Candida albicans Infection Disrupts the Metabolism of Vaginal Epithelial Cells and Inhibits Cellular Glycolysis

Vulvovaginal candidiasis (VVC) is a common gynecologic disorder caused by fungal infections of the vaginal mucosa, with the most common pathogen being Candida albicans (C. albicans). Exploring metabolite changes in the disease process facilitates further discovery of targets for disease treatment. However, studies on the metabolic changes caused by C. albicans are still lacking. In this study, we used C. albicans-infected vaginal epithelial cells to construct an in vitro model of VVC, analyzed the metabolites by UHPLC-Q-Exactive MS, and screened the potential metabolites based on metabolomics. The results showed that C. albicans infection resulted in significant up-regulation of D-arabitol, palmitic acid, adenosine, etc.; significant down-regulation of lactic acid, nicotinamide (NAM), nicotinate (NA), etc.; and disruption of amino acid metabolism, and that these significantly altered metabolites might be potential therapeutic targets of VVC. Further experiments showed that C. albicans infection led to a decrease in glycolytic enzymes in damaged cells, inhibiting glycolysis and leading to significant alterations in glycolytic metabolites. The present study explored the potential metabolites of VVC induced by C. albicans infection based on metabolomics and verified the inhibitory effect of C. albicans on vaginal epithelial cell glycolysis, which is valuable for the diagnosis and treatment of VVC.


Introduction
VVC is the second most common inflammatory disease of the vagina after bacterial vaginitis.It often presents clinically with symptoms such as vulvar itching, vaginal pain, and increased discharge.According to foreign epidemiological studies, about 75% of women of reproductive age have the disease once in their lifetime, and about 50% have it twice or more [1], which seriously affects women's health and quality of life.The main pathogens of VVC are Candida spp., including Candida albicans, Candida smoothus, and Candida tropicalis [2].C. albicans is the most common pathogen in women with VVC and can be isolated in 75% to 90% of cases [3].
Because of its high prevalence and recurrence rate, timely diagnosis of VVC is particularly important for the control of disease progression.We can determine whether a patient is infected with C. albicans by observing the clinical symptoms or performing fungal cultures [4].However, these methods often do not allow us to make a quick and accurate judgment.In recent years, metabolomics has been widely used to study key metabolites of diseases, and it is an effective tool for diagnosis and treatment of diseases [5].However, studies on disease metabolites for VVC induced by C. albicans infection are still lacking.In this study, we infected the human vaginal epithelial cells, which are commonly used for in vitro vaginitis studies, with C. albicans, the most common pathogen causing VVC, to Microorganisms 2024, 12, 292 2 of 13 construct an in vitro model of VVC, analyzed the metabolites using UHPLC-Q-Exactive MS, and explored the differential metabolites based on metabolomics.
C. albicans is a commensal and opportunistic pathogen that can cause various diseases when the body's immunity is out of balance, most commonly superficial infections of the vaginal mucosa, esophagus, and skin [6].The pathogenic mechanisms reported for C. albicans include mainly the changes in C. albicans itself during infection, as well as the effects on epithelial cells.C. albicans adhering to the vaginal mucosa changes its morphology from a free yeast state to an invasive hyphae state [7], facilitating its further colonization and adhesion to cells and penetrating the barrier into the deeper tissues.The invasion of epithelial cells by C. albicans can affect the cellular immune response by activating signaling pathways such as TLR and MAPK [8].In addition, C. albicans secretes virulence factors that enhance invasion and infection of cells, including cell wall adhesins (ALS, EPA, HWP1), secreted aspartyl proteinases (Saps), candidalysin, and phospholipases [9].Saps and candidalysin are considered the most important virulence factors.Saps promote the adhesion of C. albicans and facilitate hyphae penetration into tissues by hydrolyzing host histones.The interaction of high concentrations of candidalysin with cell membranes can cause pore-like structures in the cell membrane, leading to cell membrane damage [10,11].
In addition, previous studies have shown that during C. albicans infection of cells, the cytoarchitecture of vaginal epithelial cells is damaged by C. albicans hyphae and virulence factors [12], and it can be inferred that this would lead to a massive release of the cellular damage marker lactate dehydrogenase (LDH) [13].A large loss of LDH, an important enzyme in glycolysis, may result in the inhibition of glycolysis in vaginal epithelial cells.Glycolysis is an essential metabolic pathway for the production of ATP and the activation of immune cells [14], and its reaction intermediates are involved in various biosynthetic pathways, which are essential for maintaining normal cellular life activities [15,16].However, there is insufficient evidence that C. albicans affects the glycolysis of vaginal epithelial cells, and there is a lack of studies exploring the mechanism of action of C. albicans on vaginal epithelial cells from the metabolomic perspective.Therefore, we examined the metabolic effects of C. albicans on vaginal epithelial cells by detecting changes in cellular metabolites, key metabolites, and enzymes involved in glycolysis and observed whether cellular glycolysis levels were suppressed in infection, which could be valuable in further exploring the pathogenesis of C. albicans.

