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. 2025 Oct 22;56:234–247. doi: 10.1016/j.bioactmat.2025.10.010

A “controlled assembly” strategy for constructing Coptidis Rhizoma self-assembled nanoparticles to ameliorate inflammatory damage in Atopic dermatitis

Rui Song a,b,1, Yuwen Zhu a,1, Kailin Xue a, Xiang Deng a,b, Run Wang a, Yaya Su a, Xu Chen a,⁎⁎, Hailong Yuan a,b,⁎
PMCID: PMC12590232  PMID: 41211576

Abstract

Atopic dermatitis (AD) is a prevalent chronic inflammatory disorder requiring effective therapeutic intervention. Self-assembled nanoparticles derived from traditional Chinese medicine (TCM) decoctions have gained increasing attention for their potential clinical applications in AD treatment. However, the self-assembly process of TCM occurs simultaneously with the extraction of active ingredients, leading to poor stability and uniformity of the self-assembled nanoparticles, which results in unpredictable therapeutic efficacy. In this study, we propose an innovative “controlled assembly” strategy to improve the self-assembly process of decoction. Coptidis Rhizoma self-assembled nanoparticles (CR-SAN) were constructed via hydrochloric acid-assisted aqueous extraction followed by pH-driven assembly. CR-SAN exhibited a spherical morphology with an average particle size of 234.9 ± 5.1 nm and a polydispersity index of 0.21 ± 0.03. In vitro permeation studies demonstrated that CR-SAN enhances skin permeation and retention by disrupting the stratum corneum structure, altering lipid organization, and modifying the secondary structure of keratin. Notably, CR-SAN demonstrated remarkable anti-inflammatory efficacy in both in vitro and in vivo. NLRP3, the most distinctive member of the NOD-like receptors (NLRs) family, plays a pivotal role in inflammatory regulation. Integrative network pharmacology and transcriptomic analysis revealed that CR-SAN significantly downregulated the expression of NLRP3 pathway (including GSDMD and IL-33) in AD models, thereby alleviating the itch-scratch cycle and preserving skin barrier function. Collectively, the development of CR-SAN establishes a novel therapeutic strategy for AD management through NLRP3 pathway modulation, while also providing new insights into the advancement of self-assembled nanoparticles derived from TCM.

Keywords: Coptidis Rhizoma, Self-assembled nanoparticles, Atopic dermatitis, NLRP3 inflammasome

Graphical abstract

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Highlights

  • •

    A novel drug delivery system for AD was developed using the “controlled assembly” strategy.

  • •

    CR-SAN improves skin permeation by modulating the structural integrity of the stratum corneum.

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    The NLRP3/GSDMD-IL-33 axis constitutes the primary mechanistic basis for the therapeutic efficacy of CR-SAN.

1. Introduction

Atopic dermatitis (AD) is one of the most prevalent chronic inflammatory skin disorders worldwide, with its hallmark symptom, persistent pruritus, significantly impairing patients’ quality of life [1,2]. Current therapeutic approaches for AD primarily consist of topical agents, such as corticosteroids and immunomodulators, and systemic medications, including antihistamines and immunosuppressants [3,4]. Although these treatments can provide partial symptom relief, they are often associated with adverse effects, including skin atrophy, compromised barrier function, and drug resistance [5,6]. These limitations underscore the urgent need for innovative therapeutic strategies to improve treatment efficacy and patient well-being in AD.

In recent years, the clinical application of traditional Chinese medicine (TCM) in the topical treatment of AD has gained increasing attention. Coptidis Rhizoma, a traditional herbal medicine, has been extensively studied for its potent bioactivity, particularly in alleviating inflammation. Its major active components, such as Berberine and other alkaloids, exhibit notable anti-inflammatory [7], antimicrobial [8], and immunomodulatory effects [9,10]. However, the poor skin permeability of these components limits their clinical efficacy in topical treatments.

Recent studies have demonstrated that during decoction, molecules from Coptidis Rhizoma (Huanglian) can spontaneously form self-assembled nanoparticles through non-covalent interactions, including electrostatic forces, van der Waals forces, π–π stacking, and hydrogen bonding [[11], [12], [13]]. These nanoparticles have been shown to enhance the delivery efficiency of alkaloid compounds [14]. However, the nanoparticles commonly exhibit poor stability and are prone to aggregation, which limits their reliability and therapeutic efficacy in practical applications (Fig. S1, and Table S1). This issue arises because the self-assembly process occurs simultaneously with the extraction of herbal components, making it challenging to precisely control the assembly conditions. This “extraction-assembly integration” hinders the optimization of nanoparticle stability and delivery performance. To overcome these limitations, this study proposes an innovative “controlled assembly” strategy. By separating the extraction process from the self-assembly process, this approach allows independent optimization of key parameters at each stage. Specifically, the active components of Coptidis Rhizoma are first extracted using a hydrochloric acid aqueous solution, followed by the construction of structurally stable and size-controlled self-assembled nanoparticles (CR-SAN) using a pH-driven method. Subsequently, the physicochemical properties of CR-SAN were characterized, and its transdermal properties were evaluated to assess its potential for effective drug delivery.

