Transcriptomics analysis reveals the effect of Pulsatilla decoction butanol extract on endoplasmic reticulum and peroxisome function of Candida albicans in hyphal state.

ETHNOPHARMACOLOGICAL RELEVANCE

The traditional Chinese medicine formula known as Pulsatilla decoction was utilized to treat conditions such as bacterial dysentery, ulcerative colitis, and fungal infections like vulvovaginal candidiasis (VVC) caused by Candida albicans (C. albicans). In our prior research, it was shown that the n-butanol extract from Pulsatilla Decoction (BEPD) exhibited effective inhibition of C. albicans. Nevertheless, the exact mechanism by which BEPD hinders hyphal growth, a critical virulence factor of C. albicans, remains unclear.

AIM OF THE STUDY

In the present study, the inhibitory effect and mechanism of the BEPD on C. albicans hyphal growth was predicted by transcriptome analysis, and further verified by in vitro and in vivo experiments.

MATERIALS AND METHODS

The BEPD was prepared and C. albicans was cultured to induce the hyphal state. Transcriptome analysis was conducted to predict the significant difference in enrichment genes and signaling pathways in the inhibitory effect of BEPD on C. albicans hyphae. Various methods, such as spot assay, time-growth curve analysis, Confocal laser scanning microscope (CLSM), scanning electron microscope (SEM), transmission electron microscope (TEM), flow cytometry, and spectrophotometer, were used to assess the effect of BEPD on hyphal structure and growth activity, lipid peroxidation level, peroxidase (CAT) activity, superoxide dismutase (SOD) activity, and apoptosis of C. albicans. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was employed to examine the expression levels of genes associated with endoplasmic reticulum and peroxisome function. The VVC model was employed to evaluate the influence of BEPD on the growth of C. albicans hyphae in vivo.

RESULT

The growth of C. albicans hyphae on solid culture media was significantly inhibited by BEPD. CLSM showed that the length of C. albicans hyphae was decreased and their vitality was lowered. SEM indicated that the hyphae of C. albicans were fractured, while TEM revealed damage to the organelles within the cells. GO enrichment and KEGG pathways analysis from transcriptomic data demonstrated that BEPD effectively suppressed the functioning of the endoplasmic reticulum and peroxisomes in C. albicans hyphae. RT-qPCR verified the decreased expression of genes associated with endoplasmic reticulum and peroxisome function by BEPD. Investigation of the endoplasmic reticulum revealed that BEPD elevated levels of reactive oxygen species (ROS) and apoptosis, indicating endoplasmic reticulum stress, as well as malondialdehyde (MDA), a marker of oxidative stress. Additionally, BEPD was shown to lower the activities of catalase (CAT) and superoxide dismutase (SOD). In animal trials, BEPD effectively hindered the growth of C. albicans hyphae in the vaginas of mice with VVC, thus reducing immune inflammatory damage to the vaginal mucosa of these mice.

CONCLUSION

This study demonstrates that BEPD has an inhibitory effect on hyphae, which are an important virulence factor of C. albicans. This effect may be related to BEPD's inhibitory effect on endoplasmic reticulum (ER) and peroxisome function. The findings suggest that BEPD could potentially play a therapeutic role in C. albicans infectious diseases by inhibiting hyphae.