Groups and Treatment
Three groups were set up for this experiment: control group, model group, and amphotericin B (AmB) group.C. albicans cells were cocultured with vaginal epithelial cells for 24 h of infection to simulate an in vitro model of VVC [6].The cells were seeded in cell culture flasks and incubated at 37 • C.After 24 h, the medium was discarded, and DMEM was added to the control group; DMEM containing C. albicans was added to the model and AmB groups.After 24 h, the medium was discarded, and DMEM was added to the control and model groups; DMEM containing 0.5 µg/mL AmB was added to the AmB group.After 24 h, the supernatant was collected and stored at −80 • C for glucose, pyruvic acid, lactic acid, LDH, and inflammatory factors assay, and the cultures were washed with PBS and stored at −80 • C for metabolism analysis.

Metabolomics Analysis
The cultures were added with 1mL 80% methanol aqueous solution, ground in the freeze grinder, and centrifuged at 4 • C, 14,000 rpm for 15 min.The supernatant was evaporated with nitrogen and re-dissolved by adding 50% methanol aqueous solution to precipitate the protein fully, and then centrifuged at 4 • C, 14,000 rpm for 15 min, and collected the supernatant for UHPLC-Q-Exactive Plus Orbitrap MS (Thermo Fisher Scientific, Waltham, MA, USA) analysis.

Scanning Electron Microscopy
To further judge the damage to vaginal epithelial cells, we used scanning electron microscopy to observe the morphology of the cells.The cell crawls attached in the 12-well plates were washed with PBS, then added with electron microscope fixative (2.5% glutaraldehyde), fixed at room temperature for 2 h, and stored at 4 • C. The samples were dehydrated with different gradients of ethanol (30, 50, 70, 80, 90, 95, and 100%), dried, and sputtered with gold plating.The scanning electron microscope (JSM-IT700HR, JEOL, Tokyo, Japan) was used to observe and acquire images.

Cellular Supernatant Index Assay
In order to detect the content of glycolysis-related metabolites and LDH in the culture supernatant, we processed the supernatant after centrifugation with the glucose assay kit (glucose oxidase method), pyruvic acid, lactic acid, and LDH assay kits (NJJCBIO, Nanjing, China).The inflammatory factors were detected by human interleukin-6 (IL-6) and human interleukin-8 (IL-8) ELISA kit (4 A Biotech, Beijing, China) according to the instructions.Finally, the absorbance was detected with a microplate reader.