Substantial evidence has established that NLRP3 inflammasome activation is intimately associated with the pathogenesis of inflammatory disorders, positioning it as a promising therapeutic target for AD [[15], [16], [17], [18]]. NLRP3 activation exacerbates inflammation through Gasdermin D (GSDMD)-mediated pyroptosis [[19], [20], [21]]. Recent studies have shown that with the assembly of inflammasomes, GSDMD forms plasma membrane pores, leading to the leakage of cellular contents, the release of alarms (especially IL-33), and inducing deep pruritus and scratching behaviors [22,23]. This cascade establishes a vicious itch-scratch cycle that perpetuates skin barrier disruption and disease progression. Consequently, the NLRP3/GSDMD-IL-33 axis emerges as a critical pathogenic pathway in AD, representing a promising therapeutic target for novel intervention strategies. However, whether CR-SAN exerts its anti-inflammatory effects specifically through this pathway remains to be fully elucidated.

In conclusion, this study underscores the advantages of CR-SAN and its potential as a therapeutic approach for AD. Moreover, it offers valuable insights into the further development of self-assembled nanoparticles derived from TCM.

2. Materials and methods

2.1. Preparation and characterization of CR-SAN

Coptidis Rhizoma (30 g) was immersed in 10 times its volume of a 0.3 % hydrochloric acid aqueous solution and soaked for 30 min, followed by reflux extraction for 1 h. The resulting extract was filtered through gauze and concentrated to a final concentration of 0.25 g/mL of crude drug, yielding the pH-CR solution. The pH-CR solution was then stirred at 200 rpm on a magnetic stirrer, and its pH was adjusted to 7.0 using a 1 mol/L KOH solution, leading to the formation of the CR-SAN solution. The concentration of alkaloids in CR-SAN was quantified using the High Performance Liquid Chromatography (HPLC) method described in the Chinese Pharmacopoeia. The encapsulation efficiency of the alkaloids in CR-SAN was determined using the ultrafiltration centrifugation method.

The size distribution of CR-SAN was analyzed by dynamic light scattering (DLS) using a Nanotrac Wave Ⅱ analyzer (Microtrac Inc., USA) at 25 °C, following a 100-fold dilution with deionized water. The zeta potential was measured using a JS94 zeta potential analyzer (Shanghai Zhongchen Digital Technic Apparatus Co., Ltd., China) at 25 °C. The morphologies of CR-SAN were observed and recorded using a S-4800 scanning electron microscopy (Hitachi, Ltd., Japan) and a Tecnai G2 F30 transmission electron microscope (FEI, USA). Fourier transform infrared (FTIR) spectroscopy was performed using a Nicolet iS50 FTIR spectrometer (Thermo, USA) over a wavelength range of 4000-400 cm−1. Thermogravimetric analysis (TG) was performed using a TGA550 thermogravimetric analyzer (TA Instruments, USA) at a heating rate of 10 °C/min from 40 to 600 °C. X-ray diffraction (XRD) analysis was performed using a D8 X-ray diffractometer (Bruker, Germany) in step scan mode with Cu radiation over the 2θ range of 3–60° at a scanning rate of 2°/min.

2.2. Identification of phytochemical constituents in CR-SAN by Ultra-High Performance Liquid Chromatography electrospray ionization Quadrupole-orbitrap hybrid mass spectrometry (UHPLC-ESI-QE-orbitrap-MS)

Sample separation was performed using a Waters ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm). The mobile phase consisted of 0.1 % formic acid acetonitrile (A) and 0.1 % formic acid water (B). The gradient elution profile was as follows: 0–10 min, 100 %B; 10–20 min, 100 %-70 %B; 10–25 min, 70 %-60 %B; 25–30 min, 60 %-50 %B; 30–40 min, 50 %-30 %B; 40–45 min, 30 %-0 %B; 45–60 min, 0 %B; 60–60.1 min, 0 %-100 %B; 60.1–70 min, 100 %B. The flow rate was set at 0.2 mL/min, and the column temperature was maintained at 35 °C. Heated electrospray ionization source (HESI) was employed. The ion source parameters were configured as follows: sheath gas flow rate 40 arb, auxiliary gas flow rate 15 arb, capillary temperature 320 °C, auxiliary gas heater temperature 350 °C, positive spray voltage 3.2 kV, negative spray voltage 3.0 kV. The mass spectrometer operated at a resolution of 70,000 for MS and 17,500 for MS/MS, in full scan mode. Both positive and negative ion modes were simultaneously monitored, with the positive ion spectrum scanning range set at m/z 100–1500. Unknown compounds were identified using Compound Discoverer 3.3, mzCloud, and mzVault databases.

2.3. Ethics approval statement

Male BALB/c mice (18–20 g) were obtained from SPBF (Beijing) Biotechnology Co., Ltd. Animal experiments were carried out following the guidelines of the Animal Care and Use Committee. Approval for all animal experiments was obtained from the Ethics Committee of Air Force Medical Center, PLA of China (No. 2025-45-PJ01).

2.4. In vitro skin penetration experiments for CR-SAN

Drug permeation experiments were conducted using a Franz diffusion cell (Tianjin Jingtuo Instrument Technology Co., Ltd.) to assess the permeation characteristics of CR-SAN. The effects of CR-SAN on the skin surface microstructure were visualized using Scanning electron microscopy (SEM). Thermal analysis of the stratum corneum (SC) thermotropic phase behavior was performed using differential scanning calorimetry (DSC, Shimadzu DSC-60, Japan). The treated skin samples were weighed, placed into aluminum crimp cells under nitrogen purge, and analyzed from 25 to 200 °C at a heating rate of 10 °C/min. FTIR was used to assess the effects of CR-SAN on SC lipids and keratin. The treated skin samples were analyzed using the KBr disc technique, with spectra recorded in the range of 4000–400 cm−1 at a resolution of 4 cm−1.

