Uncovering the mechanistic basis of Rheum palmatum L. (rhubarb) in the treatment of chronic kidney disease: an integrative approach using network pharmacology, molecular docking, and experimental validation

Abstract

Context

Rheum palmatum L. (Rhubarb) has shown potential in managing Chronic Kidney Disease (CKD) but its protective mechanisms remain unclear.

Objective

This study investigates rhubarb’s therapeutic effects and underlying mechanisms in CKD.

Materials and methods

High-performance liquid chromatography (HPLC) established a rhubarb fingerprint to ensure quality control. Network pharmacology and Mendelian randomization identified primary CKD therapeutic targets. Inflammation, oxidative stress, and renal performance were assessed through ELISAs and biochemical tests. Renal structure and fibrosis were examined using hematoxylin-eosin and Masson staining. Protein expression related to fibrosis, apoptosis, and NF-κB pathway activity was measured via Western blotting. Discovery Studio 2019 (DS 2019) was used for molecular docking.

Results

HPLC fingerprinting confirmed high batch-to-batch consistency of rhubarb, identifying five key anthraquinones (aloe-emodin, rhein, emodin, chrysophanol, physcion) with similarity indices >0.91. Network pharmacology identified 19 active compounds targeting 2,597 CKD-related proteins, with 47 overlapping targets including IL6, TNF, TP53, CASP3, and IL1B as core nodes. MR analysis demonstrated a statistically significant causal association between TNF and CKD (OR = 1.02, p < 0.05), with positive trends for IL6 and CASP3. In CKD rat models, rhubarb significantly improved renal function by reducing blood urea nitrogen (BUN), serum creatinine (SCr), and uric acid (UA) levels (p < 0.05). Histopathology showed reduced glomerulosclerosis, tubular atrophy, interstitial fibrosis, and inflammatory infiltration after treatment. Rhubarb markedly decreased renal fibrosis markers including collagen I, collagen III, α-SMA, and TGF-β (p < 0.01). Pro-inflammatory cytokines IL-6, IL-1β, and TNF-α levels were significantly suppressed (p < 0.001). Antioxidant enzyme activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were restored, while lipid peroxidation (LPO) was reduced (p < 0.05). Rhubarb inhibited NF-κB pathway activation by lowering phosphorylated NF-κB and IκBα, and increasing total IκBα expression (p < 0.01). Apoptosis-related proteins showed upregulated Bcl-2 and downregulated Bax and cleaved caspase-3 expression (p < 0.05). Molecular docking confirmed strong binding affinities of rhubarb’s core compounds with key targets such as TNF and IL6, supporting their therapeutic roles.

Conclusion

Rhubarb significantly reduces renal impairment, fibrosis, inflammation, and apoptosis through the NF-κB pathway, supporting its traditional use and potential as an adjunct therapy for CKD.

Introduction

Chronic Kidney Disease (CKD) poses a substantial global health challenge, significantly increasing mortality rates worldwide (Wing-Shing Fung et al. 2024). Recent epidemiological data indicate a rising prevalence of CKD, primarily driven by risk factors such as diabetes, hypertension, and aging populations (Alanaeme et al. 2024; Scurt et al. 2024). CKD is characterized by a progressive decline in renal function, impaired sodium filtration, and dysregulated tubular reabsorption. It is also often accompanied by systemic inflammation and oxidative stress, which contribute to endothelial dysfunction, glomerular fibrosis, and mesangial expansion (Li et al. 2024; Reiss et al. 2024; Zhang et al. 2024). Despite advancements in medical research and therapeutic options, CKD frequently progresses to end-stage renal disease, necessitating dialysis or transplantation to sustain life. Consequently, current research efforts aim to develop more effective strategies to slow CKD progression.

Rhubarb (Rheum palmatum L.), a medicinal herb widely used in traditional Chinese medicine (TCM), has been used for centuries to treat various ailments, including digestive issues and inflammatory diseases (Gao et al. 2024; Sun et al. 2024). Recent pharmacological studies have identified several bioactive components in rhubarb, such as emodin and rhein, which exhibit nephroprotective effects (Luo et al. 2024). Additionally, emerging evidence suggests that rhubarb may modulate inflammatory responses, reduce oxidative stress, and ameliorate fibrotic processes in renal tissues, indicating its potential as an adjunct treatment for CKD (Wu et al. 2016; Che et al. 2024). These findings underscore the need for a deeper understanding of the underlying therapeutic mechanisms responsible for rhubarb’s beneficial effects.

Network pharmacology, a rapidly evolving field that integrates systems biology with multi-scale pharmacology, offers a powerful framework for deciphering the complex interactions between bioactive compounds and biological systems. This approach enables the exploration of drug-target interaction networks to identify potential therapeutic targets and underlying mechanistic pathways, making it particularly useful for studying traditional herbal medicines like rhubarb, which contains numerous active ingredients exhibiting diverse pharmacological activities.

Mendelian randomization (MR) is an analytical technique that uses genetic variants as instrumental variables (IVs) to estimate the causal effect of a risk factor on an outcome while controlling for confounding variables. By exploiting the natural randomization of genes at conception, this method simulates the conditions of a randomized controlled trial.

In this study, we employed MR analysis to explore the potential mechanisms and key targets of rhubarb in treating CKD from a genetic perspective. Additionally, we combined network pharmacology and molecular docking with experimental validation to elucidate the potential molecular mechanisms through which rhubarb mitigates CKD-related pathological effects and identify its potential pharmacological components (Figure 1).

Figure 1.

Experimental flowchart. This schematic depicts the comprehensive strategy to elucidate the bioactive components of rhubarb, their molecular targets, and the underlying mechanisms for CKD alleviation. Step 1: Rhubarb quality control and active component identification using high-performance liquid chromatography (HPLC) fingerprintin. Step 2: Network pharmacology analysis was applied to screen potential bioactive components, predict their targets, construct interaction networks (component-target, target-pathway), and perform enrichment analysis (GO, KEGG) to identify key targets and pathways implicated in CKD. Step 3: Experimental validation using relevant in vivo model to confirm the regulatory effects of rhubarb on these prioritized targets and pathways. Step 4: Molecular docking simulations conducted to validate and explore the binding interactions between key rhubarb components and critical targets identified in the preceding steps.

Materials and methods

Preparation of rhubarb extracts and high-performance liquid chromatography (HPLC)

Rhubarb used in this study was obtained from the Affiliated Hospital of Changchun University of Chinese Medicine. Rhubarb quality was assessed using HPLC, analysed extracts were prepared from rhubarb using standard procedures outlined in the Chinese Pharmacopoeia (Tang et al. 2021).

For the free anthraquinone assay, rhubarb was extracted with methanol, heated with refluxing for one hour, then additional methanol was added to compensate for weight loss, yielding the test sample. Creating the total anthraquinone test sample involved an extra step: the sample was treated with 8% hydrochloric acid and trichloromethane, heated and refluxed for one hour, followed by trichloromethane extraction repeated three times. The solvent was removed under vacuum until the sample was dry then the residue was dissolved in methanol to generate the final test sample. HPLC analysis was performed with a mobile phase consisting of water with 0.1% phosphoric acid aqueous solution (Solution A) and methanol (Solution B), column temperature maintained at 25 °C, a flow rate of 1 mL/min, and UV monitoring conducted at a wavelength of 254 nm.

