Aloin remodels the cell wall of 
The mortality associated with Candida infections has shown a concerning rise in recent years (Arita et al. ; Thompson et al. ). The repeated utilization of clinical antifungal drugs and the rise of drug-resistant fungi have significantly increased the challenges of clinical antifungal therapy (Kriegl et al. ). Candida albicans is the primary pathogen of oral candidiasis (d’Enfert et al. ) and one of the most abundance fungal species of intratumoral fungi in head and neck squamous cell carcinoma (HNSC) (Dohlman et al. ). C. albicans presents a round or ovoid yeast form commensally; however, it can transform to hyphal forms to invade and damage host mucosal tissues, evade immune cell phagocytosis, and express hyphal-specific virulence factors (Liang et al. ; Moyes et al. ; Witchley et al. ). The C. albicans cell wall plays an essential part in preserving cell integrity, hyphal morphogenesis, and virulence (Arita et al. ). The cell wall of C. albicans forms a viscoelastic shell that offsets the intracellular pressure (Lew ) and adapts to environmental changes by modifying hyphal bending stiffness (Couttenier et al. ). Disruption of the cell wall can negatively affect cell physiology and hyphal growth (Couttenier et al. ). Targeting β-glucan synthesis with clinical echinocandins antifungal drugs, such as caspofungin, or applying hypertonic stress to break the C. albicans cell wall results in decreased bending stiffness, reduced hyphal diameter, and altered hyphal elongation (Couttenier et al. ). Therefore, targeting the cell wall or hyphal development to disrupt fungal-host interactions and diminish fungal virulence represents a viable strategy for developing new antifungal agents (Vila et al. ; Ye et al. ). Traditional Chinese medicine (TCM) has been proved as a vast resource reservoir for the exploration and development of antifungal medications due to its diverse structures, wide sourcing, affordability, and minimal resistance (Liang et al. ; Ye et al. ; Zhu et al. ). Aloe vera (L.) Burm.f. is a traditional herb that has been extensively documented in China, Egypt, and India (Gao et al. ; Long ). The leaves of A. vera contain juice with anthraquinones, mesophyll gel filled with polysaccharides, and a green epidermis. Aloin, the primary active component of A. vera , has garnered significant attention from the scientific community in recent years for its diverse pharmacological activities, including anti-osteoporotic, immunomodulatory, and organ-protective effects, as well as antiviral, antifungal, and antibacterial (Ali et al. ; Donkor et al. ; Ushasree et al. ; Xiao et al. ; Yang et al. ). Additionally, aloin effectively regulated the blood lipid and sugar levels and neuropathic pain (Ushasree et al. ; Yang et al. ). For its antibacterial effects, aloin can bind to the cell walls of bacteria and destroy the bacterial membranes (Gokulan et al. ). Aloin showed inhibitory effects on C. albicans at relatively high dosages according to some antifungal assays in vitro (Donkor et al. ; Megeressa et al. ); however, its antifungal mechanisms, effects on pathogenic virulence, and the therapeutic potential against oral candidiasis remain unknown. In this study, we explored the antifungal properties and mechanisms of aloin against C. albicans and oral candidiasis. We found that aloin remodeled the cell wall of C. albicans to induce the formation of abnormal hyphae to reduce its virulence on oral epithelial cell damage and mice oral candidiasis. Our results indicate that aloin could serve as a promising antifungal drug candidate with novel mechanisms of action, targeting the hyphae of C. albicans as a practical strategy for developing novel antifungal drugs. 