2.5. 2,4-Dinitrochlorobenzene (DNCB)-induced AD in mice

BALB/c mice were sensitized topically with 200 μL of 1 % DNCB diluted in a mixture of acetone and olive oil (3:1) once every two days over the initial five-day period. The mice were categorized into six groups, each consisting of six mice (n = 6/group): (1)control group, (2)2,4-Dinitrochlorobenzene (DNCB), (3)DNCB + CR-SAN (0.0625 g/mL raw dose), (4)DNCB + CR-SAN (0.125 g/mL raw dose), (5)DNCB + CR-SAN (0.25 g/mL raw dose), (6)DNCB + Dexamethasone (Dexa, 2.5 mg/kg). After the initial sensitization period, the dorsal skin of the mice was stimulated with 200 μL of 0.6 % DNCB. Mice displaying DNCB-induced skin lesions were treated daily with CR-SAN and Dexa topically. Skin lesions and blood samples were collected after 15 days.

2.6. Histological evaluation (H&E)

Histological evaluation of dorsal skin tissue involved perforation with a 5 mm biopsy tool, fixation in tissue fixative, and embedding in paraffin. Skin sections embedded in paraffin were stained with H&E and toluidine blue (TB). Histological alterations were examined using a light microscope (Olympus, Tokyo, Japan). Epidermal thickness was assessed at 100× magnification using H&E staining. Mast cell infiltration was evaluated with TB staining, with four randomly chosen sections subjected to histological scrutiny.

2.7. Immunohistochemistry (IHC)

As previously described, paraffin-embedded sections were deparaffinized and rehydrated. Sections were heated in Antigen Unmasking Solution (H2002, Wuhan Servicebio Technology Co., Ltd) at 98 °C for 10 min to retrieve the antigen. Then the slices were blocked and treated with the primary antibodies with NLRP3 and Caspase-1 at 4 °C overnight. On the following day, after removing the primary antibody, immunohistochemical staining was carried out using the 3,3′-diaminobenzidine peroxidase substrate kit (H1005, Wuhan Servicebio Technology Co., Ltd). All images were captured with an upright microscope (BX53, Olympus Corporation, Tokyo, Japan), and quantitative analysis was conducted using Image J (National Institutes of Health, MD, USA).

2.8. Transcriptome sequencing

The library underwent PE150 sequencing on an Illumina high-throughput sequencing platform (HiSeqTM 250) using differently processed mouse samples (Control, Model, and CR-SAN, n = 3). The resulting sequences were aligned to reference genomes utilizing Stars (v2.52b). |log2(FoldChange)| > 1 and P < 0.05 considered differentially expressed. GOSeq (v1.22) and KOBAS (v2.0) were employed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the differentially expressed genes, with adjusted P < 0.05.

2.9. Cell culture and determination of NO and LDH

Primary bone marrow-derived macrophages (BMDMs) were isolated according to our previously established protocol [1]. BMDMs, HaCaT and RAW264.7 were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % bovine serum and 1 % penicillin/streptomycin. The cells were incubated in a Heracell™ incubator (MCO-15AC CO2, SANYO, Japan) at 37 °C with 5 % CO2. Nitrogen Monoxide (NO) and Lactate Dehydrogenase (LDH) activity were detected by following the instructions of commercially available kits.

2.10. Knock-down of NLRP3 with siRNA

NLRP3 expression was knocked down using siRNA NLRP3 (si-Nlrp3) according to the manufacturer's instructions (Suzhou Genepharma Co., Ltd). The sequence (5′-3′) is CCAACUGGUCAAGGAGCAUTT. Briefly, cells were incubated with 250 μL RNAfit and 10 μL siRNA for 0.5 h, and then, the medium was replaced by DMEM (antibiotics-free) for another 24 h. Cells transfected with non-specific scramble siRNA (si-NC) were used as controls. Cells were subjected to experiment at 48 h after transfection.

2.11. Statistical analysis

Data are presented as mean ± standard deviation (SD). The experimental data and differences were analyzed using GraphPad Prism 9.5 software (GraphPad, San Diego, CA) through one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. Significant differences among groups were indicated as ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. ns No statistical significance.

3. Results

3.1. Preparation, Characterization, and Safety Assessment of CR-SAN

CR-SAN was successfully constructed using a pH-driven method. The morphology of both pH-CR and CR-SAN was observed by TEM and SEM (Fig. 1A-B). pH-CR particles were smaller, with an average diameter of approximately 100 nm. The hydrodynamic size of pH-CR was determined to be 101.0 ± 14.2 nm, with a PDI of 0.11 ± 0.06. The Zeta potential of pH-CR was determined to be −7.1 ± 3.7 mV. In contrast, CR-SAN exhibited a spherical morphology with an average diameter centered around 200 nm and displayed high electron density, indicative of a compact internal structure. The hydrodynamic size of CR-SAN was measured to be 234.9 ± 5.1 nm, with a PDI of 0.21 ± 0.03. The Zeta potential of CR-SAN was determined to be −28.1 ± 3.9 mV, indicating its good colloidal stability. Furthermore, the encapsulation efficiency results (Table S2) indicated that alkaloids participate in the self-assembly of the nanoparticles, rather than existing solely as free molecules in the solution.

