Efficacy of Berberine‐Mediated Photodynamic Therapy Against Trichophyton rubrum Infection

ABSTRACT

Objective

To investigate the antifungal effects of berberine‐mediated photodynamic therapy (BBR‐PDT) against Trichophyton rubrum (T. rubrum) both in vitro and in vivo, and to elucidate its underlying mechanisms of action.

Methods

The minimum inhibitory concentration (MIC) of BBR‐PDT against T. rubrum was determined according to the CLSI M38‐A2 guidelines. The MTT assay was used to evaluate the effects of various BBR concentrations and light doses, with results subsequently validated by colony diameter measurements and spore germination assays. Intracellular reactive oxygen species (ROS) levels were measured using DCFH‐DA, and transmission electron microscopy (TEM) was used to assess cellular damage in T. rubrum. A guinea pig skin infection model was established, and therapeutic efficacy was evaluated using clinical skin scoring, fungal culture, and histopathological analysis (H&E staining).

Results

In vitro experiments showed that at a light dose of 96 J/cm², the MIC of BBR was 12 μg/mL. Neither light irradiation alone nor BBR monotherapy exhibited antifungal activity. Combined treatment with 12 μg/mL BBR and 72 J/cm² blue light significantly inhibited radial hyphal growth and spore germination of T. rubrum. Mechanistic investigations revealed that BBR‐PDT significantly increased ROS levels within spores, and T. rubrum exhibited extensive cellular damage, including membrane rupture, cell wall loosening, cytoplasmic vacuolization, and organelle degeneration. In the guinea pig model, BBR‐PDT significantly reduced scaling, promoted hair regrowth, and improved clinical skin scores. Fungal colony counts from skin cultures were significantly reduced, and H&E staining demonstrated restoration of normal skin structure.

Conclusion

BBR‐PDT exerts antifungal effects via ROS‐mediated oxidative stress. This oxidative stress disrupts T. rubrum ultrastructure through multiple mechanisms. The therapy is effective against T. rubrum in vitro and in vivo. Therefore, BBR‐PDT holds promise as a treatment for dermatophyte infections.

1. Introduction

Dermatophytoses are fungal infections that invade the stratum corneum, hair, and nail plates [1]. Based on the anatomical site of infection, dermatophytoses are classified into clinical forms: tinea manuum (hand), tinea pedis (foot), tinea corporis (body), tinea cruris (groin), tinea capitis (scalp), and onychomycosis (nail) [2]. According to World Health Organization statistics, dermatophytoses are the most prevalent fungal infections worldwide, affecting approximately 20%–25% of the global population [3]. Among these, Trichophyton rubrum (T. rubrum) is the predominant pathogen, accounting for about 60% of all reported cases and posing significant threats to both human and animal health [4]. Zarzeka et al. [5] reported that between 2019 and 2023, 55.6% of 3510 patients diagnosed with tinea corporis were infected with dermatophytes, with T. rubrum accounting for 76.1% of these infections. Similarly, Gupta et al. [6] demonstrated that among 710,541 onychomycosis cases, 54.3% were attributed to T. rubrum infections, followed by Aspergillus species (7.1%) and Trichophyton mentagrophytes (4.5%). Although dermatophytoses are rarely life‐threatening, their contagious nature, high recurrence rates, and emerging drug resistance significantly impair patients' quality of life, social interactions, and psychological well‐being [7]. Current clinical management of dermatophytoses primarily relies on systemic or topical antifungal pharmacotherapy. Antifungal agents are categorized into several classes, including polyenes, azoles, echinocandins, and allylamines. However, these therapeutic agents are associated with considerable limitations, including varying degrees of adverse effects, development of tolerance, potential drug interactions, and the persistent emergence of multidrug‐resistant fungal strains [8].

Photodynamic therapy (PDT) represents an innovative non‐invasive or minimally invasive therapeutic approach that has been widely applied in oncology, vascular diseases, microbial infections, and dermatology. The fundamental components of PDT include a photosensitizer (PS), molecular oxygen, and an appropriate light source [9]. The underlying principle relies on the selective accumulation of the photosensitizer in pathological tissues, where it exhibits minimal distribution in normal tissues. When the affected area is irradiated with a light source of a specific wavelength, the photosensitizer absorbs the light, transitions to an excited state, and transfers the absorbed energy to molecular oxygen. This process initiates photooxidation reactions that generate reactive oxygen species (ROS). These ROS subsequently react with cellular membranes, intracellular macromolecules (including proteins and nucleic acids), and various organelles, ultimately leading to apoptosis or necrosis of target cells [10, 11]. 5‐Aminolevulinic acid (5‐ALA) has been shown to be an effective photosensitizer, and 5‐ALA‐mediated PDT induces significant DNA damage in breast adenocarcinoma and hepatocellular carcinoma cell lines [1]. Curcumin‐mediated PDT not only inhibits and kills tumor cells, including those from lung, cervical, and liver cancers, but also exhibits potent antimicrobial activity, effectively inactivating diverse bacterial and fungal pathogens [12, 13, 14].