Immunofluorescence Staining
Immunofluorescence assays were used to detect the expression of enzymes involved in glycolysis in vaginal epithelial cells.The samples were washed with PBS and fixed in 4% paraformaldehyde solution for 20 min.The cells were placed in 0.3% Triton X-100 solution for 30 min after washing with PBS and blocked with 5% goat serum at room temperature for 1 h.The cells were incubated in rabbit anti-human LDHA antibody or mouse antihuman PKM2 antibody (1:200; Proteintech Group, Inc., Wuhan, China) overnight at 4 • C, washed with PBS, and incubated in FITC AffiniPure goat anti-rabbit IgG (H + L) secondary antibody (1:50; PUMOKE, Wuhan, China) or Alexa Fluor 594 AffiniPure goat anti-mouse IgG (H + L) secondary antibody (1:50; Yeasen Biotech Co., Ltd., Shanghai, China) for 1 h while protected from light.After washing with PBS, 30 µL of antifade mounting medium with DAPI (Beyotime Biotechnology Co., Ltd., Shanghai, China) was added.The confocal laser scanning microscope (LSM 710, ZEISS, Oberkochen, Germany) was used to observe and acquire images, and fluorescence intensity was measured using Image J 1.5.3.

Statistical Analysis
Thermo Compound Discover 2.0 and Xcalibur were used for initial identification and relative quantification of metabolites; SIMCA 14.1 software was used to perform principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and permutation tests; and bioinformatics (https://www.bioinformatics.com.cn/) was used to perform heatmap analysis of metabolites.We used one-way ANOVA and Student's t-test to analyze the experimental data and plotted them with GraphPad Prism 9.5.1.We considered a p-value of less than 0.05 a significant difference.

C. albicans Infection Disrupts the Metabolism of Vaginal Epithelial Cells
In order to study the changes in metabolites after C. albicans infection of vaginal epithelial cells, we performed metabolomics studies on the cultures.Three groups were set up to explore the disease metabolites of VVC by comparing the model group with the control group, while the target metabolites for the treatment of VVC were studied by comparing the AmB group with the model group.As shown in the PCA plot (Figure 1A) and OPLS-DA (Figure 1B), the metabolic profile data of the control, model, and AmB groups exhibited the characteristics of separation between groups and proximity within groups, which indicated that the difference between groups was obvious, and the experimental methods were stable.The OPLS-DA models were tested for 200 permutations, respectively.The results (Figure 1C) showed that the values of R2 and Q2 on the left side were consistently lower than those on the right side and that Q2 regression lines intersected the negative semiaxis of y, indicating that the models were stable and reliable and did not suffer from overfitting.

Statistical Analysis
Thermo Compound Discover 2.0 and Xcalibur were used for initial identification and relative quantification of metabolites; SIMCA 14.1 software was used to perform principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and permutation tests; and bioinformatics (https://www.bioinformatics.com.cn/；accessed on 17 January 2024) was used to perform heatmap analysis of metabolites.We used one-way ANOVA and Student's t-test to analyze the experimental data and plotted them with GraphPad Prism 9.5.1.We considered a p-value of less than 0.05 a significant difference.

C. albicans Infection Disrupts the Metabolism of Vaginal Epithelial Cells
In order to study the changes in metabolites after C. albicans infection of vaginal epithelial cells, we performed metabolomics studies on the cultures.Three groups were set up to explore the disease metabolites of VVC by comparing the model group with the control group, while the target metabolites for the treatment of VVC were studied by comparing the AmB group with the model group.As shown in the PCA plot (Figure 1A) and OPLS-DA (Figure 1B), the metabolic profile data of the control, model, and AmB groups exhibited the characteristics of separation between groups and proximity within groups, which indicated that the difference between groups was obvious, and the experimental methods were stable.The OPLS-DA models were tested for 200 permutations, respectively.The results (Figure 1C) showed that the values of R2 and Q2 on the left side were consistently lower than those on the right side and that Q2 regression lines intersected the negative semiaxis of y, indicating that the models were stable and reliable and did not suffer from overfitting.Differential metabolites were screened by setting threshold conditions (fold change value greater than 2 or less than 0.5, p-value less than 0.05, variable importance in the projection value greater than 1).As shown in Figure 2, 50 endogenous metabolites were significantly changed in the model group compared to the control group, of which 19 metabolites were up-regulated (D-arabitol, lysoPC(18:2(9z,12z)), betaine, palmitic acid, adenosine, etc.), and 31 metabolites were down-regulated (lactic acid, NAM, NA, phenylalanine, etc.).These metabolites may be potential disease biomarkers for VVC caused by C. albicans infection.