Chemicals Aloin (CAS 1415–73-2, HPLC ≥ 98%), fluconazole (FLC), and dimethyl sulfoxide (DMSO) were obtained from SolarBio (Beijing, China), APExBIO (Houston, TX, USA), and MP Biomedicals (Irvine, CA, USA), respectively. Micafungin sodium (CAS 208538–73-2), itraconazole (CAS 84625–61-6), and 5-fluorocytosine (CAS 2022–85-7) were purchased from MCE (Monmouth Junction, NJ, USA). Aloin was dissolved in DMSO to prepare a stock solution with a concentration of 100 mg/mL and was then stored at − 20 °C before use. Strains and cells C. albicans SC5314 (ATCC MYA-2876) and 11 clinical isolates (CCCC-2217, CCCC-2220, CCCC-2221, CCCC-2223, CCCC-2228, CCCC-2230, CCCC-2232, CCCC-2235, CCCC-2240, CCCC-2256, CCCC-3721) were stored in the laboratory. All strains were routinely cultured in Yeast Extract Peptone Dextrose (YPD, Sigma-Aldrich, St. Louis, MO, USA) at 30 °C for subsequent antifungal experiments. For solid medium, 2% agar (Dingguo, Beijing, China) was added. Under the conditions of 37 °C and 5% CO 2 , C. albicans hyphae were induced in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Scoresby, Victoria, Australia). Human Oral Keratinocytes (HOK) cells (ScienCell, Carlsbad, CA, USA) and macrophage RAW 264.7 cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS. Growth curve testing C. albicans monoclonal clones were selected and adjusted to 2 × 10 6 colony-forming units (CFU)/mL in YPD. Aloin was added to achieve final concentrations of 0, 50, and 100 µg/mL. A 100 µL aliquot of the mixture was placed in a 96-well plate, and the OD 600 was measured every hour at 30 °C using a microplate reader (SpectraMax ID3, Thermo Fisher, Waltham, MA, USA). The plate was shaken every 15 min for continuous detection over 24 h. Hyphal assessment C. albicans hyphae were induced with serum as previously described (Liang et al. ). Briefly, 50 µL of the fungal solution (approximately 1000 cells) and 50 µL of either aloin or DMSO were added to the 96-well plate, yielding final drug concentrations of 0, 50, and 100 µg/mL. After 6 h of culture, 10 µL of the culture was mixed and transferred to slides. Then, hyphal formation was observed using the DIC mode of a Leica DMi8 inverted fluorescence microscope (Leica, Wetzlar, Germany). At least 100 fungal cells were randomly selected from each sample to count the hyphal branching, and ImageJ software was used to quantify the diameter of C. albicans hyphae. After 24 h of culture, the cells were transferred to Cytation 5 (BioTek, Winooski, VT, USA) to observe the formation of hyphae. Scanning electron microscopy (SEM) C. albicans hyphae were induced as previously described (Liang et al. ). Briefly, 100 µg/mL aloin or an equal volume of DMSO was added, and the test tubes were incubated at 37 °C on a shaker at 220 rpm for 24 h. Subsequently, the cells were suspended in 2.5% glutaraldehyde and fixed overnight in the dark at 4 °C. Dehydration was carried out using a series of alcohol gradients (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 10 min each. Following dehydration, a small amount of the suspension was dropped onto a glass slide. The glass slide was gently affixed to a conductive adhesive, sprayed with ion sputtering, and observed using the SEM (Inspect, FEI, Beaverton, OR, USA). Assessment of antifungal sensitivity The minimum inhibitory concentration (MIC) was assessed according to previous studies (Liao et al. ). Briefly, C. albicans was diluted to a concentration of 10 4 CFU/mL using RPMI 1640 medium. Fluconazole was used as a positive control, and different concentrations of aloin were added to a 96-well plate using a twofold serial dilution method. After overnight incubation at 35 °C, fungal growth in each well was assessed. Cytotoxicity assay of the drug Approximately 5000 HOK cells were seeded into each well and incubated for 24 h. Subsequently, 10 µL of aloin was applied to achieve final concentrations of 0, 50, 100, and 200 µg/mL. After an additional 24 h of incubation, the remaining drug was washed off with PBS. Cell viability was then assessed according to the instructions provided with the CCK-8 assay kit (Dojindo, Nara, Japan). Cell adhesion assay Briefly, C. albicans was prepared in DMEM containing 10% FBS at a concentration of 2 × 10 5 cells/mL. Aloin (50 µg/mL or 100 µg/mL) or DMSO was added to the C. albicans suspension, which was then introduced to HOK cells for co-culture for 1 h. Following the co-culture, the cells were washed to remove any non-adherent fungi. Subsequently, 1 mL of sterile water was added to lyse the cells, and the mixture was incubated at 37 °C for 1 h. The resulting culture was then serially diluted and plated onto YPD plates for colony counting (Zhou et al. ). Cell invasion assay The invasion of cells by C. albicans was carried out as previously described (Zhou et al. ). Initially, HOK cells were cultured on sterile cell culture slides for 48 h. Subsequently, C. albicans was resuspended in DMEM supplemented with 10% fetal bovine serum at 10 5 cells/mL. A solution of 100 µg/mL aloin or DMSO was added to the C. albicans suspension, which was then introduced to the cells. After co-culturing for 3 h, the slides were washed with PBS to remove any non-invading C. albicans . The slides were then fixed with 4% paraformaldehyde to maintain cellular structure integrity. Staining of the slides was carried out using ConA 647 (Thermo Fisher, Waltham, MA, USA) for 45 min. Cells were permeabilized with 0.1% Triton X-100 for 15 min. After permeabilization, the slides were treated with Calcofluor White (CFW, Sigma-Aldrich, St. Louis, MO, USA) for 20 min. After three additional washes with PBS to remove excess stains, an anti-quenching agent was applied to preserve the fluorescence signal. Finally, coverslips were used to seal the slides, and they were examined using confocal microscopy (FV1000, Olympus, Tokyo, Japan). At least 100 fungal cells were randomly selected from each sample, and images were captured to calculate the ratio of fungi invading the cells to the total number of fungi. Cell damage assessment Briefly, C. albicans was prepared in DMEM at a concentration of 10 6 cells/mL, and 50 µL of the suspension was added to HOK cells cultured overnight. Subsequently, 50 µL of aloin or DMSO was added to achieve final aloin concentrations of 0, 6.25, 12.5, 25, 50, and 100 µg/mL. After 24 h of co-culture, LDH activity in the supernatant was analyzed according to the instructions provided with the Cytotoxicity Detection Kit (LDH) (Roche, Basel, Switzerland). Transcriptome sequencing and analysis C. albicans cultured overnight was resuspended in RPMI 1640 medium containing 10% FBS. Aloin (100 µg/mL) or DMSO was added to treat C. albicans , and the cells were incubated for 12 h at 37 °C in a shaker at 220 rpm. Following the respective incubation periods, the fungi were collected via high-speed centrifugation and snap-frozen in liquid nitrogen. Total RNA was then extracted, and transcriptome sequencing was performed by OE Biotech Co., Ltd. (Shanghai, China). Measurement of cell wall chitin content A 100 mL culture of C. albicans at a concentration of 10 6 CFU/mL was prepared in YPD and divided into two test tubes, to which 100 µg/mL aloin and DMSO were added, respectively. The cultures were incubated with shaking at 220 rpm and 30 °C for 24 h. The cell walls of C. albicans were isolated following the protocol provided in the Fungal/Yeast Cell Wall Isolation Kit (Jiaozuo Yunzhiyu Biotechnology Co., Ltd., Henan, China). After obtaining the cell wall samples, the chitin content was measured using the Chitin ELISA Kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. Measurement of cell wall β−1,3-glucan content The measurement of cell wall β−1,3-glucan content was constructed and conducted as previously described (Suwunnakorn et al. ). Initially, C. albicans was prepared by diluting overnight cultures to 10 8 CFU/mL in YPD. Subsequently, 10 mL of these diluted cultures was treated with either aloin or DMSO to achieve final drug concentrations of 0, 50, and 100 µg/mL, respectively. The treated cultures were incubated overnight in a shaker at 220 rpm and 30 °C. The fungal precipitate was then collected by centrifugation and washed twice with TE buffer. Subsequently, 500 µL of the fungal solution was centrifuged and resuspended in 100 µL of 6 M sodium hydroxide solution, then treated at 80 °C for 30 min. An aniline blue mixture was prepared, comprising 0.03% aniline blue, 0.18 M hydrochloric acid, and 0.49 M glycine, with the pH adjusted to 9.5 using sodium hydroxide. Following this, 2.1 mL of the aniline blue solution was introduced into the centrifuge tube containing the treated cells, which was then vortexed and shaken before being treated at 50 °C for 30 min. After cooling to room temperature, fluorescence was measured at an excitation/emission wavelength of 400/460 nm. Susceptibility of C. albicans to macrophagocyte The macrophage-mediated clearance of C. albicans was evaluated to assess the effect of aloin on macrophage phagocytosis (Zhou et al. ). Briefly, RAW cells were cultured in 96-well plates overnight, followed by the addition of C. albicans yeast cells treated with 100 µg/mL aloin or DMSO (control). The cells were incubated for 3 h at 37 °C with 5% CO 2 . C. albicans cells which were not phagocytosed were then removed by washing with PBS. After that, RAW cells were lysed by incubation in sterile double-distilled water at 37 °C for 1 h. The suspensions were then collected, diluted, and plated on YPD plates. The plates were incubated at 30 °C for 48 h to quantify fungal counts. RT-qPCR C. albicans was inoculated into RPMI 1640 medium with 10% serum. Aloin was added to achieve final concentrations of 0, 50, or 100 µg/mL for overnight treatment, with the remaining volume filled with DMSO. Fungal cells were collected and homogenized using a FastPrep-24 homogenizer (MP Biomedicals, Santa Ana, CA, USA). Total RNA was extracted and subsequently purified using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Kyoto, Japan), followed by reverse transcription to obtain cDNA. qPCR was carried out using TB Green Premix Ex Taq™ II (Takara, Kyoto, Japan) with specific primers (Supplemental Table ). Interaction of aloin with antifungal agents Fifty microliters of C. albicans (approximately 1000 cells) prepared in RPMI 1640 medium was added to each well in a 96-well plate, along with 50 µL of either aloin, antifungal agents, and their combinations. Aloin and the antifungal agents including micafungin, itraconazole, and 5-fluorocytosine (5-FC) were performed a twofold serial dilution, respectively. The final concentrations for the aloin and antifungal agents were as follows: aloin (6.25 to 100 µg/mL), micafungin (0.016 to 0.25 µg/mL), itraconazole (0.03 to 0.25 µg/mL), and 5-FC (0.125 to 1 µg/mL). For their combinations, 100 µg/mL aloin was combined with different concentrations of the three antifungal agents. The plates were incubated overnight at 35 °C, and the MICs were observed visually. The fractional inhibitory concentration index (FICI) was used to calculate the interaction between aloin and the antifungal agents (Zhu et al. ). FICI of drug combination = (MIC of antifungal agent in combination/MIC of antifungal agent alone) + (MIC of aloin in combination/MIC of aloin alone). To further investigate the antagonistic interaction between aloin and micafungin, 2 µL of fungal solution from each well was spotted on YPD plates and then incubated at 35 °C overnight. Murine model of oral candidiasis The model was conducted following previous studies (Solis and Filler ; Zhou et al. ). Female BALB/c mice, 4–6 weeks of age with an average weight of 20 g, were sourced from DOSSY Co., Ltd. (Chengdu, China). They were randomly allocated to either the DMSO control group or the 100 µg/mL aloin treatment group, with each group consisting of five mice. The mice were housed in ventilated cages at a temperature of 25 °C, with five mice per cage. Chopped wood shavings were used as bedding. Before the experiment, the mice were acclimated to the facility for at least 1 week to adjust to the environmental conditions, including temperature, light/dark cycles, and access to food. Throughout the study, all mice were maintained under consistent environmental conditions to minimize potential sources of bias. Each mouse received a subcutaneous injection of 3 mg cortisone acetate prior to and following infection. On the day of infection, the mice were anesthetized with an intraperitoneal injection of tribromoethyl alcohol at a dose of 20 µL/g. Normal saline was used to prepare a suspension containing 10 7 CFU/mL of C. albicans . A cotton ball soaked in the solution was placed on the tongue of anesthetized mice and retained for 75 min. The mice were administered 100 µg/mL aloin or an equal volume of DMSO in drinking water the day after infection. After 3 days, the mice were euthanized, and the infection area on the tongues of each group was recorded. The tongue was longitudinally divided and weighed, and half of the Candida species were homogenized and quantified on CHROMagar™ Candida plates (CHROMagar, Paris, France), while the other half underwent histopathological analysis. The mice were monitored at least once daily throughout the entire experimental process. If any mouse exhibited signs such as hunching, severe diarrhea, inability to eat, significantly reduced activity, weight loss exceeding 20% of its initial body weight, or a noticeable drop in body temperature (cold to the touch), the affected mouse was immediately euthanized using CO 2 to minimize pain and stress. Histopathological detection After longitudinally sectioning the mouse tongue tissue, half of the sample was immediately preserved in paraformaldehyde. The tissue was then dehydrated and embedded in paraffin. Paraffin sectioning was carried out using a microtome (Leica 2016, Wetzlar, Germany). Hematoxylin–eosin staining (HE) and periodic acid-Schiff staining (PAS) were conducted as previously described (Moyes et al. ). Tissue section images were acquired using a digital slicing scanner (PANNORAMIC 250, Budapest, Hungary). Statistical analysis One-way ANOVA was employed to analyze the cell adhesion rate, cell damage rate, β−1,3-glucan relative content, and relative mRNA expression levels. t -tests were used to compare the cell invasion rate and chitin relative content between the aloin-treated and control groups. For the cell survival rate and fungal load in mouse tongue tissues that did not meet the homogeneity of variances assumption, Welch’s t -test and Welch’s ANOVA were used after data correction, respectively. Hyphal branching and diameter were analyzed using the Kruskal–Wallis test, and the infection area in mouse tongue tissues was analyzed using the Mann–Whitney U test. All statistical analyses and figure constructions were conducted using GraphPad Prism version 8.0.2 (GraphPad Software, LLC, Boston, MA, USA), and statistical significance was defined as P < 0.05. 

