Evaluating the effects of metronidazole, metformin, and onion extract on GAPDH activity and nitric oxide production in Blastocystis sp. cultures: a biochemical and therapeutic approach

Abstract

Abstract Background Blastocystis sp. is a highly prevalent anaerobic intestinal protozoan increasingly associated with gastrointestinal disorders. Its survival within the gut microenvironment depends on metabolic pathways such as glycolysis, where glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a central role, alongside interactions with nitric oxide (NO) derived from host defenses and surrounding microbiota. The emergence of metronidazole resistance necessitates exploration of alternative therapeutic strategies. Objective To evaluate the in vitro effects of metronidazole, metformin, and Allium cepa (onion) extract on Blastocystis sp. growth, and GAPDH activity and NO levels within the xenic culture medium. Methods Twenty-one Blastocystis -positive stool samples were cultured in Jones’ medium under xenic conditions. Cultures were treated with graded concentrations of metronidazole, metformin, and onion extract. Parasite counts were assessed microscopically, while GAPDH activity and NO levels were quantified using gas chromatography–mass spectrometry (GC–MS)-based analysis. Results All tested agents significantly reduced Blastocystis counts compared with untreated controls ( p < 0.05), with metronidazole demonstrating the greatest antiparasitic effect. Significant reductions in NO levels and GAPDH activity were observed across all treatment groups ( p < 0.001). Metformin (1000 µg/mL) showed the strongest inhibition of GAPDH activity, whereas metronidazole (500 µg/mL) resulted in the lowest NO levels. Correlation analysis revealed a significant negative association between NO and GAPDH in metronidazole-treated cultures, while a positive correlation was observed with metformin. Conclusion Metronidazole, metformin, and onion extract exhibit significant anti- Blastocystis activity, accompanied by modulation of key metabolic and nitrosative pathways within the microbial milieu supporting the growth of Blastocystis sp. Targeting GAPDH and NO-related mechanisms represents a promising therapeutic approach, particularly in the context of emerging drug resistance and co-colonization with other gut microbiota.