27,28 When damage-induced morphological changes, such as cell shrinkage, rounding, and reduced protrusions, were observed under a microscope (Thermo Fisher Scientific), this indicated successful establishment of the ZAS model. Besides, differentiated MPC-5 cells were maintained in DMEM with 5% CO₂ at 37 °C for 14 consecutive days before receiving various concentrations (0–100 mg/L) of BYHWD for 24 hours.

For in vitro studies, differentiated MPC-5 podocytes were divided into two experimental sets according to their respective validation purposes. To investigate the effects of BYHWD on ZAS-induced model, cells were assigned to the following groups: control group, ZAS-induced group (10% ZAS, 24 h), ZAS + BYHWD-L group (10% ZAS + 1 mg/mL BYHWD, 24 h), ZAS + BYHWD-H group (10% ZAS + 10 mg/mL BYHWD, 24 h).

To elucidate the effect of BYHWD on the advanced glycation end products (AGE)/ receptor for advanced glycation end products (RAGE) signaling pathway in ZAS-induced podocytes, MPC-5 cells were divided into the following groups: control group, ZAS-induced group (10% ZAS, 24 h); ZAS + BYHWD-H group (10% ZAS + 10 mg/mL BYHWD, 24 h); and ZAS + BYHWD-H + AGE-BSA group (10% ZAS + 10 mg/mL BYHWD + 200 μg/mL AGE-BSA, AGE/RAGE pathway agonist, ab51995, Abcam, Cambridge, UK, 24 h).

In addition, the conditionally immortalized human podocyte cell line AB8/13, purchased from ATCC, was cultured as described elsewhere.²⁹ Briefly, AB8/13 podocytes were cultured at 33°C to promote proliferation and at 37°C for 14–16 days to induce differentiation. When the podocytes reached approximately 80% confluence, the cells were exposed to 100 nmol/L Angiotensin II (Ang II, Sigma, Darmstadt, Germany) for 24 h to establish Ang II–induced MN cell models.³⁰ The subsequent treatment conditions for the differentiated AB8/13 podocytes were the same as those for MPC-5 cells.

Similarly, in vitro studies, differentiated AB8/13 podocytes were divided into two groups according to the experimental purpose. Group One: Control group; Ang II–induced group (100 nmol/L Ang II, 24 hours); Ang II + BYHWD-L group (100 nmol/L Ang II + 1 mg/mL BYHWD, 24 hours); Ang II + BYHWD-H group (100 nmol/L Ang II + 10 mg/mL BYHWD, 24 hours). Another group: Control group; Ang II–induced group (100 nmol/L Ang II, 24 hours); Ang II + BYHWD-H group (100 nmol/L Ang II + 10 mg/mL BYHWD, 24 hours); and Ang II + BYHWD-H + AGE-BSA group (100 nmol/L Ang II + 10 mg/mL BYHWD + 200 μg/mL AGE-BSA).

All treatments were performed for 24 h. The research protocol was approved by the Ethics Committee of Jiaxing University (approval no. JUMC2025-044). All experiments and procedures were performed in accordance with the Declaration of Helsinki (revised in 2013).

Cell Proliferation Activity Detection

The proliferative capacity of podocytes was assessed using the Cell Counting Kit-8 (CCK-8) assay (BioSharp, Hefei, China). MPC-5 cells and AB8/13 cells at 80% confluence were cultured in DMEM for 24 h, then treated with a gradient of BYHWD concentrations (0, 0.1, 0.5, 1, 5, 10, 50, and 100 mg/mL) for an additional 24 h. According to the manufacturer’s instructions, podocytes subjected to different treatments were seeded into 96-well plates at a density of 5 × 10³ cells per well and incubated at room temperature for 24 hours. Subsequently, 10 μL of CCK-8 solution was added to each well, followed by incubation at 37°C for 2 h. During this process, wells with medium only were used as blank controls to eliminate interference from the absorbance of the culture medium. Optical density (OD) was measured at 450 nm using a microplate reader (Thermo Fisher Scientific). Changes in podocyte proliferation were evaluated based on OD values. All experiments were performed in triplicate.

