The Toxic Effect Of Lithium Chloride On Auditory Hair Cells In Vitro

Objective:To study the toxic effect and possible mechanism of lithium chloride on auditory hair cells.Method:Firstly,CCK-8 kit was used to detect the inhibitory effect of 0,25,50,75,100,125,150m M lithium chloride concentrations and 12,24,48,72 hours treatment time on HEI-OC1 cells,and the concentration value of IC₅₀ was calculated.3 days old newborn rats of Sprague Dawley were dissected and cultured for 24 hours.Then they were divided into control group（without any intervention）and experimental group(treated with IC₅₀concentration for 24 hours).After treatment,the hair cells were stained and counted,and the differences between the two groups were compared.Then the HEI-OC1 cells in logarithmic phase were divided into control group（without any intervention）and experimental group(treated with IC₅₀ concentration for 24 hours).TUNEL assay and Flow cytometry were used to analyze apoptosis rates.Expression of Caspase-3,Caspase-9,JNK,BAX,Bcl-2 m RNA were detected by RT-PCR.Expression of Cleaved-Caspase-3,Cleaved-Caspase-9,JNK,p-JNK,BAX,Bcl-2 protein were detected by Western blot.Results:The results of the CCK-8 experiment showed that with the increase of lithium chloride concentration and treatment time,the inhibition rate of lithium chloride on HEI-OC1 cells gradually increased compared with the control group,the difference was statistically significant（P<0.01）.The IC₅₀ value of lithium chloride in 24 hours was69.53m M,so the concentration of lithium chloride in the experimental group was 70m M.Compared with the control group,the basal membrane staining results of TRITC rhodamine labeled phalloidin showed that the arrangement of hair cells in the experimental group was disordered,hair cells were missing,and cilia morphology was changed.The number of hair cells in the experimental group was 7.67±1.03,which was lower than that in the control group（10.67±1.03）,the difference was statistically significant（t=4.097,P<0.01）;the number of outer hair cells in the experimental group was 30.50±1.05,which was lower than that in the control group（33.00±1.79）,the difference was statistically significant（t=3.414,P<0.01）.The results of TUNEL showed that the apoptosis rate of the experiment group（36.00±2.65%）was significantly increased than the control group（2.33±0.58%）,the difference was statistically significant（t=21.53,P<0.01）.Flow cytometry indicated that the rate of apoptosis in the experiment group（42.40±0.82%）was significantly increased than the control group（0.42±0.02%）,the difference was statistically significant（P<0.01）.The results of RT-PCR showed that,compared with the control group,the m RNA expression of Caspase-3,Caspase-9,JNK,and BAX increased in the experimental group,but Bcl-2 decreased,the difference was statistically significant（P<0.01）.The results of Western blot showed that,compared with the control group,the protein expression of Cleaved-Caspase-3,Cleaved-Caspase-9,p-JNK,and BAX increased in the experimental group,while Bcl-2,Bcl-2/BAX decreased,the difference was statistically significant（P<0.01）;compared with the experimental group,the protein expression of Cleaved-caspase-3,Cleaved-caspase-9,p-JNK,and BAX decreased in the JNK inhibited group,while Bcl-2 and Bcl-2/BAX increased,the difference was statistically significant（P<0.01）.Conclusion:Lithium chloride induce the apoptosis of auditory hair cells HEI-OC1 cells via the JNK signaling pathway in vitro.