Fig. 1.

Fig. 1

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Preparation, Characterization, and Safety Assessment of CR-SAN. (A) The TEM image of pH-CR and CR-SAN; scale bars = 100 nm. (B)The SEM image of pH-CR and CR-SAN; scale bars = 1 μm and 200 μm. (C) The XRD spectrum of pH-CR and CR-SAN. (D)The DTG spectrum of pH-CR and CR-SAN. (E) The TG spectrum of pH-CR and CR-SAN. (F)The FTIR spectrum of pH-CR and CR-SAN. (G) Representative H&E-stained images of the heart, liver, spleen, lungs, and kidneys of mice. (H) Individual blood biochemistry analysis. ns No statistical significance.

Both pH-CR and CR-SAN exhibited similar crystalline characteristics in their XRD patterns. A sharp diffraction peak was observed at 2θ = 9.5°, accompanied by a broad amorphous diffraction band at 2θ = 24.7°. These findings indicate that the pH-driven self-assembly process did not significantly alter the crystalline structure of the material (Fig. 1C). The pH-CR exhibited three distinct thermal decomposition peaks in the range of 350–550 °C, whereas CR-SAN displayed a single thermal decomposition peak around 450 °C. Compared to pH-CR, CR-SAN exhibited a more concentrated thermal degradation process with a higher onset temperature. These results suggest that the self-assembly process significantly improved the thermal stability of CR-SAN (Fig. 1D and E). The FTIR spectrum of pH-CR exhibits a hydroxyl (O–H) stretching vibration absorption peak at 3329 cm−1, a carbonyl (C=O) stretching vibration absorption peak at 1720 cm−1, and characteristic aromatic ring vibrations at 1610 cm−1 and 1508 cm−1. In contrast, the FTIR spectrum of CR-SAN shows the complete disappearance of the C=O absorption peak at 1720 cm−1, while the hydroxyl absorption peak shifts to 3412 cm−1, indicating enhanced hydrogen bonding interactions during the self-assembly process. Additionally, the aromatic ring vibration peaks shift to 1608 cm−1 and 1506 cm−1, suggesting the possible formation of π-π stacking interactions between molecule (Fig. 1F).

To evaluate the biosafety of CR-SAN, we performed histopathological analysis of major organs in mice. Heart, liver, spleen, lung, and kidney tissues were collected from the Control, pH-CR and CR-SAN groups. H&E staining (Fig. 1G) revealed no observable pathological lesions, and no significant changes in body weight were detected. Furthermore, serum biochemical analysis showed no statistically significant differences in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), or blood urea nitrogen (BUN) levels (Fig. 1H).

3.2. CR-SAN enhances penetration and retention in vitro

The transdermal characteristics of CR-SAN were assessed utilizing an advanced Franz diffusion cell, with a focus on elucidating the underlying transdermal mechanisms. Skin retention of Berberine in CR-SAN-H demonstrated a 1.83-fold and 1.92-fold increase compared to CR-SAN-L and CR-SAN-M, respectively. Additionally, the skin retention of Coptisine, Epiberberine, and Palmatine in CR-SAN-H exhibited significantly elevated levels compared to CR-SAN-L and CR-SAN-M (Fig. 2A). The transdermal characteristics of CR-SAN-L and CR-SAN-M exhibited similarity, whereas CR-SAN-H demonstrated notably higher permeability compared to the former two formulations. Following 24 h, the cumulative transdermal Berberine amount in CR-SAN-H surpassed that of CR-SAN-L and CR-SAN-M by factors of 4.00 and 4.63, respectively (Fig. 2B-E). The primary FTIR absorption peaks of skin samples are the asymmetric (2920 cm−1) and symmetric (2850 cm−1) CH2 stretching vibrations of stratum corneum lipids, along with the amide I (1640 cm−1) and amide II (1520 cm−1) peaks related to keratin. Following CR-SAN treatment, a decrease in the intensities of these characteristic peaks was observed compared to the control group, with the most pronounced reduction noted in the CR-SAN-H group. This finding indicates that CR-SAN may potentially augment transdermal drug permeation by influencing lipid arrangement and altering the secondary structure of keratin (Fig. 2F). The DSC analysis of skin samples revealed a distinctive endothermic peak within the 125–150 °C temperature range, indicative of the phase transition of stratum corneum lipids. Following treatment with CR-SAN, this peak underwent a notable leftward shift, implying the disruption of the organized structure of stratum corneum lipids by CR-SAN, thereby enhancing drug permeation (Fig. 2G). CR-SAN treatment led to significant modifications in the microstructure of the stratum corneum surface, manifesting as increased wrinkling, elevated roughness, and the development of fissures, with these effects intensifying with prolonged treatment durations and higher CR-SAN concentrations compared to the control group (Fig. 2H). These findings indicate that CR-SAN disrupts the dense structure of the stratum corneum, resulting in structural relaxation. This phenomenon enhances the penetration and retention of active compounds in the skin.

Fig. 2.