Aloin (CAS 1415–73-2, HPLC ≥ 98%), fluconazole (FLC), and dimethyl sulfoxide (DMSO) were obtained from SolarBio (Beijing, China), APExBIO (Houston, TX, USA), and MP Biomedicals (Irvine, CA, USA), respectively. Micafungin sodium (CAS 208538–73-2), itraconazole (CAS 84625–61-6), and 5-fluorocytosine (CAS 2022–85-7) were purchased from MCE (Monmouth Junction, NJ, USA). Aloin was dissolved in DMSO to prepare a stock solution with a concentration of 100 mg/mL and was then stored at − 20 °C before use. 

C. albicans SC5314 (ATCC MYA-2876) and 11 clinical isolates (CCCC-2217, CCCC-2220, CCCC-2221, CCCC-2223, CCCC-2228, CCCC-2230, CCCC-2232, CCCC-2235, CCCC-2240, CCCC-2256, CCCC-3721) were stored in the laboratory. All strains were routinely cultured in Yeast Extract Peptone Dextrose (YPD, Sigma-Aldrich, St. Louis, MO, USA) at 30 °C for subsequent antifungal experiments. For solid medium, 2% agar (Dingguo, Beijing, China) was added. Under the conditions of 37 °C and 5% CO 2 , C. albicans hyphae were induced in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Scoresby, Victoria, Australia). Human Oral Keratinocytes (HOK) cells (ScienCell, Carlsbad, CA, USA) and macrophage RAW 264.7 cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS. 

C. albicans monoclonal clones were selected and adjusted to 2 × 10 6 colony-forming units (CFU)/mL in YPD. Aloin was added to achieve final concentrations of 0, 50, and 100 µg/mL. A 100 µL aliquot of the mixture was placed in a 96-well plate, and the OD 600 was measured every hour at 30 °C using a microplate reader (SpectraMax ID3, Thermo Fisher, Waltham, MA, USA). The plate was shaken every 15 min for continuous detection over 24 h. 

