Protective Effects of Jiawei Baihu Tang in Kawasaki Disease: Omics Analysis and Mechanistic Exploration

Abstract

Purpose

Kawasaki disease (KD) is classified as a “WenBing” syndrome in traditional Chinese medicine (TCM), characterized by systemic inflammation and vasculitis. Jiawei Baihu Tang (JWBHT) is an optimized herbal formulation derived from the classical Baihu Tang (BHT), a renowned ancient TCM prescription traditionally used to treat WenBing syndromes by clearing heat, reducing fever, and promoting body fluid production. However, the precise pharmacological mechanisms underlying JWBHT’s intervention effects remain largely unexplored. We aimed to assess the protective effects of JWBHT on coronary artery inflammation in mice with KD and explore the underlying mechanisms, with a focus on the lipocalin-2/matrix metalloproteinase 9 (LCN2/MMP9) axis in vascular remodeling.

Methods

Histopathological analysis, multiplex immunofluorescence, data-independent acquisition (DIA) proteomics, and non-targeted metabolomics were employed to comprehensively measure the effects of JWBHT on coronary artery injury. The experiments were conducted using a Candida albicans water-soluble fraction (CAWS)-induced murine model of KD vasculitis. Multi-omics integration also revealed the role of JWBHT in regulating extracellular matrix (ECM)-receptor interaction and enhancing α-linolenic acid metabolism.

Results

JWBHT significantly alleviated coronary arteritis by reducing inflammatory cell infiltration, preserving ECM integrity, and alleviating fibrosis. Multi-omics analysis indicated that JWBHT regulated ECM-receptor interaction and restored lipid metabolism, particularly α-linolenic acid metabolism, which was closely linked to ECM stabilization. Mechanistically, JWBHT suppressed the LCN2/MMP9 axis, a critical mediator of vascular remodeling. Validation based on the clinical dataset revealed elevated levels of LCN2/MMP9 in patients with acute KD, which were normalized after treatment.

Conclusion

For the first time, this study unveiled the multi-target mode of action of JWBHT in KD through the crosstalk between ECM and lipid metabolism, and LCN2 was identified as a potential novel intervention target. Our findings suggest that JWBHT is a promising traditional Chinese medicine for preventing the cardiovascular complications of KD.

Graphical Abstract

Potential mechanisms through which JWBHT mitigates KD-related coronary arteritis likely include regulation of the LCN2/MMP9 pathway, ECM-receptor interactions, and α-linolenic acid metabolism. These actions collectively contribute to the attenuation of inflammatory damage and vascular remodeling, underscoring the protective role of JWBHT against vasculitic injury in KD

Supplementary Information

The online version contains supplementary material available at 10.1007/s10557-026-07849-8.

Introduction

KD is an acute febrile disease characterized by systemic vasculitis whose pathogenesis is not fully understood. KD is mainly diagnosed based on fever, non-secretory conjunctival congestion, lip changes, rash, cervical lymph node enlargement, fingertip changes, etc. The most serious complication of KD is cardiovascular injury [1, 2]. In the acute phase of KD, immune cells are overactivated and produce cytokines [3], therefore, early treatment mainly focuses on anti-inflammatory agents. The treatment of KD mainly includes aspirin and intravenous immunoglobulin (IVIG), which greatly suppresses the inflammatory response. However, even with timely and active treatment, a small number of patients may still develop coronary aneurysms [4, 5]. These patients require long-term or lifelong medications, which greatly affects their quality of life. Therefore, exploring new treatment strategies is of great clinical significance for the diagnosis and treatment of KD.

KD is categorized as a WenBing in TCM, presenting with high fever, systemic inflammatory responses, and vasculitis. The classical formula BHT, with its origins in the Eastern Han Dynasty’s “Shang Han Lun” by Zhong-Jing Zhang, has been historically employed to manage WenBing syndromes. This formulation comprises four principal herbs, Gypsum Fibrosum (Shi-Gao), Anemarrhena asphodeloides Bunge. (Zhi-Mu), Glycyrrhiza uralensis Fisch. ex DC. (Gan-Cao), Oryza sativa L. subsp. japonica Kato (Geng-Mi), collectively functioning to reduce fever and stimulate fluid production. The modified formula JWBHT represents an optimized version of BHT, specifically adapted for pediatric applications. Considering the vulnerability of children’s spleen and stomach systems to excessive cooling medications, Codonopsis pilosula (Franch.) Nannf. (Dang-Shen) was incorporated to fortify spleen function, enhance lung health, enrich blood quality, and facilitate fluid generation, thereby safeguarding digestive organs and maintaining vital qi, making it a valuable medicinal and dietary herb for both preventive and therapeutic applications [6]. Furthermore, the addition of Paeonia × suffruticosa Andrews (Dan-Pi) serves to clear heat, cool blood circulation, activate blood flow, resolve stasis, and block pathogenic invasion into the blood system, exhibits notable vascular protective effects [7]. This enhanced formulation effectively supports physiological functions while eliminating pathogenic factors and arresting disease advancement.