Network analysis for identifying potential interactions between rhubarb active ingredients and CKD targets

Identification of chemical constituents of rhubarb was achieved through network pharmacology analysis following methodologies developed by Li et al. (Li 2021) with databases such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, https://tcmspw.com/tcmsp.php) and the Bioinformatics Analysis Tool for the Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM, http://bionet.ncpsb.org/batman-tcm/) (Tang et al. 2021). Selection criteria for rhubarb compounds included an oral bioavailability (OB) threshold of at least 30% and a drug-likeness (DL) index of no less than 0.18. Interactions between rhubarb components and target genes were visualized using the UniProt database (https://www.uniprot.org/) and Cytoscape 3.9.1 software (Peng et al. 2024; Yang et al. 2024).

Potential CKD targets were identified through GeneCards (https://www.genecards.org/), Online Mendelian Inheritance in Man (OMIM, https://www.omim.org/), and DrugBank (https://go.drugbank.com/) databases (Pu et al. 2024). Overlapping targets between rhubarb components and CKD were visualized using a Venn diagram generated by the Weishengxin online platform (http://www.bioinformatics.com.cn/) and further analyzed with Cytoscape. This comprehensive approach ensured a robust compilation of potential CKD-related targets, facilitating subsequent analyses.

Comprehensive protein–protein interaction (PPI) analysis and enrichment analysis

The PPI network was constructed using the STRING database (https://string-db.org/) with a medium confidence threshold of >0.4 and output visualized as a spectrogram (Peng and Zhou 2024). The Cytoscape CytoNCA tool was employed for network visualization and analysis of key targets.

Pathway enrichment analysis was conducted to identify biological pathways and gene ontologies associated with the common target genes using the Omic Share platform (https://www.omicshare.com/), the g: Profiler platform (http://biit.cs.ut.ee/gprofiler/gost), and R programming language-based analyses (Xu et al. 2020). Enrichment criteria included a p-value ≤ 0.05 and the species filter set to ‘Homo sapiens’.

Mendelian randomization (MR) statistical analysis

Data were sourced from the genome-wide association study (GWAS) database (https://gwas.mrcieu.ac.uk/). Single nucleotide polymorphisms (SNPs) strongly associated with exposure factors were selected as IVs for correlation analysis based on a p threshold of <5e − 8. Linkage disequilibrium (LD) was analyzed to ensure independence of genetic variations (r² < 0.001, kb = 10,000). Significant SNPs were chosen as IVs, replacing key targets, and extracted from the GWAS dataset to serve as outcome variables used to finalize the IVs. An F-statistic >10 was used to remove weak IVs to minimize bias and eliminate confounding factors. MR analysis of SNPs as IVs was based on three critical assumptions: 1. SNPs must be closely associated with the exposure factor; 2. SNPs are independent of confounding factors; 3. SNPs influence clinical outcomes solely through their relationships with exposure factors.

Heterogeneity in our analysis was evaluated using inverse-variance weighted (IVW) and MR-Egger tests, with a p-value > 0.05 indicating the absence of heterogeneity. To detect pleiotropy, the MR-Egger intercept test was applied, which demonstrates no pleiotropy among the IVs when the p-value exceeds 0.05. These findings confirm that our data were free of confounding factors while maintaining both independence and exclusivity, thereby reinforcing the robustness of our results. The analysis was conducted using the TwoSampleMR and MendelianRandomization packages in R version 4.4. Our research adhered to all relevant guidelines and protocols to ensure both methodological rigor and ethical compliance. Only aggregated data from existing studies were used, negating the need for additional ethical approvals.

Rhubarb extract preparation for animal experiments

Rhubarb was extracted by macerating in an 80% (v/v) water-ethanol solution at a ratio of 20:1 (solvent to herb weight). The mixture was refluxed with heating for 1 h, and the liquid was separated from the undissolved residue by filtration. The remaining solid was re-extracted with an equal volume of 80% ethanol under the same conditions then the filtrates were combined and concentrated using a rotary film evaporator (R-300, Büchi Labortechnik AG, Canton of St. Gallen, Switzerland) to remove the ethanol, resulting in a crude extract. Rhubarb residue was subsequently decocted in water, with an initial extraction for 2 h followed by a second extraction for 1.5 h. The decoctions were combined with the filtrates then the pooled sample was freeze-dried to yield the final product.

Animal experiments and preparation of kidney tissue homogenates

All animal experiments in this study adhered to the European Community guidelines (EEC Directive of 1986; 86/609/EEC) and were approved by the Experimental Animal Committee of Changchun University of Chinese Medicine (Approval No. 2022564). A total of 50 Sprague–Dawley rats, weighing between 150 and 200 grams, were sourced from the Animal Center of Jilin University. The rats underwent a 1-week acclimatization period before they were used in experiments. They were then randomly assigned to the control group, the CKD group, and three treatment groups receiving low, medium, and high doses of rhubarb extract (CKD + rhubarb). The CKD model was induced as previously described (Calderón-Garcidueñas et al. 2023; Yang et al. 2024) by administering a daily gavage of a suspension containing 200 mg/(kg·d) adenine (HY-B0152, MedChemExpress., NJ, US) for 28 days (Neven et al. 2020); the therapeutic dosage was determined using the allometric scaling method, which equates body surface area between humans and rats. Accordingly, the three groups of rhubarb-treated model rats received low (0.45 g/kg), medium (0.9 g/kg), and high (1.8 g/kg) doses of rhubarb extract (ROL, ROM, and ROH, respectively) via gavage over 5 weeks. Upon treatment completion, the successful establishment of the CKD model and the potential therapeutic effects of the rhubarb extract were assessed by measuring serum blood urea nitrogen (BUN, C013, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and serum creatinine (SCr, C011, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) levels.

Following treatment, rats were anesthetized with 1% sodium pentobarbital. Blood was collected via the abdominal aorta, and kidneys were excised aseptically. During the surgery, a midline abdominal incision was made, followed by meticulous dissection of the surrounding renal tissues and vasculature to ensure that kidney tissue integrity was maintained. Once the kidneys were fully detached, they were rapidly placed into a pre-chilled container filled with ice-cold saline to preserve their freshness and viability. Next, the excised kidneys were rinsed on ice to remove excess blood and tissue debris. Subsequently, the kidneys and blood were processed according to experimental requirements: the collected blood was placed in tubes without anticoagulants and centrifuged at 4 °C at 10,000 ×g for 10 min. The left kidney was fixed in 4% paraformaldehyde (P0099, Beyotime Biotechnology, Shanghai, China), dried, embedded in paraffin, and sectioned for histological and immunochemical analyses as described in section 2.8. The right kidney was promptly frozen in liquid nitrogen and processed for biochemical and protein analyses.

Frozen right kidney samples were accurately weighed, finely minced, and transferred to pre-chilled grinding tubes. Next, nine sample volumes of either normal saline (for biochemical analysis) or radioimmunoprecipitation assay (RIPA (P0013, Beyotime Biotechnology, Shanghai, China) buffer containing protease inhibitors (for protein analysis) were added, achieving a ratio between the sample weight in grams (g) and the total RIPA buffer volume added in milliliters (mL) of 1:9. Thereafter, samples were homogenized, centrifuged at 10,000 ×g for 20 min at 4 °C, and supernatants were collected for further analysis. Protein concentrations in the kidney homogenates were quantified using the bicinchoninic acid (BCA) assay with a BCA Protein Assay Kit (P0009, Beyotime Biotechnology, Shanghai, China), which is essential for subsequent biochemical or Western blot analyses.