Western Blot

Podocytes were seeded into six-well plates and treated with BYHWD alone or in combination with an AGE/RAGE pathway agonist for a specified duration. Following treatment, the cells were harvested and washed with phosphate-buffered saline (PBS). Total protein was extracted from podocytes using RIPA lysis buffer (KeyGEN, Nanjing, China) supplemented with protease inhibitors, and protein concentrations were determined using a bovine serum albumin (BSA) protein assay kit (Beyotime, Shanghai, China). Equal amounts (30 μg) of protein samples were separated using 10% SDS-PAGE, transferred to PVDF membranes (Vazyme, Nanjing, China), and blocked with skim milk. The Membranes were incubated overnight at 4 °C with primary antibodies against RAGE and GAPDH. After three washes with TBST (10 minutes each), the membranes were incubated at room temperature for 1 hour with the HRP-conjugated secondary antibodies (1:10,000, 31460, Thermo Fisher Scientific). Protein bands were visualized using an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific), and band intensities were quantified using the ImageJ software. The antibodies used in this experiment were as follows: primary antibodies against RAGE (1:1000, PA1-075, Invitrogen, Carlsbad, CA, USA) and GAPDH (1:50,000, 4A9L6, Invitrogen).

ELISA Detection of Inflammatory Factors

According to the manufacturer’s protocol, ELISA was used to determine the levels of TNF-α, IL-1β, IL-6, C5b-9, and AGE in supernates from podocytes. Their performance characteristics are as follows: Mouse TNF-α (bsk12002, Bioss, Beijing, China) exhibited a linear range of 15.6–1000 pg/mL and detection limit of 7 pg/mL; Mouse IL-β (bsk12004, Bioss) exhibited a linear range of 7.8–500 pg/mL and detection limit of 4 pg/mL; Mouse IL-6 (bsk12004, Bioss) demonstrated 7.8–500 pg/mL linear range and 4 pg/mL detection limit; Mouse C5b-9 (MBS261074, MyBioSource,California, USA) showed 0.312–20 ng/mL linear range and 0.06 ng/mL detection limit; AGE (a general-purpose reagent kit, ab238539, Abcam, Cambridge, USA) showed 0.36–100 µg/mL linear range and 0.5 µg/mL detection limit.

Human TNF-α (bsk11014, Bioss) exhibited a linear range of 15.6–1,000 pg/mL and detection limit of 7 pg/mL; Human IL-β (E-EL-H0149, Elabscience, Wuhan, China) exhibited a linear range of 7.81–500 pg/mL and detection limit of 4.69 pg/mL; Human IL-6 (bsk11007, Bioss) demonstrated 4–250 pg/mL linearity and 2 pg/mL detection limit; Human C5b-9 (MBS2516230, MyBioSource) showed 1.563–100 ng/mL linear range and 100 ng/mL detection limit. All three kits maintained both intra- and inter-assay coefficients of variation below 10%, as per manufacturer’s specifications.

Statistical Analysis

Each experiment was performed at least thrice. All data were statistically analyzed using GraphPad Prism software (version 8.0) and are presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with the post-hoc Tukey’s test was used to compare multiple groups. Prior to conducting the ANOVA analyses, we formally tested for normality using the Shapiro–Wilk test. Statistical significance was set at P < 0.05.