In modern medicine, BHT is useful to relieve fever, reduce inflammation and strengthen the immune system in patients with febrile conditions, and systemic inflammation. BHT possesses significant anti-inflammatory effects [8, 9]and vascular protection [10] align with its traditional heat-clearing and detoxifying functions. Additionally, BHT improved the survival rate of rats with sepsis [11]. A modified BHT, has demonstrated confirmed efficacy and favorable safety in the treatment of influenza and viral infections, Evidence-based clinical consensus, supported by expert recommendations, has been established to guide its rational use in clinical practice [12]. The SARS-CoV-2-related multisystem inflammatory syndrome in children partially overlaps with KD in clinical and cytokine profiles [13, 14], supporting KD as manifestations of traditional Chinese WenBing, characterized by exogenous pathogenic heat toxins triggering latent pathogens and damaging the blood-nutrient system. A modified BHT demonstrates significant therapeutic efficacy in patients infected with the novel coronavirus Omicron variant, effectively alleviating symptoms and shortening disease duration, providing valuable insights for clinical management [15]. Another formula with a composition similar to our JWBHT is Qingwen Baidu Yin, which integrates BHT with two other classical formulations, exerts effects of clearing heat and cooling blood, alleviating heat-producing and dis-intoxicating in Corona Virus Disease 2019 treatment [16]. A heat-clearing and blood-cooling decoction (composed of Shi-Gao, Zhi-Mu, Dan-Pi, etc.) similar to JWBHT can effectively shorten fever duration, improve hypercoagulability, downregulate inflammatory cytokines, and demonstrate potential cardioprotective effects in KD [17]. Therefore, JWBHT demonstrates excellent anti-inflammatory and vasculoprotective effects in the treatment of KD, a febrile disorder vasculitis WenBing in TCM.

Recent pharmacological studies have provided valuable mechanistic insights into TCM interventions for KD. For instance, a monomeric component, paeoniflorin, a key bioactive compound also presents in Dan-Pi, which is included in JWBHT, was shown to alleviate coronary artery lesions in a murine KD model by activating the NRF2 antioxidant pathway and inhibiting oxidative stress [18]. Furthermore, research on another classical TCM formula, Xijiao Dihuang Tang, demonstrated its protective effects against CAWS-induced coronary arteritis by modulating the PI3K/Akt-NFκB signaling axis [19]. These findings underscore the scientific rationale for exploring TCM-based strategies and highlight critical pathways involved in KD vascular injury. Building upon this groundwork, we aimed to investigate the protective effects and the underlying multi-target mechanisms of JWBHT, an optimized formula for pediatric febrile vasculitis, utilizing an integrated multi-omics approach.

In the present study, we employed a well-described and widely accepted mouse model of KD, established by injecting water-soluble components of CAWS. This model reliably predicts the efficacy of treatment regimens for patients with KD [20, 21]. We investigated the protective effects and mechanism of JWBHT on KD-related coronary artery injury. In vivo experiments showed that JWBHT significantly reduced coronary artery inflammation, mitigated damage to vascular endothelium and elastic fibers. To investigate the pathway and potential mechanism underlying the effects of JWBHT, we studied its effects on protein and metabolite levels in the mice model of CAWS-induced KD. We used DIA proteomics to analyze changes in mouse plasma proteins and non-targeted metabolomics to analyze plasma metabolites. The results indicated that ECM and α-linolenic acid metabolism may be potential targets and metabolic pathways involved in the intervention effects of JWBHT in mice with KD, and LCN2 may be an intermediate molecule regulating fatty acid metabolism after treatment with JWBHT.

Materials and Methods

Preparation of JWBHT

The six varieties of Chinese medicinal herbs included in JWBHT were purchased from Suzhou Tianling Traditional Chinese Medicine Pieces Co., Ltd. The detailed information and ingredient ratios of the materials used in JWBHT are provided in (Table S1). The administered dose was determined by converting the original human dose recorded in “Shang Han Lun” using standard body surface area normalization [22, 23]. This classical prescription ratio(Table S1) was maintained to preserve ethnopharmacological authenticity. The classical prescription dose based on “Shang Han Lun” has been validated in contemporary studies for its anti-pyretic effects [24, 25]. The preparation process of JWBHT was as follows: (1) accurately weighing the Chinese medicinal herbs according to the prescribed amounts and placing them in a ceramic pot; (2) wrapping the gypsum in cloth and simmering it, then soaking the other medicinal herbs for 30 min, wrapping glutinous rice in cloth and simmer; (3) boiling over high heat, then reducing the temperature and simmer for 30 min; (4) filtering the completed decoction, pouring it into a container, and setting it aside; (5) adding cold distilled water to the remaining residue in the ceramic pot, boiling, and decocting for another 30 min; (6) filtering the medicinal solution and combining the two filtered decoctions; and (7) concentrating the combined decoction under reduced pressure and storing it at -80 °C for future experimental use.