Renal function testing

To evaluate the long-term therapeutic effects of rhubarb extract, urine samples were collected at the end of the treatment period from animals housed in metabolic cages (Oxylet Pro, Harvard Bioscience, Shanghai, China) for 24 h, during which all urine was collected. The samples were then centrifuged at 1,500 ×g for 20 min to obtain the supernatant. Urine analysis was performed using a urine protein assay kit (C035) and an N-acetyl-β-D-glucosaminidase (NAG, A031) assay kit (both from Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and a kidney injury molecule 1 (Kim-1) assay kit (PK683, Beyotime Biotechnology, Shanghai, China)) following the manufacturers’ instructions provided with the kits. After urine collection, blood samples were obtained via the abdominal aorta to assess the concentrations of blood urea nitrogen (BUN), serum creatinine (SCr), and uric acid (UA, BC1365, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).

Pathological morphological assessment

Kidneys were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, and deparaffinized using xylene. Tissue sections were stained with hematoxylin-eosin (HE, G1120) and Masson’s trichrome (G1340) (both from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Differentiation was achieved using a 1% hydrochloric acid-alcohol solution (G1861, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Stained sections were examined under a light microscope (Eclipse E100, Nikon Corp., Japan) to document histopathological changes and collagen fiber accumulation (Ding et al. 2021).

Inflammatory factor detection

Levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in rat renal tissue samples were measured using enzyme-linked immunosorbent assay (ELISA) kits (catalog numbers R6000B, RLB00, and RTA00, respectively; R&D Systems, MN, USA). Quantification was performed with an ELISA plate reader at 450 nm, according to the manufacturer’s instructions.

Biochemical analysis

Supernatants of renal tissue homogenates were prepared as described in section 2.6 for the control, CKD (model), and the three CKD + rhubarb experimental groups. The samples were then subjected to biochemical analyses to quantitatively assess superoxide dismutase (SOD, A001) and glutathione peroxidase (GSH-px, A005) activities and lipid peroxidation (LPO, A106) levels. All assays were performed using commercial assay kits (Nanjing Jiancheng Bioengineering Institute), following the manufacturer’s protocols.

Western blot analysis

Renal tissues were harvested from the control, CKD, and three CKD+rhubarb groups, processed for Western blot analysis, and protein concentrations were determined as described in Renal function testing. Samples containing 40 μg of total protein were loaded onto 12% SDS-PAGE gels for separation. Following electrophoresis, proteins were transferred onto polyvinylidene fluoride membranes (PVDF, 03010040001, Roche,. Basel, Switzerland) Membranes were blocked with 5% non-fat milk for 1 h then incubated overnight at 4 °C with primary antibody against TGF-β (1:1000), alpha-smooth muscle actin (α-SMA, 1:1000), Collagen I (1:1000), Collagen III (1:1000), Bcl-2 (1:1000), Bax (1:1000), Caspase 3 (1:1000), inducible nitric oxide synthase (iNOS, 1:1000), COX2 (1:1000), NF-κB (1:1000), p-NF-κB (1:1000), IκBα (1:1000), p-IκBα (1:1000). The membrane was subsequently incubated with a horseradish peroxidase-conjugated secondary antibody at a dilution of 1:5,000 for 1 h at room temperature. Protein bands were visualized using a chemiluminescence detection system and quantitative analysis of the bands was performed using Image J software (Yang et al. 2022). The following primary antibodies were used in this study: TGF-β (MA1-21595, Thermo Fisher Scientific Inc., MA, U.S.), α-SMA (14395, Proteintech Group, Inc., Wuhan, China), Collagen I (14695 Proteintech Group, Inc., Wuhan, China), Collagen III (22734, Proteintech Group, Inc. Wuhan, China), Bcl-2 (28150, Cell Signaling Technology, Inc., Boston, U.S.), Bax (2772, Cell Signaling Technology, Inc., Boston, U.S.), Caspase 3 (9662, Cell Signaling Technology, Inc., Boston, U.S.), iNOS (18985, Proteintech Group, Inc, Wuhan, China), COX2 (12282 Cell Signaling Technology, Inc., Boston, U.S.), NF-κB (8242, Cell Signaling Technology, Inc., Boston, U.S.), p- NF-κB (3033, Cell Signaling Technology, Inc., Boston, U.S.), IκBα (1842, Cell Signaling Technology, Inc., Boston, U.S.), p-IκBα (2859, Cell Signaling Technology, Inc., Boston, U.S.). The following secondary antibodies were used in this study: HRP-Goat Anti-Mouse/Rabbit Recombinant Secondary Antibody (H + L) (RGAM001/RGAG001, Proteintech Group, Inc., Wuhan, China).

Immunohistochemical (IHC) analysis

IHC techniques were applied to perform qualitative, quantitative, and spatial analyses of fibrosis-associated proteins. Tissue sections prepared from embedded wax blocks were subjected to high-pressure treatment to ensure optimal section thickness. The sections were then processed using a systematic immunohistochemical staining protocol. Targeted proteins included Collagen I (1:250, 14695 Proteintech Group, Inc., Wuhan, China), Collagen III (1:250, 22734, Proteintech Group, Inc. Wuhan, China), TGF-β (1:250, MA1-21595, Thermo Fisher Scientific Inc., MA, U.S.), α-SMA (1:250, 14395, Proteintech Group, Inc., Wuhan, China), with dilutions prepared according to the manufacturer’s specifications provided with the stains. Chromogenic detection was conducted using diaminobenzidine (DAB, P0202, Beyotime Biotechnology, Shanghai, China), and hematoxylin (C0107, Beyotime Biotechnology, Shanghai, China) was used for nuclear counterstaining. The sections were mounted onto slides using neutral resin to preserve stained tissues. Staining positivity was assessed based on the presence of brown-stained areas, and the proportion of these areas relative to the total visual field was quantified for statistical analysis (Deng et al. 2022).

Molecular docking model construction and analysis

Three-dimensional conformations of pivotal proteins were obtained from the RCSB Protein Data Bank (https://www.pdb.org/), and corresponding root mean square deviation (RMSD) values were calculated. The RMSD is a quantitative measure of the deviation of atomic positions from their original coordinates, serving as a reliable indicator of the conformational stability of proteins and ligands (Burley et al. 2023). These values were determined using DS 2019 software, with docking parameters and receptor configurations considered reliable when all complex RMSD values remained below 2.0 Å. After screening, the target crystal structures were extracted from the receptor, imported into DS 2019, and subjected to dehydration and hydrogenation processes to facilitate ligand separation. Chemical structures of key rhubarb components and routinely prescribed medications for CKD were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and docked with protein receptors. The efficacy of the molecular interactions between key targets and compounds was evaluated through a detailed analysis of the docking results.

Statistical analysis

In univariate MR analyses, several regression methods were employed, including MR-Egger, weighted median estimate (WME), IVW, simple mode, and weighted mode, to examine associations between diseases and key targets. The IVW method serves as the primary analytical approach, supplemented by MR-Egger and weighted mode for additional analyses. A p-value below 0.05 was considered statistically significant in the MR analyses. An IVW p-value greater than 0.05, combined with the MR-Egger intercept and Cochran’s Q statistic, suggests no substantial deviation from zero, indicating the presence of pleiotropy in the data. Heterogeneity was assessed using the IVW method in conjunction with Cochran’s Q statistic, where a Q p-value below 0.05 denotes heterogeneity within the study.

For experimental verification, data are presented as the mean ± standard deviation. Statistical analyses were performed using GraphPad Prism software. One-way ANOVA followed by Tukey’s post hoc test was used to determine statistical significance, with a p-value of <0.05 considered statistically significant.