Results

Enrichment and Signaling Pathway Analysis of MN-Related Target Genes of BYHWD

Based on the GeneCards database, 2,816 genes associated with MN were identified. For potential drug targets of BYHWD, 667 genes were extracted from the TCMSP database. The overlap between MN-associated genes and BYHWD’s predicted targets of BYHWD was illustrated using the Venny 2.1 online tool, revealing 230 common genes, as presented in Figure 1A. Subsequently, the top 60 genes among the 230 intersecting genes, ranked by node degree, were visualized (Figure 1B). Cytoscape software (version 3.9.1) was used to visualize the top 30 genes based on their degree ranking (Figure 1C). GO and KEGG enrichment analyses were conducted for 230 intersecting genes (Figure 1D and E). The pathogenesis of MN involves a complex interplay of aberrant immune cell proliferation and activation, cell cycle dysregulation, and altered activity of multiple protein kinases.^31–33 In this study, GO enrichment analysis indicated that these genes were significantly associated with biological processes, including immune cell proliferation and activation, regulation of the cell cycle, protein kinase activity, and signal transduction (Figure 1D). KEGG pathway analysis identified 10 enriched pathways, including the p53 signaling pathway and AGE-RAGE pathway (Figure 1E), which are involved in regulating podocyte injury and glomerular inflammatory activation.^34–36 These results indicate that BYHWD has the potential to confer therapeutic benefits in MN by regulating these signaling pathways. Furthermore, a topological network of “formula-compound-small molecule” was constructed to visualize the potential active components of the individual herbs within the BYHWD formula (Supplementary Figure 1). We selected seven herbal components of BYHWD and constructed a network map based on 187 active small molecules.

Figure 1.

Target genes of BYHWD in MN. (A) With the aid of the TCMSP database and the GeneCards database, Venn diagram illustrating the overlapping genes between the targets of BYHWD and MN-associated targets. (B) Node ranking plot of the top 60 overlapping genes shared by BYHWD and MN. (C) Top 30 overlapping genes ranked by degree centrality, indicating their relative importance in the BYHWD-MN interaction network. (D) GO enrichment analysis revealed the top five biological processes (BP), cellular components (CC), and molecular functions (MF). (E) KEGG pathway analysis identified the top ten enriched pathways using R 4.2.1, with the number of genes and pathway categories shown.

Abbreviations: BYHWD, Buyang Huanwu Decoction; MN, membranous nephropathy; TCMSP, Traditional Chinese Medicine Systems Pharmacology; GO, Gene Ontology; KEGG, KyotoEncyclopedia of Genes and Genomes; BP, Biological Process; CC, Cellular Component; MF, Molecular Function; AGE, Advanced Glycation End products; RAGE, Receptor for Advanced Glycation End products.

Network Pharmacology Map of Active Compounds, Targets, and Pathways of BYHWD

We constructed a network illustrating the relationships between BYHWD’s active constituents of BYHWD, their targets, and relevant signaling pathways (Figure 2), enabling the exploration of molecular targets and affected pathways. As depicted in Figure 2, the complex “active compound-target-pathway” network illustrates the interactions among 41 active compounds of BYHWD, 29 target genes, and 20 signaling pathways, with a clear one-to-one correspondence. This network consisted of 90 nodes, indicating that the active compounds of BYHWD may regulate intracellular signaling pathways by directly or indirectly targeting specific genes.

Figure 2.

“Active compound-target-pathway” network of BYHWD. The “active component-target-pathway” network of BYHWD was constructed using Cytoscape 3.9.1 software. Yellow rectangles represent the key active compounds of BYHWD; blue diamonds denote their corresponding target genes; purple hexagons indicate the signaling pathways regulated by these targets.

Diagnostic Value of BYHWD Core Targets in MN

ROC curve analysis was conducted to evaluate the predictive value of BYHWD’s seven key target genes of BYHWD (AKT1, IL-1β, IL6, INS, MMP9, PTGS2, and TNF) for MN (Figure 3A–G), evaluating. Among these, IL-1β exhibited a peak AUC value of 0.969 (Figure 3B), indicating its superior predictive performance in disease diagnosis. The AUC values for AKT1, IL6, INS, MMP9, and TNF were approximately 0.7, reflecting a moderate diagnostic value and suggesting their potential utility as biomarkers for assessing MN progression. Conversely, PTGS2 showed an AUC of only 0.620, indicating a limited predictive significance for MN diagnosis.

Figure 3.

ROC curve analysis of the core targets of BYHWD for diagnostic evaluation in MN. (A–G) Receiver Operating Characteristic (ROC) curves were plotted to assess the diagnostic performance of core targets regulated by BYHWD in MN. ROC curves of the core targets of BYHWD. ROC curves for AKT1 (A), IL-1β (B), IL6 (C), INS (D), MMP9 (E), PTGS2 (F), and TNF (G). BYHWD, Buyang Huanwu Decoction; MN, membranous nephropathy.