Characterization of Constituents in JWBHT by UHPLC-MS/MS

Sample Preparation. A 200 mg aliquot was vortexed with 10 mL of methanol: water (50:50, v/v) in 15 mL polypropylene tubes. After 30 min of ultrasonication, 1 mL of supernatant was centrifuged (14,000 × g, 5 min), filtered (0.22 μm), and transferred to vials for analysis.

Chromatography. Separation was conducted on ACQUITY UPLC HSS T3 (2.1 × 100 mm, 1.8 μm) at 35 °C with 10 µL injection. Gradient elution (0.3 mL/min) consisted of (A) 0.1% formic acid/H₂O and (B) 0.1% formic acid/ACN.

Mass spectrometry. Q Exactive Orbitrap HRMS operated in Full MS/ddMS2 mode (± ESI). The parameters were as follows: m/z 100–1500; resolutions MS1/MS2 = 70,000/17,500; spray voltage 3.2 kV; capillary 320℃; aux gas 15 L/min (350℃); sheath gas 40 L/min; AGC 1e6; TopN = 5 with stepped NCE (30–50). Data were processed using Compound Discoverer 3.3.

Animal Experiments

CAWS preparation. CAWS was extracted from Candida albicans (NBRC1385) following the established protocols [26]. Briefly, Candida albicans was cultured in carbon-limited medium (48 h, 26℃, 250 rpm), followed by sequential ethanol precipitation (equal volume) and overnight incubation at 4℃. Pellets were resuspended in water (2 h stirring) and subjected to repeated centrifugation and subsequent supernatant ethanol precipitation (1:1). The final precipitate was acetone-desiccated (48 h) and reconstituted in PBS before the experiment.

The mouse model of KD. Male C57BL/6 mice (3–4 weeks, JOINN Lab, Suzhou) were acclimatized under controlled conditions (25 ± 2℃/50 ± 5% humidity). After 7 days of acclimatization, the animals were randomly assigned to three cohorts (n = 4–5, a small sample size per group was used, which is common for initial exploratory histopathological and omics studies in this field [19]): PBS control, CAWS (5-day; 4 mg/mouse i.p), and CAWS+JWBHT (supplementary i.g TCM, 13.91 g/kg/day crude drug × 14). Terminal sampling (peripheral blood/cardiac tissue) was conducted 14 days after CAWS via isoflurane euthanasia. All methods in this study were carried out following relevant guidelines and regulations.

Histological and Immunohistochemical Staining

Histological and immunohistochemical staining. Mouse heart was dissected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 0.5 μm thick slices. As previously described, the sections were stained with hematoxylin and eosin (HE), elastic van Gieson (EVG) [27], Masson and Sirius Red staining. Images were captured using a microscope (Nikon DS-Ri2, Tokyo, Japan). The severity of inflammatory infiltration was measured using the cardiovascular inflammation score [28].

Immunofluorescence staining. Mouse heart samples were incubated with lipocalin-2/NGAL recombinant antibody (83853-2-RR, Invitrogen), rabbit anti-MMP9 (YM8019, ImmunoWay Biotechnology Co., USA), rabbit anti-Ly6G (YM8307, ImmunoWay Biotechnology Co., USA), rabbit anti-CD86 (91882, Cell Signaling Technology, USA), rabbit anti-CD31 (YM8207, ImmunoWay Biotechnology Co., USA), rabbit anti-COL1 (PA5-29569, Thermo Fisher Scientific, USA), and rabbit anti-α-SMA (55135-1-AP, SanYing Biotechnology, China). Alexa Fluor 488 goat anti-rabbit/mouse was used as the secondary antibody (GB25303/GB21301; Servicebio, Wuhan, China). Images were captured using a laser scanning confocal microscope (Olympus FV1200, Tokyo, Japan), and the nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI).

Proteomics Study

Sample processing. Plasma samples underwent centrifugation (12,000 × g, 10 min, 4 °C) for clarification and high-abundance protein depletion. Protein quantification (BCA assay) was conducted before acetone precipitation (5:1 v/v, -20℃, 16 h). The pellets were washed with 80% acetone (12,000 rpm, 4℃), dissolved via sonication, processed through sequential reduction (10 mM DTT, 55℃, 20 min), alkylation (15 mM IAA, in the dark, 30 min), and tryptic digestion (1:50, 37℃).

LC-MS/MS analysis. Peptides (200 ng/sample) were separated on a nanoElute2 UPLC system (Bruker) using PepSep C18 column (75 μm × 15 cm, 1.9 μm) with 0.1% FA/ACN mobile phases (5%-35% B over 44 min, 400 nL/min). DIA-PASEF acquisition was conducted on timsTOF Pro2 covering m/z 350–1250, with collision energy ramping from 20 eV (1/K0 0.6 Vs/cm²) to 59 eV (1/K0 1.6 Vs/cm²).

