Abstract

Background

   Radiation-induced skin injury (RISI) is a notable complication of
   cancer radiotherapy, impacting patients’ quality of life. Existing
   interventions mainly address symptoms, with limited success in
   targeting the fundamental mechanisms. Berberine (BBR), a bioactive
   compound recognized for its anti-inflammatory, antioxidant, and
   anti-fibrotic characteristics, presents a compelling option for
   treating RISI.

Methods

   The molecular targets of BBR and RISI were identified using Swiss
   Target Prediction and GeneCards databases. A protein-protein
   interaction (PPI) network was then constructed, and core targets were
   screened with the Cytoscape plug-in. Molecular functions and pathways
   were analyzed through GO and KEGG pathway enrichment analyses.
   Proteomic analysis identified differential protein expression following
   BBR treatment. Molecular docking validated BBR’s binding to core
   targets PRKACA and PIK3CB. Finally, the therapeutic efficacy of BBR was
   confirmed in irradiated cell and animal models.

Results

   BBR is pivotal in modulating molecular pathways linked to inflammation,
   oxidative stress, and tissue repair. Protein histology indicates a
   marked increase in epithelial migration and proliferation markers
   (KRT14, KRT16) and a decrease in inflammatory markers (IL6ST,
   TNFRSF10B). Enrichment of pathways like the MAPK cascade and epithelial
   development highlights BBR’s role in skin regeneration. Molecular
   docking confirms BBR’s stable binding to key targets PRKACA and PIK3CB,
   essential for cell proliferation and inflammation control. Moreover,
   BBR treatment promoted the proliferation of irradiated cells and
   accelerated wound healing in irradiated animal models.

Conclusion

   Berberine demonstrates multi-target therapeutic potential in managing
   RISI by modulating inflammation, oxidative stress, and cellular repair
   processes. These findings provide a foundation for future clinical
   studies to optimize its dosage and delivery, aiming to improve
   treatment outcomes for RISI.

   Keywords: berberine, radiation-induced skin injury (RISI), proteomics,
   network pharmacology, wound healing

Introduction

   Radiation therapy is a cornerstone of oncology, effectively targeting
   malignant cells ([50]Liangliang et al., 2024). However, it often leads
   to radiation-induced skin injury (RISI), affecting 85%–95% of patients
   ([51]Yan et al., 2024). RISI manifests as erythema ([52]Abdlaty, 2016),
   xerosis, severe desquamation, ulceration, and late-stage fibrosis,
   significantly impairing quality of life and causing treatment
   interruptions in 10%–15% of cases ([53]Yiren et al., 2024). The
   pathophysiology involves DNA strand breaks, ROS-induced oxidative
   stress, activation of pro-inflammatory cytokines (notably TGF-β and
   IL-6), and dysregulated fibroblast proliferation ([54]Tao et al.,
   2024). Current treatments, such as topical corticosteroids, hydrogel
   dressings, and barrier creams, offer only palliative relief and do not
   effectively prevent chronic radiation dermatitis ([55]Deng et al.,
   2024). This highlights the urgent need for targeted interventions that
   address the molecular mechanisms of RISI while promoting tissue
   regeneration.

   Berberine (BBR), a natural isoquinoline alkaloid found in Berberis
   species and other traditional medicinal plants, has gained attention as
   a potential therapeutic agent for RISI management due to its diverse
   mechanisms of action ([56]Amin et al., 2023). Historically used in
   traditional medicine to treat gastrointestinal disorders, diabetes, and
   infections, berberine has shown radioprotective properties against
   radiation-induced organ damage in recent studies ([57]Xiao-Yu et al.,
   2024). Experimental models indicate its ability to lower the incidence
   of radiation pneumonitis, improve pulmonary oxygenation, and
   downregulate fibrogenic markers such as TGF-β1 in irradiated lung
   tissue ([58]Yunfang et al., 2008). By targeting oxidative stress,
   inflammatory signaling, and tissue repair processes, berberine offers a
   promising multimodal approach for managing RISI.

   This study utilized network pharmacology to explore the therapeutic
   mechanisms and key targets of BBR in managing RISI. Quantitative
   proteomic analysis demonstrated BBR’s influence on protein expression
   in irradiated cells, pinpointing two core targets validated via
   molecular docking (binding energies ≤−7.8 kcal/mol). BBR treatment
   significantly improved cell proliferation and reduced cell death in
   irradiated cells. Additionally, oral administration of BBR (50 mg/kg)
   in an animal model significantly mitigated RISI progression,
   underscoring BBR’s potential as a multifunctional phytotherapeutic
   agent for RISI prevention and treatment.