Changes in Metabolites after Administration of Antifungal Therapy
Amphotericin B is a polyene antifungal drug commonly used in clinical practice to treat vaginitis, endocarditis, and other fungal infections caused by C. albicans infection [17,18].In this study, amphotericin B was used as an anticandidal-infection strategy to observe whether there is a tendency for the changes in metabolites to return to the control

Changes in Metabolites after Administration of Antifungal Therapy
Amphotericin B is a polyene antifungal drug commonly used in clinical practice to treat vaginitis, endocarditis, and other fungal infections caused by C. albicans infection [17,18].In this study, amphotericin B was used as an anticandidal-infection strategy to observe whether there is a tendency for the changes in metabolites to return to the control group after controlling the course of the fungal infection and whether the glycolysis of vaginal epithelial cells can return to normal.After administration of AmB for antifungal treatment, 28 metabolites (Table 1) tended to revert to the control group, and we considered these 28 reverse-regulated metabolites as potential biomarkers representing effective control of the VVC pathologic process.Of these 28 metabolites, 15 metabolites (lysoPC(18:2(9z,12z)), adenylsuccinic acid, indole, palmitic acid, 2-furoic acid, guanosine monophosphate, betaine, cysteinylglycine, pipecolic acid, guanine, pyruvic acid, ophthalmic acid, D-arabitol, diacetyl, cis-aconitic acid) were positively correlated with C. albicans infection, and 13 metabolites (thiamine, NAM, NA, hypoxanthine, N-acetyl-L-aspartic acid, inosine, uracil, allantoic acid, Beta-citryl-L-glutamic acid, lactic acid, 2-phosphoglyceric acid, phosphoenolpyruvic acid, octadecanamide) were negatively correlated with infection status.Previous studies have shown that decreased lactic acid was a common feature of reproductive tract infections [19].In our experiment, the lactic acid content of the model group was significantly reduced, along with significant changes in the levels of pyruvic acid, phosphoenolpyruvic acid, 2-phosphoglyceric, NAM, NA, etc. (Figure 3).The significant changes in the levels of these substances, which were essential metabolites involved in glycolysis, suggested that C. albicans infection may cause a disturbance in the glycolysis response of vaginal epithelial cells.

C. albicans Infection Disrupted Glycolysis-Related Metabolites and Enzymes
To verify the inhibitory effect of C. albicans on cellular glycolysis, we observed the damage to cells' membranes by scanning electron microscopy (Figure 4) and detected the contents of glucose, pyruvic acid, lactic acid, and LDH in the supernatant with a microplate reader (Figure 5).From the scanning electron microscopy results, it can be seen that the cells in the control group had a full and naturally spreading morphology, while the cells in the model group were penetrated and invaded by hyphae, and their surfaces were severely damaged, with ruptured cell membranes, wrinkled morphology, and disappeared pseudopods.In addition, the experimental results showed that the glucose content in the supernatant was significantly reduced compared with the control group.After administration of antifungal therapy, the growth of C. albicans was inhibited, and the glucose content was increased considerably.In addition, the pyruvic acid content was elevated, and the lactic acid content was decreased in the model group, which was consistent with the results of metabolomics.LDH, an important metabolic enzyme for glycolysis, as a marker of cell damage, was significantly elevated in the supernatant of the model group, indicating that the cell damage was severe.A large amount of leakage of LDH in the cytoplasm occurred, and the glycolysis of the cells was inhibited.