C. albicans hyphae were induced with serum as previously described (Liang et al. ). Briefly, 50 µL of the fungal solution (approximately 1000 cells) and 50 µL of either aloin or DMSO were added to the 96-well plate, yielding final drug concentrations of 0, 50, and 100 µg/mL. After 6 h of culture, 10 µL of the culture was mixed and transferred to slides. Then, hyphal formation was observed using the DIC mode of a Leica DMi8 inverted fluorescence microscope (Leica, Wetzlar, Germany). At least 100 fungal cells were randomly selected from each sample to count the hyphal branching, and ImageJ software was used to quantify the diameter of C. albicans hyphae. After 24 h of culture, the cells were transferred to Cytation 5 (BioTek, Winooski, VT, USA) to observe the formation of hyphae. 

C. albicans hyphae were induced as previously described (Liang et al. ). Briefly, 100 µg/mL aloin or an equal volume of DMSO was added, and the test tubes were incubated at 37 °C on a shaker at 220 rpm for 24 h. Subsequently, the cells were suspended in 2.5% glutaraldehyde and fixed overnight in the dark at 4 °C. Dehydration was carried out using a series of alcohol gradients (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 10 min each. Following dehydration, a small amount of the suspension was dropped onto a glass slide. The glass slide was gently affixed to a conductive adhesive, sprayed with ion sputtering, and observed using the SEM (Inspect, FEI, Beaverton, OR, USA). 

The minimum inhibitory concentration (MIC) was assessed according to previous studies (Liao et al. ). Briefly, C. albicans was diluted to a concentration of 10 4 CFU/mL using RPMI 1640 medium. Fluconazole was used as a positive control, and different concentrations of aloin were added to a 96-well plate using a twofold serial dilution method. After overnight incubation at 35 °C, fungal growth in each well was assessed. 

Approximately 5000 HOK cells were seeded into each well and incubated for 24 h. Subsequently, 10 µL of aloin was applied to achieve final concentrations of 0, 50, 100, and 200 µg/mL. After an additional 24 h of incubation, the remaining drug was washed off with PBS. Cell viability was then assessed according to the instructions provided with the CCK-8 assay kit (Dojindo, Nara, Japan). 

Briefly, C. albicans was prepared in DMEM containing 10% FBS at a concentration of 2 × 10 5 cells/mL. Aloin (50 µg/mL or 100 µg/mL) or DMSO was added to the C. albicans suspension, which was then introduced to HOK cells for co-culture for 1 h. Following the co-culture, the cells were washed to remove any non-adherent fungi. Subsequently, 1 mL of sterile water was added to lyse the cells, and the mixture was incubated at 37 °C for 1 h. The resulting culture was then serially diluted and plated onto YPD plates for colony counting (Zhou et al. ). 

The invasion of cells by C. albicans was carried out as previously described (Zhou et al. ). Initially, HOK cells were cultured on sterile cell culture slides for 48 h. Subsequently, C. albicans was resuspended in DMEM supplemented with 10% fetal bovine serum at 10 5 cells/mL. A solution of 100 µg/mL aloin or DMSO was added to the C. albicans suspension, which was then introduced to the cells. After co-culturing for 3 h, the slides were washed with PBS to remove any non-invading C. albicans . The slides were then fixed with 4% paraformaldehyde to maintain cellular structure integrity. Staining of the slides was carried out using ConA 647 (Thermo Fisher, Waltham, MA, USA) for 45 min. Cells were permeabilized with 0.1% Triton X-100 for 15 min. After permeabilization, the slides were treated with Calcofluor White (CFW, Sigma-Aldrich, St. Louis, MO, USA) for 20 min. After three additional washes with PBS to remove excess stains, an anti-quenching agent was applied to preserve the fluorescence signal. Finally, coverslips were used to seal the slides, and they were examined using confocal microscopy (FV1000, Olympus, Tokyo, Japan). At least 100 fungal cells were randomly selected from each sample, and images were captured to calculate the ratio of fungi invading the cells to the total number of fungi. 

Briefly, C. albicans was prepared in DMEM at a concentration of 10 6 cells/mL, and 50 µL of the suspension was added to HOK cells cultured overnight. Subsequently, 50 µL of aloin or DMSO was added to achieve final aloin concentrations of 0, 6.25, 12.5, 25, 50, and 100 µg/mL. After 24 h of co-culture, LDH activity in the supernatant was analyzed according to the instructions provided with the Cytotoxicity Detection Kit (LDH) (Roche, Basel, Switzerland). 

C. albicans cultured overnight was resuspended in RPMI 1640 medium containing 10% FBS. Aloin (100 µg/mL) or DMSO was added to treat C. albicans , and the cells were incubated for 12 h at 37 °C in a shaker at 220 rpm. Following the respective incubation periods, the fungi were collected via high-speed centrifugation and snap-frozen in liquid nitrogen. Total RNA was then extracted, and transcriptome sequencing was performed by OE Biotech Co., Ltd. (Shanghai, China). 

A 100 mL culture of C. albicans at a concentration of 10 6 CFU/mL was prepared in YPD and divided into two test tubes, to which 100 µg/mL aloin and DMSO were added, respectively. The cultures were incubated with shaking at 220 rpm and 30 °C for 24 h. The cell walls of C. albicans were isolated following the protocol provided in the Fungal/Yeast Cell Wall Isolation Kit (Jiaozuo Yunzhiyu Biotechnology Co., Ltd., Henan, China). After obtaining the cell wall samples, the chitin content was measured using the Chitin ELISA Kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. 

The measurement of cell wall β−1,3-glucan content was constructed and conducted as previously described (Suwunnakorn et al. ). Initially, C. albicans was prepared by diluting overnight cultures to 10 8 CFU/mL in YPD. Subsequently, 10 mL of these diluted cultures was treated with either aloin or DMSO to achieve final drug concentrations of 0, 50, and 100 µg/mL, respectively. The treated cultures were incubated overnight in a shaker at 220 rpm and 30 °C. The fungal precipitate was then collected by centrifugation and washed twice with TE buffer. Subsequently, 500 µL of the fungal solution was centrifuged and resuspended in 100 µL of 6 M sodium hydroxide solution, then treated at 80 °C for 30 min. An aniline blue mixture was prepared, comprising 0.03% aniline blue, 0.18 M hydrochloric acid, and 0.49 M glycine, with the pH adjusted to 9.5 using sodium hydroxide. Following this, 2.1 mL of the aniline blue solution was introduced into the centrifuge tube containing the treated cells, which was then vortexed and shaken before being treated at 50 °C for 30 min. After cooling to room temperature, fluorescence was measured at an excitation/emission wavelength of 400/460 nm. 