Fig. 2

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CR-SAN enhances penetration and retention in vitro. (A) The drug retention amounts on the skin of different treatment groups at 24 h (n = 3). (B–E) In vitro transdermal permeation curves of Berberine, Coptisine, Palmatine, and Epiberberine in isolated rat skin tissue for 24 h (n = 3). (F) The DSC spectra of 24 h skin treated with different concentrations of CR-SAN. (G) The FTIR spectra of skin at different concentrations of CR-SAN at 24 h. (H) The SEM images of skin treated with different concentrations of CR-SAN for 2 h, 8 h, and 24 h; scale bars = 1 μm (n = 3).

3.3. CR-SAN ameliorates DNCB-induced AD-like symptoms in mice

To evaluate the therapeutic potential of CR-SAN in AD, we established a DNCB-induced AD-like mouse model (Fig. 3A). DNCB challenge elicited characteristic AD symptoms in the Model group, including erythema, scaling, erosion, and exudation (Fig. 3B). The weight changes and dermatitis scores of the mice in each group within 15 days are shown in Fig. 3C. Moreover, serum IgE levels and pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) were significantly suppressed (Fig. 3D–F, S2). Histopathological analysis revealed that the CR-SAN-treated group exhibited a reduction in epidermal and dermal thickness comparable to that observed in the Dexa, in contrast to the AD group (Fig. 3G–S3). Notably, high-dose CR-SAN demonstrated superior efficacy in cytokine inhibition compared to the low-dose regimen. Furthermore, this study investigated the impact of CR-SAN on oxidative stress in AD mice. The CR-SAN treatment significantly elevated serum levels of the antioxidant enzyme total superoxide dismutase (SOD) while reducing malondialdehyde (MDA) and reactive oxygen species (ROS) levels (Fig. 3H and I). Taken together, our findings demonstrate that CR-SAN ameliorates skin lesions by mitigating immune cell infiltration and oxidative stress, both of which play a pivotal role in the inflammatory progression of AD.

Fig. 3.

Fig. 3

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Effect of CR-SAN on DNCB-induced AD in BALB/c mice. (A) Schematic diagram of experimental procedure. (B) Back skin images were obtained at the same magnification and representative images were obtained on day 15. (C) Weight changes (n = 6); Dermatitis score (n = 6). Levels of IgE (D), TNF-α (E), and IL-1β (F) in serum were detected by ELISA assay (n = 6). (G) H&E and TB staining of the mouse skin; scale bars = 50 μm; 100 μm (n = 6). (H) The release levels of SOD and MDA were detected by ELISA (n = 6). (I) ROS level as assessed by DHE staining in skin tissues of mice; scale bars = 100 μm (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. Model group. ns No statistical significance.

3.4. Network pharmacology and transcriptome analysis results targeting NLRs signaling pathways

To elucidate the precise mechanism by which CR-SAN alleviates AD, we performed transcriptome sequencing on skin tissues from AD mice. Principal component analysis (PCA) demonstrated notable differences among the groups, indicating that model establishment and drug intervention could impact transcriptional profiles (Fig. 4A). Reveals 2118 differentially expressed genes in the Model compared to the Control, with 1739 up-regulated and 379 down-regulated genes (Fig. 4B). In contrast, the CR-SAN group exhibited 766 differentially expressed genes compared to the Model, comprising 532 up-regulated and 234 down-regulated genes (Fig. 4C). GO enrichment analysis revealed that the therapeutic effects of CR-SAN were linked to multiple biological processes (Fig. 4D) and KEGG enrichment analysis identified the top 20 enriched pathways (Fig. 4E). Furthermore, integrated network pharmacology (Fig. S4) and transcriptomic analysis revealed that the NOD-like Receptors (NLRs) signaling pathway serves as a key mechanistic pathway underlying CR-SAN-mediated amelioration of AD (Fig. 4F). Subsequent Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) validation confirmed that CR-SAN exerts therapeutic effects on the NLRs signaling pathway particularly the NLRP3 inflammasome (Fig. 4G–N). Based on these findings, we hypothesized that the therapeutic effects of CR-SAN may be linked to NLRP3 pathway modulation, prompting further experimental investigation.

Fig. 4.

Fig. 4

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Network pharmacology and transcriptomics reveal the mechanism of CR-SAN treatment of AD. (A) PCA mapping of Control, Model, and CR-SAN treated samples. (B) Volcano map of DEGs up and down between Control and Model groups. (C) Volcanic map of DEGs up-regulation and down-regulation between Model and CR-SAN group. (D) GO enrichment analysis revealed the anti-AD effects of CR-SAN. (E) KEGG analysis of differentially expressed genes between CR-SAN and Model group. (F) Schematic diagram of the NLRs signaling pathway co-targeted by network pharmacological screening and transcriptomic analysis. (G–N) Quantitative analysis of representative mRNA associated with NLRPs by RT-qPCR (n = 6). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. Model group. ns No statistical significance.