Results

Rhubarb quality control: HPLC fingerprinting and chemometric-based approaches for Q-marker discovery

The retention times of key rhubarb compounds were evaluated using reference standards, including aloe-emodin, rhein, emodin, chrysophanol, and physcion (Figure 2(A)). By comparing these retention times with those of the standards, we identified five characteristic peaks corresponding to bound and free anthraquinones in rhubarb (Figure 2(B and C)). Subsequently, an analysis of rhubarb’s HPLC fingerprints was conducted using the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, Version 2012 (Ding et al. 2021). Examination of 12 different batches showed similarity indices exceeding 0.91 for total anthraquinones (Figure 2(D)) and 0.95 for free anthraquinones (Figure 2(E)), demonstrating a high degree of sample reproducibility.

Figure 2.

Chromatographic analysis of a mixture containing five standard references: aloe-emodin, rhein, emodin, chrysophanol, and physcion. Separation was achieved using an agilent ZORBAX eclipse plus C18 column (4.6 mm × 250 mm, 5 μm) with a mobile phase of 0.1% phosphoric acid aqueous solution and methanol at a flow rate of 1 mL/min. Separation parameters were set to measure absorbance units (mAU) and detection was performed at 254 nm. HPLC chromatograms showing spectra of total anthraquinones (A), free anthraquinones (B), and rhubarb (C). (D–E) Fingerprint profiles of twelve batches of rhubarb samples (S1–S12), with (D) representing total anthraquinones and (E) representing free anthraquinones. The x-axis denotes retention time (min), and the y-axis represents signal intensity (mV).

Network pharmacology analysis of rhubarb against CKD

A total of 19 active compounds related to rhubarb were identified from the published literature (Table 1, Supplementary Table 1). Additionally, 70 rhubarb targets were retrieved from the TCMSP and BATMAN-TCM databases. Furthermore, 2,597 disease targets for CKD were compiled from the OMIM, DrugBank, and GeneCards databases. A comparative analysis of rhubarb and CKD targets using VENNY identified 47 overlapping targets (Figure 3(A)). An interaction network was then constructed using Cytoscape software to elucidate the relationships between rhubarb compounds and CKD therapeutic targets, comprising 62 nodes and 110 edges.

Table 1.

List of core components of rhubarb.

Compounds name	Degree centrality	Betweenness centrality	Closeness centrality	Neighborhood connectivity
Emodin	37	0.13	0.51	4.7
Aloe-emodin	19	0.04	0.42	6.05
Beta-sitosterol	18	0.04	0.42	5.94
Physcion	18	0.03	0.42	7.39
Eupatin	15	0.02	0.41	7.33
Crysophanol	12	0.01	0.4	7.83
Physcion-9-O-beta-D-glucopyranoside_qt	9	0	0.39	10.78
(-)-Catechin	9	0.01	0.39	9.89
Rheochrysin	9	0.01	0.39	5.11
Rhein	8	0	0.38	10.75
Toralactone	8	0.01	0.38	9.38
Chrysophanol glucoside	6	0	0.38	11.17
(+)-Catechin-pentaacetate	4	0	0.37	12
Daucosterol_qt	3	0	0.37	11
Anthraglycoside B	2	0	0.36	14.5
Physciondiglucoside	2	0	0.36	14.5
Rheinoside A_qt	2	0	0.36	10.5
Torachrysone-8-O-beta-D-(6′-oxayl)-glucoside	2	0	0.36	14.5
Emodin-1-O-beta-D-glucopyranoside	2	0	0.36	14.5

Figure 3.

Network pharmacology analysis of rhubarb against CKD. (A) Venn diagram illustrating the overlap between rhubarb targets (yellow) and CKD-associated targets (purple). Rhubarb active compounds are colored blue, and target proteins associated with CKD are colored yellow. Each edge identifies the relationship between the active ingredient molecule and the target protein. (B) PPI network and highly connected hubs. The PPI network of 47 rhubarb anti-CKD targets was constructed using the STRING database (nodes = proteins, edges = interactions). The right panel highlights a radial bar plot of nodes with connections >40, indicating potential key genes. (C) Key target identification. Key targets network filtered by degree centrality (DC), betweenness centrality (BC), closeness centrality (CC), eigenvector centrality (EC), local average connectivity (LAC), and network centrality (NC), highlighting key targets in the network. The chart sequentially displays the filtered nodes, corresponding to 47, 19, and 15 nodes from left to right.

Target genes identified from interaction network analysis were uploaded to the STRING database to construct a PPI network (Figure 3(B)). A confidence threshold above 0.4 was selected to establish indirect links among the target proteins, resulting in a PPI network comprising 47 nodes. Radial columnar statistical graphs were used to analyze the relationships between neighboring nodes. Among the genes, IL6 and TNF each displayed connections with 74 adjacent nodes, identifying them as the most critical genes within the PPI network. Figure 3(C) illustrates these nodes, specifically emphasizing those with more than 40 connections. Topological analysis identified the top 15 key targets, some of which overlap with those identified in the PPI analysis. By synthesizing findings from the PPI and key target studies, we propose that the highly interconnected nodes IL6, TNF, TP53, CASP3, and IL1B may play key roles in the rhubarb protective mechanisms against CKD.

Causal association of IL6, TNF, and CASP3 with chronic kidney disease (CKD) using MR

In this study, a total of 76 SNPs were selected as IVs for MR analysis. The IVW method was employed as the primary analytical technique. The analysis indicated causal relationships between IL6 (OR: 1.02, 95% CI: 0.90–1.28), TNF (OR: 1.02, 95% CI: 1.00–1.03), and CASP3 (OR: 1.45, 95% CI: 0.96–1.36) and CKD. While only TNF showed statistical significance (p < 0.05), the results from IVW, MR-Egger, WME, simple mode, and weighted mode analyses for IL6 and CASP3 all indicated OR > 1 and β > 0 (Supplementary Table 2). Furthermore, a comprehensive analysis of forest plots (Figure 4(A–C)) and scatter diagrams (Figure 4(D–F)) demonstrated a positive correlation between these three key targets and CKD, as suggested by both IVW and MR–Egger results.

Figure 4.

Causal association of IL6, TNF, CASP3, and CKD by the MR method. (A–C) Forest plots: (A) CASP3, (B) IL6, (C) TNF. The vertical axis in the graph represents SNPs used as IVs, while the horizontal axis shows the 95% confidence interval of each SNP’s effect size. (D–F) Scatter plots: (D) CASP3, (E) IL6, (F) TNF. The horizontal axis represents the effect values of the SNPs on key targets (IL6, TNF, CASP3). The vertical axis indicates the effect of the SNPs on CKD. (G–I) Leave-one-out: (G) CASP3, (H) IL6, (I) TNF. The vertical axis represents SNPs used as IVs, and the horizontal axis shows the 95% confidence interval of each SNP’s effect size.

We further conducted a series of sensitivity analyses, including assessments for pleiotropy, heterogeneity, and leave-one-out sensitivity. The intercepts from MR–Egger regression were close to zero for IL6 (Egger = 0.007, p = 0.91), CASP3 (Egger = −0.04, p = 0.51), and TNF (Egger = 0.008, p = 0.47), suggesting no evidence of horizontal pleiotropy among the IVs. Heterogeneity among IVs was examined using both IVW and MR-Egger regression methods. MR-Egger results indicated no significant heterogeneity for CASP3 (Cochran’s Q = 2.71, Q-pval = 0.26), IL6 (Cochran’s Q = 0.16, Q-pval = 0.69), and TNF (Cochran’s Q = 48.88, Q-pval = 0.95). Similarly, IVW results showed no significant heterogeneity for CASP3 (Cochran’s Q = 3.55, Q-pval = 0.31), IL6 (Cochran’s Q = 0.18, Q-pval = 0.91), and TNF (Cochran’s Q = 49.40, Q-pval = 0.96), indicating an absence of heterogeneity among the IVs. The leave-one-out sensitivity analysis showed no substantial differences in effect estimates before and after the exclusion of individual SNPs, suggesting stability in the causal relationships (Figure 4(G–1)). These findings indicate that IL6, TNF, and CASP3 have relatively stable causal associations with CKD.