The macrophage-mediated clearance of C. albicans was evaluated to assess the effect of aloin on macrophage phagocytosis (Zhou et al. ). Briefly, RAW cells were cultured in 96-well plates overnight, followed by the addition of C. albicans yeast cells treated with 100 µg/mL aloin or DMSO (control). The cells were incubated for 3 h at 37 °C with 5% CO 2 . C. albicans cells which were not phagocytosed were then removed by washing with PBS. After that, RAW cells were lysed by incubation in sterile double-distilled water at 37 °C for 1 h. The suspensions were then collected, diluted, and plated on YPD plates. The plates were incubated at 30 °C for 48 h to quantify fungal counts. 

C. albicans was inoculated into RPMI 1640 medium with 10% serum. Aloin was added to achieve final concentrations of 0, 50, or 100 µg/mL for overnight treatment, with the remaining volume filled with DMSO. Fungal cells were collected and homogenized using a FastPrep-24 homogenizer (MP Biomedicals, Santa Ana, CA, USA). Total RNA was extracted and subsequently purified using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Kyoto, Japan), followed by reverse transcription to obtain cDNA. qPCR was carried out using TB Green Premix Ex Taq™ II (Takara, Kyoto, Japan) with specific primers (Supplemental Table ). 

Fifty microliters of C. albicans (approximately 1000 cells) prepared in RPMI 1640 medium was added to each well in a 96-well plate, along with 50 µL of either aloin, antifungal agents, and their combinations. Aloin and the antifungal agents including micafungin, itraconazole, and 5-fluorocytosine (5-FC) were performed a twofold serial dilution, respectively. The final concentrations for the aloin and antifungal agents were as follows: aloin (6.25 to 100 µg/mL), micafungin (0.016 to 0.25 µg/mL), itraconazole (0.03 to 0.25 µg/mL), and 5-FC (0.125 to 1 µg/mL). For their combinations, 100 µg/mL aloin was combined with different concentrations of the three antifungal agents. The plates were incubated overnight at 35 °C, and the MICs were observed visually. The fractional inhibitory concentration index (FICI) was used to calculate the interaction between aloin and the antifungal agents (Zhu et al. ). FICI of drug combination = (MIC of antifungal agent in combination/MIC of antifungal agent alone) + (MIC of aloin in combination/MIC of aloin alone). To further investigate the antagonistic interaction between aloin and micafungin, 2 µL of fungal solution from each well was spotted on YPD plates and then incubated at 35 °C overnight. 

The model was conducted following previous studies (Solis and Filler ; Zhou et al. ). Female BALB/c mice, 4–6 weeks of age with an average weight of 20 g, were sourced from DOSSY Co., Ltd. (Chengdu, China). They were randomly allocated to either the DMSO control group or the 100 µg/mL aloin treatment group, with each group consisting of five mice. The mice were housed in ventilated cages at a temperature of 25 °C, with five mice per cage. Chopped wood shavings were used as bedding. Before the experiment, the mice were acclimated to the facility for at least 1 week to adjust to the environmental conditions, including temperature, light/dark cycles, and access to food. Throughout the study, all mice were maintained under consistent environmental conditions to minimize potential sources of bias. Each mouse received a subcutaneous injection of 3 mg cortisone acetate prior to and following infection. On the day of infection, the mice were anesthetized with an intraperitoneal injection of tribromoethyl alcohol at a dose of 20 µL/g. Normal saline was used to prepare a suspension containing 10 7 CFU/mL of C. albicans . A cotton ball soaked in the solution was placed on the tongue of anesthetized mice and retained for 75 min. The mice were administered 100 µg/mL aloin or an equal volume of DMSO in drinking water the day after infection. After 3 days, the mice were euthanized, and the infection area on the tongues of each group was recorded. The tongue was longitudinally divided and weighed, and half of the Candida species were homogenized and quantified on CHROMagar™ Candida plates (CHROMagar, Paris, France), while the other half underwent histopathological analysis. The mice were monitored at least once daily throughout the entire experimental process. If any mouse exhibited signs such as hunching, severe diarrhea, inability to eat, significantly reduced activity, weight loss exceeding 20% of its initial body weight, or a noticeable drop in body temperature (cold to the touch), the affected mouse was immediately euthanized using CO 2 to minimize pain and stress. 

After longitudinally sectioning the mouse tongue tissue, half of the sample was immediately preserved in paraformaldehyde. The tissue was then dehydrated and embedded in paraffin. Paraffin sectioning was carried out using a microtome (Leica 2016, Wetzlar, Germany). Hematoxylin–eosin staining (HE) and periodic acid-Schiff staining (PAS) were conducted as previously described (Moyes et al. ). Tissue section images were acquired using a digital slicing scanner (PANNORAMIC 250, Budapest, Hungary). 

One-way ANOVA was employed to analyze the cell adhesion rate, cell damage rate, β−1,3-glucan relative content, and relative mRNA expression levels. t -tests were used to compare the cell invasion rate and chitin relative content between the aloin-treated and control groups. For the cell survival rate and fungal load in mouse tongue tissues that did not meet the homogeneity of variances assumption, Welch’s t -test and Welch’s ANOVA were used after data correction, respectively. Hyphal branching and diameter were analyzed using the Kruskal–Wallis test, and the infection area in mouse tongue tissues was analyzed using the Mann–Whitney U test. All statistical analyses and figure constructions were conducted using GraphPad Prism version 8.0.2 (GraphPad Software, LLC, Boston, MA, USA), and statistical significance was defined as P < 0.05. 