3.5. CR-SAN ameliorates skin inflammatory damage in AD mice via NLRP3/GSDMD-IL-33 axis

To investigate the role of the NLRP3 inflammasome in AD, we analyzed mRNA expression levels of Nlrp3, Pycard, Caspase-1, and Gsdmd in DNCB-induced dorsal lesions. The AD group exhibited a significant increase in mRNA expression, which was markedly reduced in mice treated with CR-SAN (Fig. 5A). IL-33, an alarmin predominantly localized in the nuclear compartment of epithelial cells, is passively released during GSDMD-mediated pyroptosis, analogous to classical secretory signal peptides [25]. We therefore hypothesize that the NLRP3/GSDMD-IL-33 axis may serve as the primary pathogenic driver underlying cutaneous inflammatory responses and the consequent pruritic behaviors and excoriation lesions. In our study, we observed a significant increase in Il-33 mRNA expression in the AD group, which was markedly reduced in CR-SAN (Fig. 5B). Additionally, we assessed the mRNA levels of inflammatory factors Il-18, Il-1β, Tnf-α, Il-6, and Il-4. Our findings demonstrated a significant decrease in the mRNA expression of Tnf-α, Il-6, and Il-4 in CR-SAN (Fig. 5C). Immunohistochemical analysis revealed reduced protein expression levels of NLRP3 and Caspase-1 in the CR-SAN treatment group compared to the Dexa and AD groups (Fig. 5D). Furthermore, Western blot analysis validated dose-dependent alterations in the protein expression of NLRP3, ASC, Caspase-1, GSDMD, and IL-33 (Fig. 5E). Collectively, these findings demonstrate that CR-SAN mitigates symptoms of AD by regulating the NLRP3/GSDMD-IL-33 axis in DNCB-induced skin lesions on the dorsal area.

Fig. 5.

Fig. 5

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CR-SAN ameliorates skin inflammatory damage in AD mice via the NLRP3/GSDMD-IL-33 axis. (A–C) RT-qPCR were used to detect the mRNA expression levels of Nlrp3, Pycard, Caspase-1, Gsdmd, Il-33, Il-1β, IL-18, Tnf-α, Il-6 and Il-4 (n = 6). (D) Immunohistochemical staining analysis of NLRP3 and Caspase-1 expression; scale bars = 50 or 100 μm (n = 6). (E) Western blots were used to detect the expression of NLRP3, ASC, Caspase-1, GSDMD and IL-33 (n = 3). ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. Model group. ns No statistical significance.

3.6. CR-SAN mitigates lipopolysaccharide (LPS)-induced cellular damage and oxidative stress in vitro

CR-SAN demonstrates favorable biosafety and anti-inflammatory activity in HaCaT cells, BMDMs, and RAW264.7 cells. To evaluate its macrophage-targeting efficacy, LPS-stimulated macrophages were employed to assess anti-inflammatory and antioxidant effects. Notably, CR-SAN reduced NO and LDH release in a dose-dependent manner compared to the LPS group (Fig. 6A–C). Furthermore, CR-SAN treatment significantly elevated SOD and decreased the level of MDA in macrophage supernatant (Fig. 6D). Consistent with flow cytometry data, DCFH-DA staining demonstrated that CR-SAN significantly attenuated LPS-induced intracellular ROS generation (Fig. 6E and F). Collectively, these findings demonstrate CR-SAN's ability to protect macrophages against inflammatory injury, with 200 μg/mL established as the optimal concentration for subsequent mechanistic evaluation.

Fig. 6.

Fig. 6

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CR-SAN reduces LPS-induced cellular damage and oxidative stress in vitro. (A–C) Cytotoxicity evaluation and inflammatory marker analysis (NO/LDH) of CR-SAN in HaCaT cells, BMDMs, and RAW264.7 cells. (n = 6). (D) Levels of SOD and MDA release in macrophage supernatants were assessed by ELISA assay (n = 6). (E–F) Levels of ROS in macrophages were observed and quantified by fluorescence upright microscopy and flow cytometry, scale bar = 200 μm (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. LPS group. ns No statistical significance.

3.7. si-Nlrp3 partly erased the protective effect of CR-SAN on LPS-induced macrophages

Mitochondrial dysfunction and the assembly of components of the NLRP3 inflammasome are the main signs of the activation of the NLRP3 inflammasome [26]. To investigate the therapeutic effect of CR-SAN, cells were co-treated with si-Nlrp3. Remarkably, si-Nlrp3 mitigated the release of NO, LDH, and MDA while increasing SOD and ATP release compared to the LPS group partly (Fig. 7A–E). Mito-Tracker and Mito-SOX staining revealed that LPS stimulation led to an elevation in mitochondrial-dependent ROS production. Conversely, CR-SAN reduced the levels of LPS-induced mitochondrial superoxide anions (Fig. 7F). Immunofluorescence analysis demonstrated that CR-SAN decreased the expression of NLRP3 and ASC compared to LPS treatment. Si-Nlrp3 weakened the expression of NLRP3 and ASC (Fig. 7G). Western blot results confirmed that in the group of si-Nlrp3, the inhibitory effect of CR-SAN on the accumulation of LPS-induced NLRP3 inflammasome activation-related proteins was partly erased (Fig. 7H). In conclusion, the findings indicate that CR-SAN has the potential to mitigate the inflammatory response in AD by inhibiting the progression of pyroptosis through the suppression of NLRP3 inflammasome assembly and activation in LPS-stimulated cells.

Fig. 7.

Fig. 7

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si-Nlrp3 partly erased the protective effect of CR-SAN on LPS-induced macrophages. (A–E) Levels of NO, LDH, SOD, MDA, and ATP release were detected by ELISA assay (n = 6). (F) The oxidative stress level was assessed by Mito-Tracker, Mito-SOX staining; scale bar = 10 μm (n = 3). (G) NLRP3 and ASC protein expression was detected by immunofluorescence; scale bar = 10 μm (n = 3). (H) Western blots were used to detect the expression of NLRP3 and Caspase-1 (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. LPS group. ns No statistical significance.