Analysis of GO and KEGG pathways related to rhubarb’s therapeutic effects against CKD

Gene Ontology (GO) enrichment analysis was used to extract biological information from gene datasets to study the functionalities of genes and their associated proteins across Cellular Components (CC), Molecular Functions (MF), and Biological Processes (BP) (Chen et al. 2022). As shown in Figure 5(A), we identified 3,296 GO terms, of which 1,932 demonstrated specific differential expression (CC: 34.81%, BP: 60.94%, MF: 50.18%). The most prominent enrichments were observed in areas such as DNA-binding transcription factor binding, response to oxidative stress, response to drugs, cellular response to chemical stress, and response to oxygen levels.

Figure 5.

Analysis of GO and KEGG pathways in rhubarb therapeutic effects against CKD. (A) Gene ontology (GO) enrichment analysis across three ontologies: biological process (BP), cellular component (CC), and molecular function (MF). The x-axis represents the enrichment score, reflecting the statistical significance of each term, while the y-axis lists the most significantly enriched GO terms. Bars are color-coded by ontology (BP: yellow, CC: green, MF: blue). (B) KEGG pathway enrichment analysis. The x-axis indicates the number of genes mapped to each pathway, and the y-axis displays KEGG pathway terms. Key pathways (e.g., NF-κB) are labeled. (C) KEGG pathway annotation. The x-axis shows the gene count, and the y-axis provides annotated KEGG pathway terms. Bars are color-coded to represent six major functional categories. (D) NF-κB signaling pathway map. Genes overlapping with the 47 key targets identified in the enrichment analysis are highlighted in red. Nodes represent pathway components (e.g., cytokines, kinases).

Concurrently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed clusters of pathway maps based on the latest molecular interaction networks. Our analysis identified 210 key pathways associated with 47 genes, with 62.86% showing differential expression profiles. Analysis of the top 20 identified pathways suggests that rhubarb’s therapeutic effects against CKD may involve the modulation of inflammatory processes. Figure 5(C) indicates that, within the domains of cellular processes, organismal systems, and human diseases, rhubarb’s effects are particularly linked to cell growth and death, as well as immune system diseases. These findings align with the pathways shown in Figure 5(B), including those related to human cytomegalovirus infection, the phosphatidylinositol 3-kinase-Akt (PI3K-AKT) signaling pathway, the TNF signaling pathway, and the NF-kappa B (NF-κB) signaling pathway. Notably, the NF-κB signaling pathway, a classical pathway involved in regulating inflammation, exhibited significant overlap with the 47 targeted genes under investigation (Figure 5(D)).

Rhubarb reduces renal damage in CKD rats

Figure 6(A–C) demonstrates the effects of various concentrations of rhubarb extract on blood biochemistry in rats with CKD. Compared to the untreated CKD group, the rhubarb-treated groups exhibited significantly lower levels of BUN, SCr, and UA. Urine analysis showed elevated levels of uroprotein (Upro), NAG, and β2-microglobulin (β2-MG) in the CKD group, which were markedly reduced following rhubarb treatment (Figure 6(D–F)).

Figure 6.

Rhubarb reduces renal damage in CKD rats. (A–C) Serum biochemical markers of renal function measured by ELISA: (A) blood urea nitrogen (BUN), (B) Serum creatinine (SCr), and (C) Uric acid (UA) levels showing significant elevation in the model group compared to controls, indicating impaired renal function. (D–F) Urinary biomarkers of renal tubular injury quantified by ELISA: (D) Uroprotein (upro), (E) N-acetyl-β-D-glucosaminidase (NAG), and (F) β2-microglobulin (β2-MG) levels, reflecting glomerular permeability and tubular damage. (G) HE staining of rat kidney tissue (20 × and 40 × magnification): the black arrow indicates dilated renal tubules with flattened epithelium; the red arrow points to connective tissue proliferation and inflammatory cell infiltration in the renal interstitium; the green arrow shows degeneration of renal tubular epithelial cells with loose and pale cytoplasm. *p < 0.05, ***p < 0.001, vs. control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. model group. n = 8/group.

HE staining revealed multiple histopathological changes in the kidneys of CKD rats, including mesangial cell proliferation, dilation or atrophy of renal tubules, glomerulosclerosis with irregular structures, adhesions within Bowman’s capsules, increased fibrotic tissue deposition, widened interstitial areas, and inflammatory cell infiltrates (Figure 6(G)). These pathological features were significantly improved following rhubarb treatment, suggesting that rhubarb effectively mitigates renal tissue damage and enhances renal function in CKD rats.

Rhubarb inhibits renal fibrosis in CKD rats

To assess the anti-fibrotic effects of rhubarb in CKD rats, Masson’s trichrome staining and Western blotting were employed to examine the accumulation of renal collagen fibers and expression levels of key fibrotic proteins in kidney tissues. Masson’s trichrome staining revealed pronounced thickening of the renal mesentery, widespread myofibroblast infiltration, and enhanced collagen fiber deposition in the renal tissues of CKD rats. Treatment with rhubarb markedly attenuated these changes, significantly reducing both myofibroblast proliferation and collagen fiber accumulation in renal tissues (Figure 7(A)). Additionally, Western blotting and immunohistochemistry analyses demonstrated that rhubarb administration led to substantial decreases in the levels of collagen I, collagen III, α-SMA, and TGF-β (Figure 7(B–D)). These findings collectively suggest that rhubarb effectively mitigates renal fibrosis in the CKD rat model.

Figure 7.

Rhubarb inhibits renal fibrosis in CKD rats. (A) Masson staining was performed to detect collagen deposition in renal tissue (scale bar = 100 μm and 50 μm). Quantitative analysis of positive staining area in renal tissue on the right side. (B) The expression levels of collagen I, collagen III, α-SMA, and TGF-β (brown DAB) were observed through immunohistochemical staining (scale bar = 100 μm), with relative expression levels quantified on the right. (C) The levels of collagen I, collagen III, α-SMA, and TGF-β were analyzed by Western blotting. (D) Quantitative analysis of target protein expression. ***p < 0.001, vs. control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. model group. n = 8/group.

Rhubarb inhibits inflammation and oxidative damage in CKD rats

Network pharmacology analysis indicated that three of the top five key genes encoded inflammatory mediators. To validate these findings, ELISAs were performed to quantify the concentrations of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α. The assays showed that the levels of these cytokines were significantly elevated in the CKD group compared to the control group. However, following rhubarb treatment, levels of all three cytokines were markedly reduced as shown in Figure 8(A–C), indicating the potent anti-inflammatory properties of rhubarb.

Figure 8.

Rhubarb inhibits inflammation and oxidative damage in CKD rats. Levels of TNF-α (a), IL-6 (B), and IL-1β (C) in renal tissue were measured using ELISA kits. Histograms showing SOD activity (D), GSH-px activity (E), and LPO (F) levels in renal tissue. ***p < 0.001, vs. control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. model group. n = 8/group.

In addition, oxidative stress plays a critical role in the pathogenesis of CKD, potentially aggravating inflammatory responses and promoting cellular apoptosis. This study further assessed oxidative stress markers and antioxidant capacity in renal cells following rhubarb treatment. As shown in Figure 8(D–F), the activities of SOD and GSH-px were significantly diminished in CKD rats but were substantially restored following rhubarb treatment. Concurrently, LPO levels, which were significantly elevated in CKD rats, were notably decreased after rhubarb administration. These findings demonstrate that rhubarb effectively attenuates renal inflammation and oxidative damage in CKD rat models.