Aloin-induced C. albicans to form abnormal hyphae without growth inhibition Aloin at both 50 and 100 µg/mL had no significant effect on the growth of C. albicans SC5314 (Fig. a, b), and the MIC was higher than 800 µg/mL (Table ), while the MIC of fluconazole was only 0.25 µg/mL (Table ). Then, RPMI 1640 medium with the addition of 10% serum was employed to induce the formation of C. albicans hyphae. According to the live cell imaging and scanning electron microscopy, C. albicans formed typical hyphae; however, the C. albicans hyphae were abnormal with smaller diameters and fewer branches in the 100 µg/mL aloin-treated group (Fig. c, d). When calculating the hyphal diameters and branching, both 50 and 100 µg/mL of aloin significantly inhibited the diameter and branching of C. albicans hyphae (Fig. e, f). Eleven clinical isolates of C. albicans obtained from oral candidiasis patients were then employed to assess the impact of aloin on the growth and hyphae of clinical strains. The clinical isolates exhibited susceptibility to fluconazole (MIC = 0.25 ~ 0.5 µg/mL), while aloin showed no growth inhibition on these isolates (MIC > 800 µg/mL) (Table ). However, aloin induced abnormal hyphal formation in all 11 clinical isolates, similar to that observed in C. albicans SC5314, indicating its potential clinical applications (Fig. ). Aloin inhibited cell damage caused by C. albicans To further validate whether aloin could repress the virulence of C. albicans due to its induction of the abnormal hyphae, HOK cells were infected with C. albicans . Aloin showed no cytotoxicity to cells ranging from 50 to 200 µg/mL (Fig. a) and also had no significant effects on fungal adhesion and invasion into HOK cells (Fig. b, c, and d). However, aloin significantly reduced damage to HOK cells caused by C. albicans in a dose-dependent manner (Fig. e), indicating that aloin induced the formation of avirulent hyphae in C. albicans to repress its pathogenicity to host cells. The link between abnormal hyphae and virulence was confirmed by the expression of the hyphal-specific toxin candidalysin (encoded by the ECE1 gene), as aloin significantly reduced the expression of genes associated with ECE1 and its upstream CBK1 - BCR1 pathway (Fig. f, Supplemental Table ). Aloin remodeled the cell wall of C. albicans To further explore the mechanism of aloin against C. albicans , the transcriptomes of C. albicans exposed to 100 µg/mL aloin or an equal amount of DMSO (as control) for 12 h were sequenced and analyzed (Fig. a). Aloin treatment led to the significant upregulation of 59 genes and the downregulation of 201 genes (Fig. b). The GO enrichment analysis (top 30) revealed that the term related to the fungal-type cell wall was significantly changed (Fig. c), indicating that aloin could remodel the cell wall of C. albicans . The expression of genes associated with the fungal cell wall was significantly reduced (Fig. d), including the key genes associated with C. albicans hyphal development and virulence, such as BMT9 , CSA1 , YWP1 , SOD5 , PGA7 , RBT5 , CHT2 , ECM21 , TPS2 , and BIG1 (Fig. d). To confirm the impact of aloin in the C. albicans cell wall, we measured the levels of chitin and β−1,3-glucan. Aloin showed no significant effect on chitin, but significantly increased the content of β−1,3-glucan (Fig. e). The expression of genes responsible for the biosynthesis of β−1,3-glucan (Supplemental Table ) was then measured. Aloin significantly upregulated GSL1 , GSC1 (also known as FKS1 ), and RHO1 (Fig. f). Micafungin was then employed to validate the combinational effects with aloin since micafungin targets at FKS1 to inhibit β−1,3-glucan biosynthesis and aloin could upregulate FKS1 to increase the β−1,3-glucan contents. Two other antifungal agents, itraconazole and 5-FC, were also employed as control. Aloin showed no direct or combinational antifungal effects with itraconazole and 5-FC, but significantly antagonized with micafungin (Fig. g and Supplemental Table ), furtherly confirming that aloin increased β−1,3-glucan biosynthesis to remodel the C. albicans cell wall. Since dectin-1 from macrophage could recognize β-glucan from the C. albicans cell wall to regulate antifungal immunity (Quintin et al. ), we next evaluated the effects of aloin on macrophage phagocytosis. Interestingly, aloin did not significantly affect the macrophage phagocytosis rate (Supplemental Fig. ), indicating that the exposure of β-glucan on the cell wall was not changed although aloin upregulated β−1,3-glucan content. Aloin inhibited oral candidiasis in mice The antifungal efficacy of aloin in vivo was validated using a mouse oral infection model. Infected mice exhibited distinct white patches on the surface of the tongue (Fig. a). Treatment with 100 µg/mL of aloin resulted in a significant reduction in leukoplakia area ( P = 0.0238), with a median infection area of 25% in the aloin-treated group ( n = 5) compared to 50% in the control group ( n = 5). These findings indicate a notable reduction in the infection area attributable to aloin treatment (Fig. a, b). Regarding fungal burden, the mean fungal load was significantly lower in the aloin-treated group (144.6 ± 268.3 CFU/mg tissue) compared to the control group (843.2 CFU/mg tissue), indicating the significant impact of aloin treatment on reducing the fungal load (Fig. c). HE staining of the tongue revealed that C. albicans triggered epithelial inflammation and substantial tissue damage in the control group, whereas aloin treatment helped preserve the normal morphology of the tongue epithelium and significantly repressed the epithelial inflammatory infiltration and local tissue damage (Fig. d). PAS staining revealed extensive invasion of C. albicans hyphae into the mucosa in the control group, while no typical hyphal invasion was observed in the aloin-treated group (Fig. d), in line with the observed effects of aloin on the development of C. albicans hyphae. 

Aloin at both 50 and 100 µg/mL had no significant effect on the growth of C. albicans SC5314 (Fig. a, b), and the MIC was higher than 800 µg/mL (Table ), while the MIC of fluconazole was only 0.25 µg/mL (Table ). Then, RPMI 1640 medium with the addition of 10% serum was employed to induce the formation of C. albicans hyphae. According to the live cell imaging and scanning electron microscopy, C. albicans formed typical hyphae; however, the C. albicans hyphae were abnormal with smaller diameters and fewer branches in the 100 µg/mL aloin-treated group (Fig. c, d). When calculating the hyphal diameters and branching, both 50 and 100 µg/mL of aloin significantly inhibited the diameter and branching of C. albicans hyphae (Fig. e, f). Eleven clinical isolates of C. albicans obtained from oral candidiasis patients were then employed to assess the impact of aloin on the growth and hyphae of clinical strains. The clinical isolates exhibited susceptibility to fluconazole (MIC = 0.25 ~ 0.5 µg/mL), while aloin showed no growth inhibition on these isolates (MIC > 800 µg/mL) (Table ). However, aloin induced abnormal hyphal formation in all 11 clinical isolates, similar to that observed in C. albicans SC5314, indicating its potential clinical applications (Fig. ). 

To further validate whether aloin could repress the virulence of C. albicans due to its induction of the abnormal hyphae, HOK cells were infected with C. albicans . Aloin showed no cytotoxicity to cells ranging from 50 to 200 µg/mL (Fig. a) and also had no significant effects on fungal adhesion and invasion into HOK cells (Fig. b, c, and d). However, aloin significantly reduced damage to HOK cells caused by C. albicans in a dose-dependent manner (Fig. e), indicating that aloin induced the formation of avirulent hyphae in C. albicans to repress its pathogenicity to host cells. The link between abnormal hyphae and virulence was confirmed by the expression of the hyphal-specific toxin candidalysin (encoded by the ECE1 gene), as aloin significantly reduced the expression of genes associated with ECE1 and its upstream CBK1 - BCR1 pathway (Fig. f, Supplemental Table ). 

