All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Enzyme-linked immunosorbent assay (ELISA) kits were obtained from Enzyme Immunoassay Co., Ltd. (Jiangsu, China). The Calcein/PI Live/Dead Viability/Cytotoxicity Assay Kit, Cell Counting Kit-8 (CCK-8), and ROS detection kit were acquired from Beyotime Biotechnology Co., Ltd. (Shanghai, China). PCR reagents were provided by Accurate Biotechnology Co., Ltd. (Changsha, China). CAT, MDA, SOD, glutathione (GSH), and nicotinamide adenine dinucleotide (NADH) assay kits were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Anti-PI3K (AF6241) and anti-phospho-PI3K (AF3242) antibodies were sourced from Affinity Biological Research Center (Jiangsu, China). Anti-β-actin (66009-1), anti-NF-κB p65 (66535-1), anti-phospho-NF-κB p65 (82335-1), anti-AKT (10176-2), and anti-phospho-AKT (66444-1-Ig) antibodies were obtained from Proteintech Group, Inc. (Wuhan, China). Staphylococcus aureus (S. aureus, ATCC 43300) and Escherichia coli (E. coli, ATCC 25923) were obtained from the Institute of Burn Research, Southwest Hospital, Third Military Medical University.

Fifty female BALB/c mice, aged 6–8 weeks, were used in this study. After one week of acclimatization, the animals were randomly assigned to five experimental groups: control group, AD model group, blank hydrogel group (HG), BHG group, and dexamethasone group (Dex). One day prior to the experiment, hair was removed from a 2 cm × 3 cm area on the dorsal skin of each mouse using an electric shaver followed by depilatory cream. The control group received vehicle solution of the 2,4-dinitrofluorobenzene DNFB inducer (a 4:1 acetone/olive oil mixture). On days 1 and 3 of the experiment, 100 μL of 0.5% DNFB solution was administered via micropipette and evenly applied to the dorsal skin, with an additional 20 μL applied to the ears. Starting on day 5, mice were treated every other day with 0.2% DNFB solution, while HG, BHG, and Dex administrations were initiated concurrently and administered once daily for two consecutive weeks. Successful induction of the AD model was confirmed by the presence of clinical signs including erythema, mild erosion, edema, crusting, and increased scratching frequency. All animal procedures were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Chongqing Medical University (IACUC-CQMU) (approval number: IACUC-CQMU-2024–0579).

Equal volumes of 1% sodium hyaluronate solution and 10% gelatin solution were mixed and stirred in a 55°C water bath to form a homogeneous composite hydrogel matrix. Berberine (40 mg/mL) was added proportionally to the hydrogel and continuously stirred under the same temperature conditions, followed by repeated freeze-thaw cycles until complete incorporation and uniform distribution of berberine within the hydrogel matrix were achieved.

The morphology and pore size of the hydrogel were examined using scanning electron microscopy (HITACHI SU-X650, Tokyo, Japan). The hydrogel was freeze-dried under vacuum for 48 hours prior to analysis. Following gold sputter coating, the sample was imaged under scanning electron microscopy (SEM).

To evaluate the antimicrobial activity of the hydrogel, samples were aseptically treated with UV irradiation and subsequently incubated with Staphylococcus aureus and Escherichia coli. Bacterial cultures of S. aureus and E. coli were grown in Luria-Bertani (LB) broth and maintained at 37°C under shaking conditions at 160 rpm. A bacterial suspension with an initial concentration of 10⁶ CFU/mL was incubated with the hydrogel for 12 hours, after which the inhibition rate was determined using a microplate reader (BioTek, USA) by measuring optical density at 600 nm. Additionally, the bacterial suspension was adjusted to an OD₆₀₀ of approximately 0.6 before being inoculated into sterile LB liquid medium. Sterile hydrogel specimens were introduced into test tubes containing 5 mL of the bacterial suspension and incubated at 37°C for 12 hours. Subsequently, 10 μL of the resuspended culture was plated onto solid agar medium, followed by incubation at 37°C for 12 hours to assess antibacterial efficacy. The resulting colonies were documented by photography.

The HaCaT cell line was purchased from Hysigen Bio. Cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. The BHG was evenly spread onto the bottom of 96-well or 24-well plates and sterilized by UV irradiation for 12 hours prior to use.