Berberine (BBR) is a natural isoquinoline alkaloid. It is extracted from several medicinal plants, including Coptis chinensis Franch., Phellodendron amurense, and Berberis species. Traditionally, BBR has been used to treat bacterial gastroenteritis, dysentery, and other gastrointestinal disorders [15]. Research has shown that berberine modulates interactions among enteric glial cells, epithelial cells, and immune cells. This provides protective effects against ulcerative colitis [16]. Furthermore, berberine has significant antibacterial properties. It accumulates within bacterial cells and binds to both single‐ and double‐stranded DNA. This binding disrupts DNA structure and function, leading to bacterial death [17]. Berberine exhibits diverse pharmacological activities and possesses promising photosensitizer properties. Following photoexcitation, the relaxation process of berberine involves intramolecular electron transfer, which may lead to quenching of its excited state. Modulating this electron transfer pathway can prolong the excited‐state lifetime. The extended excited‐state lifetime of berberine subsequently results in substantially increased ROS generation efficiency [18]. Lopes et al. [19] demonstrated that berberine generates ROS under blue light excitation, promoting autophagy and apoptosis in renal cancer cells. Similarly, Meschi et al. [20] reported that MDA‐MB‐231 breast cancer cell growth was significantly inhibited following treatment with PDT combining berberine and valproic acid. Therefore, berberine represents a promising photosensitizer candidate for PDT by facilitating cell apoptosis.

In recent years, numerous studies have investigated BBR‐PDT for oncology applications [10, 21]. However, no study to date has specifically targeted T. rubrum using BBR‐PDT. Investigating BBR‐PDT for dermatophytosis caused by T. rubrum is therefore essential and clinically significant, representing a promising research direction with potential therapeutic applications.

2. Material and Methods

2.1. Main Materials and Instruments

2.1.1. Two Strains of T. rubrum

Two strains of T. rubrum were utilized in this study: a reference strain (ATCC 28188) obtained from the Shaanxi Provincial Center for Disease Control and Prevention (China), and a clinical strain isolated from a toenail of a patient with onychomycosis at the Department of Dermatology, The Second Affiliated Hospital of Xi'an Jiaotong University (China). The clinical strain was identified as T. rubrum based on morphological characteristics and comparison with the reference strain. Notably, only the clinical strain was employed for the in vivo experiments.

2.1.2. Reagents

Berberine (BBR, 98% purity) was purchased from Adamas Reagent Co. Ltd. (Shanghai, China). Sabouraud Dextrose Agar (SDA) solid medium, Sabouraud Dextrose Broth (SDB) liquid medium, Potato Dextrose Agar (PDA) solid medium, and the Reactive Oxygen Species (ROS) Assay Kit were obtained from the following respective suppliers: Haibo Biotechnology Co. Ltd. (Qingdao, China) for SDA, Luqiao Technology Co. Ltd. (Beijing, China) for SDB, Hangzhou Microbial Reagent Co. Ltd. (China) for PDA, and Nanjing Jiancheng Institute of Bioengineering (China) for the ROS Assay Kit.

2.1.3. Instruments

Full‐wavelength microplate reader (TECAN Infinite 200 PRO, Beijing World Trade Far East Instrument Co. Ltd., China), simulated daylight xenon lamp (PL‐X500D, Beijing Princess Technology Co. Ltd., China), recording solar meter (TES‐132, Taisi Electronics Industry Co. Ltd.), fluorescence inverted microscope (Nikon ECLIPSE Ti2‐U, Nanjing Sigao Information Co. Ltd., China), and transmission electron microscope (HITACHI HT7700, HITACHI, Japan).

2.1.4. Animals

Twenty‐four male guinea pigs, weighing 250 ± 20 g, were obtained from Changsha Tianqin Biotechnology Co. Ltd., which holds production license No. SCXK (Xiang) 2019‐0015. All animal experiments were approved by the Animal Ethics Committee of Yichun University (Jiangxi Province, China), with the approval number (2024) 032.