C. albicans Infection Disrupted Glycolysis-Related Metabolites and Enzymes
To verify the inhibitory effect of C. albicans on cellular glycolysis, we observed the damage to cells' membranes by scanning electron microscopy (Figure 4) and detected the contents of glucose, pyruvic acid, lactic acid, and LDH in the supernatant with a microplate reader (Figure 5).From the scanning electron microscopy results, it can be seen that the cells in the control group had a full and naturally spreading morphology, while the cells in the model group were penetrated and invaded by hyphae, and their surfaces were severely damaged, with ruptured cell membranes, wrinkled morphology, and disappeared pseudopods.In addition, the experimental results showed that the glucose content in the supernatant was significantly reduced compared with the control group.After administration of antifungal therapy, the growth of C. albicans was inhibited, and the glucose content was increased considerably.In addition, the pyruvic acid content was elevated, and the lactic acid content was decreased in the model group, which was consistent with     To further demonstrate the inhibition of glycolysis in vaginal epithelial cells, we examined the key glycolytic metabolizing enzymes, LDHA and PKM2, using immunofluorescence.As shown in Figure 6, the green fluorescence intensity represented the expression degree of LDHA in cells, the red fluorescence intensity represented the expression degree of PKM2 in cells, and the blue fluorescence represented the nucleus.Compared with the control group, the levels of LDHA and PKM2 were significantly lower in the model group, indicating that the glycolysis of infected cells was inhibited; after the administration of antifungal treatment, the levels of these two enzymes were close to those of the control group, indicating that the antifungal treatment restored the cells to the normal level of glycolysis.To further demonstrate the inhibition of glycolysis in vaginal epithelial cells, we examined the key glycolytic metabolizing enzymes, LDHA and PKM2, using immunofluorescence.As shown in Figure 6, the green fluorescence intensity represented the expression degree of LDHA in cells, the red fluorescence intensity represented the expression degree of PKM2 in cells, and the blue fluorescence represented the nucleus.Compared with the control group, the levels of LDHA and PKM2 were significantly lower in the model group, indicating that the glycolysis of infected cells was inhibited; after the administration of antifungal treatment, the levels of these two enzymes were close to those of the control group, indicating that the antifungal treatment restored the cells to the normal level of glycolysis.

Discussion
Exploring metabolites of diseases is vital for disease prevention, diagnosis, and tre ment.Metabolomics, as an important method for detecting endogenous small molec metabolites, can be used to detect metabolites in body fluids, tissues, and cells for effici and accurate diagnosis of diseases, and it is often used in the study of various diseas including cancer, cardiovascular disease, genital inflammation, etc. Gynecological d eases are often closely linked to metabolic disorders, such as cervical cancer, cervici and vaginitis [20,21].
Camilla Ceccarani et al. performed a metabolomic study of vaginal secretions fr VVC patients using 1 H-NMR.They found more than 10 differential metabolites, includ lactic acid, phenylalanine, taurine, etc., in samples from VVC patients compared with healthy group [19].In our study, we performed metabolomic analysis of an in vitro mo of VVC using UHPLC-Q-exact MS, and we found that a total of 50 metabolites were s nificantly altered in the model group, and these metabolites may be disease biomark of VVC.Specifically, we found that lactic acid and phenylalanine levels were also sign cantly down-regulated in the model group.This further suggested that lactic acid a phenylalanine could be used as disease markers for VVC.In addition, previous stud have shown that D-arabitol is the biomarker for C. albicans infection, and GC-MS was u to detect the D-arabitol and L-arabitol ratio in urine to determine the presence of C. a cans infection [22].In our experiments, the D-arabitol level was significantly elevated