3.8. CR-SAN disrupts the itch-scratch cycle in atopic dermatitis by targeting NLRP3 inflammasome assembly

Subsequently, we further investigated the potential mechanisms by which CR-SAN inhibits NLRP3 inflammasome activation. ASC oligomerization is an essential step for NLRP3 inflammasome assembly, leading to pro-caspase-1 auto-cleavage and mature IL-1β release [[27], [28], [29]]. We therefore examined the effect of CR-SAN on ASC oligomerization using the NLRP3 inhibitor MCC950 as a control. Western blot analysis revealed that 200 μg/mL CR-SAN significantly suppressed LPS/ATP-induced ASC oligomerization in BMDMs (Fig. 8A), demonstrating a direct inhibitory effect of CR-SAN on this process. This finding was further corroborated by caspase-1 activity assays (Fig. 8B). Next, we sought to determine whether CR-SAN directly interferes with inflammasome assembly. Our results showed that CR-SAN treatment markedly reduced the interaction between NLRP3 and ASC/Caspase-1 upon LPS/ATP stimulation (Fig. 8C). Given the multi-component nature of CR-SAN, we employed UHPLC-ESI-QE-Orbitrap-MS to analyze its potential bioactive constituents (Fig. S6, total ion chromatogram; 169 distinct phytochemicals identified shown in Table S3). Molecular docking studies were performed to evaluate the binding patterns of CR-SAN-derived alkaloids (Berberine, Coptisine, Epiberberine, and Palmatine) to NLRP3, using MCC950 as a positive control. The docking scores (−9.4, −9.1, −8.9, −7.6, and −9.8 kcal/mol, respectively) indicated strong binding affinities, particularly for berberine and coptisine (Fig. 8D–E, S7). These interactions were further validated by molecular dynamics simulations, providing robust support for the proposed mechanism (Fig. 8F–S8).

Fig. 8.

Fig. 8

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CR-SAN suppresses NLRP3 inflammasome assembly. (A) Western blot analysis of ASC oligomerization (n = 3). (B) The activity of Caspase-1 was detected by ELISA assay (n = 3). (C) Co-IP was applied to analyze the interaction between ASC, Caspase-1, and NLRP3 (n = 3). Three-dimensional and two-dimensional docking patterns and interactions of NLRP3 with Berberine (D) and Coptisine(E). (F) The results of molecular dynamics simulation analysis showed RMSD, RMSF, and the number of hydrogen bonds for three different proteins and their corresponding complex with covalent ligands. ∗∗∗P < 0.001 vs. LPS-ATP group. ns No statistical significance.

4. Discussion

AD has become one of the most challenging conditions in dermatology due to its increasing prevalence and the limited availability of effective treatments. We successfully constructed CR-SAN using a “controlled assembly” strategy and systematically evaluated its physicochemical properties and transdermal permeation characteristics. Subsequently, we demonstrated CR-SAN's therapeutic efficacy in ameliorating AD mice. Through integrated cellular and molecular analyses, we further elucidated that CR-SAN exerts its anti-AD effects via dual anti-inflammatory and antioxidant mechanisms, primarily through modulation of the NLRP3/GSDMD-IL-33 axis. Collectively, these comprehensive findings provide compelling scientific evidence supporting CR-SAN as a promising therapeutic candidate for AD management.

The self-assembled nanoparticles in traditional Coptidis Rhizoma decoction play a crucial role in the absorption and permeation of alkaloids. However, the concurrent processes of extraction and nanoparticle assembly often result in uncontrolled self-assembly, leading to nanoparticles with large particle sizes and poor stability. In this study, we propose an innovative “controlled assembly” strategy. By precisely controlling a series of steps, including both extraction and self-assembly processes, we successfully constructed CR-SAN with a uniform particle size distribution and enhanced stability. FTIR analysis revealed that hydrogen bonding and π-π stacking interactions played a key role in the self-assembly process of CR-SAN. The resulting CR-SAN exhibited a high Zeta potential, which effectively prevents particle aggregation by promoting electrostatic repulsion, thus maintaining the colloidal stability of the system [30]. This enhanced stability provides a solid foundation for the potential application of CR-SAN in transdermal drug delivery systems.

In subsequent transdermal studies, FTIR analysis revealed a significant reduction in the peak areas corresponding to stratum corneum lipids and keratin, suggesting that CR-SAN may enhance transdermal drug permeation by influencing lipid arrangement and altering the secondary structure of keratin [31]. Furthermore, DSC analysis demonstrated a notable leftward shift in the endothermic peak associated with stratum corneum lipids, indicating a disruption of their highly ordered structure [32]. These structural changes are likely to compromise the integrity of the skin barrier, thereby facilitating the diffusion of drugs through the stratum corneum. The observed alterations in the microstructure of the stratum corneum, such as increased wrinkling, surface roughness, and the formation of fissures, further support the hypothesis that CR-SAN disrupts the compact architecture of the stratum corneum. This disruption enhances the penetration and retention of active compounds within the skin, thereby contributing to the improved efficacy of transdermal drug delivery systems.