Rhubarb inhibits renal cell apoptosis in CKD rats

Network pharmacology analysis revealed enrichment of apoptosis-associated genes, including those encoding caspase-3, Bcl-2, and Bax, suggesting that rhubarb may protect renal tissues by inhibiting apoptosis. Notably, rhubarb treatment showed a strong regulatory effect on key apoptotic factors, particularly in the high-dose group, where it significantly upregulated Bcl-2 expression while downregulating Bax and cleaved CASP3 expression compared to the CKD group (Figure 9(A and B)). These results suggest that rhubarb effectively suppresses apoptosis in the renal tissues of CKD rats, thereby enhancing renal protection.

Figure 9.

Rhubarb inhibits renal cell apoptosis in CKD rats. (A) Western blot analysis was conducted to examine the expression levels of Bcl-2, Bax, and cleaved caspase-3 proteins in renal tissue samples. GAPDH was utilized as an internal control. (B) Quantification of relative expression levels of proteins in (A). ***p < 0.001, vs. control group; ^##p < 0.01, ^###p < 0.001 vs. model group. n = 8/group.

Rhubarb inhibits renal inflammation in CKD rats by regulating the NF-κB pathway

Network pharmacology analysis revealed significant enrichment of genes encoding inflammatory mediators, such as NFKBIA (encoding nuclear the inhibitor of kappa B alpha protein, IκBα) and PTGS2 (encoding cyclooxygenase-2, COX-2), both critically associated with inflammation. Additionally, KEGG pathway analysis demonstrated alterations in several key proteins within the NF-κB pathway, suggesting that this pathway might be a crucial mechanism through which rhubarb mitigates renal injury in CKD. To explore this possibility, we examined rhubarb’s influence on NF-κB activation by assessing the expression of related proteins.

Compared to the control group, kidneys from the CKD group exhibited significant upregulation of iNOS and COX2 expression, which was markedly reduced by rhubarb treatment (Figure 10(A and B)). Furthermore, the CKD group showed elevated expression of p-NFκB/NFκB and p-IκBα, with increases of approximately 1.55-fold and 1.57-fold, respectively, compared to the control group; these increases were significantly attenuated following rhubarb treatment. Additionally, the expression of IκBα, which was significantly diminished in the CKD group, was markedly enhanced following rhubarb treatment (Figure 10(C and D)). These findings suggest that rhubarb mitigates renal inflammation in CKD rats primarily by suppressing NF-κB activation.

Figure 10.

Rhubarb inhibits renal inflammation in CKD rats by regulating the NF-κB pathway. (A) Western blot analysis of iNOS and COX2 protein levels in renal tissue. (B) Semi-quantitative analysis of relative expression for each protein in (A). (C) Western blot showing levels of p-NFκB, NFκB, p-IκBα, and IκBα proteins in renal tissue. (D) Quantified relative levels from (C) as determined using ImageJ. *p < 0.05, **p < 0.01, ***p < 0.001, vs. control group; ^##p < 0.01, ^###p < 0.001 vs. model group. n = 8/group.

Molecular docking analysis

The application of molecular docking techniques facilitated the identification of potential pharmacologically active compounds. Based on the results for key targets and core components, ten key genes (IL6, TNF, TP53, CASP3, IL1B, BCL2, NFKB1, PTGS2, NFKBIA and BAX, where NFKBIA encodes IκBα and five components (emodin, aloe-emodin, β-sitosterol, physcion, eupatin) were selected for molecular docking. The results demonstrated that these biologically active compounds exhibited significant binding affinities with BCL2, BAX, PTGS2, TP53, TNF, and IL6.

Libdock scores were utilized to quantify binding efficiencies, with higher scores indicating stronger and more stable protein–ligand interactions. The five main active components showed exemplary binding affinities with the proteins encoded by the target genes. The most notable binding efficiencies were observed between β-sitosterol and TNF (Libdock Score = 131.29), β-sitosterol and TP53 (Libdock Score = 119.79), β-sitosterol and PTGS2 (Libdock Score = 118.09), aloe-emodin and TNF (Libdock Score = 113.656), and aloe-emodin and NFKB1 (Libdock Score = 110.858), as illustrated in Figure 11(A–J). Furthermore, Libdock-generated heatmaps provided a clear and intuitive visualization of binding capacities, as shown in Figure 11(K).

Figure 11.

Molecular docking results. (A–J) Two-dimensional molecular docking models of key compounds with core targets. The following panels depict the 2D molecular docking models of key compounds interacting with their core targets: (A) Eupatin-TNF; (B) Physcion-NFKBIA; (C) Physcion-BAX; (D) Aloe_emodin-TNF; (E) Aloe_emodin-NFKB1; (F) Beta_sitosterol-BAX; (G) Beta_sitosterol-TNF; (H) Beta_sitosterol-BCL2; (I) Emodin-PTGS2; (J) Emodin-NFKB1. Lines of different colors represent various interactions. (K) Heatmap of molecular docking scores. A heatmap is provided to visualize the molecular docking scores. Different colors indicate the range of libdock scores, with deeper shades of blue representing higher libdock scores.

Discussion

Rhubarb has demonstrated effectiveness in “Tongfu Xiezhuo” (purging turbidity and facilitating bowel clearance) and has shown therapeutic benefits as a treatment for CKD in recent years (Wojcikowski et al. 2004; Peng et al. 2005). Studies indicate that rhubarb can reduce BUN, enhance renal function, and facilitate the excretion of toxins or mitigate their harmful effects (Huang et al. 2023). As a result, rhubarb has become a specialized medicine for treating uremia (Zou et al. 2020).

In recent years, network pharmacology, as an innovative approach integrating omics data with systems biology analyses, has become a crucial tool for elucidating the synergistic mechanisms of traditional Chinese medicine (TCM) characterized by multiple components, targets, and pathways. By constructing complex drug-target-disease networks, this method systematically deciphers the potential mechanisms underlying the effects of herbal formulations in disease intervention, attracting widespread attention (Han et al. 2024). Meanwhile, molecular docking technology serves as an important complement to the predictions made by network pharmacology. It simulates the binding modes between active compounds and key target proteins, effectively validating predicted targets and significantly improving the accuracy and reliability of mechanistic studies (Li et al. 2023; Chen et al. 2024).

In this study, network pharmacology analysis revealed that IL6, TNF, TP53, CASP3, and IL1B are key targets implicated in rhubarb’s efficacy for treating CKD. MR analysis suggests that IL6, TNF, and CASP3 have relatively stable causal associations with CKD. GO and KEGG pathway analysis indicated that the NF-κB signaling pathway mediates the anti-inflammatory effects of rhubarb. Using a CKD rat model, we validated rhubarb’s anti-inflammatory activity, demonstrating that it reduces renal inflammation, oxidative damage, and cellular apoptosis in rats with CKD by modulating the NF-κB signaling pathway. Molecular docking analysis identified active components, such as emodin, aloe-emodin, β-sitosterol, physcion, and eupatin, which may contribute to rhubarb’s nephroprotective effects. Our findings underscore rhubarb’s potential as a therapeutic agent in ameliorating CKD, consistent with its historical use in TCM. By integrating network pharmacology, MR, molecular docking, and experimental validation, we provide a comprehensive analysis of rhubarb’s impact on CKD.

