Candida albicans SC5314 was kindly provided by Professor Jiang Yuanying from the College of Pharmacy, Second Military Medical University (Shanghai, China). The glycerol-preserved fungal strain taken out from the −80°C refrigerator was initially streaked onto Sabouraud’s agar. By using a single colony, the strain was routinely inoculated for three generations in Sabouraud’s agar plates. The strain was then activated and propagated in liquid Sabouraud medium (HopeBio-Tech Co., Qingdao, China) at 37°C for 12–16 h until exponential growth phase was achieved. Revived C. albicans cells were pooled by centrifugation at 3,000 ×g. After two washing steps using sterile PBS (Punuosai Life-Tech Co., Wuhan, China), cells were resuspended in RPMI-1640 medium (LifeTechnologies Corporation, United States) and adjusted to a defined cell density using a hemocytometer prior to subsequent tests.

A431 human vaginal epidermoid carcinoma cell line (Shanghai Cell Bank of the Chinese Academy of Sciences) was cultured at 37°C with 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (Hyclone, United States) supplemented with 10% fetal bovine serum (Gibco, United States) and 1% penicillin-streptomycin (Biyuntian Biotechnology Co., Ltd., Shanghai, China). At 24 h prior to experimentation, the culture medium was replaced with serum-free medium and maintained until cell harvest.

The minimum inhibitory concentration (MIC) of BBR (Chengdu Munster Biotech Co., Ltd., China; purity: 98.38% by HPLC) against C. albicans SC5314 was evaluated in 96-well microplates (Corning, United States) using the broth microdilution method described by the Clinical and Laboratory Standard Institute (CLSI) M27-A3 document (CLSI, 2008) (Pierce et al., 2008). Initial yeast inoculum was adjusted to1-5× 10³ CFU/ml in PRMI-1640 medium buffered to pH 7.0 with MOPS. The inoculum size was confirmed by plating serially diluted yeast-containing dilutions on Sabouraud agar plates. Each well that contains 100 μl of yeast inoculum and a similar volume of two-fold diluted BBR (16–1,024 μg/ml) was incubated at 37°C for 48 h. In addition, a drug-free control with fungal cells was included. Fluconazole was included as quality control. MIC of BBR was defined as the lowest drug concentration that caused no visible cell growth.

XTT reduction assay was carried out to assess C. albicans viability. Briefly, in a 96-well plate, 100 μl of C. albicans (2 × 10⁶ CFU/ml) and 100 μl of BBR (32–256 μg/ml) were added into each well and incubated at 37°C for 1, 2 and 3 h. Following two washing steps using sterile PBS, the OD value was detected by XTT reduction assay at 492 nm in a microplate reader (Manoharan et al., 2017; Rossoni et al., 2019).

In order to detect the toxicity of BBR to A431 cells, we treated A431 cells (2 × 10⁵ cells/ml) with BBR at different concentrations. Cells were inoculated in 96-well plates and cultured at 37°C for 24 h before treatment with the same volume of BBR (8–128 μg/ml). Finally, 10 μl of CCK8 solution (Biyuntian Biotechnology Co., Ltd., Shanghai, China) was added into each well and incubated for 1 h, and the absorbance of each well was measured at 450 nm using a microplate reader.

Gram staining, an established staining method for bacteria, was used to observe the adherence of C. albicans. Briefly, in a 6-well plate, 2 ml of cells (2 × 10⁵ cells/ml) were cultured for 24 h at 37°C and 5% CO₂ before incubation with C. albicans diluted by the same volume of DMEM culture medium to different concentrations (2 × 10⁴, 2 × 10⁵, 2 × 10⁶, 2 × 10⁷, 2 × 10⁸ CFU/ml, respectively) for 1, 2 and 3 h. At designated time points, cells were fixed with 4% paraformaldehyde at room temperature for 15 min, and washed 3 times with PBS. After that, enhanced Gram staining solution (Beijing Regen Biotechnology Co., Ltd., China) was applied. Finally, oil immersion lens (OLYMPUS BX51, Tokyo) was used for cell imaging.