Inflammation plays a crucial role in the pathogenesis of AD, where impaired skin barrier function and immune system dysregulation synergistically cause inflammatory cell infiltration and the overproduction of inflammatory mediators [[30], [31], [32], [33]]. However, the specific mechanisms underlying these processes require further elucidation. Our study demonstrates that CR-SAN exerts its therapeutic effects on AD primarily through its anti-inflammatory properties, manifested by a reduction in skin inflammation, stratum corneum thickness, and mast cell infiltration [[34], [35], [36]]. IgE, IL-1β, TNF-α, and IL-6 are widely recognized as biomarkers of AD severity [[37], [38], [39], [40], [41]]. Consistent with prior studies, our findings indicate that CR-SAN effectively lowers serum IgE levels and inhibits the production of inflammatory mediators such as IL-1β, TNF-α, and IL-6. Furthermore, CR-SAN mitigates oxidative stress by enhancing SOD activity and reducing MDA levels. Moreover, through network pharmacology and transcriptomic analysis validated by RT-qPCR, Western blot, and IHC, we confirmed that CR-SAN significantly downregulates the protein expression of NLRP3, ASC, Caspase-1, GSDMD, and IL-33. These results demonstrate that CR-SAN's therapeutic effects are mediated through the NLRP3-GSDMD-IL-33 axis, wherein GSDMD post-translationally regulates IL-33 release, as established in prior studies [24].

To further elucidate the mechanism of CR-SAN in AD, we evaluated its biosafety and anti-inflammatory efficacy using three in vitro models. To assess macrophage-targeting effects, an AD-mimicking pathological model was established via LPS-stimulated macrophages. [[42], [43], [44], [45]]. LDH and NO are commonly recognized as pivotal markers of cellular damage, which exacerbate inflammatory responses and oxidative stress in AD [41,[46], [47], [48], [49]]. Our findings indicate that CR-SAN reduced the release of NO and LDH, increased SOD activity, and decreased MDA levels in the supernatant of LPS-induced macrophages. Moreover, flow cytometry analysis revealed a significant reduction in intracellular ROS levels following CR-SAN treatment compared with the LPS group. Subsequently, we conducted co-treatment experiments with si-Nlrp3 on RAW264.7 cells for further verification. The results showed that si-Nlrp3 decreased the release of NO, LDH, and MDA while increasing SOD and ATP levels. Immunofluorescence and Western blot analyses further supported the finding that CR-SAN increased the accumulation of NLRP3 inflammasome activation-related proteins induced by LPS in si-Nlrp3. These results provide further evidence supporting the anti-inflammatory mechanism of CR-SAN through the inhibition of the NLRP3/GSDMD-IL-33 axis. Notably, our study provides the first experimental evidence that CR-SAN exerts potent inhibitory effects on NLRP3 inflammasome activation. Mechanistically, CR-SAN not only directly targets NLRP3 but also suppresses ASC speck formation and oligomerization, consequently attenuating caspase-1 activity and ultimately blocking inflammasome assembly. This multi-targeted action likely stems from the synergistic effects of bioactive components within CR-SAN, particularly its characteristic alkaloids (e.g., berberine and coptisine). Our computational analyses, including molecular docking and dynamic simulations, further substantiate this mechanism by demonstrating high-affinity binding between these alkaloids and critical domains of NLRP3 (binding energy < −9 kcal/mol).

Collectively, this study integrated transcriptomics and molecular analyses to elucidate the mechanism of CR-SAN. Our in vivo and in vitro experiments demonstrate that CR-SAN disrupts the itch-scratch cycle and preserves skin barrier function by directly inhibiting NLRP3 inflammasome assembly, thereby suppressing GSDMD-IL-33 axis activation. However, the molecular dynamics underlying interactions between CR-SAN's bioactive components require further investigation to delineate their synergistic effects.

5. Conclusion

This study develops an innovative "controlled assembly" strategy to fabricate CR-SAN nanoparticles from Coptidis Rhizoma decoction, which exhibit enhanced skin permeation and potent NLRP3 inflammasome inhibition. CR-SAN alleviates AD symptoms by specifically suppressing NLRP3-mediated ASC oligomerization, caspase-1 activation, and GSDMD-IL-33 signaling, while improving skin barrier function. These findings establish CR-SAN as a promising NLRP3-targeted therapeutic for AD, while providing new insights for developing self-assembled nanoparticles from TCM.

CRediT authorship contribution statement

Rui Song: Writing – original draft, Validation, Software, Resources, Investigation, Formal analysis, Data curation. Yuwen Zhu: Methodology, Investigation, Formal analysis, Data curation. Kailin Xue: Methodology, Investigation, Formal analysis. Xiang Deng: Project administration, Investigation, Formal analysis. Run Wang: Investigation, Formal analysis, Conceptualization. Yaya Su: Investigation. Xu Chen: Writing – review & editing, Supervision, Project administration. Hailong Yuan: Validation, Supervision, Data curation, Conceptualization.

Ethics approval and consent to participate

All animal experiments received approval from the Ethics Committee of Air Force Medical Center, PLA of China (No. 2025-45-PJ01).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by grants from the Special Plan for Cultivating and Enhancing the Medical and Pharmaceutical Service Capacity of the Military(2023ZY031).

Footnotes

Peer review under the responsibility of editorial board of Bioactive Materials.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioactmat.2025.10.010.

Contributor Information

Xu Chen, Email: dr-chenxu@fmmu.edu.cn.

Hailong Yuan, Email: yuanhailong@fmmu.edu.cn.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.docx (5.2MB, docx)

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