Protein identification and quantification. Proteomic data were processed using Spectronaut v18.2 with its integrated Pulsar search engine for spectral analysis. MS/MS spectra were compared to the Mus musculus UniProtKB/Swiss-Prot reference proteome (Release 2022_11, Taxon ID 10090) using the following parameters: Cysteine carbamidomethylation as the fixed modification, with methionine oxidation and N-terminal acetylation designated as post-translational modifications. Trypsin specificity was enforced with a maximum of two missed cleavages. Mass tolerance thresholds were established at 20 ppm for precursor ions and 20 ppm for fragment ions. Statistical validation was conducted employing a 1% false discovery rate threshold at both peptide-spectrum match and peptide levels. All remaining analytical configurations were conducted using the default parameters of the software.

Bioinformatics workflow. Proteins with ≥ 1 unique peptide (FDR < 1%) were retained. Before log transformation, the missing values were imputed using the half-minimum method. Multivariate analyses in R 3.6.3/SIMCA 16.0.2 were conducted to identify differentially expressed proteins (|FC|≥1.2, p < 0.05) for subsequent PCA and Kyoto encyclopedia of genes and genomes (KEGG) pathway mapping. In total, 802 proteins were retained.

Metabolomics Study

Sample preparation. The samples were extracted with four volumes of ice-cold MeOH: ACN (1:1) containing deuterated internal standards. After vortexing (30 s), cold-bath sonication (10 min, 4℃) and protein precipitation (-40℃, 1 h), the supernatant (12,000 rpm, 4℃, 15 min) was collected. QC samples comprised pooled equal aliquots of all specimens.

LC-MS/MS analysis. Chromatographic separation and mass spectrometry were conducted using an integrated ultra-high-performance liquid chromatography system (Vanquish UHPLC, Thermo Scientific) interfaced with an Orbitrap Exploris 120 mass analyzer. A Waters ACQUITY BEH Amide column (2.1 × 50 mm, 1.7 μm) maintained at 4 °C with 2 µL injection volume was employed. The mobile phase comprised 25 mM ammonium acetate/ammonium hydroxide aqueous solution (pH 9.75) as component A and acetonitrile as component B under gradient elution conditions. Mass spectral acquisition was conducted using the information-dependent acquisition method with dual electrospray ionization modes (± 3.8 kV/-3.4 kV). Optimized parameters were used for mass spectral acquisition, including sheath gas at 50 arbitrary units, auxiliary gas at 15 arbitrary units, capillary temperature at 320℃, full scan resolution of 60,000, MS/MS resolution of 15,000, and stepped normalized collision energy of 20/30/40 eV.

Data processing. Raw data were converted into the mzXML format using ProteoWizard software, Data were processed (peak detection, peak extraction, peak alignment, and peak area integration) using the XCMS toolkit developed in the R environment. Metabolite identification was conducted by integrating R computational packages with the BiotreeDB bioinformatics database (version 3.0).

Correlation Analysis

Data were analyzed using R (version 4.4.2). KEGG pathway enrichment analysis was conducted on metabolomics and proteomics data using the clusterProfiler package [29]. Significantly enriched pathways (p < 0.05) were visualized using ggplot2. Pearson correlation coefficients were calculated between metabolites and proteins. A heatmap was established using the pheatmap package of R to display the resulting correlation matrix. Relationships between metabolites, proteins, and pathways were visualized using Sankey diagrams (networkD3), while gene-metabolite-pathway interactions were visualized as network graphs using igraph and ggraph. Pearson correlation, KEGG pathway enrichment (ClusterProfiler 4.0), and network construction (Cytoscape 3.10.3) were employed for multi-omics integration. For clinical validation, we retrieved the publicly available gene expression dataset GSE18606 from the GEO database (http://www.ncbi.nlm.nih.gov/geo) This dataset is based on the GPL6480 Agilent 014850 Whole Human Genome Microarray 4 × 44 K G4112F platform. This includes data from 9 healthy and age matched control children, as well as 20 children with KD in the acute and recovery phases. Differential expression analysis of LCN2 and MMP9 was conducted using GEO2R tool, and statistical comparison was performed.

Statistical Analysis

Statistical analyses were conducted using GraphPad Prism 9 Software. One-way ANOVA was used to compare groups. All results were derived from at least three independent experiments and are presented as mean ±standard deviation (SD). p < 0.05 was considered statistically significant.