Molecular Docking of the Key Components and Targets of BYHWD

To further validate the molecular mechanisms by which BYHWD intervenes in MN, molecular docking was performed using cetylic acid, β-sitosterol, quercetin, stigmasterol, and myristic acid as ligands, with the key target proteins ALB, IL-6, TNF, and AKT1 as receptors to characterize the interaction patterns between bioactive compounds and targets. Lower binding energies indicate stronger ligand-receptor interactions and higher stability of the complex. Given that ALB is primarily localized to the cell membrane and lacks broad significance as a drug target, the strongest docking interactions between each compound and IL-6, TNF, or AKT1 were selected for visualization. The findings demonstrated that cetylic acid, stigmasterol, and myristic acid exhibited strong interactions with AKT1, and that cetylic acid (−7.269 kcal/mol) and stigmasterol (−7.030 kcal/mol) had lower binding energies than myristic acid (−4.508 kcal/mol); β-sitosterol showed a strong interaction with IL-6, exhibiting a binding energy of −7.037 kcal/mol; and quercetin showed the strongest interaction with TNF, with a binding energy of −7.060 kcal/mol (Figure 4A). Hydrogen bonds were observed between the cetylic acid and the key active-site residues Gln43, Gln47, and Arg41 of AKT-1. However, an unfavorable acceptor-acceptor interaction was identified with Gln47, which likely weakened its overall binding affinity. Additionally, an alkyl interaction was identified between cetylic acid and Ala50 of AKT-1 (Figure 4B). Stigmasterol formed alkyl and π-alkyl interactions with Pro139, Tyr97, Pro65 and Leu147 of IL-6 (Figure 4C). In addition, π-alkyl interactions were observed between quercetin and Ala50 and Ala38 in TNF. Multiple hydrogen bonds mediated the binding of quercetin to TNF, whereas the unfavorable interaction with Leu37 might have reduced its effectiveness (Figure 4D). Moreover, β-sitosterol displayed π-sigma interactions with Trp11 of AKT1, and multiple hydrogen bonds mediated the binding of myristic acid to AKT1 (Figure 4E and F). Based on binding energy values, cetylic acid, β-sitosterol, quercetin, and stigmasterol can form stable complexes with their respective target proteins. These molecular docking results further substantiated the predictions made by network pharmacology.

Figure 4.

Molecular docking between key active compounds of BYHWD and critical target proteins. (A) Heatmap of binding affinities between selected active compounds (cetylic acid, stigmasterol, quercetin, β-sitosterol, and myristic acid) and target proteins IL-6, TNF, and AKT1. (B) Molecular docking model of Cetylic Acid with AKT1, binding energy −7.269 kcal/mol. (C) Molecular docking model of Stigmasterol with IL-6, binding energy −7.030 kcal/mol. (D) Molecular docking model of Quercetin with TNF, binding energy −7.060 kcal/mol. (E) Molecular docking model of β-Sitosterol with AKT1, binding energy −7.037 kcal/mol. (F) Molecular docking model of Myristic Acid with AKT1, binding energy −4.508 kcal/mol.

BYHWD Relieves Podocyte Damage

To assess how BYHWD protected podocytes from MN-induced injury, a mouse podocyte model was created in vitro using ZAS treatment. Initially, MPC-5 cells (mouse podocytes) were treated with BYHWD at 0, 0.1, 0.5, 1, 5, 10, 50, and 100 mg/mL for 48 h. CCK-8 assay was performed to assess the viability of the cells. As shown in Figure 5A, BYHWD exhibited marked cytotoxicity at concentrations of 50 and 100 mg/mL. Therefore, 1 mg/mL and 10 mg/mL were selected for subsequent experiments as the low-(BYHWD-L) and high-dose (BYHWD-H) groups, respectively, for intervention following ZAS-induced podocyte injury. As illustrated in Figure 5B, ZAS exposure significantly impaired cell viability relative to that in the control group (P < 0.001). However, treatment with BYHWD notably restored cell proliferation, with the high-dose group demonstrating superior efficacy compared to the low-dose group (P < 0.01).

Figure 5.

