XIAP inhibits gentamicin-induced hair cell damage and ototoxicity through the caspase-3/9 pathway

Gentamicin (GM), an aminoglycoside antibiotic, is one commonly used clinical drugs with ototoxic side effects. One of the most principal mechanisms of its ototoxicity is that GM can activate caspase-mediated cell death pathways in the cochlea. Since the anti-apoptotic protein known as X-linked Inhibitor of Apoptosis Protein (XIAP) has been reported to directly bind to activated caspase protein and inhibit their activities, we hypothesized that it might protect cochlea hair cells from GM ototoxicity. To evaluate this hypothesis, postnatal day 2-3 (P2-3) transgenic (TG) mice, in which XIAP gene is over-expressed under a pure C57BL/6J genetic background was constructed. We first extracted the cochlea tissue of normal mice and treated them with different concentrations of GM, and the number of hair cells were observed to determine the concentration of GM used in subsequent experiments. Next, we used Western Blot experiment to examine the effect of GM on XIAP protein expression in normal mouse cochlea, and then Western Blot and RT-PCR experiments were used to identify the transgenic mice. Finally, immunofluorescence assays were used to detect the effect of GM on the expression of caspase protein and verify the protective effect of XIAP. We found that GM at a concentration of 0.5 mM significantly affected the function of cochlea hair cells, up-regulating the expression of cleaved-caspase-3 and cleaved-caspase-9 protein but down-regulating XIAP protein. In the cochlea tissues of TG mice, this effect of GM was suppressed, and the destruction of hair cells was significantly reduced, and the cleaved-caspase-3 and cleaved-caspase-9 proteins were significantly suppressed. These results suggested that XIAP reduces GM-induced ototoxicity and caspase-3/9 pathway is associated with this process.