Discussion
Exploring metabolites of diseases is vital for disease prevention, diagnosis, and treatment.Metabolomics, as an important method for detecting endogenous small molecule metabolites, can be used to detect metabolites in body fluids, tissues, and cells for efficient and accurate diagnosis of diseases, and it is often used in the study of various diseases, including cancer, cardiovascular disease, genital inflammation, etc. Gynecological diseases are often closely linked to metabolic disorders, such as cervical cancer, cervicitis, and vaginitis [20,21].
Camilla Ceccarani et al. performed a metabolomic study of vaginal secretions from VVC patients using 1 H-NMR.They found more than 10 differential metabolites, including lactic acid, phenylalanine, taurine, etc., in samples from VVC patients compared with the healthy group [19].In our study, we performed metabolomic analysis of an in vitro model of VVC using UHPLC-Q-exact MS, and we found that a total of 50 metabolites were significantly altered in the model group, and these metabolites may be disease biomarkers of VVC.Specifically, we found that lactic acid and phenylalanine levels were also significantly down-regulated in the model group.This further suggested that lactic acid and phenylalanine could be used as disease markers for VVC.In addition, previous studies have shown that D-arabitol is the biomarker for C. albicans infection, and GC-MS was used to detect the D-arabitol and L-arabitol ratio in urine to determine the presence of C. albicans infection [22].In our experiments, the D-arabitol level was significantly elevated in the model group and significantly decreased after drug administration, suggesting that D-arabitol can be used as one of the biomarkers for diagnosing VVC.
In addition, these significantly altered potential metabolites we have identified are closely linked to disease.Lysophosphatidylcholine is considered a deleterious pro-inflammatory mediator, with levels elevated considerably when inflammation occurs in the body [23], and has been implicated as a biomarker for cervical cancer [24].Palmitic acid, a saturated fatty acid, activates toll-like receptors to promote inflammation [25].Under hypoxic and inflammatory conditions, adenosine levels are significantly elevated to inhibit inflammation [26], but persistently high adenosine levels favor tumor cell growth, promoting cancer [27].Thiamine is a kind of B vitamin that has an important effect on cellular energy metabolism, and a deficiency can cause a decrease in ATP production and lead to cell death [28].Betaine (trimethylglycine) is an osmoprotective agent that is anti-inflammatory and regulates energy metabolism [29], but a positive correlation has been reported between betaine concentrations and reproductive inflammation [21].Maintaining normal levels of amino acids is important for health, and disturbances in amino acid metabolism often accompany genital inflammation.In the present study, significant changes in amino acids were observed in the model group, including phenylalanine, L-homoserine, cysteinylglycine, L-glutamine, L-arginine, N-acetyl-L-aspartic acid, Beta-citryl-L-glutamic acid, and L-aspartic acid.This may be due to the secretion of the virulence factor SAPs by C. albicans, which allows the hydrolysis of various proteins in the vaginal epithelium, leading to disturbed amino acid metabolism in the VVC [19].
In particular, we found that metabolites involved in the glycolysis, nicotinate, and nicotinamide metabolism pathways were significantly altered in the VVC model, including 2-phosphoglyceric acid, phosphoenolpyruvic acid, pyruvic acid, lactic acid, NAM, and NA.
Lactic acid is essential for maintaining the stability of the intravaginal environment.Lactic acid produced by vaginal epithelial cells and lactic acid produced by Lactobacillus maintains the pH of the vaginal environment against the growth of harmful microorganisms [30].Some studies have shown that lactic acid inhibits the growth of C. albicans [31].The innate immune mechanism of vaginal epithelial cells plays a crucial role in antibacterial infections [32].Notably, lactic acid can further modulate local immunity within the vagina by stimulating the production of anti-inflammatory factors by human vaginal epithelial cells and inhibiting the production of inflammatory factors (IL-6 and IL-8) triggered by toll-like receptors [33].This was corroborated by the ELISA results (Figure 7), which showed that the levels of the inflammatory factors IL-6 and IL-8 in the cell supernatants were significantly lower in the control and AmB groups, which had normal lactic acid levels, than in the model group, which had lower lactic acid levels.
reported between betaine concentrations and reproductive inflammation [21].Maintaining normal levels of amino acids is important for health, and disturbances in amino acid metabolism often accompany genital inflammation.In the present study, significant changes in amino acids were observed in the model group, including phenylalanine, Lhomoserine, cysteinylglycine, L-glutamine, L-arginine, N-acetyl-L-aspartic acid, Beta-citryl-L-glutamic acid, and L-aspartic acid.This may be due to the secretion of the virulence factor SAPs by C. albicans, which allows the hydrolysis of various proteins in the vaginal epithelium, leading to disturbed amino acid metabolism in the VVC [19].
In particular, we found that metabolites involved in the glycolysis, nicotinate, and nicotinamide metabolism pathways were significantly altered in the VVC model, including 2-phosphoglyceric acid, phosphoenolpyruvic acid, pyruvic acid, lactic acid, NAM, and NA.
