Study On The Mechanism Of PINK1 In Regulating Cisplatin And Gentamicin-induced Ototoxity

PART 1:Study of the Mechanism underlying the actions of PINK1 in Cisplatin-induced OtotoxityObjective:Cisplatin,a clinically effective and widely used chemotherapeutic drug,produces side effects mainly including tinnitus and deafness,which are irreversible,thus greatly limiting its clinical application.The ototoxic mechanism of cisplatin is complex.Studies have shown that it mainly involves the accumulation of reactive oxygen species(ROS),mitochondrial dysfunction,autophagy,apoptosis and so on,the exact mechanism by which cisplatin exerts ototoxic effects is still unknown.Phosphatase and tensin homologue(PTEN)-induced putative kinase 1(PINK1),a serine/threonine protein kinase,acts against oxidative stress and widely takes part in the regulation of mitochondrial homeostasis,reduction of endoplasmic reticulum stress,manipulation of intracellular-transport and so forth.However,the function of PINK1 in the field of otology has not been studied yet.This study was aimed to explore the possible expression of PINK1 in C57BL/6 mouse cochlea and HEI-OC1 cell line,as well as try to elucidate its possible mechanisms underlying the process of cisplatin-induced ototoxicity,so as to discover new mechanisms of cisplatin-induced ototoxicity,thereby providing new ideas for its prevention and treatment.Methods:(1)The location and expression of PINK1 in HEI-OC1 cells,P4 and 2 months old(2 M)C57BL/6 mouse cochlea were shown by immunofluorescence and WB,which was positively controlled by brain tissues.(2)CCK8 method was used to measure the viability of HEI-OC1 cells after treatment of different concentrations of cisplatin,so as to select the effective drug concentration and half death time.(3)The levels of ROS induced by cisplatin and cisplatin combined with ROS inhibitor NAC or PEG-catalase in cells were detected by use of DCFH-DA and Mito-SOX Red through flow cytometry and fluorescence under microscope.(4)WB assay was used to detect the changes of expression of PINK1 in HEI-OC1 cells after cisplatin stimulation or co-treatment of cisplatin and ROS inhibitors.The distribution and number of parkin particles regulated by PINK1 were detected by immunofluorescence.Rhol23 and TMRM staining were used to measure the mitochondrial membrane potential through flow cytometry and fluorescence under microscope.(5)Co-localization of Tom 20,Mitotracker-deep Red and Lamp-1 was used to identify mitophagy.WB,immunofluorescence,TUNEL and flow cytometry were used to measure the JNK-related apoptosis.(6)The expression of PINK1 in HEI-OC1 was silenced by using PINK1-siRNA.The changes of autophagy and JNK-related apoptosis induced by cisplatin were detected by WB,immunofluorescence and flow cytometry.(7)The cell viability of PINK1-silenced cells stimulated by cisplatin or combined with autophagy or JNK signaling pathway inhibitors,3-MA or SP were detected by CCK8 method.(8)WB,immunofluorescence and TUNEL assay were used to research the changes of PINK1 expression,parkin distribution,ROS levels,mitochondrial membrane potential,autophagy and apoptosis in cochlear HCs after stimulus of cisplatin and cisplatin combined with ROS inhibitor NAC or PEG-catalase in vitro.(9)WB,immunofluorescence and TUNEL assay were used to research parkin distribution,ROS levels,mitochondrial membrane potential,autophagy and apoptosis in SGNs after stimulus of cisplatin and cisplatin combined with ROS inhibitor NAC or PEG-catalase in vitro.Results:(1)PINK1 is widely expressed in the cytoplasm of C57BL/6 mouse cochlear HCs,SGNs,stria vascularis and HEI-OC1 cells.It is worth noting that the expression level of PINK1 is higher in P4 mouse cochlear HCs and SGNs than that of adult mice,and we would choose the P4 mouse cochlea and HEI-OC1 cells as the main research materials.(2)Treatment of HEI-OC1 cells with 30 ?M cisplatin resulted in the time-dependent formation of reactive oxygen species(ROS),which can be effectively or partially inhibited by the co-treatment of ROS scavenger,NAC,or H2O2 inhibitor,PEG-catalase.(3)Treatment of HEI-OC1 cells with 30 p.M cisplatin can cause damage to mitochondrial membrane potential,which can be alleviated by co-treatment of NAC or PEG-catalase.