Materials and methods

Target prediction of BBR

   The molecular structure of BBR was obtained from the PubChem database
   ([59]https://pubchem.ncbi.nlm.nih.gov/) ([60]Sunghwan et al., 2022).
   The compound then submitted to the Swiss Target Prediction platform
   ([61]http://www.swisstargetprediction.ch/) for potential target
   screening ([62]Antoine et al., 2019), with a probability threshold set
   at >0. Using the UniProt database ([63]https://www.uniprot.org/), we
   systematically mapped the corresponding protein identifiers to their
   respective gene names (UniProt: the Universal Protein Knowledgebase).
   This integrated bioinformatics approach successfully identified a
   comprehensive set of potential molecular targets for BBR.

RISI target prediction

   The target genes associated with radiation-induced skin injury (RISI)
   were retrieved from the GeneCards database
   ([64]https://www.genecards.org/) using the search term
   “radiation-induced skin injury” ([65]Gil et al., 2016). After
   consolidating the identified targets, their gene symbols were
   standardized using the UniProt database. These RISI-related targets
   were then cross-referenced with the potential therapeutic targets of
   berberine (BBR). The overlapping genes, which may represent BBR’s
   mechanistic targets against RISI, were visualized using a Venn diagram
   for further functional analysis.

Protein–protein interaction network construction

   The intersecting target genes were analyzed using the STRING database
   (version 11.5; [66]http://string-db.org/) with the species limited to
   Homo sapiens and an interaction confidence score threshold of <0.4
   ([67]Damian et al., 2022). The resulting protein-protein interaction
   (PPI) network was exported in TSV format and visualized using Cytoscape
   3.9.0 for topological analysis ([68]Paul et al., 2003). Densely
   interconnected network modules were identified with the MCODE plugin
   (v2.0.0), which implements a graph-theoretical clustering algorithm
   specifically optimized for biological networks. Nodes with the highest
   network centrality (degree centrality >15) were designated as hub
   targets for further functional validation.

GO and KEGG pathway enrichment analysis

   Functional annotation and pathway enrichment analyses were performed
   using the clusterProfiler R package (version 3.14.3) to elucidate the
   biological processes and signaling pathways associated with BBR’s
   therapeutic targets in RISI. Statistical significance thresholds were
   set at p < 0.01 with a false discovery rate (FDR) < 0.05 ([69]The Gene
   Ontology Consortium, 2018). For Gene Ontology (GO) annotation, GO IDs
   were extracted from the identified proteins using eggnog-mapper
   software based on the EggNOG database ([70]Jaime et al., 2018).

Cell processing and protein extraction

   Human umbilical vein endothelial cells (HUVECs) were cultured according
   to a previously described protocol ([71]Bahare et al., 2014). The cells
   were seeded in flat-bottom 6-well plates and allowed to grow until they
   reached 80%–90% confluence using an optical microscope and an eyepiece
   graticule. The culture medium was then replaced with fresh medium.
   After an additional 24 h of cultivation, the cells were exposed to 5 Gy
   of 160 kVp X-ray irradiation using a RADSOURCERS 2000 X-ray machine
   (United States) at a dose rate of 1.20 Gy/min. Concurrently or
   separately, the cells were treated with 5 µM berberine (BBR) and
   incubated at 37.8°C in a 5% CO[2] atmosphere for another 24 h. The
   sample was grinded with liquid nitrogen into cell powder and then
   transferred to a 5-mL centrifuge tube. After that, four volumes of
   lysis buffer (8 M urea, 1% protease inhibitor cocktail) were added to
   the cell powder, followed by sonication 3 minutes on ice using a high
   intensity ultrasonic processor (Scientz) (Note: For PTM experiments,
   inhibitors were also added to the lysis buffer, e.g., 3 μM TSA and
   50 mM NAM for acetylation, 1% phosphatase inhibitor for
   phosphorylation). The remaining debris was removed by centrifugation at
   12,000 g at 4°C for 10 min. Finally, the supernatant was collected and
   the protein concentration was determined with BCA kit according to the
   manufacturer’s instructions.