To evaluate the effect of the hydrogel on HaCaT cell viability and proliferation, cells were seeded at a density of 6,000 cells per well in 96-well plates and incubated for 24 hours. Subsequently, the culture supernatant was replaced with fresh medium containing 10% CCK-8 solution, and the cells were further incubated for 2 hours. Then, 100 μL of the resulting solution was transferred to a new 96-well plate, and the optical density (OD) was measured at 450 nm using a microplate reader (BioTek, USA). For fluorescence live/dead staining, cells were seeded at a density of 10,000 cells per well in 24-well plates and incubated for 24 hours. After one wash with phosphate-buffered saline (PBS), 250 μL of working solution containing calcein acetoxymethyl ester (Calcein AM) and propidium iodide (PI) was added to each well and incubated at 37°C for 30 minutes in the dark. Fluorescence images were captured using a fluorescence microscope (Leica, Germany).

To assess the in vitro hemocompatibility of the hydrogel, whole blood was collected from healthy mice via orbital sinus puncture. An equal volume of normal saline was added to dilute the blood, followed by centrifugation at 3,000 rpm for 10 minutes. The supernatant was discarded, and the erythrocyte pellet was washed 2–3 times with normal saline until the supernatant became colorless. A 2% (v/v) red blood cell (RBC) suspension was prepared in normal saline. Normal saline served as the negative control, and distilled water served as the positive control. Hydrogel samples were cut into 5 × 5 mm² pieces, thoroughly mixed with the RBC suspension, and incubated in a constant-temperature water bath at 37°C for 1 hour. After centrifugation under the same conditions, the appearance of the supernatant in each tube was visually inspected, and 100 μL of the supernatant was transferred to a 96-well plate for OD measurement at 540 nm.

The severity of AD lesions was clinically assessed based on four parameters: edema, erythema, erosion, and scaling or dryness. Severe manifestations were assigned a score of 3, moderate lesions a score of 2, mild lesions a score of 1, and absence of lesions was scored as 0. The total dermatitis score for each subject ranged from 0 to 12. Prior to the end of the experiment, spontaneous scratching behavior was observed and recorded over a 10-minute period, with continuous scratching episodes counted as a single event.

At the conclusion of the experiment, heart, liver, spleen, kidney, dorsal skin, and ears were harvested from mice and fixed in 4% paraformaldehyde for 24 hours. Subsequently, paraffin-embedded tissue blocks were sectioned at a thickness of 4 μm using a microtome. Following deparaffinization and rehydration, the sections were stained with Hematoxylin-eosin (H&E), and histological architecture and cellular morphology were examined under a light microscope (Leica, Germany).

To evaluate mast cell infiltration in skin tissues, sections were stained with 0.25% toluidine blue solution for 20 minutes. The number of positively stained mast cells (appearing dark purple-blue) was quantified by light microscopy.

The tissue sections were immersed in antigen retrieval solution at 95°C for 20 minutes, then rinsed with phosphate-buffered saline (PBS). Subsequently, 5% normal serum blocking solution was applied dropwise and incubated at room temperature for 30 minutes to minimize nonspecific binding. Primary antibodies against Filaggrin, Occludin, ZO-1, and MMP-9, appropriately diluted in buffer, were added dropwise and incubated overnight at 4°C. After thorough washing with PBS, biotin-conjugated secondary antibodies were applied dropwise and incubated for 30 minutes at room temperature. Next, horseradish peroxidase (HRP)-labeled streptavidin was added dropwise and incubated for an additional 30 minutes at room temperature. Thereafter, DAB (3,3′-diaminobenzidine) chromogen solution was applied, and the slides were monitored under a microscope for color development. The reaction was terminated by rinsing with distilled water when distinct positive staining was observed. Finally, nuclei were counterstained with hematoxylin, and the tissue sections were dehydrated, cleared, and mounted for examination under a light microscope.

Fresh skin lesion tissues were used to prepare frozen sections. A working solution of the fluorescent probe DCFH-DA (10 μM) was applied dropwise to the tissue sections and incubated in the dark at 37°C for 30 minutes. After washing to remove excess probe, the slides were mounted with a DAPI-containing mounting medium and sealed. Fluorescence intensity was then visualized and recorded using a fluorescence microscope.

The frozen skin lesion samples were weighed, and the levels of CAT, MDA, SOD, GSH, and the NAD⁺/NADH ratio were quantitatively determined according to the manufacturer’s instructions for the respective assay kits.

Skin tissue samples were homogenized, lysed, and centrifuged to collect the supernatant. Protein concentration was determined using the BCA assay. Protein samples were prepared by addition of protein loading buffer, boiled for 10 minutes, and stored at -80°C. Equal amounts of protein were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 hours at room temperature (RT). Thereafter, membranes were incubated overnight at 4°C with appropriate primary antibodies to ensure specific binding to target proteins. On the following day, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system (Bio-Rad, USA), and band intensities were quantified by densitometry using ImageJ software.