To further explore the mechanism of aloin against C. albicans , the transcriptomes of C. albicans exposed to 100 µg/mL aloin or an equal amount of DMSO (as control) for 12 h were sequenced and analyzed (Fig. a). Aloin treatment led to the significant upregulation of 59 genes and the downregulation of 201 genes (Fig. b). The GO enrichment analysis (top 30) revealed that the term related to the fungal-type cell wall was significantly changed (Fig. c), indicating that aloin could remodel the cell wall of C. albicans . The expression of genes associated with the fungal cell wall was significantly reduced (Fig. d), including the key genes associated with C. albicans hyphal development and virulence, such as BMT9 , CSA1 , YWP1 , SOD5 , PGA7 , RBT5 , CHT2 , ECM21 , TPS2 , and BIG1 (Fig. d). To confirm the impact of aloin in the C. albicans cell wall, we measured the levels of chitin and β−1,3-glucan. Aloin showed no significant effect on chitin, but significantly increased the content of β−1,3-glucan (Fig. e). The expression of genes responsible for the biosynthesis of β−1,3-glucan (Supplemental Table ) was then measured. Aloin significantly upregulated GSL1 , GSC1 (also known as FKS1 ), and RHO1 (Fig. f). Micafungin was then employed to validate the combinational effects with aloin since micafungin targets at FKS1 to inhibit β−1,3-glucan biosynthesis and aloin could upregulate FKS1 to increase the β−1,3-glucan contents. Two other antifungal agents, itraconazole and 5-FC, were also employed as control. Aloin showed no direct or combinational antifungal effects with itraconazole and 5-FC, but significantly antagonized with micafungin (Fig. g and Supplemental Table ), furtherly confirming that aloin increased β−1,3-glucan biosynthesis to remodel the C. albicans cell wall. Since dectin-1 from macrophage could recognize β-glucan from the C. albicans cell wall to regulate antifungal immunity (Quintin et al. ), we next evaluated the effects of aloin on macrophage phagocytosis. Interestingly, aloin did not significantly affect the macrophage phagocytosis rate (Supplemental Fig. ), indicating that the exposure of β-glucan on the cell wall was not changed although aloin upregulated β−1,3-glucan content. 

The antifungal efficacy of aloin in vivo was validated using a mouse oral infection model. Infected mice exhibited distinct white patches on the surface of the tongue (Fig. a). Treatment with 100 µg/mL of aloin resulted in a significant reduction in leukoplakia area ( P = 0.0238), with a median infection area of 25% in the aloin-treated group ( n = 5) compared to 50% in the control group ( n = 5). These findings indicate a notable reduction in the infection area attributable to aloin treatment (Fig. a, b). Regarding fungal burden, the mean fungal load was significantly lower in the aloin-treated group (144.6 ± 268.3 CFU/mg tissue) compared to the control group (843.2 CFU/mg tissue), indicating the significant impact of aloin treatment on reducing the fungal load (Fig. c). HE staining of the tongue revealed that C. albicans triggered epithelial inflammation and substantial tissue damage in the control group, whereas aloin treatment helped preserve the normal morphology of the tongue epithelium and significantly repressed the epithelial inflammatory infiltration and local tissue damage (Fig. d). PAS staining revealed extensive invasion of C. albicans hyphae into the mucosa in the control group, while no typical hyphal invasion was observed in the aloin-treated group (Fig. d), in line with the observed effects of aloin on the development of C. albicans hyphae. 