   The ratio of the relative quantitative values of proteins in two
   samples was used as the fold change (FC). The fold change of proteins
   between sample A and sample B was calculated. A fold change greater
   than 1.5 was set as the threshold for significant upregulation, while a
   fold change less than 1/1.5 was set as the threshold for significant
   downregulation.

   For the CCK-8 assay, HUVECs were seeded in 96-well plates at a density
   of 1.5 × 10^3 cells/cm^2 and divided into the following groups: a
   control group and BBR-treated groups with different concentrations
   (5 μM, 10 μM, and 20 μM). After 24 h of culture, cell proliferation was
   assessed using the CCK-8 kit, and the optical density (OD) at 450 nm
   was measured with a Bio-Rad 450 microplate reader. To evaluate the
   effects of BBR on irradiated cells, the same experimental procedure was
   repeated under 20 Gy irradiation. Additionally, cytotoxicity was
   determined by measuring LDH (Lactate Dehydrogenase) release in HUVECs
   treated with the aforementioned concentrations of BBR (5 μM, 10 μM, and
   20 μM).

Irradiated animal model and normal wound data

   To investigate single-cell transcriptional changes in irradiated skin,
   rats were anesthetized via intraperitoneal injection of ketamine
   (75 mg/kg) and medetomidine (10 mg/kg), followed by shaving of the
   gluteal hair using a razor. A 3-cm-thick lead shield was positioned to
   delineate a 3 cm × 4 cm radiation field. Radiation was delivered using
   a 6-MeV electron beam accelerator at a dose rate of 1,000 cGy/min, with
   a total dose of 30 Gy administered to the target area. Skin tissues
   were harvested from irradiated regions at 7, 14, 28, and 60 days
   post-exposure ([72]Xiaowu et al., 2019), with non-irradiated skin
   samples serving as controls. The resulting sequencing data have been
   deposited in the GEO database under accession number [73]GSE193564
   ([74]Fenghao et al., 2024).

   To further evaluate the therapeutic effects of berberine (BBR) on
   radiation-induced skin injuries, we established an irradiated murine
   model. Mice in the BBR treatment group received intragastric
   administration of BBR (50 mg/kg/day) for three consecutive days prior
   to irradiation. The radiation-induced skin ulcer model was established
   as previously described ([75]Sanath et al., 2008). Briefly, mice were
   anesthetized with 1% pentobarbital sodium (30 mg/kg), after which their
   hind limbs were exposed to 160 kVp X-ray irradiation (RADSOURCERS 2000
   X-ray machine, United States) at a dose rate of 1.20 Gy/min for
   8–9 min, delivering a total dose of 20 Gy (Precision X-ray). Lead
   shielding was used to protect other body parts. Skin wound healing was
   monitored for 24 days post-irradiation in both BBR-treated and
   irradiation-only (IR) groups, with photographic documentation of wound
   sites. Skin injury severity was graded according to a five-point scale
   previously established by our research group ([76]Rhonda et al., 2016).

   Furthermore, transcriptomic data from normal wound healing at day 1,
   day 7, and chronic ulcer wounds were obtained from the GEO database
   (accession number [77]GSE174661) ([78]Zhuang et al., 2022). By
   integrating these datasets with transcriptomic data from irradiated
   animal skin, we analyzed the expression dynamics of the core genes
   PRKACA and PIK3CB under both irradiated and normal wound healing
   conditions.

Molecular docking and molecular dynamic simulations

   The 3D structures of BBR’s core targets in RISI were retrieved from the
   PDB database ([79]https://www.rcsb.org/) ([80]Damiano, 2024). We
   obtained the crystal structures of key targets from the PDB library,
   including PRKACA (PDB ID: 8X5L) and PIK3CB (UniProt ID: [81]P42338).
   Using AutoDock Tools, we performed protein preparation by removing
   water molecules, adding hydrogen atoms, and assigning charges.
   Subsequently, molecular docking with BBR was conducted ([82]Garrett et
   al., 2008). For molecular dynamics analysis, the IMOD server
   ([83]http://imods.chaconlab.org) was employed to assess the stability
   and molecular mobility of the BBR-core target complexes. The IMODS
   platform additionally enabled structural dynamics analysis of the
   docking complexes. Elastic networks, deformability, B-factors,
   eigenvalues, variance, and covariance maps were all used to illustrate
   the stability of the BBR-core target complex.