Results

JWBHT Ameliorated Coronary Artery Inflammation in the Mice Model of CAWS-induced KD

Experimental workflow is shown in Fig. 1a. UHPLC-MS/MS was used to identify the pharmacologically active components of JWBHT in positive and negative ion modes (Fig. 1b). Detailed compound information is shown in (Table S2), containing the names of monomeric compounds, molecular formula, m/z, etc. HE staining revealed distinct morphological differences between the experimental groups. The normal group exhibited intact and smooth endothelium in cardiac blood vessels with no inflammatory cell infiltration. In contrast, the CAWS group demonstrated significant vascular abnormalities characterized by irregular endothelial surface, structural abnormalities, and disorganized cellular arrangement. Numerous inflammatory cells were observed in both the vasculature and surrounding myocardial tissues. The JWBHT treatment group showed a significant improvement compared to the model group, with better preservation of endothelial architecture and reduced abundance of inflammatory cells in cardiac tissues. EVG staining revealed distinct structural patterns in myocardial vasculature across experimental cohorts. The normal group exhibited well-organized elastic fibers demonstrating characteristic parallel alignment and physiological helical conformation. In contrast, the CAWS group showed significant disruption of the elastin matrix, with fibers displaying disrupted organization, including fragmented strands and reduced structural continuity, compared to physiological controls. Therapeutic intervention analysis demonstrated partial restoration of the elastin architecture in the JWBHT group, with improved fiber alignment and maintenance of structural integrity compared to the modeling group (Fig. 1c-d). To further characterize the inflammatory infiltrate, immunofluorescence staining for neutrophil (Ly6G) and macrophage (CD86) markers was performed. The results demonstrated a significant increase in perivascular Ly6G⁺ and CD86⁺ cells in the CAWS group, while JWBHT treatment led to a marked reduction in the abundance of both cell types (Fig. 1e).

Fig. 1.

JWBHT ameliorated coronary arteritis in CAWS-induced KD mice. (a) Experimental design of CAWS-induced vasculitis model with JWBHT treatment timeline. (b) LC-HRMS total ion chromatogram of JWBHT in positive/negative ionization modes. (b) Semiquantitative inflammation scores of coronary and aortic tissues. (d) Representative H&E staining and EVG staining showing vascular inflammation and elastin degradation. (e) Immunofluorescence analysis of inflammatory cell infiltration. Representative images showing perivascular infiltration of Ly6G⁺ neutrophils (red) and CD86⁺ macrophages (green) in cardiac tissues

These data supported the multimodal protective efficacy of JWBHT against experimental coronary arteritis through inhibition of inflammatory cell infiltration, stabilization of vascular matrix.

Treatment with JWBHT Modulated Proteome Profiles in CAWS-Induced KD

Proteomic profiling revealed the systemic regulatory effects of JWBHT on CAWS-induced vasculitis. Principal components analysis (PCA) demonstrated orthogonal separation among the control, CAWS, and JWBHT groups (Fig. 2a). Comparative analysis identified 161 CAWS-dysregulated serum proteins (73 upregulated and 88 downregulated) and pharmacological intervention normalized 106 proteins (75 upregulated and 31 downregulated) (Fig. 2b-c). Co-regulated proteins (n = 39) across disease and treatment groups (Fig. 2d) represent potential intervention targets, supported by conserved expression patterns in hierarchical clustering (Fig. 2e).

Fig. 2.

Proteomic profiling of JWBHT-mediated modulation of signaling pathways. (a) PCA of proteomes across groups. (b) Volcano plot of differentially expressed proteins (DEPs, |fold change| ≥1.2). (c) Quantification of upregulated (red) and downregulated (blue) DEPs. (d) Venn diagram of overlapping targets between CAWS/Control (161 proteins) and JWBHT/CAWS (106 proteins) comparisons. (e) Heatmap of DEP expression patterns. KEGG pathway analysis for (f) CAWS vs Control and (g) JWBHT vs CAWS (ECM-receptor interaction, complement/coagulation cascades, and focal adhesion) FDR-adjusted p-values shown by color gradient

Pathway analysis of CAWS-associated proteins identified ECM-receptor interaction, focal adhesion, and hemostasis pathways (Fig .2f). Bioinformatic pathway enrichment analysis indicated that JWBHT treatment was associated with the modulation of ECM dynamics, with suggested involvement of the PI3K/Akt signaling and cardiomyopathy-related pathways (Fig. 2g), showing synergistic regulation of ECM components (collagen, elastin and laminin) and vascular homeostasis mediators. This multi-target engagement suggests that JWBHT preserves endothelial function by maintaining ECM integrity, a critical determinant involved in the stability of the vascular structure.

JWBHT Restored Metabolic Homeostasis in KD by Regulating Lipid Metabolism

Metabolomic profiling defined the lipid-modulatory effects of JWBHT in CAWS-induced vasculitis. Partial Least Squares Discriminant Analysis (PLS-DA) revealed distinct metabolic fingerprints across groups (Fig. 3a). Differential analysis (p < 0.05 & VIP > 1) identified 214 CAWS-associated metabolic alterations (40 upregulated and 174 downregulated), among which 91 were restored by JWBHT (38 upregulated and 53 downregulated) (Fig. 3b-d). Cross-comparison mapping demonstrated treatment-specific metabolite clusters (Fig. 3e-f).