During the validation phase, we confirmed that rhubarb administration mitigates renal damage and fibrosis in CKD rats. We quantified serum BUN, SCr, and UA levels, as these indicators reliably gauge renal impairment. Our findings revealed that rhubarb therapy reduced BUN, SCr, and UA levels. We also analyzed the levels of Upro, NAG, and β2-MG, three pivotal urinary biomarkers of renal damage, and found that these biomarkers were markedly elevated in the CKD group but significantly attenuated by rhubarb treatment.

Fibrosis represents a critical pathological hallmark in multiple organs across various diseases and is often linked to reduced renal function in CKD (Yamashita and Kramann 2024). Studies indicate that CKD rats exhibit progressive proteinuria, expansion of the glomerular mesangial matrix, glomerulosclerosis, mesangial dilation, and elevated levels of type I and type III collagen and fibronectin proteins in the kidneys (Zhang et al. 2020). Consistent with these observations, our study demonstrated significant upregulation of fibrotic proteins, including type I and III collagen, in CKD rats with renal fibrosis, as evidenced by Masson’s trichrome staining showing elevated levels of fibroblast-specific activation markers α-SMA and TGF-β; expression of these proteins is upregulated when fibroblasts differentiate into myofibroblast, a central process in renal fibrosis development (Piera-Velazquez et al. 2011; Miao et al. 2022). Our findings indicated that rhubarb treatment significantly inhibited the expression of both of these biomarkers in the kidneys, supporting rhubarb’s protective role in mitigating renal failure and highlighting its potential clinical utility.

Oxidative stress is a pivotal pathway in the development of CKD. Our GO enrichment analysis suggested that rhubarb exerts beneficial effects in CKD through key biological processes, such as cellular responses to oxidative stress, chemical stress, and oxygen levels, suggesting that rhubarb may mitigate renal tissue damage and restore renal function by counteracting excessive oxidative stress. Overproduction of reactive oxygen species (ROS) has been linked to renal dysfunction in studies showing that reducing ROS levels is crucial for minimizing renal injury (Pieniazek et al. 2021; Khanahmad et al. 2022). SOD and GSH-px are primary endogenous antioxidant enzymes that constitute the initial defense against ROS. Products of LPO reactions damage cellular membranes and organelles, leading to cellular dysfunction and potentially cell death (Punziano et al. 2024). Our research indicated that rhubarb administration enhances SOD and GSH-px activities and decreases LPO levels in CKD rats, emphasizing the potential antioxidant-mediated protective mechanisms of rhubarb. Thus, rhubarb alleviates renal inflammation and oxidative damage, exerting a protective effect.

This study integrates genetic epidemiological causal inference, network pharmacology, and in vivo experiments to establish a multi-level mechanistic framework for rhubarb treatment of CKD. Network pharmacology PPI analysis revealed enrichment of apoptosis-related proteins such as caspase-3, Bcl-2, and Bax. MR results also highlighted a positive correlation between the pro-apoptotic factor caspase-3 and the increased risk of CKD, suggesting that rhubarb possesses potential anti-apoptotic properties. Observed anti-apoptotic effects, including the upregulation of Bcl-2 and downregulation of Bax and cleaved caspase-3, suggest that rhubarb may protect renal cells by inhibiting pathways associated with apoptosis. These results illustrate that rhubarb mitigates oxidative damage and apoptosis in renal tissues, confirming its protective activity against CKD-induced renal injury.

Analysis of the “Rhubarb Targets CKD” network identified several key targets. Further examination of the relationships between key proteins and CKD pathophysiological features identified five critical core elements: IL-6, TNF-α, p53, caspase-3, and IL-1β. Among these, IL-6, TNF-α, and IL-1β are significant pro-inflammatory cytokines involved in inflammatory responses. Chronic inflammation, a prominent feature of CKD, is characterized by enhanced inflammatory responses (Kadatane et al. 2023), with proinflammatory cytokines such as IL-6, IL-1β, and TNF-α positively correlated with CKD severity (CKD) (Stefania et al. 2024; Tanaka 2024), and elevated serum inflammatory marker levels frequently observed in CKD patients (Zhao et al. 2024). We further utilized MR to investigate the core targets derived from network pharmacology analyses, revealing that IL6 and TNF were positively associated with an increased CKD risk, with TNF showing particularly significant effects. Although results obtained for IL6 did not reach statistical significance, its OR > 1 and β > 0 indicated a positive causal relationship with CKD, consistent with our network pharmacology and MR findings revealing markedly elevated levels of TNF-α, IL-6, and IL-1β in kidney tissues of CKD rats that were significantly attenuated after rhubarb treatment.

KEGG pathway enrichment analysis identified critical pathways, including the PI3K-AKT, TNF, and NF-κB signaling pathways. Notably, the NF-κB signaling pathway, a key regulator of inflammatory responses, has been shown to play a regulatory role in renal tissue injury (Ouyang et al. 2023). Under quiescent conditions, NF-κB is sequestered by IκB. Upon exposure to diverse intracellular and extracellular stimuli, IκB kinase becomes activated, initiating the phosphorylation of the IκB protein, triggering IκB degradation and subsequent NF-κB activation. Following activation, NF-κB translocates to the nucleus, where it orchestrates the release of inflammatory mediators, a process essential for regulating inflammatory responses (Wu et al. 2019). Studies have demonstrated that activation of the NF-κB signaling pathway affects crucial cellular functions needed for survival, proliferation, and apoptosis (Wang et al. 2015). Therefore, key targets in the NF-κB signaling pathway have garnered significant attention, aligning with our results showing that these intersecting core targets constitute more than 85% of the dataset.

Numerous studies have demonstrated that inhibition of NF-κB contributes to renal protection (Zhang et al. 2024). Consistent with these observations, our results showed that CKD is associated with increased NF-κB activation, while rhubarb treatment suppresses this activation and reduces the expression of p-IκBα and inflammatory chemokines such as iNOS and COX2. This aligns with findings from other studies showing that CKD triggers an inflammatory response in renal tissue (Ji et al. 2020; Gu et al. 2022). Our results indicate that rhubarb ameliorates inflammatory damage by modulating the NF-κB signaling pathway. Additionally, several active constituents in rhubarb, including rhein, chrysophanol, and aloe-emodin, provide corroborative regulatory effects (Pooja and Karunagaran 2014; Hu et al. 2021). These findings suggest that rhubarb may alleviate inflammatory processes in CKD by regulating the NF-κB pathway, thereby slowing disease progression.

Integrating significant target information from the “component-disease” network with PPI data, we identified emodin, aloe-emodin, β-sitosterol, physcion, and eupatin as the core active components in rhubarb’s therapeutic effects against CKD. These components, which primarily include anthraquinones, phytosterols, and flavonoids, are associated with promoting extracellular matrix synthesis, tissue repair, antimicrobial activity, and anti-inflammatory and antioxidative effects (Li et al. 2019; Sun et al. 2022). Notably, anthraquinones are characteristic components described in the Chinese Pharmacopoeia that are used to conduct rhubarb quality control assessments and are considered its core biologically active components. In our study, we selected IL6, TNF, TP53, CASP3, IL1B, BCL2, NFKB1, PTGS2, NFKBIA, and BAX for docking with the five core rhubarb components. The results demonstrated that all biologically active components exhibited good binding affinities, particularly anthraquinones. The strongest binding affinities were observed for aloe-emodin with TNF (Libdock Score = 113.656) and aloe-emodin with NFKB1 (Libdock Score = 110.858). Additionally, aloe-emodin, a characteristic component identified by HPLC, serves as an indicator of rhubarb’s medicinal value. Our findings suggest that rhubarb ameliorates CKD primarily by inhibiting inflammation, thereby regulating apoptosis and mitigating renal tissue damage, with anthraquinones and flavonoids playing pivotal roles in these beneficial effects.

