Research On Cisplatin Induced Apoptosis Of Cochlear Spiral Ganglion Neurons By Cav1.2

Objective:Cisplatin is a broad spectrum drug commonly used in the clinical treatment of cancer,but its ototoxicity is currently no effective measures to prevent or treat.Spiral ganglion neurons are an important hub for the transmission of sound information to the central nervous system,and the basic structure for maintaining normal auditory function.Once damaged,it is difficult to self-repair.In this study,C57/6J mice were used to establish a cisplatin ototoxicity model to observe the relationship between cisplatin induced cochlear spiral ganglion cell apoptosis and Cav1.2 in C57BL/6J mice,and to explore whether Cav1.2 is an important target of cisplatin induced cochlear spiral ganglion cell apoptosis.Methods:Animal experiment:8W male C57BL/6J mice were divided into Control group（Control）.One-cycle cisplatin administration group（Cycle 1）,two-cycle cisplatin administration group（Cycle 2）,three-cycle cisplatin administration group（Cycle 3）.Auditory brainstem response（ABR）was used to detect the changes of Auditory threshold in each group.Superoxide dismutase（SOD）and Malonaldehyde（MDA）kits were used to detect SOD activity and MDA content in cochlea tissues of each group.Morphological changes of spiral ganglion of cochlea in each group were observed by hematoxylin-eosin HE staining.Cochlear spiral ganglion apoptosis was detected by TUNEL staining.The distribution and expression of Cav1.2 and Caspase-3 were observed by immunofluorescence.Spiral ganglion cells were cultured and divided into five groups:Control group（Control）,solvent group（DMSO）,Cav1.2 blocker group（N）,Cisplatin group and Cisplatin+N group.Hoechst33342 staining was used to observe the apoptosis of each group.Flow cytometry was used to detect the apoptosis rate of each group.Western Blot was used to detect the expression of Cav1.2 protein and related apoptotic proteins.Mito SOX detected the ROS release of mitochondria.Results:1.Animal experiments:（1）ABR test results showed that the hearing threshold in Cycle 2 group and Cycle 3 group was significantly increased（P<0.01,n=6）;（2）The results of SOD activity and MDA content in cochlear tissue showed that SOD activity was decreased in Cycle 2 and Cycle 3groups（P<0.05,n=3）,and MDA content was increased in Cycle 3 groups（P<0.05,n=3）.（3）HE staining results showed that vacuoles and cytoplasmic swelling were observed in Cycle 2 group,and cell density was decreased compared with Control group（P<0.05,n=4）.Injury was aggravated in Cycle 3 group,and the density was significantly decreased（P<0.01,n=4）.（4）TUNEL staining showed that the apoptosis rate of SGNs in Cycle 2 group and Cycle 3 group was increased（P<0.05,P<0.01,n=4）.（5）Immunohistochemical results showed that the expression of Caspase-3 in SGNs was significantly increased in Cycle 2 and Cycle 3 groups（P<0.01,n=4）,and the expression of Cav1.2 was significantly increased in Cycle 3 group（P<0.01,n=4）.2.Cell experiments:（1）culture 5μmol/L for 12h by CCK8 for subsequent intervention;（2）Western blot results showed that cisplatin up-regulated the expression of Cav1.2 protein in SGNs（P<0.05,n=3）;（3）Hoechst33342 staining and flow cytometry showed that cisplatin significantly increased the apoptosis rate of SGNs（P<0.01,n=3）,while Cav1.2 blocker decreased the apoptosis rate of SGNs（P<0.05,n=3）.（4）Western blotting results showed that:Cisplatin up-regulated the levels of pro-apoptotic protein Cleaved caspase-3 and Bax protein in SGNs（P<0.01,P<0.001,n=3）,down-regulated the levels of anti-apoptotic protein Bcl-2 protein（P<0.01,n=3）,When Cav1.2 blocker was added,Cleaved caspase-3 and Bax protein levels were decreased（P<0.05,n=3）,and bcl-2 protein levels were increased（P<0.05,n=3）;（5）Calcium probe results showed that cisplatin up-regulated intracellular Ca²⁺concentration of SGNs（P<0.01,n=3）,and Cav1.2 blocker decreased intracellular Ca²⁺concentration（P<0.05,n=3）.（6）Mito SOX results showed that cisplatin up-regulated mitochondrial ROS release of SGNs（P<0.001,n=3）,and Cav1.2 blocker down-regulated mitochondrial ROS release of SGNs（P<0.05,n=3）.（7）JC-1 results showed that cisplatin significantly down-regulated SGNs mitochondrial membrane potential（P<0.01,n=3）,and the SGNs mitochondrial membrane potential increased after Cav1.2 blocker was added（P<0.05,n=3）.Conclusion:Cisplatin may promote calcium influx through up-regulation of Cav1.2,thereby increasing mitochondrial ROS,causing oxidative stress damage of SGNs and inducing apoptosis of mitochondrial pathway.