Gram staining was performed to evaluate the adherence of C. albicans with or without BBR treatment. Briefly, in a 6-well plate, 2 ml of A431 cells (2 × 10⁵ cells/ml) were cultured for 24 h at 37°C and 5% CO₂, then the culture supernatant was removed. Cells receiving different treatments were grouped as follows: Blank group (A431 cells added in DMEM culture medium), Control group (A431 cells added in DMEM culture medium plus C. albicans), Treatment group (A431 cells added in DMEM culture medium plus C. albicans and BBR), Negative control group (A431 cells added in DMEM medium containing BBR without C. albicans). Cells were cultured at 37°C with 5% CO₂ for 1, 2 and 3 h. At corresponding time points, cells were fixed with 4% paraformaldehyde, followed by washing with PBS and staining with enhanced Gram staining solution. Finally, oil immersion lens (OLYMPUS BX51, Tokyo, Japan) was used for cell imaging.

To further detect the adhesion of C. albicans to vaginal epithelial cells, samples of fungi-cells adhesion were prepared by using round cover slides (Bioshrp, China) in a 12-well plate for visualization using SEM. Cell grouping and BBR intervention were performed as described above. Samples were fixed by 2.5% glutaraldehyde overnight, and dehydrated by 30, 50, 70, 90, and 100% ethanol for 10 min. Samples were air-dried before sputter coating with gold in a vacuum evaporator. Morphological observation and image acquisition were performed using SEM at ×2000 magnification (SU8100, HITACHI, Japan).

Calcofluor white (CFW) (Sigma, United States) was used to determine the adhesion of C. albicans to vaginal epithelial cells. Samples were prepared in 6-well plates as described above. Briefly, A431 cells were cultured for 1, 2, and 3 h after BBR treatment. At corresponding time points, cells were washed with sterile PBS before staining with 5% CFW (1 mg/ml) at room temperature for 10 min. Cells were observed using DMI8 fluorescence microscope at ×200 magnification (Leica, GER).

To further determine the possible role of intercellular adhesion molecule ICAM-1 in C. albicans adhesion to vaginal epithelial cells, cellular localization of expressed ICAM-1 was analyzed. Briefly, cell sample preparation was performed as described above, following which coverslips were taken out and washed 3 times with PBS. Cells were then fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 (Beijing Soleibao Company, China) for 30 min before blocking with 1% BSA (Beijing Soleibao Company, China) for 1 h. After 3 washing steps with PBS, cells were incubated with ICAM-1 primary antibody (1:200 diluted in 1% BSA) overnight at 4°C, and then with FITC-conjugated Goat Anti-Rabbit IgG (Haul) secondary antibody (1:150 diluted in 1% BSA) in the dark at room temperature for 1 h. Finally, 50 μl of anti-fluorescence attenuation sealing agent (including DAPI) (Beijing Soleibao Company, China) was added and incubated at room temperature in the dark for 20 min for cell imaging and quantification using a DMI8 inverted fluorescence microscope (Leica, GER).

Western blotting was performed to detect the expression of ICAM-1, mucin1 and mucin4. At corresponding time points, culture medium was removed and cells were washed 6 times with PBS before harvest using a cell scraper. After treatment with protein extract solution containing protease inhibitor, total cell protein extract solution was centrifuged at 12,000 rpm, 4°C for 5 min. The protein concentration of cells in each treatment group was determined using BCA Protein Assay Kit. Proteins were separated by SDS-PAGE gel electrophoresis before being transferred onto PVDF membranes (Milipore, United States). Transferred membranes were blocked for 2 h in 5% non-fat dry milk in TBST before incubation overnight at 4°C with one of the following primary antibodies respectively: rabbit anti-β-actin (1:5,000; Abbkine, United States), rabbit anti-ICAM-1 antibody (1:1,000; Zhengneng Biotech Co., Chengdu, China), rabbit anti-Mucin-1 antibody (1:900; Boaosen Biotech Co., Beijing, China) and rabbit anti-Mucin-4 antibody (1:1,000; Zhengneng Biotech Co., Chengdu, China). After three 10-min washing steps with Tween20/TBS, the membranes were incubated with secondary antibody (1:10,000; Affinity, United States) at room temperature for 2 h. After washing three times with Tween20/TBS, the membranes were treated with ECL reagent (Advansta, United States) and visualized using FluorChem M Imaging System (ProteinSimple, United States). Data were quantified by automated densitometry using ImageJ. Densitometric data were normalized against β-actin in triplicates.

The levels of cytokines (IL-2 and IL-4) from vaginal epithelial cells were measured by enzyme-linked immunosorbent assay kit. Cell grouping and BBR treatment were performed as described above. At corresponding time points, cell culture supernatant was collected and the levels of IL-2 and IL-4 were analyzed by ELISA (ElabScience, Wuhan, China) according to manufacturer’s instructions.