BYHWD alleviates injured podocytes. (A) CCK-8 assay evaluating cell proliferation in MPC-5 cells and AB8/13 cells treated with various concentrations of BYHWD for 48 hours. (B) Cell viability was assessed using the CCK-8 assay following ZAS treatment and Ang II stimulation, with subsequent intervention by low- and high-dose BYHWD. NS represents no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001 vs 0 mg/mL or Control group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 vs ZAS/Ang II group. n = 3 independently biological replicates.

Abbreviations: BYHWD, Buyang Huanwu Decoction; MN, membranous nephropathy; CCK-8, cell counting kit-8; ZAS, zymosan-activated serum; Ang, Angiotensin II.

AB8/13 podocytes were used for validation. BYHWD showed significant cytotoxicity at concentrations of 50 mg/mL and 100 mg/mL (Figure 5A). For subsequent experiments, 1 mg/mL and 10 mg/mL were selected as the low-(BYHWD-L) and high-dose (BYHWD-H) groups, respectively, following Ang II–induced injury. Ang II significantly reduced cell activity compared to that in the control (P < 0.001), but BYHWD treatment restored proliferation, with the high-dose group showing greater efficacy than the low-dose group (Figure 5B, P < 0.01).

BYHWD Inhibits the Expression of Pro-Inflammatory Factors and the Deposition of C5b-9 in Injured Podocytes

The overexpression of pro-inflammatory factors and deposition of C5b-9 are key contributors to podocyte injury and are central to the advancement of MN. The role of BYHWD was examined by measuring TNF-α, IL-1β, IL-6, and C5b-9 levels in podocytes using ELISA under different treatment conditions. The results showed that ZAS or Ang II treatment led to a significant increase in TNF-α, IL-1β, IL-6, and C5b-9 expression relative to the control group (P < 0.001, Figure 6A–D). In contrast, both BYHWD-L and BYHWD-H treatments effectively attenuated the levels of pro-inflammatory factors and C5b-9 in the two MN cell models (P < 0.05, Figure 6A–D), achieving a stronger inhibitory effect than the low-dose group (Figure 6A–D). These results indicated that BYHWD markedly alleviated the inflammatory response, inhibited complement activation in damaged podocytes, and exerted a dose-dependent effect on the MN cell model.

Figure 6.

BYHWD attenuates inflammatory responses and complement activation in injured podocytes. (A) ELISA quantification of TNF-α levels in MPC-5 cells and AB8/13 cells from different groups. (B) ELISA quantification of IL-1β levels in MPC-5 cells and AB8/13 cells from different groups. (C) ELISA quantification of IL-6 levels in MPC-5 cells and AB8/13 cells from different groups. (D) ELISA quantification of C5b-9 levels in MPC-5 cells and AB8/13 cells from different groups. ***P < 0.001 vs Control group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 vs ZAS/Ang II group. n = 3 independently biological replicates.

Abbreviations: BYHWD, Buyang Huanwu Decoction; MN, membranous nephropathy; ZAS, zymosan-activated serum; Ang, Angiotensin II; ELISA, enzyme-linked immunosorbent assay; TNF-α, tumor necrosis factor α; IL-1β, interleukin 1β; IL-6, interleukin 6; C5b-9, complement membrane attack complex, MAC.

BYHWD Inhibits AGE/RAGE Signaling Pathway

Numerous studies have demonstrated that the AGE/RAGE signaling pathway is closely associated with podocyte injury and glomerular disease pathogenesis.^37,38 Furthermore, activation of the AGE/RAGE signaling pathway is positively correlated with the inflammatory response during chronic inflammation.³⁹ Meanwhile, our result also indicated that the AGE/RAGE signaling pathway is a potential pathway for BYHWD to intervene in MN (Figure 1E). Therefore, we propose that BYHWD exerts its therapeutic effects in MN by modulating the AGE/RAGE pathway. The results showed that ZAS and Ang II treatment significantly elevated AGE levels compared with the control group, whereas BYHWD at 10 mg/mL attenuated this increase, reducing AGE levels (P < 0.01, Figure 7A). Consistent with AGE, RAGE protein expression was markedly upregulated following ZAS treatment and Ang II treatment but was suppressed upon high-dose BYHWD intervention (P < 0.001, Figure 7B). These findings imply that BYHWD suppresses activation of the AGE/RAGE signaling pathway. To corroborate this mechanism, we performed a rescue experiment by introducing AGE-BSA, an AGE/RAGE pathway agonist, following BYHWD treatment. As illustrated in Figure 7C, AGE-BSA abolished the BYHWD-induced inhibition of the AGE/RAGE pathway and restored RAGE protein expression (P < 0.01). Collectively, these results indicate that the therapeutic effect of BYHWD on the MN cell model may involve the modulation of the AGE/RAGE pathway.

