Cisplatin Induced Apoptosis Of Cochlear Pericytes And The Protective Mechanism Of Quercetin On Cisplatin Ototoxicity

Objective:Cisplatin（CDDP）is widely used in the treatment of tumors,but its ototoxic effect has been lack of effective prevention and treatment measures due to unclear mechanism.In this study,male C57 BL/6J mice were used to investigate whether CDDP induced apoptosis in the mitochondrial pathway of cochlear stria capillary pericytes（PCs）by increasing the level of oxidative stress,and whether quercetin（QU）reduced the permeability of cochlear blood labyrinth barrier（BLB）by inhibiting CDDP induced apoptosis of PCs,so as to play a protective role on hearing loss caused by cisplatin ototoxicity.Method:Animal experiments:normal saline group（NS）,cisplatin group（CDDP）,10%dimethyl sulfoxide group（10%DMSO）,cisplatin+（50 mg/kg）quercetin group（Low-dose）and cisplatin+（100 mg/kg）quercetin group（High-dose）.Cell experiment:primary culture and identification of stria vascularis PCs and endothelial cells（ECs）in the cochlea of C57 BL/6J mice;In vitro BLB model was constructed by cell co-culture.Divided into（1）Control group and CDDP group;（2）Control group,CDDP group,CDDP+QU（10,20,40μM）Group,N-acetylcysteine（NAC）+CDDP group;（3）EC group,EC+PC group,EC+CDDP group,EC+PC+CDDP group,EC+PC+CDDP+QU（10,20,40μM）Group and EC+PC+CDDP+NAC group.Auditory brainstem response（ABR）was used to detect the hearing changes of mice;The morphological changes of stria vascularis in mouse cochlea were observed by H&E staining;CCK-8 was used to detect the cell viability of each group;The activity of SOD（superoxide dismutase）and the content of malondialdehyde（MDA）were detected by WST-1 and TBA respectively;The content of reactive oxygen species（ROS）on PCs was detected by DCFH-DA fluorescent probe;Hoechst 33342 and flow cytometry were used to detect the apoptosis rate of PCs;Immunofluorescence technique was used to detect the coverage of PCs on the stria vascularis of cochlea,and the distribution and expression of apoptosis related proteins and junction related proteins in PCs;The expressions of apoptosis related proteins,mitochondrial related proteins and tight junction proteins were detected by immunohistochemistry and Western blot;Mito SOX^TM-red and JC-1 were used to detect the mitochondrial function of PCs;Evans blue（EB）staining was used to observe the permeability of blood labyrinth barrier（BLB）;The permeability of endothelial barrier was measured by Millipore Millicell ERS-2 resistance meter,Na-Flu（376.27 Da）and FITC-dextran（4 k Da）.Result:Animal experiment:（1）In CDDP group,the hearing threshold increased and the latency of wave I prolonged（P<0.01）;QU could reduce the hearing threshold and shorten the latency of wave I in CDDP model mice in a concentration dependent manner（P<0.05 or P<0.01）.（2）The results of WST-1 and TBA showed that the activity of SOD in cochlea of CDDP group decreased and the content of MDA increased（P<0.01）;QU concentration dependently increased SOD activity and decreased MDA content in the cochlea of CDDP model mice（P<0.05,P<0.01）.（3）H&E staining showed that the stria vascularis in CDDP group was disordered and shrunk,and QU could improve this phenomenon in a concentration dependent manner.（4）The results of immunohistochemistry,immunofluorescence and Western blot showed that the expression of apoptosis related proteins c-Caspase-3 and Bax increased and the expression of anti-apoptotic protein Bcl-2 decreased in CDDP group;QU down regulated the expression of c-Caspase-3 and Bax in stria vascularis of CDDP model mice and up regulated the expression of Bcl-2（P<0.01）;（5）The results of immunofluorescence showed that the coverage of PCs on stria vascularis in CDDP group decreased（P<0.01）,and QU could increase the coverage of PCs on stria vascularis in CDDP model mice（P<0.05）.（6）EB staining showed that the permeability of BLB in CDDP group increased and QU concentration dependent decreased the permeability of BLB in mouse cochlea（P<0.05）;The expressions of ZO-1 and VE-cad decreased in CDDP group（P<0.01）;QU could up regulate the expression of ZO-1 and VE-cad in stria vascularis of CDDP model mice（P<0.01）.Cell experiment:（7）The results of WST-1,TBA and DCFH-DA fluorescence probe showed that the activity of SOD decreased and the contents of MDA and ROS increased in PCs in CDDP group（P<0.01）;After NAC pretreatment,the activity of SOD increased and the contents of MDA and ROS decreased in PCs（P<0.01）;After QU intervention,SOD activity increased and MDA and ROS contents decreased in PCs,which were dose-related（P<0.05 or P<0.01）;（8）The results of immunofluorescence and Western blot showed that the expression of c-Caspase-3 and Bax increased and the expression of Bcl-2 decreased in CDDP group（P<0.01）;NAC could partially reverse the effect of CDDP on apoptosis related proteins（P<0.01）;QU could partially reverse the effect of CDDP on apoptosis related proteins and was dose-dependent（P<0.05 or P<0.01）;In CDDP group,AIF and Cyt-c in mitochondria were released to the cytoplasm（P<0.01）,and QU and NAC decreased the release of AIF and Cyt-c protein in mitochondria induced by CDDP;（9）The results of Mito SOX^TM-red and JC-1 showed that the content of ROS in PCs mitochondria increased and the membrane potential decreased in CDDP group（P<0.01）;NAC decreased the content of ROS in mitochondria of PCs and increased membrane potential;QU concentration dependently decreased the content of ROS in mitochondria of PCs and increased membrane potential（P<0.05 or P<0.01）;（10）The results of Na-Flu,FITC-dextran permeability and TEER resistance showed that the permeability of endothelial barrier in EC+PC group was lower and the resistance was higher than that in EC group（P<0.01）;CDDP significantly increased the permeability of endothelial barrier and decreased the resistance in EC+PC group（P<0.01）;QU reversed CDDP induced endothelial barrier permeability and resistance（P<0.05 or P<0.01）;（11）The expression of ZO-1 and VE-cad protein in EC+PC group was higher than that in EC group（P<0.05）;CDDP significantly decreased the expression of ZO-1 and VE-cad protein in EC+PC group;QU concentration dependent upregulation of CDDP induced ZO-1 and VE-cad protein expression（P<0.05 or P<0.01）.In addition,CDDP significantly decreased the expression of anti-apoptotic protein Bcl-2 and increased the expression of apoptosis related proteins c-caspase-3 and Bax in EC+PC group（P<0.01）;QU reversed this change in a concentration dependent manner（P<0.05 or P<0.01）.Conclusion:CDDP induce the increase of oxidative stress in the cochlea and cause the apoptosis of PCs mitochondrial pathway in cochlear stria capillaries,while QU may reduce the permeability of BLB by inhibiting the apoptosis of PCs induced by CDDP,so as to play a protective role against cisplatin ototoxic hearing loss.