Differences between experimental groups were assessed for significance using a two-tailed unpaired Student’s t-test and Two-way ANOVA analysis with GraphPad Prism 5 software. All experiments were repeated at least three times. Data are expressed as mean ± standard deviation and p < 0.05 is considered to be statistically significant.

To determine the inhibitory effect of BBR on C. albicans, the MIC of BBR against C. albicans SC5314 was examined. The MIC of BBR for this strain was 64 µg/ml according to the result of two-fold dilution assay, and the MIC of fluconazole against C. albicans is 1 µg/ml. To further investigate the effect of BBR on the viability of this strain, an XTT reduction assay was performed. After this strain was treated with different concentrations of BBR ranging from 32 µg/ml to 256 µg/ml for 1, 2, 3 h, the OD value was measured at a wavelength of 492 nm. Our results showed that this compound had no effects on the viability of C. albicans at the sub-inhibitory concentrations of 16 and 32 µg/ml (Figures 1A,B), while it could significantly reduce this fungal viability in a dose and time-dependent manner at concentrations higher than 32 µg/ml (Figure 1C). Taken together, our findings indicate that although it shows a certain inhibitory effect at high concentrations, the inhibitory effect of BBR on the growth of C. albicans can be neglected when its concentration is lower than 32 µg/ml.

To determine the toxicity of this compound to vaginal epithelial cells, the viability of these cells after being treated with BBR at different doses and times was measured. The untreated cell group with a cell survival rate of 100% was used as a control for relative comparison. As shown in Figure 2A, there was no change in cell viability after treatment with 8–128 μg/ml BBR for 1 h. However, when they were exposed to 128 μg/ml BBR for 2 h (Figure 2B) or 3 h (Figure 2C) or 64 μg/ml for 3 h (Figure 2C), the viability of these cells was significantly reduced. Furthermore, when the cells were exposed to BBR for 24 h, each dose group could show cytotoxicity (Figure 2D). The above results indicate that the cell survival rate is related to the time and concentration of the compound BBR to treat the cells. For this reason, 32 μg/ml was determined as the appropriate concentration of BBR in the following adhesion experiment.

To explore the appropriate condition of the adhesion of C. albicans to vaginal epithelial cells, the concentration of C. albicans and the exposure time of A431 cells to C. albicans were considered as two crucial factors. The results were measured by the Gram staining. As shown in Figure 3A, the adhered number of yeasts to A431 cells increased with the increase of C. albicans concentrations from 2×10⁴ to 2 × 10⁷ CFU/ml, while the adhesion capability significantly reduced when the concentration of this fungi increased to 2 × 10⁸ CFU/ml. On the other hand, there were no significant differences for the adhesion capability of C. albicans to vaginal epithelial cells at different exposure times (1, 2 and 3 h) when the concentration of C. albicans was 2 × 10⁴ to 2 × 10⁵ CFU/ml(Figure 3B). However, the adhesion capability significantly increased with the increase of exposure time when the concentration of C. albicans increased to 2 × 10⁷ CFU/ml, in which the adhesion number of C. albicans to cells peaked with a mean of 75 C. albicans per cell (Figure 3B). Therefore, 2 × 10⁷ CFU/ml was considered to be the adhesion concentration of C. albicans.

To determine the inhibitory effect of BBR on the adhesion of C. albicans to vaginal epithelial cells, 32 μg/ml BBR and 2 × 10⁷ CFU/ml C. albicans were added into 2 × 10⁵ A431cells in a 6-well plate. After treatment with different exposure time, the plate was stained by the Gram staining assay. As shown in Figure 4A, compared with the control, the adhesion number of C. albicans treated with BBR significantly reduced. It is worth noting that the inhibitory effect of BBR on adhesion of C. albicans to A431 cells was time-dependent. Compared with 1 h, the inhibitory effect on adhesion of C. albicans to cells was much stronger after treatment with BBR for 2–3 h (Figure 4B). To further observe the inhibitory effect of BBR, the adhesion of C. albicans after BBR treatment was visualized by SEM. A similar result with the Gram staining assay was obtained (Figure 4C).

Furthermore, CFW staining was used to evaluate the inhibitory effect of BBR on adhesion of C. albicans to A431 cells. The adhesion number of C. albicans was counted and the mean fluorescence intensity (MFI) was determined under an inverted fluorescence microscope. As shown in Figure 4D, compared with the control, the adhesion number (right-up panel) and MFI (right-low panel) of C. albicans to A431 cells in the group treated with BBR significantly decreased. Also, a stronger inhibitory effect was observed after treatment with BBR for 2 or 3 h, which is similar to the result of the Gram staining assay. Taken together, our data revealed that BBR may play an important role in regulation of adhesion of C. albicans to vaginal epithelial cells.