2.2. In Vitro Effects of BBR‐PDT on T. rubrum

2.2.1. Preparation of Fungal Spore Suspensions

Spore suspensions were prepared as described previously with minor modifications [22]. Two strains stored in glycerol at −80°C were revived at room temperature for 30 min. Next, 100 μL of the revived suspension was spread onto PDA plates. The plates were incubated at 28°C for 10–12 days until spores formed. Then, 5 mL of sterile PBS was added to each plate. Mycelia and spores were scraped off and transferred to 15 mL centrifuge tubes. The mixture was vortexed for 1 min and then filtered through a cell strainer to collect the spore suspensions.

2.2.2. Absorption Spectrum of Berberine

The absorption spectrum of berberine (12 μg/mL) was measured in the wavelength range of 300–600 nm using a full‐wavelength microplate reader. The experiment was repeated three times.

2.2.3. Determination of the Minimum Inhibitory Concentration (MIC) of BBR‐PDT Against T. rubrum

The MIC assay was performed following the Clinical and Laboratory Standards Institute (CLSI) M38‐A2 guidelines with minor modifications. BBR was diluted in PBS to concentrations of 3, 6, 12, 24, 48, 96, and 192 μg/mL. Spore suspensions were diluted to 1 × 10⁵ CFU/mL using the BBR solutions at varying concentrations. For the negative control group, the spore suspension was diluted with PBS to 1 × 10⁵ CFU/mL. Samples were divided into two groups according to light exposure conditions: BBR‐PDT and BBR. Spore suspensions from each group were added to 35 mm sterile Petri dishes. The dishes were incubated in a constant‐temperature shaking incubator at 28°C and 150 rpm for 2 h. The BBR‐PDT group was then exposed to 420 nm light at 96 J/cm². The BBR group received no light exposure. Following irradiation, suspensions from each group were transferred to 96‐well plates (three replicates per concentration, with three independent repetitions) and incubated at 28°C for 3 days. The MIC was defined as the lowest drug concentration at which fungal growth was completely inhibited, as determined by observation using a fluorescence inverted microscope (Nikon ECLIPSE Ti2‐U). Terbinafine (TBF) and amphotericin B (AmB; 0.5–32 μg/mL) served as positive controls.

2.2.4. Dose‐Response Relationship of BBR‐PDT against T. rubrum

2.2.4.1. Concentration‐Dependent Effects

BBR was diluted in PBS to seven concentrations: 64, 32, 16, 8, 4, 2, and 1 μg/mL. Each concentration was mixed with a spore suspension (1 × 10⁷ CFU/mL). The negative control group received PBS instead of BBR. The mixtures were transferred to 35 mm Petri dishes. They were incubated in the dark at 28°C with gentle shaking for 2 h. After incubation, they were exposed to 420 nm light at 96 J/cm². Following irradiation, 100 μL of suspension from each group was seeded into 96‐well plates, and 100 μL of SDB was added. Three replicate wells were established for each group. According to the MTT assay kit instructions, the supernatant was removed by centrifugation, cells were resuspended in 200 μL of PBS, and 20 μL of MTT solution was added to each well. Plates were incubated at 37°C for 4 h. The supernatant was then removed by centrifugation. Next, 200 μL of DMSO was added, and the mixture was shaken for 15 min. Absorbance was measured at 492 nm. Spore survival rates were calculated using the following formula:

$$\mathrm{Survival}\mathrm{rate}(\%)=\frac{{\mathrm{OD}}_{492\mathrm{nm}}\mathrm{value}\left(\mathrm{treated}\mathrm{groups}\right)-{\mathrm{OD}}_{492\mathrm{nm}}\mathrm{value}\left(\mathrm{blank}\right)}{{\mathrm{OD}}_{492\mathrm{nm}}\mathrm{value}\left(\mathrm{control}\mathrm{groups}\right)-{\mathrm{OD}}_{492\mathrm{nm}}\mathrm{value}\left(\mathrm{blank}\right)}\times 100\%$$

2.2.4.2. Light Dose‐Dependent Effects

Spore suspensions were diluted with 12 μg/mL BBR to 1 × 10⁷ CFU/mL and divided into groups receiving light doses of 0, 2.4, 24, 72, 96, and 144 J/cm². The light doses (24, 72, 96, and 144 J/cm²) were selected based on preliminary experiments and previous literature on berberine‐mediated photodynamic therapy. Light dose was measured using a solar meter (TES‐132, Taisi Electronics Industry Co. Ltd.), and irradiation time was adjusted accordingly to achieve the desired fluence. The exposure time was calculated using the following formula: Time (s) = Irradiance (J/cm²)/Irradiance (W/cm²). In this study, the irradiance was maintained at 0.1 W/cm². Accordingly, when the light dose was 24, 72, 96, and 144 J/cm², the exposure times were 4 min, 12 min, 16 min, and 24 min, respectively. Add the suspensions from each group to 35‐mm culture dishes, incubate in the dark for 2 h, and then expose them to the corresponding light dose. Following irradiation, 100 μL of spore suspension from each group was inoculated into 96‐well plates with 100 μL of SDB medium added (three replicate wells per group). Spore survival rates were assessed via MTT assay and calculated using the above formula.

