Effect Of Actinomycin D On Cochlear Hair Cells And Its Mechanism

Part Ⅰ Effects of Actinomycin D on Cochlear Hair Cells and Spiral Neurons in Vitro Objective:To investigate the potential toxic effects of actinomycin-D on the Cochlear hair cells and Spiral Neurons in vitro.Methods:Cochlear explant cultures from 3days SD rats were isolated and incubated with various concentrations of actinomycin-D(5,10 and 20 μM)and for different durations(24,36 and 48 h).Morphology and quantity of cochlear tissue including hair cells(HCs)and spiral ganglion neurons(SGNs)were evaluated using immunofluorescence staining.Mitosox red fluorescence probe method for reactive oxygen species(ROS)and TUNEL assays were also performed to cell apoptosis in cochlear with actinomycin D of different concentrations(0,5,10 and 20 μM)after 36hours.Results:We observed the cochlear hair cells were arranged neatly and in good shape in the control group in vitro.Under 24-hour culture,a small amount of hair cells were lost and arranged appropriately in the actinomycin D treatment group of 5μM.A large number of inner and outer hair cells were lost with the concentration of 10 and 20μM.The damage was more serious in the 20μM group than that in the 10μM group.With the extension of incubation time to 36 hours and 48 hours,the damage of hair cells became more serious,the arrangement was disordered and the number was less.The cochlear hair cells almost died with the 20μM actinomycin D for 48 hours.The damaged hair cells first appeared in the basilar membrane and gradually turned to the top with the increase of concentration.Under the same treatment conditions,there were no significant changes in cochlear spiral neurons and nerve fiber bundles.TUNEL assay showed that a large number of TUNEL-positive cells could be found of 5 and 10μM actinomycin D with basilar membrane for 36 hours.TUNEL-positive cells in 20μM actinomycin D treatment group were fewer than those in the other two groups and located close to supporting cells.MitoSOX Red staining showed that the fluorescence intensity of each group increased with the increase of concentration.Conclusions:For the first time,it was proved that actinomycin D had toxic effect on cochlear hair cells in vitro,and thedamage was aggravated with the increase of concentration and time,but did not cause spiral neuron injury.It was found that the damage mechanism of actinomycin D on hair cells may be related to the increase of apoptosis and ROS production.Part Ⅱ Protective effect of z-VAD-FMK on the damage of cochlear hair cells induced by actinomycin D.Objective:To investigate the effect of anti-apoptotic factor z-VAD-FMK on the damage of cochlear hair cells induced by actinomycin D in rats,and to further explore the mechanism of the damaged cochlear hair cells by actinomycin D.Methods:The cochlear basilar membrane was treated with apoptosis inhibitor z-VAD-FMK,actinomycin D and actinomycin D combined with z-VAD-FMK in vitro.The morphological changes of hair cells and the number of hair cells were observed by immunofluorescence staining.To verify whether z-VAD-FMK has a protective effect on hair cell injury induced by actinomycin DResults:The results of immunofluorescence assay showed that there was no damage to cochlear hair cells by z-VAD-FMK in a certain concentration range.Different concentrations of anti-apoptotic factor z-VAD-FMK could reduce the loss of cochlear hair cells induced by actinomycin D,and the protective effect was more obvious with the increase of concentration.The number of TUNEL staining positive cells in 40mg/ml group of z-VAD-FMK+actinomycin D was lower than that in the group of actinomycin D,and the number of C-caspase staining positive cells in the group of z-VAD-FMK FMK+actinomycin D was also lower than that in the group of actinomycin D.Conclusions:Apoptosis inhibitor z-VAD-FMK has a protective effect on cochlear hair cell injury induced by actinomycin D.Actinomycin D mainly induces cochlear hair cell injury through apoptosis.Part Ⅲ:The effect of NAC on the damage of cochlear hair cells induced by actinomycin DObjective:To investigate the effect of antioxidant NAC on the damage of cochlear hair cells and HEI-OC1 cells induced by actinomycin D.Methods:The effect of NAC on damaged hair cells induced by actinomycin D was studied by immunofluorescence staining,cell survival rate was assessed using CCK-8 and cell proliferation rate by DAPI staining.The mechanism of cochlear hair cell injury induced by actinomycin D was discussed.Results:Compared with the normal control group,the number and arrangement of hair cells in 10 μM actinomycin D treated group were lost and disordered when the basialrmembrane was cultured in vitro for 24 hours.However,the hair cells in the l0mM NAC+10 μM actinomycin D group were more seriously damaged than those in the actinomycin D group.The fluorescence of MitoSOX Red staining was stronger than that in the actinomycin D group.In the HEI-OC1 cells,CCK-8 assay and DAPI staining were consistent with those of basilar membrane culture.NAC and actinomycin D combined treatment of cells made more cell damage.Conclusions:Antioxidant NAC has no protective effect on injuried hair cell induced by actinomycin D,which indirectly suggests that the increase of ROS in the intracellular may not be the main mechanism of hair cell apoptosis induced by actinomycin D.