Fig. 3.

Metabolic remodeling by JWBHT in KD mice. (a) PLS-DA plot of metabolic profiles. (b, c) Volcano plots of differential metabolites (p<0.05, VIP>1) in CAWS vs Control (b) and JWBHT vs CAWS (c). (d) The number of dysregulated metabolites across comparisons. (r) Shared metabolic targets between groups. (f) Heatmap of metabolite expression trajectories. (g, h) Pathway enrichment analysis highlighting α-linolenic acid metabolism

Pathway enrichment revealed seven disease-associated pathways, particularly α-linolenic acid metabolism, JWBHT selectively normalized five of the modulated pathways (Fig. 3g-h), reversed CAWS-driven suppression of essential ω-3/ω-6 fatty acids (α-linolenic/γ-linolenic acids, cis-4,10,13,16-docosatetraenoic acid (Fig. 4a), and restored the homeostasis of lipid and organic acid (Fig. 4b). It is noteworthy that these observed shifts in the plasma lipid profile likely reflect systemic modulation of lipid metabolism in major metabolic organs, such as the liver, rather than indicating a direct effect on vascular endothelial cells.

Fig. 4.

Lipidomic validation of pathway regulation. (a) Relative abundance of α-linolenic acid, γ-linolenic acid, cis-4,10,13,16-docosatetraenoic acid, and SM(d18:1/18:0). (b) Altered levels of additional lipid species. n=4; *p<0.05, **p<0.01, ***p<0.001

Integrated Multi-omics Analysis Reveals Dual Regulatory Axis: ECM and Lipid Metabolism

Cross-omics integration via Pearson correlations (91 metabolites × 106 proteins, p < 0.05) identified 17 lipid mediators and 26 proteins co-regulated by JWBHT. Six structurally defined glycerophospholipids, including PC (22:4/P-18:1) and PC (20:4/20:1) subclasses, exhibited pathway-centric enrichment (KEGG: glycerophospholipid/linoleic/α-linolenic acid metabolism). Proteomics highlighted the role of ECM remodeling (COL1A1, COL1A2 and ITGA2) via three interactive axes including ECM-receptor signaling, focal adhesion kinetics, and PI3K/Akt pathway activation (Fig. 5a).

Fig. 5.

Cross-omics integration of therapeutic mechanisms. (a) Pearson correlation network (26 proteins and 17 metabolites) with KEGG annotations. (b) Co-expression modules linking therapeutic targets to pathway reactivation

Functional mapping identified 12 pathways modulated by JWBHT, with four fatty acid hubs, including Myristic acid, α-/γ-linolenic acids and cis-9-palmitoleic acid. Cytoscape network topology (v3.10.3) integrated 26 proteins, 17 metabolites, and 23 pathways and revealed PI3K as the central protein-metabolite interaction node (Fig. 5b). Three persistently dysregulated pathways emerged as intervention pivots, including ECM receptor interaction, focal adhesion and complement-coagulation cross-talk. Taken together, these integrated data suggest that JWBHT may alleviate vasculitis through a coordinated lipid metabolism-systemic inflammation-vascular ECM axis, whereby systemic restoration of lipid homeostasis contributes to the stabilization of the vascular extracellular matrix.

LCN2/MMP9 Axis as an Intervention Target for JWBHT in KD

Metabolomic profiling revealed CAWS-induced perturbations in fatty acid metabolism, characterized by a marked reduction in the levels of myristic acid, cis-9-palmitoleic acid, α-linolenic acid, and γ-linolenic acid. Treatment with JWBHT effectively restored these metabolic perturbations. Association network analysis identified LCN2 as a prominent protein displaying robust negative correlations with all four dysregulated fatty acids (Fig. 5a). Spatial colocalization analysis showed perivascular enrichment of LCN2 and MMP9 in CD31⁺ vascular beds (Fig. 6a), suggesting a potential functional interplay between lipid metabolism and ECM regulation.

Fig. 6.

Expression level of the LCN2/MMP9 axis. (a) CD31/LCN2/MMP9 co-localization in cardiac tissues. (b, c) LCN2 and MMP9 expression in the GSE18606 dataset. (d) Plasma LCN2 levels across experimental groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Clinical validation using the GSE18606 dataset revealed that LCN2 and MMP9 were significantly upregulated in patients with acute KD(Fig. 6b-c). The diagnostic and disease relevance of LCN2 and MMP9 in KD is further supported by independent bioinformatics analyses identifying them as hub immunoregulatory genes [30]. Mirroring clinical dynamics, JWBHT suppressed CAWS-induced overexpression of both LCN2 and MMP9 (Fig. 6d), with an effect comparable to IVIG treatment.