Total RNA was extracted from dorsal skin lesion tissues using the TRIzol reagent method, and RNA concentration and purity were assessed with a NanoDrop spectrophotometer. The extracted RNA was reverse transcribed into complementary DNA (cDNA) using a reverse transcription kit according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed using SYBR^® Green Pro Taq HS Premix II on a Bio-Rad CFX96 Real-Time System (Bio-Rad Laboratories, USA). The relative mRNA expression levels were calculated using the 2^-ΔΔC_T method and normalized to the expression of the housekeeping gene Gapdh. Primer sequences used in this study are provided in Supplementary Table S1.

Disease-related targets were retrieved from public databases including GeneCards, OMIM, and DisGeNET. Berberine-associated targets were collected from ChEMBL, CTD, and PubChem. Target genes were then standardized using UniProt, and non-protein-coding or unverified entries were removed to ensure data accuracy. The overlapping targets between berberine and AD were identified through cross-referencing, and a Venn diagram was generated using R software to visualize the intersection. To further investigate the protein-protein interaction (PPI) network underlying the therapeutic mechanism, the shared target genes were uploaded to the STRING database for PPI network construction. Subsequently, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the intersected gene set. The results were visualized to predict the potential molecular mechanisms by which berberine exerts therapeutic effects in AD.

Data were analyzed using GraphPad Prism (version 9.5.0), and one-way ANOVA was employed to assess differences among groups. All data are presented as mean ± standard error of the mean (SEM). Statistical significance was considered at P < 0.05.

Hydrogels are a class of drug delivery systems characterized by excellent water retention capacity and biocompatibility. Berberine was uniformly incorporated into the hydrogel matrix, and the structural morphology was examined using SEM. As shown in Figure 2A, both the blank and BHG exhibited a well-defined three-dimensional porous network structure. At 12 hours, the transdermal concentration of berberine reached 1.5 μg/mL and remained stable up to 24 hours (Figure 2B), demonstrating sustained release properties. Chromatograms of berberine standards, blank hydrogels, and BHGs are presented in Supplementary Figure S1.

To evaluate the safety profile of the BHG, cell viability assays using HaCaT keratinocytes and erythrocyte hemolysis tests were performed. CCK-8 assay results showed no significant reduction in HaCaT cell viability after exposure to the hydrogel (Figure 2C). Consistently, live/dead staining using Calcein AM and PI revealed minimal cell death across all treatment groups (Figure 2D), further confirming low cytotoxicity. In the in vivo experiments, H&E staining results indicated that the morphological and structural features of the liver, heart, kidney, and spleen of mice in each group were generally consistent. These results are presented in Supplementary Figure S2.

In addition, hemolysis testing demonstrated that the BHG induced a hemolysis rate of 0.28 ± 0.09% (n = 4), significantly lower than that of the positive control (>95%) and well below the 2% threshold for non-hemolytic biomaterials. Compared with the blank hydrogel (0.53 ± 0.2%), berberine incorporation did not significantly increase hemolytic potential, indicating favorable blood compatibility (Figure 2E).

In vitro antibacterial assays revealed that the BHG exerted inhibitory effects against both Staphylococcus aureus and Escherichia coli. The antimicrobial activity was more pronounced against Escherichia coli (Figures 2F, G), suggesting broad-spectrum antibacterial functionality.

A mouse model of AD induced by DNFB exhibited characteristic symptoms including skin erythema, scaling, exudation, and epidermal thickening. Starting on day 5, the BHG was applied topically to the dorsal and auricular skin of mice to evaluate its therapeutic effects on dermatitis. Mice in the model group developed severe crust formation, scaling, and erosion on both dorsal and ear skin. In contrast, dermatitis symptoms in the BHG group were significantly alleviated, with a marked reduction in clinical dermatitis scores. In the Dex treatment group, extensive crusting covered with regrowing hair was observed (Figures 3A, B). No significant differences in body weight were observed among the groups; however, a decreasing trend was noted in both the model and Dex groups (Figure 3C). Compared with the control group, ear thickness and weight were significantly increased in the model group, indicating inflammatory swelling, which was markedly reduced following treatment with either BHG or dexamethasone (Figure 3D).

Pruritus is a hallmark symptom of atopic dermatitis; therefore, scratching behavior was recorded over a 10-minute observation period. The results showed no scratching episodes in the control or Dex groups, whereas model mice exhibited an average of six scratching bouts. Treatment with BHG reduced the average scratching frequency by approximately 50% (Figure 3E).