Statistical analysis

   For comparisons between two groups, Student’s t-test or Mann-Whitney U
   test was applied based on data distribution. One-way analysis of
   variance (ANOVA) followed by Tukey’s post hoc test was used for
   multiple group comparisons. Two-way ANOVA was employed to analyze the
   interaction effects between different treatment conditions. In all
   tests, a p-value <0.05 or a false discovery rate (FDR) < 0.05 was
   considered the threshold for statistical significance.

Results

Targets of BBR and RISI

   The potential targets of BBR were explored by inputting its chemical
   structure ([84]Figure 1A) in SMILES format. An initial examination
   identified 121 potential BBR-associated targets, offering initial
   understanding of its mechanism of action. Subsequently, 6,880
   associated targets were discovered according to the GeneCards database.
   After integrating the data and applying stringent threshold-based
   filtering, a final cohort of 2,921 RISI-related target genes was
   defined for subsequent analysis ([85]Figure 1B). Through Venn diagram
   analysis, we identified 82 overlapping targets. These candidate targets
   were prioritized based on their dual involvement as both molecular
   targets of berberine and genetic markers associated with RISI
   ([86]Figure 1B).

FIGURE 1.

   [87]FIGURE 1
   [88]Open in a new tab

   Structure and potential targets of BBR. (A) Structure of BBR. (B)
   Overlapping targets between RISI and BBR. (C) PPI network of
   overlapping targets. (D,E) GO and KEGG pathway enrichment analysis.
   (F–H) Construction of PPI network, subnetwork 1 and subnetwork 2 via
   Cytoscape.

GO and KEGG results

   Functional enrichment analysis (GO/KEGG) of the 82 overlapping targets
   (FDR <0.05) revealed 1,838 significant terms. The analysis encompassed
   1,644 biological processes, including peptidyl-serine phosphorylation,
   xenobiotic metabolic response, cell cycle regulation (particularly G2/M
   transition), and MAPK cascade activation. Cellular component analysis
   revealed 71 terms, primarily involving membrane structures (rafts,
   microdomains, caveolae) and kinase complexes (PI3K, cyclin-dependent,
   serine/threonine). Molecular function analysis identified 123 terms,
   predominantly related to kinase activities (serine/threonine/tyrosine,
   histone), binding functions (heme, tetrapyrrole, insulin receptor
   substrate) and enzymatic activities (PI3K, PIP2 kinase) ([89]Figure
   1D). KEGG pathway enrichment analysis identified 161 significant
   pathways ([90]Figure 1E), including the AGE-RAGE signaling pathway in
   diabetic complications, endocrine resistance, progesterone-mediated
   oocyte maturation, lipid metabolism and atherosclerosis, Kaposi
   sarcoma-associated herpesvirus infection, neurotrophin signaling
   pathway, VEGF signaling pathway, cellular senescence, Yersinia
   infection, and prolactin signaling pathway.

PPI network construction and hub targets selection

   Subsequent STRING database analysis demonstrated significant functional
   interactions among proteins encoded by the 82 overlapping targets
   ([91]Figure 1C). The network contained 81 nodes and 488 edges
   ([92]Figure 1F). MCODE plugin analysis identified two functionally
   significant subnetworks: Subnetwork 1 (23 nodes, 105 edges) contained
   core targets including ATR, CDK4, ABL1, JAK2, RAC1, PIK3CB, PIK3CA, AR,
   PLK1, CDK2, AURKB, MET, CSF1R, CYP19A1, PGR, PIK3CD, PIK3CG, RPS6KB1,
   LCK, TYMS, PIK3R1, MAPK14, and CHEK2 ([93]Figure 1G), while Subnetwork
   2 (13 nodes, 30 edges) featured key targets such as NTRK1, CHEK1,
   AURKA, PRKACA, ESR1, ROCK1, SRC, KIT, CDC42, ESR2, CCNB1, PTGS1, and
   NR3C2 ([94]Figure 1H). Targets from both subnetworks were prioritized
   as hub targets for subsequent mechanistic investigations.