2.2.5. Effects of BBR‐PDT on Radial Growth and Spore Germination of T. rubrum

A light dose of 72 J/cm² was selected based on dose‐response results (Section 3.1.3 and Figure 1C), which demonstrated that this dose significantly inhibited spore survival while avoiding the maximal dose of 96 J/cm² to minimize potential photothermal effects.

Figure 1.

The effect of BBR‐mediated PDT on the survival of T. rubrum spores. (A) Survival rate of control T. rubrum strain incubated with different concentrations of BBR and irradiated with 96 J/cm² energy doses of light. (B) Survival rate of clinical T. rubrum strain incubated with different concentrations of BBR and irradiated with 96 J/cm² energy doses of light. (C) Survival rate of two T. rubrum strains incubated with 12 μg/mL of BBR and irradiated with different energy doses of light. (D) Absorption spectra of BBR. Each value refers to mean standard deviation (SD) (n = 3) (ns: no significance, ****p < 0.0001).

2.2.5.1. Radial Growth

Based on the MIC result (12 μg/mL), this concentration was employed in subsequent experiments along with two additional concentrations (6 and 24 μg/mL) for comparison. BBR solutions at 6, 12, and 24 μg/mL were prepared and used to dilute spore suspensions to 1 × 10⁷ CFU/mL. For the negative control group, spore suspensions were diluted with PBS. Spore suspensions were transferred to 35 mm sterile Petri dishes and incubated in the dark for 2 h. Samples were then exposed to 72 J/cm² light, as determined in Section 2.2.4. After irradiation, the spore suspensions were serially diluted 10‐fold. Then, 10 μL aliquots of each dilution were spotted onto PDA plates. The plates were incubated at 28°C for 1–7 days. Colony diameters were measured daily using a vernier caliper. The experiment was repeated three times.

2.2.5.2. Spore Germination

The procedure for photodynamic therapy was identical to that described in the assessment of radial growth. After irradiation, the spore suspension was incubated at 28°C for 24 h. Subsequently, 50 μL of the spore suspension was placed on a microscope slide, and the number of germinating spores and the total spore count were recorded under a microscope; three fields of view were observed for each sample. The spore germination rate was calculated as the percentage of germinating spores relative to the total spore count.

2.3. Mechanism of Action of BBR‐PDT Against T. rubrum

2.3.1. Effect of BBR‐PDT on ROS Production by T. rubrum Spores

For the BBR‐PDT group, T. rubrum spore suspensions were prepared by diluting BBR solutions (6 and 12 μg/mL) to 1 × 10⁷ CFU/mL. The negative control group was diluted with PBS, and the positive control group was diluted with 8 μg/mL terbinafine. The suspensions in each group were incubated in the dark for 2 h, then exposed to 72 J/cm² of light. After irradiation, the 10 μM DCFH‐DA fluorescent probe was added, and the samples were incubated at 37°C for 30 min (with shaking every 5–10 min). Wash three times with PBS to remove unbound probe. Finally, measure the fluorescence intensity at the FITC wavelength using a fluorescence microplate reader. Each group in this experiment was conducted four times.

2.3.2. Ultrastructural Analysis of T. rubrum Spores

The photodynamic treatment procedure for T. rubrum suspensions was identical to that described in Section 2.3.1. Treated suspensions were centrifuged at 4000 rpm for 10 min, and supernatants were discarded. For spore suspension resuspension, 1 mL of PBS was added, followed by 2.5% glutaraldehyde fixative. Samples were fixed overnight at 4°C (with 4 samples per group). After fixation, they underwent dehydration, then were soaked in a 1:1 mixture of propylene oxide and epoxy resin for 2 h, followed by pure epoxy resin for 3 h. The samples were then sectioned into 70 nm‐thick slices, stained, and examined under a transmission electron microscope.