Detailed insights obtained in this study regarding the mechanisms by which rhubarb exerts its renoprotective effects not only validate its traditional use but also offer a compelling basis for its integration into modern strategies for treating CKD. Rhubarb’s efficacy in regulating key pathways involved in inflammation, fibrosis, oxidative stress, and apoptosis highlights its potential as a multifaceted therapeutic agent (Hu et al. 2013; Lu et al. 2015; Liu et al. 2021). While our study had certain limitations, we demonstrated significant therapeutic effects of rhubarb extracts in the context of renal failure when administered at dosages employed in clinical settings. Our preliminary toxicological assessment, based on a review of existing literature and key biomarker assessments in CKD rats in this study, is only applicable to short-term experimental studies. Nevertheless, these experimental outcomes will pave the way for future research to critically evaluate the impact of various doses of rhubarb extracts on specific organ systems as an approach for providing necessary clinical data toward increasing the precision of rhubarb administration in clinical practice.

Given modern medicine’s growing interest in the detailed study of TCM, the active constituents within rhubarb will be a central focus of future investigations. In this study, we used molecular docking to preliminarily validate the targeting potential of active rhubarb components; however, this approach does not fully determine their safety and efficacy in practical applications. Future studies will involve an in-depth exploration of individual compounds, accompanied by rigorous testing, to strengthen the scientific foundation for the broader use of rhubarb.

In recent years, significant progress has been made in elucidating the pharmacological mechanisms of Rhubarb and its major active constituents. Recent studies have reported that emodin and aloe-emodin regulate tumor cell behavior through dual mechanisms, including inhibition of the Wnt/β-catenin signaling pathway and activation of mitochondrial-dependent apoptotic pathways, thereby significantly suppressing proliferation and migration of various tumor cells (Tsai et al. 2013). Furthermore, emodin upregulates Smad7 expression, negatively regulating the TGF-β/Smad signaling pathway, effectively reducing myofibroblast activation and collagen deposition, demonstrating promising anti-fibrotic potential. These mechanistic insights provide a solid theoretical foundation for the development of rhubarb-related anti-tumor and anti-fibrotic therapeutics (Tian et al. 2018).

Clinically, an increasing body of evidence from evidence-based medicine supports the adjunctive use of Rhubarb and its active components. For example, combined with conventional therapy, Rhubarb significantly reduces serum amylase levels in patients with severe acute pancreatitis and shortens their hospital and ICU stays (Zhou et al. 2016; Hu et al. 2018). In septic patients, treatment with Rhubarb leads to significant reductions in pro-inflammatory cytokines IL-6 and TNF-α, with marked alleviation of clinical symptoms (Fang et al. 2007; Zhang et al. 2015). Additionally, in patients with chronic renal failure, retention enema therapy using Rhubarb has been shown to delay renal function deterioration and improve proteinuria indicators (Khan et al. 2014). Moreover, systematic identification of Rhubarb’s main chemical constituents by ultra-high-performance liquid chromatography coupled with mass spectrometry provides a robust scientific basis for quality control (Jin et al. 2006; VanMen et al. 2012). However, current clinical studies still face limitations such as small sample sizes, inconsistent efficacy evaluation criteria, and insufficient long-term safety data. Therefore, large-scale, multicenter, randomized controlled trials are urgently needed in the future. These should incorporate modern biomarkers and imaging technologies to comprehensively and systematically evaluate the clinical efficacy and safety of Rhubarb and its derivatives, thereby promoting their standardized application and clinical translation. In summary, by deeply integrating the latest basic and clinical research findings, this manuscript further highlights the multi-target pharmacological value and application potential of Rhubarb in disease prevention and treatment, providing important references for related research and clinical practice.

In addition, our forthcoming research will integrate HPLC analysis with more advanced analytical techniques, such as mass spectrometry (MS), to further validate and expand our compound library. This integration will enable the construction of a more precise network of drug-target interactions, which is essential for developing targeted therapeutics and elucidating pharmacological mechanisms. By leveraging the complementary strengths of HPLC and MS, we aim to enhance the resolution and sensitivity of our analytical assays, allowing for a deeper understanding of the complex chemical constituents within rhubarb and their potential applications in medicine.

Although this study systematically explored the potential mechanisms of Rhubarb and its main active components in chronic kidney disease (CKD) with multi-dimensional validation, certain limitations remain. Firstly, while anthraquinones exhibit pharmacological activity, their potential hepatotoxicity, nephrotoxicity, and gastrointestinal irritation warrant careful consideration. Integrated pharmacokinetic and toxicological studies are needed to thoroughly evaluate safety. Secondly, more in-depth functional validations such as interventions in cell signaling pathways, gene regulation, proteomics, and animal models are still lacking, which are essential to fully clarify mechanisms of action and pathological relevance. Moreover, due to the complex and heterogeneous nature of CKD, current target identification is mainly based on existing databases and may miss novel targets. Future work should incorporate single-cell sequencing, metabolomics, and multi-omics integration for more comprehensive analysis. Finally, the interactions and synergistic effects among Rhubarb formula components have not been systematically assessed; thus, further research should emphasize holistic pharmacodynamics to elucidate the molecular basis of multi-component synergy.

Addressing the aforementioned limitations and considering the dosing flexibility of rheum in traditional Chinese medicine theory can help provide more scientific guidance for its rational clinical use. As one of the widely used and clinically effective herbs, Rheum officinale demonstrates considerable flexibility in dosing within traditional practice. Although the high-dose group in this study slightly exceeds the pharmacopoeial recommended range after conversion, this actually reflects the core characteristic of traditional Chinese medicine (TCM)—syndrome differentiation and individualized treatment, where different diseases and pathological conditions correspond to adjusted dosages (Zha et al. 2015). The dose-effect relationship of herbal formulas has become a significant focus in contemporary TCM research, providing a promising direction for further exploration based on our experimental findings (Chen et al. 2020). The rational use of herbal medicine is grounded both in clinical efficacy and scientific standardization. The results of this study not only offer a novel perspective and scientific evidence for the clinical application of Rheum officinale but also contribute to advancing the value and innovation of TCM in modern clinical practice.

Conclusions

In this investigation, HPLC fingerprint analysis was employed to detect and characterize the primary chemical constituents of rhubarb. Network pharmacology analysis suggested that rhubarb’s therapeutic action in CKD primarily involves modulation of the NF-κB signaling pathway, with IL6, IL1β, TNF, PTGS2, and NFKBIA identified as key targets. Within these targets, MR analysis identified IL6, TNF and CASP3 as having relatively stable causal associations with chronic kidney disease. Furthermore, in vivo studies have shown that rhubarb markedly decreased renal functional impairment and attenuated renal fibrosis. Additionally, Rhubarb regulated the NF-κB signaling pathway, which led to the suppression of renal inflammation and the modulation of apoptosis. From the result of molecular docking, we found anthraquinones, such as aloe-emodin and β-sitosterol, were identified as key active compounds in these effects. These findings corroborate the traditional application of Rhubarb in the management of renal disorders and support its integration as an adjunctive therapy in CKD treatment.

Funding Statement

This work was financially supported by the Jilin Provincial Department of Science and Technology Project [YDZJ202301ZYTS460], Jilin Provincial Department of Education Project [JJKH20230991KJ].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The raw datasets supporting the conclusions of this article will be available from the authors, without undue reservation, to any qualified researcher.