Proteomics analysis and subcellular localization

   We conducted comparative proteomic analysis to characterize protein
   expression changes in irradiated cells with and without berberine
   treatment. Quantitative analysis revealed that BBR treatment
   significantly modulated the irradiated cell proteome, upregulating 199
   proteins (KRT14, KRT16 and PRKACA) while downregulating 485 proteins
   (notably LRP10 and GJC1) ([95]Figures 2A,B). Subcellular localization
   profiling demonstrated distribution of these differentially expressed
   proteins, with predominant nuclear localization (32.31%), followed by
   cytoplasmic (21.35%), extracellular (13.74%), mitochondrial (13.45%),
   and plasma membrane (11.55%) compartments ([96]Figure 2C).

FIGURE 2.

   [97]FIGURE 2
   [98]Open in a new tab

   Proteomics analysis of BBR treatment. (A) Statistical analysis of
   differential proteins. (B) Top differential proteins with significant
   upregulation and downregulation. (C) Subcellular location of
   differential proteins. (D) Top 20 Downregulated KEGG pathways by BBR.
   (E) Biological processes of proteins upregulated by BBR.

Functional annotation of differentially expressed proteins

   BBR significantly upregulated proteins involved in critical biological
   processes, including mitochondrial RNA metabolism, peptidyl-serine
   phosphorylation, keratinocyte activation, interleukin-5 (IL-5)
   production, skin barrier formation, and the cellular response to
   epinephrine ([99]Figure 2D). Conversely, it downregulated pathways
   linked to inflammatory responses and radiation-induced damage, such as
   oxidative phosphorylation, p53 signaling, chemical carcinogenesis
   (reactive oxygen species, ROS), diabetic cardiomyopathy, JAK-STAT
   signaling, and oncogenic pathways ([100]Figure 2E).

Core targets selection

   Intersection analysis revealed proteins differentially regulated by
   irradiation and BBR treatment, including those downregulated by
   irradiation but upregulated by BBR, and vice versa ([101]Figures 3A–C).
   These proteins are potential key regulatory targets of BBR. Among 182
   candidate regulatory proteins, we cross-referenced 82 hub targets from
   network pharmacology screening with proteomics data, identifying two
   core targets: PRKACA and PIK3CB ([102]Figures 3D–F). Notably, their
   roles in longevity regulation, growth hormone signaling, and thyroid
   hormone pathways highlight their critical importance in fundamental
   cellular processes and associated disease mechanisms ([103]Figure 3G).

FIGURE 3.

   [104]FIGURE 3
   [105]Open in a new tab

   Core targets screening of BBR in RISI treatment. (A–C) Identification
   of potential targets regulated by BBR in RISI through proteomics
   analysis. (D) Intersection of proteomics targets and hub targets. (E,F)
   Protein expression differences of core targets between irradiation and
   BBR treatment groups. (G) Key pathways involved by core targets (PRKACA
   and PIK3CB).

Expression validation and co-expression analysis

   In rat skin tissues, PIK3CB expression decreased after irradiation,
   reaching its nadir on day 14 ([106]Figure 4A). PRKACA expression showed
   a similar decline by day 14 ([107]Figure 4B). During normal wound
   healing, these genes exhibited a non-significant upregulation following
   acute tissue injury when compared to control groups ([108]Figure 4C).
   Importantly, PRKACA and PIK3CB levels were significantly elevated in
   normal wounds compared to chronic ulcers ([109]Figure 4C).
   Co-expression network analysis revealed that PRKACA, PIK3CB, and their
   associated genes were primarily involved in epithelial cell migration,
   activation of the innate immune response, tissue migration, cellular
   response to peptide hormone stimulation, and protein kinase A binding
   ([110]Figures 4D–F).

FIGURE 4.

   [111]FIGURE 4
   [112]Open in a new tab

   Expression validation and co-expression analysis of core targets. (A)
   Expression changes of PIK3CB in animal skin tissues at different time
   points (7, 14, 28, 60 days) after irradiation. (B) Expression changes
   of PRKACA in animal skin tissues after irradiation. (C) Expression
   changes of core targets during normal wound healing and in chronic
   ulcers. (D) Co-expression network and functional analysis of core
   targets.

Molecular docking and molecular dynamic simulations results

   Molecular docking simulates ligand-protein interactions, with binding
   energy (affinity) determining docking quality. Lower affinity values
   indicate stronger binding, where values < −7 kcal mol^−1 typically
   suggest effective interactions. PRKACA and PIK3CB exhibited
   particularly stable binding, with energies ranging from −7.723 to
   −8.409 kcal mol^−1 ([113]Table 1). [114]Figures 5A,B details the
   docking targets and their corresponding active compounds.

