Biologichni Studii / Studia Biologica

Background. One of the major complications of COVID-19 is pulmonary fibrogenesis with an incompletely understood etiology, which is initiated by the activation of epithelial-mesenchymal transition (EMT) in the alveolar epithelium. Given that the lea­ding principle of medicine remains “primum non nocere” (“first, do no harm”), the search for substances with a broad therapeutic potential combined with an exceptional safety profile is highly relevant. Such compounds include secondary plant metabolites, among which multifunctional citrus flavonoids have recently attracted considerable interest.
The aim of this study was to investigate whether flavonoids can modulate TGF-β₁-induced metabolic, lysosomal, and epithelial-mesenchymal remodeling in epithelial cells by assessing relevant molecular markers, as well as their antimicrobial activity in an experimental EMT model.
Materials and Methods. The inhibitory effects of hesperidin and hesperetin on EMT were investigated in vitro using the human type II alveolar epithelial cell line A549. EMT was induced by TGF-β₁ at concentrations of 2.0 and 10 ng/mL. Morphological changes in the cells were assessed by light microscopy. The expression of mesenchymal markers in cell lysates and supernatants was determined by Western blot analysis. Total protein content was measured using the Bradford assay. The antioxidant activity of the flavonoids was evaluated using the DPPH assay, whereas β-glucosidase activity (β-GA) in cell lysates was determined spectrophotometrically based on the hydrolysis of pNPG. The antimicrobial and antifungal activity of the flavonoids was assessed by determining their minimum inhibitory concentrations (MICs) against Candida albicans ATCC 90028, Escherichia coli ATCC 25922, and Streptococcus agalactiae ATCC 13813 using the broth microdilution method. Microbial growth was evaluated by changes in the turbidity of the medium after incubation in the appropriate culture media.
Results. Hesperidin caused a more pronounced reduction in the level of phosphory­lated SMAD3 (p-SMAD3), an early molecular marker of EMT (P** < 0.05). Both flavonoids, particularly hesperetin, significantly (P* < 0.05) reduced the expression of mesenchymal proteins (collagen І and α-SMA), thereby inhibiting the myofibroblastic differentiation of epithelial cells. In addition, the aglycone, owing to its higher antioxidant activity, more markedly (P** < 0.05) affected the expression of PAI-1 – a marker associated with oxidative stress and EMT. The flavonoids modulated the enhanced TGFβ₁-induced lysosomal metabolism of transformed cells, significantly (P* < 0.05) reducing the lysosomal remodeling marker – β-GA. Hesperidin and hesperetin exhibited low to moderate antimicrobial and antifungal activity, which may be considered one of the factors potentially contributing to the maintenance of microbial homeostasis and the functional integrity of the epithelial barrier.
Conclusions. The obtained results indicate the ability of the citrus flavonoids hesperidin and hesperetin to suppress the expression of early signaling molecules and late markers in an experimental model of EMT. Hesperidin and its aglycone hesperetin modulate the TGF-β/SMAD3 signaling pathway and inhibit the metabolic and lysosomal remodeling associated with EMT,  demonstrating their antimicrobial potential.