Subsequently, serum levels of key inflammatory markers were analyzed. ELISA results demonstrated that IL-4, IL-13, IgE, and histamine levels were significantly lower in both the BHG and Dex groups compared to the model group (Figures 3F–I), indicating effective suppression of systemic allergic inflammation.

Patients with atopic dermatitis often exhibit skin thickening; therefore, dorsal and auricular skin thickness in mice was measured using a vernier caliper. Results showed that skin thickness was significantly increased in the model group, and treatment with BHG led to a significant reduction (Figures 4A, B). Histopathological changes in ear and dorsal skin were further evaluated by H&E staining. In both the model and blank hydrogel groups, marked epidermal and dermal thickening was observed, accompanied by hyperkeratosis and parakeratosis in the epidermis, as well as inflammatory cell infiltration in the dermis. Following treatment with BHG, skin thickness decreased and inflammatory cell infiltration was reduced. In contrast, although dexamethasone treatment reduced skin thickness, it was associated with persistent inflammatory cell infiltration and increased tissue rigidity, which may reflect adverse effects related to long-term corticosteroid use (Figures 4C, D).

Mast cell accumulation is closely associated with Th2-type immune responses, and represents a key immunopathological feature of atopic dermatitis. Toluidine blue staining of dorsal skin lesions revealed a significant increase in dermal mast cells in the model group, while mast cell infiltration was markedly reduced following treatment with BHG, indicating alleviation of allergic inflammation (Figure 4E).

A total of 147 target genes were commonly identified in both berberine and AD, with the top four being AKT1, TNF-α, IL-6, and IL-1β (Figures 5A, B). GO biological process enrichment analysis revealed that these shared targets were predominantly involved in oxidative stress and inflammatory response pathways (Figure 5C). KEGG pathway enrichment analysis indicated that the PI3K-AKT signaling pathway is a key potential mechanism underlying the therapeutic effects of berberine in AD (Figure 5D). Previous studies have demonstrated that the PI3K/AKT pathway regulates the activation of downstream NF-κB (19, 20). As a central transcriptional regulatory hub, the NF-κB signaling pathway promotes the expression of various pro-inflammatory mediators, including cytokines such as IL-6, TNF-α, and IL-1β, chemokines such as IL-8 and MCP-1, and matrix metalloproteinase MMP-9 (21). This pathway is critically involved in immune responses, inflammatory processes, and cell survival, highlighting its significance in the pathogenesis of AD.

ROS levels in dorsal skin tissues of mice were assessed using immunofluorescence staining. As shown in Figure 6A, ROS fluorescence intensity was significantly elevated in the AD model group compared to the control group. Fluorescence intensity decreased in both the HG and BHG groups, with the lowest ROS levels observed in the BHG group. Subsequently, the expression of oxidative stress-related enzymes was evaluated. Compared with the control group, the levels of GSH, CAT, and the NAD⁺/NADH ratio were significantly reduced in the AD model group, and treatment with BHG restored these parameters toward normal levels, although the differences did not reach statistical significance (Figures 6B–D). In contrast, the BHG group exhibited a significant increase in SOD activity compared to the AD model group (Figure 6E), along with a significant reduction in MDA levels (Figure 6F). These findings collectively indicate that the BHG ameliorates oxidative stress in the skin tissue of a mouse model of AD.

Barrier dysfunction is one of the core pathogenic mechanisms of AD, contributing to a vicious cycle with disease progression. Therefore, the expression of skin barrier-related proteins was evaluated using immunohistochemical staining. As shown in Figures 7A–C, the model group exhibited significantly reduced staining intensity of filaggrin, occludin, and ZO-1 in the epidermis, which was markedly restored following treatment with BHG. In contrast, MMP-9 protein expression was significantly upregulated in AD mice but downregulated in the BHG group (Figure 7D).

Finally, the expression of inflammation-related mRNAs in dorsal skin tissue of mice was analyzed by quantitative PCR. In the model group, mRNA levels of IL-4, IL-13, IL-6, IL-1β, IL-10, and TNF-α were significantly upregulated compared to the control group. Following BHG treatment, the expression of these inflammatory factors was significantly downregulated, whereas no significant improvement was observed in the dexamethasone group (Figures 8A–F). Subsequently, protein expression in mouse skin tissues was assessed by Western blotting. Compared with the control group, phosphorylated PI3K, AKT, and NF-κB proteins were significantly elevated in the model group, while their expression was markedly reduced following treatment with BHG (Figures 8G–J).