2.4. In Vivo Effects of BBR‐PDT Against T. rubrum Infection

2.4.1. Establishment and Grouping of Guinea Pig Dermatophytosis Model

A guinea pig dermatophytosis model was established according to existing protocols with minor modifications [23, 24]. Guinea pigs were housed at 25°C with ad libitum access to food and water. A 3 cm × 3 cm area of dorsal fur was shaved using electric clippers, residual hair was removed with depilatory cream, and the area was washed and dried. Following anesthesia with etamate, the dorsal skin was abraded with sterile coarse sandpaper until exudate appeared (care was taken to avoid causing bleeding). Guinea pigs were randomly divided into four groups: control, model, TBF, and BBR‐PDT groups (n = 6 per group). The control group was treated with saline solution, whereas the remaining groups were inoculated with 100 μL of T. rubrum spore suspension (2 × 10⁸ CFU/mL). Following inoculation, dexamethasone sodium phosphate injection (7.5 mg/kg) was administered intramuscularly for 4 consecutive days. On day 4 post‐inoculation, skin scrapings were collected for microscopic examination and culture. Successful model establishment was confirmed by observing T. rubrum growth.

2.4.2. BBR‐PDT Treatment

Following the successful establishment of the guinea pig skin infection model, the TBF group was treated with terbinafine (2.0 mg/cm²) twice daily, and the BBR‐PDT group was treated with 100 μL of BBR (12 μg/mL) once daily. Both the control and model groups were treated with 100 μL of PBS; 2 h after application, samples were exposed to 420 nm blue light at 72 J/cm².

2.4.3. Effects of BBR‐PDT on Skin Lesions and Clinical Scores in Guinea Pigs

Therapeutic effects were clinically assessed and documented according to previously reported studies [24]. Detailed scoring criteria are presented in Table 1.

Table 1.

Clinical scoring criteria for guinea pig dermatophytosis.

Score	Description
0	No skin lesions, hair growth
1	A small amount of mild erythema on the skin
2	Marked redness and swelling
3	Obvious redness, scabby, and sparse hair
4	Partial skin damage, severe scab, and hair loss
5	Large area of skin damage and hair loss

2.4.4. Effect of BBR‐PDT on Fungal Colony Counts in Guinea Pig Skin

To quantify T. rubrum fungi on guinea pig skin, a sterile PBS‐moistened swab was gently rubbed across the skin surface. The swab was then placed in a centrifuge tube with 1 mL of sterile PBS and vortexed to suspend the fungi. The suspension was filtered through a cell strainer, serially diluted six times, and 100 μL of each dilution was evenly spread on SDA plates. After incubating at 28°C for 72 h, colonies were counted.

2.4.5. Effect of BBR‐PDT on Histopathological Analysis (H&E Staining) of Guinea Pig Skin Tissue

Animals were euthanized 12 days after treatment. Skin tissue was excised from the dorsal area where T. rubrum had been inoculated. Tissues were fixed in 4% paraformaldehyde for 48 h. They were then dehydrated, embedded, sectioned, dewaxed, and stained with hematoxylin and eosin. After staining, the tissues were dehydrated using an ethanol gradient, cleared with xylene, and embedded in neutral resin. Finally, they were examined under a light microscope, and images were captured for documentation.

2.5. Statistical Analysis

Data analysis was performed using GraphPad Prism 8.0 software. Following normality testing, comparisons were conducted using one‐way analysis of variance (ANOVA). Pairwise comparisons were performed using Welch's test. Differences were considered statistically significant at p < 0.05.

3. Results

3.1. In Vitro Effects of BBR‐PDT on T. rubrum

3.1.1. Absorption Spectrum of Berberine

The absorption spectrum of berberine (12 μg/mL) exhibited characteristic peaks at approximately 340 nm and 420 nm (Figure 1D).

3.1.2. Minimum Inhibitory Concentration (MIC) of BBR‐PDT Against T. rubrum

The minimum inhibitory concentrations (MICs) of BBR, BBR‐PDT, TBF, and AmB against T. rubrum are presented in Table 2. BBR alone exhibited weak antifungal activity, with an MIC of 192 μg/mL. In contrast, following photodynamic activation, the MIC of BBR‐PDT was substantially reduced to 12 μg/mL. The standard antifungal agents TBF and AmB demonstrated potent activity, with MIC values of 8 μg/mL and 4 μg/mL, respectively.

Table 2.

The minimum inhibitory concentrations (MICs) of BBR, BBR‐PDT, terbinafine, and amphotericin B against T. rubrum.