Lactic acid is essential for maintaining the stability of the intravaginal environment.Lactic acid produced by vaginal epithelial cells and lactic acid produced by Lactobacillus maintains the pH of the vaginal environment against the growth of harmful microorganisms [30].Some studies have shown that lactic acid inhibits the growth of C. albicans [31].The innate immune mechanism of vaginal epithelial cells plays a crucial role in antibacterial infections [32].Notably, lactic acid can further modulate local immunity within the vagina by stimulating the production of anti-inflammatory factors by human vaginal epithelial cells and inhibiting the production of inflammatory factors (IL-6 and IL-8) triggered by toll-like receptors [33].This was corroborated by the ELISA results (Figure 7), which showed that the levels of the inflammatory factors IL-6 and IL-8 in the cell supernatants were significantly lower in the control and AmB groups, which had normal lactic acid levels, than in the model group, which had lower lactic acid levels.As shown in Figure 8, in the hypoxic vaginal cavity, vaginal epithelial cells can produce ATP by anaerobic glycolysis and produce lactic acid to diffuse into the vaginal cavity.Briefly, glucose is metabolized to a series of glycolytic intermediates such as glucose- As shown in Figure 8, in the hypoxic vaginal cavity, vaginal epithelial cells can produce ATP by anaerobic glycolysis and produce lactic acid to diffuse into the vaginal cavity.Briefly, glucose is metabolized to a series of glycolytic intermediates such as glucose-6-phosphate, glyceraldehyde 3-phosphate, 2-phosphoglyceric acid, etc. 2-phosphoglyceric acid is converted to phosphoenolpyruvic acid, which PKM2 catalyzes to produce pyruvic acid, and eventually pyruvic acid is catalyzed by LDH to produce lactic acid [15,33].Compared with the control group, the glucose content in the supernatant of the model group was significantly lower, which implied that C. albicans infection depleted a large amount of glucose in the culture system, which may lead to a decrease in the amount of glucose that enters vaginal epithelial cells to participate in glycolysis.In addition, after the cells were attacked by C. albicans, the expression of LDHA and PKM2 was inhibited, which prevented normal glycolysis in the cells.Thus, the production of metabolites of glycolysis was reduced.The metabolomics results showed a significant decrease in 2-phosphoglyceric acid, phosphoenolpyruvic acid, and lactic acid and an increase in pyruvic acid in the model group, which was consistent with the results of pyruvic acid and lactic acid detection in the culture medium.The increased pyruvic acid content in the model group may be due to the accumulation of pyruvic acid as a result of the significant reduction in LDH preventing its normal conversion to lactic acid.
glycolysis was reduced.The metabolomics results showed a significant decrease in phosphoglyceric acid, phosphoenolpyruvic acid, and lactic acid and an increase in py vic acid in the model group, which was consistent with the results of pyruvic acid a lactic acid detection in the culture medium.The increased pyruvic acid content in model group may be due to the accumulation of pyruvic acid as a result of the signific reduction in LDH preventing its normal conversion to lactic acid.NADH is oxidized to nicotinamide adenine dinucleotide (NAD+) during the LDH catalyzed conversion of pyruvic acid to lactic acid [34].NAD+ is an essential coenzyme cellular metabolism and is indispensable for glycolysis.NAM and NA are both precurs of NAD+, producing NAD+ to maintain cellular homeostasis.NAM and NA are both vitamins necessary for living cells, and NA deficiency can lead to mucosal inflammat [35].NAM and glycolysis are closely linked.As an inhibitor of PARP-1, it avoids the blo ing effect of glycolysis caused by PARP-1.In addition, glycolysis produces NAD+, wh produces NAM in the presence of enzymes [36].NAM has been shown to reduce oxidat stress and has anti-inflammatory effects [37].NAM is reported to have significant anti albicans activity and can synergize with amphotericin B to play a common antifungal r [38,39].In our study, the levels of NAM and NA were significantly lower in the mo group, which may be attributed to the reduced expression of LDHA in the cells and inhibition of anaerobic glycolysis, reducing NADH conversion to NAD+.
It can be seen that metabolites such as lactic acid, NAM, and NA, which are clos related to glycolysis, are essential for maintaining the health of the human intravagi environment and that C. albicans infection causes disruptions in cellular glycolysis, wh disrupts the balance of these natural protective factors, allowing the body's biological p tective effects to be adversely affected.NADH is oxidized to nicotinamide adenine dinucleotide (NAD+) during the LDHAcatalyzed conversion of pyruvic acid to lactic acid [34].NAD+ is an essential coenzyme in cellular metabolism and is indispensable for glycolysis.NAM and NA are both precursors of NAD+, producing NAD+ to maintain cellular homeostasis.NAM and NA are both B vitamins necessary for living cells, and NA deficiency can lead to mucosal inflammation [35].NAM and glycolysis are closely linked.As an inhibitor of PARP-1, it avoids the blocking effect of glycolysis caused by PARP-1.In addition, glycolysis produces NAD+, which produces NAM in the presence of enzymes [36].NAM has been shown to reduce oxidative stress and has anti-inflammatory effects [37].NAM is reported to have significant anti-C.albicans activity and can synergize with amphotericin B to play a common antifungal role [38,39].In our study, the levels of NAM and NA were significantly lower in the model group, which may be attributed to the reduced expression of LDHA in the cells and the inhibition of anaerobic glycolysis, reducing NADH conversion to NAD+.
It can be seen that metabolites such as lactic acid, NAM, and NA, which are closely related to glycolysis, are essential for maintaining the health of the human intravaginal environment and that C. albicans infection causes disruptions in cellular glycolysis, which disrupts the balance of these natural protective factors, allowing the body's biological protective effects to be adversely affected.