TABLE 1.

   Docking scores between component and core targets.
   Protein Ligand Binding energy (kcal·mol^–1) Binding site Applied force
   PRKACA Berberine −7.723 ARG97 Cation-Pi interaction
   PIK3CB Berberine −8.409 LYS169 Hydrogen bond
   [115]Open in a new tab

FIGURE 5.

   [116]FIGURE 5
   [117]Open in a new tab

   Molecular docking and dynamic simulation. (A) Binding mode of PIK3CB to
   berberine. (B) Binding mode of PRKACA to berberine. Three-dimensional
   structures of the binding pockets were showed by Pymol software. (C–F)
   deformability and B-factor plots. (G,H) eigenvalue plots (Colored bars
   show the individual (purpule). (I,J) covariance map.

   The peaks in [118]Figures 5C,D indicate regions of protein
   deformability (backbone distortion). Protein hinges, which are not
   structurally significant, contribute to overall stability, while
   high-deformability regions correspond to chain hinge locations. In this
   study, the B-factor quantifies residue-specific molecular deformability
   ([119]Figures 5E,F). Molecular motion stiffness is characterized by
   eigenvalues associated with each normal mode, with calculated values of
   5.456307 × 10^−5 and 2.103388 × 10^−5 in this analysis ([120]Figures
   5G,H). Higher eigenvalues indicate greater variance, while lower values
   correspond to simpler deformations. The covariance map ([121]Figures
   5I,J) displays residue motion correlations: red represents correlated
   motion, blue indicates anti-correlated motion, and white shows no
   correlation.

BBR promotes cell proliferation and mitigates radiation-induced damage

   Our analysis identified a strong link between core targets and wound
   healing, especially in pathways related to longevity regulation and
   epithelial cell migration. Protein expression analysis showed that BBR
   treatment increased proliferation and remodeling markers (MKI67,
   COL1A1, COL1A2) and epithelial migration markers (KRT9, 14/16/17),
   while decreasing aging-related proteins (CDKN1A, CDKN2A) and
   inflammatory markers (IL6ST, TNFRSF10B) ([122]Figures 6B–L).
   Importantly, BBR reversed the irradiation-induced decline of the
   longevity marker SIRT3 ([123]Figure 6A).

FIGURE 6.

   [124]FIGURE 6
   [125]Open in a new tab

   BBR regulates markers associated with cellular inflammation and
   proliferation. (A) BBR upregulated longevity-associated protein SIRT3.
   (B–D) BBR upregulated skin repair-associated protein COL1A1, COL1A2 and
   MKI67. (E,F) BBR downregulated aging-related proteins such as CDKN1A
   and CDKN2A. (G,H) BBR downregulated inflammatory markers like IL6ST and
   TNFRSF10B. (I–L) BBR upregulated key markers of keratinocyte activation
   and epithelial migration (KRT9, KRT14, KRT17 and KRT16).

   CCK-8 assays assessed BBR’s impact on HUVEC proliferation, revealing
   that both 5 μM and 20 µM BBR significantly promoted cell proliferation
   in non-irradiated conditions ([126]Figure 7A). Under irradiated
   conditions, 20 µM BBR notably enhanced proliferation ([127]Figure 7B).
   Consistent with these results, LDH activity assays showed that 20 µM
   BBR significantly lowered LDH levels, indicating reduced cell death
   ([128]Figure 7C).

FIGURE 7.

   [129]FIGURE 7
   [130]Open in a new tab

   Therapeutic effect of BBR on irradiated cells and animal models. (A)
   CCK-8 were used to assess cell proliferation after BBR treatment. (B)
   CCK-8 were used to assess irradiated cell proliferation after BBR
   treatment. (C) detection of LDH level under different concentrations of
   BBR treatment. (D) pictures showing the scoring curves of the whole
   course of radiogenic injury in mice with different BBR treatment
   conditions.

BBR accelerated the skin healing process in irradiated animals

   After exposing the hind legs of mice to 20 Gy irradiation, we
   systematically tracked skin damage progression using continuous
   observation (two experienced observers examined the skin damage of the
   animal models under blinded conditions for 24 days) and photographic
   documentation. Skin damage scores steadily increased from day 4 to day
   11 post-irradiation, then gradually declined. Comparison between the
   irradiation-only group and the BBR-treated group indicated that oral
   BBR significantly accelerated skin healing, with marked improvements
   noted at 17 and 24 days post-irradiation ([131]Figure 7D).