	MIC (μg/mL)
	BBR	BBR‐PDT	TBF	AmB
T. rubrum control stain	192	12	8	4
T. rubrum clinical stain	192	12	8	4

Abbreviations: AmB, amphotericin B; BBR, berberine; BBR‐PDT, berberine‐mediated photodynamic therapy; TBF, terbinafine.

3.1.3. Dose‐Response Relationship of BBR‐PDT Against T. rubrum

As shown in Figure 1A–C, no significant differences were observed in T. rubrum spore survival rates among the various concentration groups treated with BBR alone. Compared with BBR treatment alone, the combination of 8 μg/mL BBR and 420 nm blue light irradiation (96 J/cm²) significantly reduced spore survival rates (p < 0.0001; Figure 1B). The inhibitory effect of BBR‐PDT on T. rubrum was concentration‐dependent; as BBR concentration increased, spore survival rates gradually decreased. At a BBR concentration of 12 μg/mL with light doses ranging from 24 to 96 J/cm², the inhibitory effect of BBR‐PDT was light‐dose‐dependent (p < 0.0001; Figure 1C), with the lowest spore survival rate observed at 96 J/cm².

3.1.4. Effects of BBR‐PDT on Radial Growth and Spore Germination of T. rubrum

3.1.4.1. Radial Growth

As shown in Figure 2A,B, on day 7 of colony culture, T. rubrum colony diameters were significantly reduced in all BBR‐PDT treatment groups compared with the untreated control group (p < 0.0001), and the inhibitory effect was concentration‐dependent. When BBR concentration reached 24 μg/mL, virtually no colony growth was observed.

Figure 2.

The impact of BBR‐mediated PDT on the growth inhibition of T. rubrum. (A) Diameters of BBR‐mediated PDT control T. rubrum colonies after incubation at 28°C for 0–7 days (irradiated with 72 J/cm² light). (B) Diameters of BBR‐mediated PDT clinical T. rubrum colonies after incubation at 28°C for 0–7 days (irradiated with 72 J/cm² light). (C) Bourgeon rate of control T. rubrum strain spore irradiated with 72 J/cm² energy doses of light. (D) Bourgeon rate of clinical T. rubrum strain spore irradiated with 72J/cm² energy doses of light. Each value refers to mean standard deviation (SD)(n=3) (****p<0.0001).

3.1.4.2. Spore Germination

As shown in Figure 2C,D, T. rubrum spore germination rates were significantly reduced in all BBR‐PDT treatment groups (6–24 μg/mL) compared with the untreated control group (p < 0.0001).

3.2. Mechanism of Action of BBR‐PDT Against T. rubrum

3.2.1. Effect of BBR‐PDT on ROS Production by T. rubrum Spores

As shown in Figure 3A,B, at a light dose of 72 J/cm², ROS levels within T. rubrum spores in all BBR‐PDT treatment groups were significantly higher than those in the untreated control group (p < 0.01). Furthermore, ROS production was concentration‐dependent, with ROS levels in the MIC group being significantly higher than those in the 0.5×MIC group (p < 0.001).

Figure 3.

The mechanism of action of BBR‐mediated PDT. (A) Reactive oxygen species content of the control T. rubrum spores. (B) Reactive oxygen species content of the clinical T. rubrum spores. (C) Transmission electron microscope images of T. rubrum spores. Each value refers to the mean standard deviation (SD) (n = 4). The “0.5MIC” corresponds to 6 μg/mL BBR‐PDT, and the “MIC” corresponds to 12 μg/mL BBR‐PDT. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

3.2.2. Effect of BBR‐PDT on Ultrastructure of T. rubrum Spores

As shown in Figure 3C, transmission electron microscopy revealed that in the untreated control group, T. rubrum spore cell membranes and cell walls were intact, with clear internal structures including mitochondria, cytoplasm, and vacuoles. In contrast, spores in the BBR‐PDT‐treated group exhibited marked morphological damage, characterized by disruption of cell membranes and cell walls, disintegration of organelles, and disordered cytoplasmic distribution.

3.3. In Vivo Effects of BBR‐PDT Against T. rubrum Infection

3.3.1. Successful Establishment of Guinea Pig Skin Infection Model

On day 4 post‐infection, skin scrapings were collected from the guinea pig infection sites and examined microscopically, revealing extensive hyphal growth consistent with T. rubrum (Figure 4A). Concurrently, skin scrapings were inoculated onto SDA plates; after incubation at 28°C for 3 days, white, villous colonies were observed, consistent with the typical morphological characteristics of T. rubrum (Figure 4B). These results indicate the successful establishment of the guinea pig skin infection model.

Figure 4.