Figure 2 .
Figure 2. Heatmap of 50 differential metabolites in the model group compared to the control group.

Figure 2 .
Figure 2. Heatmap of 50 differential metabolites in the model group compared to the control group.

Microorganisms 2024 ,
12,  x FOR PEER REVIEW 7 of 14 in glycolysis, suggested that C. albicans infection may cause a disturbance in the glycolysis response of vaginal epithelial cells.

Figure 3 .
Figure 3.There were significant changes in glycolysis-related metabolites in the model group compared to the control group, and the levels returned to normal after administration.*** p < 0.001.

Figure 3 .
Figure 3.There were significant changes in glycolysis-related metabolites in the model group compared to the control group, and the levels returned to normal after administration.*** p < 0.001.

Figure 4 .
Figure 4. C. albicans infection leads to cell membrane damage.Figure 4. C. albicans infection leads to cell membrane damage.

Figure 4 .
Figure 4. C. albicans infection leads to cell membrane damage.Figure 4. C. albicans infection leads to cell membrane damage.

Figure 4 .
Figure 4. C. albicans infection leads to cell membrane damage.

Figure 8 .
Figure 8. Schematic representation of the metabolic effects of C. albicans on vaginal epithelial ce The green borders represent down-regulation of metabolite levels and red borders represent regulation of metabolite levels; red arrows represent the glycolysis and blue arrows represent nicotinate and nicotinamide metabolism.

Figure 8 .
Figure 8. Schematic representation of the metabolic effects of C. albicans on vaginal epithelial cells.The green borders represent down-regulation of metabolite levels and red borders represent upregulation of metabolite levels; red arrows represent the glycolysis and blue arrows represent the nicotinate and nicotinamide metabolism.
• C) was used to detect the samples (injection volume: 4 µL).

Table 1 .
Significant reversal of regulation of 28 differential metabolites after administration of antifungal therapy.(The "↑" symbol represents an increase in metabolite levels and the "↓" symbol represents a decrease in metabolite levels.)