Discussion
VVC is a mucocutaneous mycosis caused by Candida that has a significant impact on women’s quality of life and leads to increased healthcare costs due to high recurrence rates and increased antifungal resistance[34]. C. albicans is the most common opportunistic pathogen in VVC and is present in the respiratory, gastrointestinal, and genitourinary tract of more than 30 percent of healthy individuals during their lifetime[2]. Usually, yeast-phase C. albicans is tolerated by the vaginal epithelium. However, in VVC, C. albicans exhibits an aggressive form of hyphae, producing various extracellular enzymes, such as secretory aspartate protease (SAP), phospholipase, and hemolysin, which invade tissues through adhesion, penetration, hyphae invasion, and endothelial colonization of vaginal epithelial cells[35].
An increasing number of metabolites have been discovered and defined with the development of metabolomics research. Metabolomics studies can investigate a variety of biological processes and phenotypes by means of metabolites[36], such as amino acid and lipid metabolites[37]. Metabolomics has been extensively studied in inflammation-related diseases such as hyperuricemia[38], inflammatory bowel disease[39], rheumatoid arthritis[40], sepsis[41], liver failure[42], and endometriosis[43]. Research evaluating cervical and vaginal lavage fluids in patients with inflammatory vaginal disease has found associations between the composition of vaginal microflora and metabolite profiles[32]. Furthermore, a lipidomics study of VVC and cellolytic vaginopathy (CV) have shown significant differences in lipid composition. Lipids play an essential role in maintaining the homeostasis of the vaginal microenvironment[44]. However, few studies have comprehensively studied the metabolomics profile of VVC.
In the present study, untargeted metabolomic analysis was performed to further explore the potential biological functions of these metabolites and their role in VVC genesis following C. albicans infection, and to determine the differential metabolic profiles between the VVC and CTL groups. The metabolic profiles of patients with VVC were significantly different from those without. In total, 211 differential metabolites were identified, including 128 upregulated metabolites and 83 downregulated metabolites in VVC patients compared with healthy individuals.
Among these differential metabolites, significant increases in sugar alcohols, amino acids, unsaturated fatty acids, and pyrimidines were observed in the VVC group. Conversely, higher levels of organic acids and fatty acyl groups were observed in the CTL group. An analysis of the diagnostic capability of well-characterized biomarkers (ROC) showed that L-(-)-arabitol, L-tyrosine, D-(+)-arabitol, and psychosine could effectively distinguish VVC from healthy people (AUC>0.97). Additionally, more than ten metabolites (AUC>0.8), including linoleic acid, arachidonic acid, L-pyrrolysine, and uracil, were found to have good discrimination capacity, which are potential biomarkers to distinguish VVC from CTL patients. Tyrosine was thought to be neurotoxic and capable of causing neurological disorders such as encephalitis[45]. Arabitol in serum[46] and urine[47] has been used as a biomarker to rapidly diagnose invasive candidiasis and can be used to inform antifungal therapy and prognosis.
Amino acid metabolism plays an important role in the activation of immune cells and the production of antibodies, and excess amino acids may be detrimental to the immune system[48]. In this study, amino acid metabolism was one of the major metabolic pathways, including phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine metabolism, and taurine metabolism. Similarly, one research reported that the same metabolic pathways were enriched in sepsis[41], indicating that amino acid metabolism was inextricably linked to the occurrence of inflammation. Moreover, phenylalanine metabolism promotes the neutrophil-evasive state[49], and excess L-phenylalanine was found to affect antibody production by inhibiting protein synthesis[50]. Similar metabolic changes were observed in our study, and excess L-phenylalanine may contribute to the development of VVC.
Furthermore, linoleic acid metabolism also played a key role, as it showed the most obvious and broadest variation in the VVC group. Linoleic acid and arachidonic acid in the VVC group both increased significantly. Linoleic acid (LA; ω-6,18:2) and arachidonic acid (AA; ω-6,20:4) are essential fatty acids for the human body and are two of the most abundant polyunsaturated fatty acids (PUFAs). AA can be obtained directly from the diet or can be synthesized from LA. One study found that a larger amount of AA was synthesized from LA than from dietary sources[51]. Therefore, the significant elevation of LA caught our attention. Although LA was associated with a reduced risk of cardiovascular disease[52], studies have shown that excess LA can promote the occurrence of inflammatory events[53, 54]. Similarly, metabolites of LA mediated inflammation through oxidative stress, and oxidized linoleic acid (LA) metabolites (OXLAMs) induced mitochondrial dysfunction, apoptosis, and activation of NLRP3 inflammasome by means of oxidative stress, promoting the development of nonalcoholic steatohepatitis (NASH)[55]. LA-derived hydroxyoctadecenoic acids (HODEs) have been found to contribute to the progression of atherosclerosis as inflammatory regulators[56]. Additionally, the LA metabolites epoxyoctadecenoic acids (EpOMEs) and dihydroxyoctadecenoic acids (DiHOMEs) activate NF-κB and AP-1 transcription factors to induce an inflammatory reaction[57]. Therefore, some lipid metabolites of VVC may be related to inflammation, which was consistent with previous findings[44]. LA, found in the VVC group, may act as a pro-inflammatory factor in the response of the vaginal mucosa to C. albicans.
IPA network analysis was performed to further explore the potential role of metabolites in VVC, revealing that the changes in differential metabolites were mainly related to MAPK signaling, NF-κB signaling, Dectin-1/Syk signaling, and IL-17 signaling pathways. The study found that C. albicans significantly activated the epidermal growth factor receptor (EGFR) in human vaginal epithelial cells, activating the inflammatory response through several pathways, including mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB)[58, 59]. Among them, Candida mainly initiated the mucosal immunity of the vaginal epithelium through the MAPK signaling pathway and activated MAPK phosphatase 1 (MKP1) and c-Fos by activating MAPK-associated proteins (p38 and ERK1/2)[60, 61]. The activation of MKP1 marked the transition of Candida from the colonizing yeast phase to the invasive toxin-producing hyphal phase.
Further molecular docking experiments showed that LA could bind to ACSL1, suggesting that ACSL1 may be the target protein of LA. As a member of the family of long-chain acyl-CoA synthetase genes, ACSL1 acted on long-chain fatty acids (FAs), shuttling FAs into mitochondria in heart and adipose tissue for β-oxidation and lipid synthesis[62, 63]. Moreover, the potential role of ACSL1 in sepsis has been reported[64]. One study confirmed that inhibition of ACSL1 activity attenuated phosphorylation of p38 MAPK, ERK1/2, and NF-κB, suggesting that ACSL1 was an upstream regulator of MAPK and NF-κB signaling pathways to mediate inflammation[65]. LA may promote the occurrence of VVC by binding to ACSL1 to regulate the MAPK and NF-κB signaling pathways. Therefore, LA has the potential to become a new method for the clinical treatment of VVC, and further research should explore its function and mechanism. This study had certain limitations. In the future, we will further validate cell and animal samples and refine in vivo and in vitro experiments to confirm our findings.