Unravelling the dual role of naringenin in response to cytotoxicity caused by indomethacin and diclofenac

Background: Indomethacin and diclofenac exhibited similar cytotoxic mechanisms, involving the generation of cell stress, disruption of mitochondria, and lysosome functions. Recently, we reported that naringenin, a natural bioactive flavonoid, elicited cytoprotection against indomethacin but not diclofenac in Caco-2 cells. Objectives: This study investigated whether these divergent effects were associated with lysosomal integrity and autophagy. Methods: Caco-2 cells were treated with naringenin (100 μM; non-cytotoxic concentration) for 24 h, followed by either indomethacin or diclofenac (1000 μM). After 72-h treatment, cell viability, apoptosis and lysosomal integrity were measured by methylthialazole tetrazolium, neutral red uptake, annexin V/propidium iodide staining, and acridine orange (AO) uptake assays. Protein expression was assessed by Western blot analysis. Results: Naringenin protected Caco-2 cells from indomethacin toxicity, evidenced by increased cell viability, reduced apoptosis, and heightened red fluorescence of AO compared to indomethacin-alone. In addition, it suppressed mammalian target of rapamycin (mTOR) expression, accompanied by a further increased light chain 3 (LC3) II/I ratio. Hence, its cytoprotection might link to inhibition of lysosomal membrane permeabilization (LMP) and induction of autophagy with autophagosome formation in indomethacin-treated cells. Conversely, in combination with diclofenac, naringenin elevated cytotoxicity and apoptosis, accompanied by a further decrease in AO signal compared to diclofenac alone. In addition to mTOR inhibition, it reduced LC3 II/I ratio and damage-regulated autophagy modulator level. Thus, despite autophagy activation, naringenin enhanced diclofenac toxicity by promoting the loss of LMP and a decrease in autophagosomes. Conclusion: The distinctive responses of naringenin to indomethacin and diclofenac could be attributed to its capacity to preserve lysosome integrity while modulating autophagosome dynamics.