These findings collectively suggest the protective effects of JWBHT on the vasculature may involve the simultaneous modulation of lipid metabolism and ECM stability via the LCN2/MMP9 axis, providing a mechanistic hypothesis for its anti-fibrotic effects.

We conducted histopathological and immunohistochemical analyses to investigate the protective effects of JWBHT on endothelial injury via ECM modulation. Histological analysis revealed CAWS-induced pan-cellular fibrotic remodeling in coronary arteries. Endothelial cells (ECs) revealed collagen I deposition and vascular smooth muscle cells (VSMCs) showed αSMA overexpression compared to controls. Multiplexed immunofluorescence mapping confirmed pathological EC dedifferentiation characterized by CD31 reduction coupled with vimentin upregulation. Treatment with JWBHT suppressed collagen deposition in Masson and Sirius Red staining, abrogated fibrosis, restored CD31⁺ EC coverage, and reduced the abundance of αSMA+/COL1 + VSMCs (Figs. 7 and 8). These data are consistent with the interpretation that JWBHT preserves vascular architecture by simultaneously modulating endothelial phenotypic stability and VSMC contractile function, potentially linked to its ECM homeostatic activity. This dual-regulatory effect on ECM may contribute to the protective effects of JWBHT on the vasculature in KD.

Fig. 7.

JWBHT alleviated vascular fibrosis. Masson and Sirius Red staining showing the resolution of vascular inflammation

Fig. 8.

Restoration of endothelial integrity by JWBHT. Immunofluorescence staining showing (a) CD31/αSMA and (b) CD31/COL1 interactions in cardiac tissues

Discussion

As an acute systemic vasculitis, the etiology and pathogenesis of KD yet remain unclear. Although IVIG can improve prognosis in most patients with KD, it is still ineffective for some refractory cases [31]. Therefore, it is necessary to explore new and potential strategies for the clinical treatment of KD. Traditional Chinese medicine offers a comprehensive treatment strategy with a long history, which has been used for centuries to prevent, treat, and cure diseases. The present study provides comprehensive evidence supporting the protective potential of JWBHT, an herbal formulation, that can ameliorate coronary arteritis and vascular dysfunction in experimental KD. By integrating histopathological, proteomic, metabolomic, and bioinformatic approaches, we elucidated a multi-targeted mechanism through which JWBHT exerts its protective effects on the vasculature. It possesses anti-inflammatory properties, stabilizes ECM, reprograms lipid metabolism, and suppresses pathological fibrosis. These findings align with the holistic protective principles of phytomedicines, which synergistically modulate interconnected biological pathways.

Our histopathological and immunofluorescence analyses revealed that JWBHT can significantly attenuate CAWS-induced coronary inflammation and preserving elastin matrix integrity. This dual regulation of inflammatory signaling and structural stabilization mirrors the pleiotropic effects reported for other herbal medicines with protective properties on the vasculature, such as resveratrol and berberine [32, 33]. The observed protective effects on the elastic laminae architecture suggest that JWBHT can improve vascular resilience against inflammatory damage, a critical factor in preventing coronary aneurysm, the hallmark complication of KD [34].

Proteomic profiling uncovered the profound effect of JWBHT on ECM-receptor interaction and focal adhesion pathways, with coordinated regulation of collagen isoforms (COL1A1/1A2) and integrins (ITGA2). This aligns with the emerging concept regarding the role of ECM dysregulation as the key driver of vasculitis in KD [35, 36]. The bioinformatic association of JWBHT treatment with the PI3K/Akt signaling pathway suggests its potential role in bridging biomechanical and biochemical signaling, a putative mechanism that warrants further experimental validation and remains to be functionally confirmed. This capacity to engage multiple targets may represent a therapeutic advantage of herbal medicines over single-target agents. Notably, the reversal of serum proteome perturbations exerts systemic effects consistent with the traditional Chinese medicine theories, which considers the human body as a complex dynamic system and focuses on the balance of the human body with the external environment [36].

Metabolomic insights revealed that JWBHT robustly restored lipid homeostasis, particularly by activating of α-linolenic acid metabolism. These fatty acids may suppress inflammation by inhibiting CAWS-induced metabolic suppression. Considering the systemic nature of herbal medicine, the observed plasma lipidomic changes most plausibly originate from the modulation of key metabolic organs. This systemic restoration of anti-inflammatory fatty acids, such as α-linolenic acid, is proposed to create a favorable metabolic milieu that secondarily supports vascular repair. The strong correlation between the restored lipid species and the normalized ECM proteins supports the existence of a lipid metabolism-systemic inflammation-vascular ECM regulatory axis. Through this axis, JWBHT may indirectly confer endothelial protection and stabilize the vascular wall.