The adhesion of C. albicans to vaginal epithelial cells involves a complex interaction between the fungi and the cells, which depends not only on the ligands on the surface of C. albicans, but also on receptors or adhesion molecules on the surface of host cells. ICAM-1, a cell adhesion molecule, is known to mediate the adhesion of C. albicans to vaginal epithelial cells (Youn et al., 2008). To explore the detailed mechanisms that BBR regulates the adhesion of C. albicans to vaginal epithelial cells, the expression of ICAM-1 in the A431 cells exposed to C. albicans was detected after treatment with BBR. As shown in Figure 5A, compared with A431 cells without any treatment, C. albicans could strongly induce the expression of ICAM-1 in A431 cells in a time-dependent manner of C. albicans exposure, while its expression significantly reduced after treatment with BBR (p < 0.01), which indicates BBR may regulate the adhesion of C. albicans to vaginal epithelial cells by blocking ICAM-1. To further verify this regulation, the A431 cells exposed to C. albicans were performed an immunofluorescence (IF) analysis after treatment with or without BBR. A similar result was obtained. The IF analysis visualized that the expression of ICAM-1 in the A431 cells exposed to C. albicans was significantly higher than that in the A431 cells without C. albicans infection with the increase of C. albicans adhesion time, while its expression was significantly decreased after treatment with BBR (Figures 5D,E; p < 0.0001).

Mucin1 and Mucin4 are another two important molecules that mediate the adhesion of pathogens to vaginal epithelial cells (Moncla et al., 2016). To further verify our finding that BBR can block adhesion of C. albicans to vaginal epithelial cells, the expressions of mucin1 and mucin4 were detected by Western blot. Our results showed that the expression levels of mucin1 and mucin4 in the A431 cells exposed to C. albicans significantly increased in a time-dependent manner of C. albicans exposure (Figures 5B,C; p < 0.01). After treatment with BBR, however, the expression of mucin1 and mucin4 in these cells exposed to C. albicans significantly decreased, indicating that BBR may block adhesion of C. albicans by regulating the expression of mucin1 and mucin4.

Taken together, our findings indicate BBR may block the adhesion of C. albicans to vaginal epithelial cells by modulating ICAM-1, mucin1, and mucin4.

When VVC occurs, C. albicans often induces vaginal epithelial cells to produce a large number of cytokines, including pro-inflammatory factors and anti-inflammatory factors, in which IL-2 and IL-4 are considered as the representative of pro-inflammatory and anti-inflammatory members, respectively. During VVC pathogenesis, the local immune homeostasis of the vagina is often broken. To explore whether the effect of the compound BBR on the local immune homeostasis of the vagina, the levels of IL-2 and IL-4 in A431 cells exposed to C. albicans were measured by ELISA assay after treatment with BBR. Our results showed that the levels of IL-2 and IL-4 in A431 cells exposed to C. albicans were significantly higher compared with those from A431 cells without C. albicans exposure (p < 0.05), indicating the local homeostasis of vagina can be broken by C. albicans exposure. In the presence of BBR, however, the level of IL-2 significantly decreased and the level of IL-4 was significantly increased (Figure 6; p < 0.01). It is worth noting that exposure time of A431 cells to C. albicans could affect the expression of IL-2 and IL-4. With extension of exposure time to C. albicans, the level of IL-2 secreted by A431 cells significantly increased, while the level of IL-4 significantly decreased (Table 1).

The ratio of IL-2 and IL-4 is usually considered as a balance of locally vaginal homeostasis. For this reason, the ratio of IL-2/IL-4 in A431 cells exposed to C. albicans was calculated after treatment with or without BBR. Our results showed that the proportion of IL-2/IL-4 in A431 cells exposed to C. albicans significantly increased with the extension of exposure time (Table 1; p < 0.001), while it is worth noting that the increase in the ratio of IL-2/IL-4 was much smaller after treatment with BBR. At the same time point, the proportion of IL-2/IL-4 in A431 cells exposed to C. albicans after treatment with BBR was significantly lower than that in A431 cells only exposed to C. albicans without BBR treatment (Table 1; p < 0.001), indicating BBR could regulate the locally vaginal homeostasis by balancing the level of IL-2 and IL-4.