The therapeutic effects of BBR‐mediated PDT for tinea corporis in vivo. (A) Skin scrapings under microscopy after T. rubrum infection. (B) Colony culture of skin scrapings after T. rubrum infection. (C) Results of the skin score of guinea pigs in each group on the 5th day of treatment. (D) Results of the skin score of guinea pigs in each group on the 12th day of treatment. (E) Skin colony counts of guinea pigs in each group after treatment. (F) Photographs of the infected dorsal skin of guinea pigs on day 5 of treatment. (G) H&E staining images of guinea pig skin after treatment, 1: epidermides, 2: stratum granulosum, 3: stratum spinosum, 4: sebaceous gland, and 5: hair follicle. Each value refers to mean standard deviation (SD) (n = 6) (^#### p < 0.0001, ****p < 0.0001).

3.3.2. Effects of BBR‐PDT on Skin Lesions and Clinical Scores in Guinea Pigs

On day 5 of treatment, guinea pigs in the control group exhibited severe erythema, profuse scaling, and crusting. In contrast, skin condition improved markedly in the TBF and BBR‐PDT groups, with reduced erythema and edema, decreased scaling, and visible new hair growth at inoculation sites. During the subsequent 7‐day observation period, skin lesions in the control group continued to worsen, with persistent erythema and increased scaling. The TBF group exhibited only mild erythema, while the skin condition in the BBR‐PDT group showed no significant changes compared to day 5 (Figure 4F).

As shown in Figure 4C,D, skin scores in the model group were significantly higher than those in the control group (p < 0.0001), indicating successful establishment of the infection model. Compared with the model group, both TBF and BBR‐PDT groups showed significantly lower skin scores (p < 0.0001), suggesting that BBR‐PDT effectively improves skin lesions caused by T. rubrum infection.

3.3.3. Effect of BBR‐PDT on Fungal Colony Counts in Guinea Pig Skin

As shown in Figure 4E, the colony count in the model group was significantly higher than that in the control group (p < 0.0001). The colonies were densely distributed and larger in diameter, indicating abundant fungal presence at the infection site. After treatment, both the TBF and BBR‐PDT groups showed significantly lower colony counts compared with the model group (p < 0.0001). These results suggest that BBR‐PDT exerts a strong inhibitory effect against T. rubrum in vivo.

3.3.4. Effect of BBR‐PDT on Histopathological Analysis (H&E Staining) of Guinea Pig Skin Tissue

As shown in Figure 4G, histopathological examination revealed that in the model group, the epidermal stratum corneum was damaged, the stratum spinosum was irregularly thickened with partial dermal invasion, and inflammatory cell infiltration was observed in the superficial dermis. In contrast, the TBF and BBR‐PDT groups showed clear epidermal and dermal structures. The stratum corneum and stratum spinosum returned to normal thickness. These findings indicate substantial recovery of skin tissue integrity.

4. Discussion

This study systematically evaluated the antifungal activity of BBR as a photosensitizer in photodynamic therapy against T. rubrum. Both in vitro and in vivo effects were assessed. The potential mechanisms of action were also investigated. In vitro results indicate that BBR exhibits limited inhibitory effects against T. rubrum in the absence of light; however, when combined with light of a specific wavelength, its antifungal activity is significantly enhanced, with a clear dose‐response relationship observed. MIC testing was performed at an irradiance of 96 J/cm². The MIC of BBR‐PDT against T. rubrum was 12 μg/mL. Under these experimental conditions, BBR‐PDT showed slightly lower antifungal efficacy than terbinafine. Nevertheless, BBR‐PDT offers an advantage due to its distinct mechanism of action. Terbinafine is a first‐line antifungal agent. It targets a single mechanism of action. Resistance has been reported after long‐term use [25]. BBR‐PDT, however, generates ROS via photochemical reactions. This ROS production damages fungal structures at multiple sites, such as cell membranes, cell walls, and organelles. This multi‐target mechanism is theoretically unlikely to induce resistance in fungi and may remain effective against strains that are already resistant to terbinafine [26]. Furthermore, this study demonstrated that BBR‐PDT also significantly inhibits the radial growth of hyphae and spore germination. Hyphae are key structures for tissue invasion and biofilm formation by T. rubrum. Spore germination causes recurrent infections [27, 28]. BBR‐PDT inhibits these key processes. This suggests that BBR‐PDT may offer advantages for treating dermatophytosis.