Figure 7.

BYHWD inhibits the AGE/RAGE signaling pathway. (A) ELISA analysis of AGE expression levels in MPC-5 cells and AB8/13 cells from the control group, ZAS-/Ang II-treated group, and ZAS/Ang II + high-dose BYHWD (BYHWD-H) treatment group. (B) Western blot analysis of RAGE protein expression in MPC-5 cells and AB8/13 cells across different treatment groups. (C) Western blot analysis of RAGE expression in MPC-5 cells and AB8/13 cells from the control group, ZAS-/Ang II–induced group, high-dose BYHWD treatment group after ZAS/Ang II induction, and the group treated with an AGE/RAGE pathway agonist. ***P < 0.001 vs Control group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 vs ZAS/Ang II group; ^##P < 0.01 vs ZAS/Ang II+BYHWT-H group. n = 3 independently biological replicates.

Abbreviations: BYHWD, Buyang Huanwu Decoction; MN, membranous nephropathy; ZAS, zymosan-activated serum; Ang, Angiotensin II; ELISA, enzyme-linked immunosorbent assay; AGE, Advanced Glycation End products; RAGE, Receptor for Advanced Glycation End products.

BYHWD Mediates AGE/RAGE Signaling Pathway to Alleviate Podocyte Injury, Inflammatory Response and Complement Activation

Finally, we verified whether BYHWD alleviated podocyte injury, excessive inflammatory responses, and complement activation by modulating the AGE/RAGE pathway in two damaged podocyte models. The proliferative capacity of podocytes in each group was measured using the CCK-8 assay (Figure 8A). Consistent with previous findings, BYHWD treatment restored the impaired proliferative activity of ZAS-induced podocytes and Ang II–induced podocytes, thereby promoting cell growth (P < 0.001). However, co-treatment with AGE-BSA abrogated the protective effect of BYHWD, significantly suppressing podocytes proliferation (P < 0.01). As depicted in Figure 8B–E, ZAS stimulation and Ang II stimulation markedly increased TNF-α, IL-1β, IL-6, and C5b-9 levels in podocytes compared with the control group (P < 0.001). In contrast, BYHWD administration attenuated the ZAS- and Ang II–induced inflammatory responses and complement activation, leading to decreased levels of these pro-inflammatory cytokines and C5b-9 (P < 0.001). Notably, consistent with the proliferation assay results, the addition of AGE-BSA negated the inhibitory effect of BYHWD on inflammation and complement activation, resulting in the elevated production of pro-inflammatory cytokines and C5b-9 (P < 0.05). Overall, these observations suggest that the protective effect of BYHWD on podocyte injury, inflammatory response, and complement activation in the MN cell model may be achieved through inhibition of AGE/RAGE pathway activation.

Figure 8.

BYHWD attenuates podocyte injury and inflammation by inhibiting activation of the AGE/RAGE signaling pathway. (A) CCK-8 assay to assess the proliferation of MPC-5 cells and AB8/13 cells in the control, ZAS-/Ang II–induced, ZAS/Ang II + BYHWD-H, and ZAS/Ang II + BYHWD-H + AGE-BSA groups. ELISA quantification of TNF-α (B), IL-1β (C), IL-6 (D), and C5b-9 (E) levels in MPC-5 cells and AB8/13 cells across the four treatment groups described above. ***P < 0.001 vs Control group; ^&&&P < 0.001 vs ZAS/Ang II group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs ZAS/Ang II+BYHWT-H group. n = 3 independently biological replicates.