Identification of the LCN2/MMP9 axis as a major intervention target provides a correlative framework to explain the anti-fibrotic effects of JWBHT. LCN2, a siderophore-binding protein implicated in iron-mediated oxidative damage [37], showed robust inverse correlations with essential fatty acids. JWBHT-mediated suppression of LCN2/MMP9 co-expression is associated with reduced matrix degradation, and may ameliorate abnormal iron metabolism, and mitigates endothelial injury. This aligns with MMP9 upregulation in acute KD and MMP9 downregulation after treatment with IVIG, supporting the potential of JWBHT as the conventional treatment of KD. Thus, targeting this axis represents a promising hypothesis derived from our integrated data.

Our multi-omics findings that JWBHT modulates inflammation, ECM homeostasis, and lipid metabolism extend the mechanistic understanding of TCM in KD. The identified suppression of the LCN2/MMP9 axis and the bioinformatically suggested involvement of PI3K/Akt signaling find context and support in prior studies. For instance, the protective role of the PI3K/Akt pathway has been documented for another TCM formula, Xijiao Dihuang Tang, in the same CAWS-induced KD model [19]. Furthermore, the observed restoration of lipid homeostasis aligns with the reported efficacy of paeoniflorin, a constituent of JWBHT, via the NRF2 antioxidant pathway [18]. While these cited studies elucidate specific pathways or components, our work demonstrates that the optimized formula JWBHT can potentially orchestrate a broader network of effects, simultaneously engaging the LCN2/MMP9 axis, lipid metabolic reprogramming, and ECM preservation. This systems-level effect underscores the investigative value and potential advantage of multi-herbal formulations for complex vasculopathies like KD.

Crucially, our histopathological and immunofluorescence analyses suggest that JWBHT may mitigate CAWS-induced endothelial phenotypic changes and modulate VSMC characteristics, processes implicated in vascular remodeling [38]. The observed pattern, concurrent restoration of CD31, an endothelial marker, expression and suppression of vimentin, a mesenchymal marker, and collagen I, indicates that JWBHT treatment was associated with the preservation of endothelial integrity alongside an attenuation of pro-fibrotic responses. This multifaceted effect aligns with the multi-component nature of JWBHT, wherein various phytochemicals could potentially target distinct pathways involved in endothelial dysfunction and fibrotic transformation.

While this study provides robust preclinical evidence, several limitations should be considered when interpreting the findings. First, regarding experimental design, the relatively small animal sample size, though adequate for exploratory omics analyses, may limit statistical power for some endpoints; future confirmatory studies would benefit from larger cohorts. Additionally, the lack of a dose-response study precludes the determination of the optimal therapeutic window for JWBHT. Second, concerning mechanistic validation, the suggested involvement of the PI3K/Akt signaling pathway is based on proteomic enrichment analysis and requires direct biochemical confirmation. Similarly, while strongly supported by correlation and clinical data, the functional role and causal relationship of the identified LCN2/MMP9 axis remain to be conclusively established through targeted interventional studies. Furthermore, although UHPLC-MS/MS characterized the chemical profile of JWBHT and its systemic exposure, the specific bioactive constituents responsible for the observed effects remain to be isolated and functionally validated. Third, in terms of model and translational relevance, the CAWS-induced murine model of KD vasculitis, while widely used, may not fully recapitulate the complex immunopathology of the human disease, the temporal dynamics of proteomic and metabolomic changes induced by JWBHT also remains to be characterized. Ultimately, human endothelial cell models and clinical trials are essential to verify the translational relevance. Future studies should explore the effects of JWBHT on macrophage polarization, platelet activation, and other pathways related to KD in omics analyses.

Conclusions

This multi-omics study identified JWBHT as a promising herbal medicine for treating vasculitis in KD. Our data suggest it acts through coordinated regulation of inflammatory signaling, ECM homeostasis, and lipid metabolism. The discovery of the LCN2/MMP9 axis as a key regulatory node underscores the value of integrative approaches in deciphering complex mechanisms underlying the protective effects of herbal medicines. Our findings provide supporting evidence for traditional applications of botanical formulations in vascular disorders and provide a scientific framework for developing multi-targeted therapies against KD and related vasculopathies.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

All authors contributed to the study conception and design. Jin Ma: Writing – review & editing, Writing – original draft, Visualization, Data curation. Xiudao Song: Project administration, Methodology, Validation, Investigation. Xuan Li: Resources, Methodology. Yunjia Tang: Project administration, Formal analysis. Haitao Lv: Supervision, Conceptualization. Yexing Chen: Supervision, Software. Peng Cao: Funding acquisition, Conceptualization.

Funding

This work was supported by the National Key R&D Program of China (2023YFC2308200) and Gusu Health Talent Project (GSWS2024093).

Data Availability

The datasets are currently under restricted access as they are part of an ongoing collaborative project. Data sharing requires approval from all participating institutions. Original files can be provided by the corresponding author upon reasonable request.

Declarations

Ethics Approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Soochow University (Ethics No. SUDA20220906A01).

Consent to Publish

Not applicable.

Competing Interests

The authors declare no competing interests.