PDT is widely used for superficial infections. It has high efficacy and a low risk of inducing resistance [29, 30]. The core principle is as follows: a photosensitizer is excited by specific wavelengths of light. This excitation generates ROS, which then destroys target cells [31]. Previous studies have shown that photoexcited BBR produces singlet oxygen and superoxide radicals. These are the primary components of ROS [32, 33, 34]. Oliveira et al. [35] reported that BBR irradiation with 447 ± 10 nm light (96 J/cm²) increased ROS levels and caspase‐3 activity, thereby killing cervical cancer cells and immortalized keratinocytes. Meschi et al. [20] demonstrated that in the presence of blue LED irradiation and propionate, combined BBR treatment not only reduced MDA‐MB‐231 cell survival rates but also decreased the required drug dose. The BBR absorption spectrum has two distinct peaks: 340 nm and 420 nm (Figure 1D). UV radiation at 340 nm lies in the UVA range (320–400 nm). UVA penetrates the dermis. It also causes skin damage via ROS generation, DNA damage, photoaging, and increased skin cancer risk. A 340 nm light source may cause skin damage. Therefore, we selected a 420 nm light source for this study. To elucidate the mechanism of action of BBR‐PDT, we used the DCFH‐DA probe to detect changes in intracellular ROS levels after treatment. The results showed that ROS fluorescence intensity within spores was significantly higher in the BBR‐PDT group than in other groups. This confirms efficient ROS generation. Excessive ROS then attacks fungal cell lipids, proteins, and nucleic acids. This leads to structural damage and functional impairment [36]. Transmission electron microscopy revealed that BBR‐PDT‐treated spores exhibited typical signs of oxidative damage, characterized by severe cell membrane and cell wall damage, destruction of numerous organelles, and cytoplasmic disorganization. Impairment of this ultrastructural integrity caused metabolic dysregulation and loss of physiological function in spores, leading to effective inhibition of their germination capacity. Based on these findings, we propose the following mechanism of action for BBR‐PDT against fungi. Upon entering or binding to fungal cells, BBR is activated by light and generates substantial amounts of ROS. The severe oxidative stress induced by ROS causes irreversible damage to cellular ultrastructure. This ultimately kills T. rubrum and halts its reproductive cycle.

The in vitro mechanism has been validated in an in vivo model. In guinea pig skin infection experiments, BBR‐PDT effectively eradicated T. rubrum from guinea pig skin, as evidenced by significant reductions in colony counts. Dynamic observation of clinical scoring revealed that BBR‐PDT treatment significantly improved erythema, scaling, and crusting at lesion sites. H&E staining results from infected guinea pig skin tissue further confirmed that inflammatory cell infiltration was significantly reduced in the treatment group, with epidermal structure returning to normal. These results provide strong evidence for the efficacy and safety of BBR‐PDT in vivo.

Although this study has yielded positive findings, several limitations remain. The absorption peak of BBR as a photosensitizer may not be optimally matched to light sources currently in clinical use. Future studies should explore the optimization of light source parameters or the development of targeted delivery systems to further enhance therapeutic efficacy. Reverse validation experiments were not included in the mechanistic investigation. Future studies incorporating ROS scavengers could be conducted to further validate the proposed mechanism. In summary, this study demonstrates that BBR‐mediated photodynamic therapy exhibits significant antifungal activity against T. rubrum both in vitro and in vivo by inducing ROS generation, disrupting fungal ultrastructure, and inhibiting hyphal growth and spore germination. As a naturally occurring photosensitizer, BBR offers the advantages of low dark toxicity and high photosensitizing activity [35]. Consequently, BBR‐PDT holds promise as a novel, non‐invasive therapeutic strategy for treating dermatophytosis, particularly against drug‐resistant strains and recurrent infections.

Author Contributions

Ruijuan Qiu, Xiaomei Wu, and Xiao Chen: conceptualization, data curation, formal analysis, investigation, writing (original draft), visualization, project administration, methodology. Jiangfeng Wen, Xiaofeng Duan, and Yu Yi: investigation, methodology, validation. Chunlin Chen: conceptualization, funding acquisition, resources, project administration, writing (review and editing). Linhua Zhou: resources, writing (review and editing), funding acquisition.

Ethics Statement

Approval was obtained from the Animal Protection Committee of Yichun University, China. The procedures used in this study adhere to the tenets of the Declaration of Helsinki.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Colony culture of guinea pig skin after 5 days of treatment. Figure S2: Absorption spectra of Berberine.

Qiu R., Wu X., Chen X., et al., “Efficacy of Berberine‐Mediated Photodynamic Therapy Against Trichophyton rubrum Infection,” Lasers in Surgery and Medicine 58 (2026): 672‐682. 10.1002/lsm.70152.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.