Abbreviations: BYHWD, Buyang Huanwu Decoction; MN, membranous nephropathy; ZAS, zymosan-activated serum; Ang, Angiotensin II; CCK-8, cell counting kit-8; ELISA, enzyme-linked immunosorbent assay; AGE, Advanced Glycation End products; RAGE, Receptor for Advanced Glycation End products; TNF-α, tumor necrosis factor α; IL-1β, interleukin 1β; IL-6, interleukin 6; C5b-9, complement membrane attack complex, MAC.

Discussion

MN has a high and rising incidence in China, imposing a significant public health burden.⁴⁰ Current treatments are often limited by toxicity incomplete remission, highlighting the need for novel therapies.⁴¹ BYHWD, a classical TCM formulation, is known to ameliorate the development of multiple kidney diseases. However, its therapeutic potential in MN remains unclear. This study suggests that BYHWD promotes cell proliferation and reduces the expression of proinflammatory factors in ZAS-induced MN model cells (MPC-5 cells) and Ang II–induced MN model cells (AB8/13 cells). By integrating network pharmacology and two cell-based MN models, we discovered that the AGE/RAGE signaling pathway might be a potential mediator of BYHWD’s protective effects of BYHWD.

BYHWD is a traditional Chinese medicinal formula traditionally applied in the management of stroke.⁴² Previous studies have highlighted the renoprotective effects of BYHWD. For instance, BYHWD has been reported to significantly suppress monocyte/macrophage infiltration, thereby alleviating glomerulosclerosis and tubulointerstitial injury in a hypertension-induced renal damage model.²⁸ Moreover, calycosin-7-glucoside (CG), a principal active component of BYHWD, has been shown to attenuate inflammation and fibrotic responses in mesangial cells exposed to high glucose, leading to improved renal function in diabetic nephropathy mouse models.¹³ Additionally, BYHWD was found ameliorated kidney injury in diabetic nephropathy by modulating lipid metabolism and reducing triglyceride levels.¹⁴ These findings collectively suggest that BYHWD alleviates renal injury through multiple mechanisms, including anti-inflammatory, anti-fibrotic, and metabolic regulatory pathways, indicating its potential for improving kidney function. In this study, we demonstrated that BYHWD treatment enhanced the proliferative capacity of ZAS-treated MPC-5 cells and Ang II-treated AB8/13 cells. However, the pharmacological mechanisms by which BYHWD exerts the renoprotective effects of BYHWD in MN require further investigation.

MN is an autoimmune glomerular pathology characterized by membrane attack complex accumulation (C5b-9) in podocytes, which significantly contributes to podocyte damage.^40,43 Numerous studies have demonstrated that inflammatory pathways are excessively activated in both patients with MN and animal models.⁴⁴ Consistently, our findings showed that ZAS and Ang II stimulation markedly elevated the production of pro-inflammatory cytokines and C5b-9. Furthermore, we observed that BYHWD substantially lowered the expression of TNF-α, IL-6, IL-1β, and C5b-9 in the MN cell models. Our results provide evidence that BYHWD protects damaged podocytes by suppressing ZAS- and Ang II–induced inflammatory responses and C5b-9 accumulation.