Discussion

   Radiation-induced skin injury (RISI) is a frequent complication of
   radiation therapy, presenting symptoms from erythema and desquamation
   to severe ulceration and fibrosis ([132]Tao et al., 2024; [133]Patricia
   et al., 2024). These effects significantly impair patients’ quality of
   life and can interrupt crucial cancer treatments ([134]Xiaojing et al.,
   2020). Current management strategies focus on symptom relief and wound
   healing, yet definitive curative interventions are lacking ([135]Manni
   et al., 2016). This underscores the urgent need for more effective
   therapeutic approaches.

   Our study highlights berberine’s therapeutic potential in mitigating
   radiation-induced skin injury (RISI). Utilizing network pharmacology,
   proteomics, animal models, and molecular docking analyses, we
   demonstrate that berberine effectively targets key molecular pathways
   and proteins linked to inflammation, oxidative stress, and tissue
   repair. These results provide a detailed understanding of berberine’s
   promise as a therapeutic agent for RISI.

   Proteomic analysis identified notable changes in proteins related to
   skin repair, inflammation regulation, and cellular aging. Berberine
   specifically upregulated key markers of keratinocyte activation and
   epithelial migration, such as KRT14 and KRT16, while downregulating
   inflammatory markers like IL6ST and TNFRSF10B ([136]Xiaowei et al.,
   2019; [137]Minseok et al., 2024). This dual action indicates that
   berberine mitigates radiation-induced inflammation while promoting skin
   regeneration and enhancing barrier function.

   GO and KEGG pathway enrichment analyses elucidated the molecular
   mechanisms of berberine’s effects. Significant enrichment was observed
   in biological processes such as MAPK cascade regulation, epithelial
   cell development, and the G2/M transition of the mitotic cell cycle,
   highlighting berberine’s potential in cell cycle progression and tissue
   regeneration ([138]Maryam et al., 2025). Additionally, downregulated
   pathways like oxidative phosphorylation and p53 signaling correspond
   with reduced oxidative stress and DNA damage following berberine
   treatment ([139]Zaigang et al., 2024; [140]Anh Phong et al., 2022).

   Our findings highlight berberine’s potential to modulate aging-related
   pathways. The marked downregulation of senescence-associated proteins,
   such as CDKN1A and CDKN2A, suggests berberine’s efficacy in mitigating
   radiation-induced aging in skin cells ([141]Zhangyang et al., 2024;
   [142]Grace et al., 2024). Moreover, the upregulation of SIRT3, a key
   protein associated with longevity, supports berberine’s role in
   enhancing cellular resilience against radiation-induced damage
   ([143]Wei et al., 2024; [144]Zhang et al., 2024).

   The identification of PRKACA and PIK3CB as key targets underscores
   berberine’s molecular specificity in modulating vital biological
   processes. Both targets are integral to pathways involved in cellular
   repair, proliferation, and inflammation ([145]Youyi et al., 2024;
   [146]Wencui et al., 2024). Molecular docking and dynamics analyses
   confirmed strong binding affinities between berberine and these
   targets, suggesting stable interactions that likely drive its
   therapeutic effects. In irradiated animal models, downregulation of
   PRKACA and PIK3CB expression was noted, correlating with the
   development of chronic ulcers during wound healing. Thus, BBR has the
   potential to ameliorate radiation-induced damage by upregulating the
   expression of these key targets. The co-expression network of PRKACA
   and PIK3CB underscores their involvement in pathways related to
   epithelial migration, innate immune activation, and peptide hormone
   signaling ([147]Xiong et al., 2024). These interactions highlight
   berberine’s complex role in modulating local skin repair and systemic
   responses to radiation-induced injury.

   Finally, the in vivo and in vitro effects of different concentrations
   of BBR were validated. We developed irradiated animal and cell models
   to examine BBR’s therapeutic effects on radiation-induced injuries. Our
   results indicate that BBR significantly boosts the proliferation of
   irradiated cells and speeds up skin damage recovery in irradiated
   animals. These findings suggest BBR as a promising candidate for the
   clinical prevention and treatment of radiation injuries.

   Despite these promising results, several limitations warrant
   acknowledgment. First, the in vivo relevance of the identified targets