Recently, there has been a huge rise in mortality rates associated with Candida infections (Arita et al. ; Thompson et al. ). However, the available antifungal agents are limited, raising concerns about their toxic side effects and the growing prevalence of antifungal resistance (Howard et al. ; Kriegl et al. ; Lockhart et al. ). Therefore, the development of novel antifungal agents with low toxicity and a reduced risk of drug resistance is of critical importance (Howard et al. ). The hyphae of C. albicans are proven to be the most important virulence factor for its pathogenesis (Chen et al. ; Liao et al. ), while targeting its hyphal development is a practical strategy to inhibit its virulence and pathogenesis (Liang et al. ; Wang et al. ; Zhou et al. ). Hyphal branching is a key characteristic of C. albicans morphogenesis and pathogenicity, regulated by multiple factors, including the cell cycle, vacuolar dynamics, cell wall remodeling, and signaling pathways (Jacobsen and Hube ; Sudbery ). Under nutrient-rich conditions, C. albicans typically forms linear hyphae through apical growth, while nutrient deprivation or environmental stress induces branching to enhance its adaptability (Harris ; Jakab et al. ). Vacuole inheritance and volume changes play a crucial role in regulating hyphal branching, as vacuole size and distribution influence the branching frequency and hyphal elongation (Veses et al. ). In mutants with smaller vacuoles, the branching frequency is higher, whereas mutants with larger vacuoles exhibit reduced branching frequency (Veses et al. ). Vacuoles also regulate cytoplasmic volume ratios and cell cycle progression, controlling the timing and frequency of branch initiation (Tong et al. ). The cell wall is equally essential in hyphal branching, as its synthesis and remodeling at branching points provide the structural foundation for branch formation (Harris ; Sudbery ). Vacuolar expansion reduces the biosynthetic demands on the cytoplasm, thereby facilitating hyphal growth and branching (Veses et al. ). Notably, vacuolar dynamics are closely linked to cell wall remodeling (Jia et al. ; Patenaude et al. ), and together, they determine the branching patterns of hyphae, ultimately influencing the adaptability and pathogenic potential of C. albicans (Barelle et al. ; Harris ). Therefore, the precise interplay between vacuoles, cell wall dynamics, and cell cycle regulation is critical to understanding the mechanisms of hyphal branching and its role in C. albicans pathogenicity. TCM emerges as a promising candidate for anti-virulence therapy, owing to its diverse chemical composition, safety profile, low toxicity, and limited propensity for resistance (Wu et al. ; Ye et al. ). Clinical trials have shown that aloe toothpaste and A. vera juice are effective in reducing the total number of C. albicans in the mouth or dental plaque of subjects (Kupnik et al. ; Pradeep et al. ). However, the specific monomeric constituents responsible for its antifungal activity and their underlying mechanisms of antifungal actions remain unclear. Our study found that aloin could target virulence factors (e.g., hyphal formation) rather than directly killing the pathogen, indicating that it was a practical anti-virulence agent. Although aloin did not show direct inhibition of C. albicans growth in vitro, it significantly reduced fungal burden in vivo. The hyphal formation is critical for the mucosal colonization of C. albicans (Liang et al. ), and the hyphal-inhibitory effects of aloin may directly reduce fungal colonization. Additionally, the host immune system has played a pivotal role in fungal clearance in vivo. Non-virulent hyphae formed by aloin treatment may be more susceptible to clearance by the host immune response. Furthermore, aloin may also enhance fungal clearance by modulating immune cell function or promoting immune-mediated mechanisms. Previous studies have shown that anti-virulence agents, such as deferasirox, an iron chelator approved by the US Food and Drug Administration (FDA), do not affect the hyphal morphology of C. albicans but significantly reduce its survival rate within neutrophils, ultimately leading to a significant reduction in fungal burden in murine models of oropharyngeal candidiasis (Puri et al. ). Similarly, our prior research showed that arteether significantly reduced fungal burden in murine models of oropharyngeal candidiasis without C. albicans growth inhibition in vitro (Liang et al. ). These findings suggest that anti-virulence agents not only suppress pathogenic virulence but also enhance host clearance, thereby inhibiting infections. Moreover, these non-lethal mechanisms minimize selection pressure for antifungal resistance and could have potent effects against infections caused by drug-resistant isolates (Liang et al. ). Nonetheless, the clinical application of anti-virulence therapies also faces challenges, including drug administration time to target the active expressions of virulence factors and the potential adaptive resistance to anti-virulence agents from pathogens. Further investigations are needed to address these challenges and optimize the clinical utility of anti-virulence strategies. Aloin was proved to inhibit the expressions of the genes associated with ECE1 , the first fungal toxin in C. albicans , and its upstream CBK1 - BCR1 pathway, in line with its virulence and pathogenesis to oral epithelial cells and mice. ECE1 is a hyphal-dependent virulence gene, and its expression is upregulated during hyphal growth, but it does not participate in the regulations of hyphal formation as the hyphal development of C. albicans cells with ECE1 deletion remains unchanged (Birse et al. ). However, it is an important virulence factor from C. albicans hyphae and lost of ECE1 significantly reduce the pathogenic abilities of C. albicans (Moyes et al. ). In our study, aloin was proved to remodel the cell wall of C. albicans and downregulated ECE1 expression, which contributed to the repressed virulence in vitro and in vivo models. Transcriptomic analysis revealed that aloin caused significant perturbations in pathogenesis, fungal-type cell wall, extracellular region, plasma membrane, metal ion binding, and other crucial biological processes of C. albicans . Aloin downregulated the expressions of several cell wall-associated genes, including CHT2 , ENO1 , PGA7 , SOD5 , TPS2 , and BIG1 (Fig. ). These genes play key roles in the virulence of C. albicans . Specifically, CHT2 , involved in chitin degradation, contributes to cell wall remodeling and enhanced pathogen fitness (Witchley et al. ). ENO1 , essential for glycolysis, is a key molecule in cell division and cell wall remodeling during the transition from yeast to hyphal form (Reyna-Beltrán et al. ). PGA7 , a member of the common fungal extracellular membrane (CFEM) family of cell wall proteins, participated in heme acquisition (Kuznets et al. ). SOD5 protects C. albicans from reactive oxygen species (ROS) produced by the host’s immune response, regulates the transition from yeast to hyphal form, and maintains its virulence (Martchenko et al. ). TPS2 is involved in the synthesis of trehalose, which protects the pathogen from various environmental stresses (Miao et al. ). BIG1 is involved in β−1,6-glucan synthesis, regulating hyphal formation and adhesion and influencing virulence (Tong et al. ). The roles of these genes in virulence have been previously confirmed in a mouse model of disseminated candidiasis caused by C. albicans (Arita et al. ), further supporting the effect of aloin on C. albicans virulence. The fungal cell wall is crucial to the pathogenicity of C. albicans . Its inner layer is composed of cross-linked chitin and glucan, while the outer layer consists of glycosylated mannoproteins covalently attached to the inner layer (Hall and Gow ). Chitin, despite comprising only a small fraction of the cell wall, is essential for maintaining the overall wall integrity of C. albicans (Ren et al. ). β-Glucan, accounting for a substantial portion of the yeast cell wall, is the main structural polysaccharide (Denning ). β−1,3-Glucan, specifically, represents a prominent component of the C. albicans cell wall (Denning ). In C. albicans , β−1,3-glucans are largely concealed within the cell wall, shielded by the outer mannan layer, but some are exposed to the cell surface (de Assis et al. ), where they are recognized by the host antifungal defense system as pathogen-associated molecular patterns (PAMPs) (Cohen et al. ). The stresses induced by lactate (Ballou et al. ), hypoxia (Avelar et al. ), or iron limitation (Pradhan et al. ) could activate distinct signaling pathways associated with β−1,3-glucans (Childers et al. ). Notably, alterations in iron levels have a significant effect on β−1,3-glucan levels (de Assis et al. ), with elevated iron concentrations increasing β−1,3-glucan content and decreasing C. albicans survival after macrophage phagocytosis (Tripathi et al. ). We found that aloin antagonized the effect of micafungin, an antifungal drug targeting the β−1,3-glucan synthetase subunit Fks1 (Arendrup et al. ; Hou et al. ). This antagonistic combination indicated that when echinocandins, such as micafungin were used in clinical, aloin could not be taken to avoid reducing the antifungal activities of echinocandins (Wambaugh et al. ). Meanwhile, the rational selection of antagonistic drug combinations can potentially reduce adverse effects associated with current therapies and delay the onset of fungal resistance. Our RT-qPCR results revealed that aloin upregulated the genes responsible for β−1,3-glucan synthesis, particularly GSC1 and GSL1 , both of which are subunits of β−1,3-glucan synthetase, also known as Fks1, in C. albicans (Mio et al. ; Niimi et al. ), and we also demonstrated that aloin upregulated β−1,3-glucan synthesis to increase its contents in the C. albicans cell wall. These findings indicated that aloin remodeled the fungal cell wall and induced the development of abnormal hyphae to inhibit the virulence of C. albicans by increasing the β−1,3-glucan biosynthesis. The upregulation of β−1,3-glucan content by aloin in C. albicans was possibly linked to the disruption in metal ion homeostasis, as aloin significantly downregulated the genes associated with iron acquisition, such as the gene for the Ftr1 iron receptor and the RBT5 , CSA1 , CSA2 , and PGA7 genes of the cell wall-anchored hemoglobin receptor gene family involved in heme iron acquisition. Therefore, the remodeling of the cell wall by aloin may be mediated through the disruptions in iron ion levels, and the detailed mechanisms will be validated in the future. In conclusion, our study demonstrated that aloin could remodel the cell wall of C. albicans by increasing the β−1,3-glucan to induce the formation of avirulent hyphae against its infections to oral epithelial cells and oral mucosa of mice without cytotoxicity, indicating that aloin was a promising novel antifungal agent with therapeutic potential against C. albicans infections. 

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