Network pharmacology, a multidisciplinary field integrating computational science and systems biology, serves as a powerful method to investigate the action mechanisms of traditional medicines and facilitating novel drug discovery.^45,46 For instance, some research has discovered through network pharmacology that BYHWD regulates multiple targets in chronic cerebral ischemia.⁴⁷ To explore the mechanistic basis of BYHWD in MN treatment, we implemented network pharmacology and constructed a “compound–target–pathway” network topology map. GO and KEGG analyses revealed that the active ingredients in BYHWD potentially participate in modulating the AGE/RAGE signaling pathway. Advanced glycation end products (AGEs) and their receptors (RAGE) are widely expressed on the surface of various cell types.⁴⁸ Crosstalk between AGEs and RAGE is known to elevate intracellular oxidative stress and activate multiple signaling cascades, such as the JAK-STAT and PI3K-AKT pathways, ultimately leading to increased apoptosis.^49–51 Emerging evidence suggests that AGE–RAGE interaction plays a pivotal role in triggering inflammatory reactions.^39,52 In kidney-related diseases, the AGE/RAGE signaling axis has been found to be aberrantly activated. For example, buildup and engagement of AGE and RAGE in glomerular mesangial cells is associated with the pathogenesis of diabetic nephropathy.⁵³ Previous work established that DPHC, a natural bioactive molecule obtained from edible marine brown algae, exerts renoprotective and anti-glycation effects by preventing AGE generation and its interaction with collagen in methylglyoxal (MGO)-stimulated mouse mesangial cells.⁵⁴ In addition, RAGE is up-regulated in most cell types following cellular damage, hypoxic exposure, or inflammatory stimulation, particularly in the kidney, including proximal tubular cells,⁵⁵ podocytes,⁵⁶ and mesangial cells.⁵⁷ Most studies have demonstrated that the deletion of RAGE in mice can protect the kidneys by inhibiting oxidative stress and pro-fibrotic pathways, whereas RAGE overexpression accelerates nephropathy in mouse models.^58,59 Moreover, the AGE-RAGE axis has been shown to induce endoplasmic reticulum (ER) stress through disruption of ER homeostasis, thereby exacerbating renal injury in diabetic nephropathy.⁶⁰ Collectively, multiple studies have confirmed that AGE/RAGE pathway activation promotes inflammatory responses in damaged podocytes.

Based on in vitro experiments with two MN cell models, we demonstrated that ZAS/Ang II treatment induced hyperactivation of the AGE/RAGE signaling pathway. Moreover, we observed that treatment with BYHWD effectively inhibited the AGE/RAGE pathway activation. Notably, the protective effects of BYHWD on damaged podocytes, including enhanced cell proliferation and inhibition of the expression of pro-inflammatory factors and C5b-9, were reversed after activation of the AGE/RAGE pathway. These results suggest that BYHWD may alleviate podocyte injury and inflammation by suppressing AGE/RAGE pathway activation. Thus, BYHWD may serve as a targeted intervention for the AGE-RAGE axis, providing a preliminary experimental basis for subsequent preclinical animal studies and small-scale clinical explorations (eg, in patients with MN characterized by AGE/RAGE accumulation and complement activation).

However, there are still limitations in this study between the in vitro mechanism research results and the therapeutic potential: (1) the lack of in vivo validation; we will validate the findings in the MN animal model to confirm in vivo efficacy and pathway regulation; and (2) the focus on the AGE-RAGE pathway without fully exploring other top-ranked pathways from the network analysis. We will explore other possible top-ranked pathways in the future. (3) While relevant for inflammation, the ZAS model does not replicate the specific autoimmune etiology of MN. We will use PLA2R-antibody to stimulate podocytes to simulate the classic model of MN and analyze our research results through small-scale clinical samples. (4) The precise molecular step of BYHWD’s intervention on the AGE-RAGE axis (eg, effects on downstream NF-κB signaling) remains uncharacterized. In the future, we will conduct an in-depth analysis of the role of the AGE-RAGE axis in BYHWD’s intervention on MN. (5) This work does not provide clinical evidence for BYHWD’s contribution of BYHWD in delaying MN advancement and enhancing prognosis in patients. Further research is necessary to confirm these observations and to determine their clinical applicability.

Conclusion

Taken together, our work demonstrates that BYHWD may improve podocyte injury and inhibit the levels of pro-inflammatory cytokines and C5b-9 in MN cell models via blockade of the AGE/RAGE pathway activation. These results lay a theoretical foundation for a deeper understanding of the molecular mechanism BYHWD’s therapeutic effects of BYHWD and provide a reference for its potential clinical application in the treatment of MN in the future.

Funding Statement

This work is supported by the grant from the Zhejiang Province Traditional Chinese Medicine Science and Techology Plan Project [Approval number: 2025ZL564].

Data Sharing Statement

All the results are presented in the article. Further inquiries can be directed to the corresponding authors.

Ethics Statement

The research protocol was approved by by the Ethics Committee of Jiaxing University (Approval No. JUMC2025-044). All experiments and procedures were performed according to the Declaration of Helsinki (as revised in 2013).

Disclosure

The authors report no conflicts of interest in this work.