(4)Treatment of HEI-OC1 cells with 30 ?M cisplatin can cause fluctuations in the expression of PINK1,aggregation of downstream parkin molecules,formation of mitophagy and p-JNK associated apoptosis in cells.Inhibition of ROS levels can reduce the number of parkin particles,mitophagy,and degree of p-JNK associated apoptosis.(5)Interfering the expression of PINK1 in HEI-OC1 cells with PINK1-specific siRNA can attenuate the activation of PINK1 signaling pathway and autophagy induced by cisplatin,but the level of p-JNK-related apoptosis is increased.(6)Inhibition of JNK signaling pathway by use of SP leaded to the increase of cell viability.However,inhibition of autophagy by 3-MA caused the decrease of cell viability.(7)Cisplatin could cause the increase of ROS,mitochondrial membrane potential damage,activation of PINK1/parkin pathway,autophagy and apoptosis in C57BL/6 murine cochlear HCs in vitro,which can be diminished by NAC or PEG-catalase.(8)Cisplatin could cause the increase of ROS,mitochondrial membrane potential damage,activation of PINK1/parkin pathway,autophagy and apoptosis in C57BL/6 murine cochlear SGNs in vitro,which can be alleviated by inhibiting the accumulation of ROS.Conclusions:We find,for the first time,that PINK1 is localized in the cochlea of C57BL/6 mice,and the regulatory function of PINK1 in cisplatin-induced ototoxicity is preliminarily investigated.Results show that PINK1 signaling pathway could protect against cisplatin-induced ototoxicity through activation of mitophagy and inhibition of JNK-related apoptosis.What's more,the levels of activation of PINK1 signaling pathway is affected by the levels of cisplatin-induced ROS.PART 2:Study of the Mechanism of PINK1 in Gentamicin-induced OtotoxityObjective:Aminoglycoside antibiotics including gentamicin are widely used in clinical practice because of their broad antibacterial spectrum and low price.But their ototoxic side effects reduce the life quality of patients and limit their clinical application.The damage of sensorineural deafness caused by aminoglycoside antibiotics mainly focuses on cochlear HCs,the mechanism of which mainly involves oxidative stresses,autophagy,apoptosis and so on.Our previous studies have shown that PINK1 signaling pathway has a certain protective effect on ototoxicity induced by cisplatin,and whether PINK1 has universal protection in common ototoxic drugs-related damage is unknown.Therefore,here we would explore the possible regulatory mechanism of PINK1 signaling pathway in GM-induced hair cell damge to provide a new idea for prevention and cure for common drugs-related ototoxiciy.Methods:(1)The levels of ROS in cells induced by 400 ?M GM or GM combined with ROS inhibitor NAC were detected by use of DCFH-DA through flow cytometry and fluorescence under microscope.(2)WB assay was used to detect the changes of expression of PINK1 in HEI-OC1 cells after GM stimulation or co-treatment of GM and NAC.The distribution and number of parkin particles regulated by PINK1 were detected by immunofluorescence.Rho123 staining was used to measure the mitochondrial membrane potential through flow cytometry and fluorescence under microscope.(3)Co-localization of Tom 20 and Lamp-1 was used to identify mitophagy.WB,immunofluorescence and CCK8 were used to measure the expression of p53 pathway and cell viability.(4)The expression of PINK1 in HEI-OC1 was silenced by using PINK1-siRNA.The changes of ROS levels,mitochondrial membrane potential,activation of PINKl/parkin pathway,autophagy and p53-related apoptosis induced by GM were detected by WB,immunofluorescence,flow cytometry and TUNEL.(5)The viability of PINK1-silenced cells stimulated by GM or combined with autophagy or p53 signaling pathway inhibitors,3-MA or PFTa,were detected by CCK8 method.(6)WB assay was used to research the changes of PINK1 expression in P4 cochlear HCs after stimulus of GM or combined with NAC in vitro.(7)Immunofluorescence was used to research the changes of parkin distribution,ROS levels and mitochondrial membrane potential in P4 cochlear HCs after stimulus of GM or combined with NAC in vitro.(8)Immunofluorescence,WB and TUNEL assay were used to detect the changes of autophagy and apoptosis in cochlear HCs after stimulus of GM or combined with NAC in vitro.Results:(1)Treatment of HEI-OC1 cells with 400 ?M GM resulted in the time-dependent formation of ROS which was effectively inhibited by GM combined with ROS scavenger NAC.(2)Treatment of HEI-OC1 cells with 400 ?M GM can cause damage to mitochondrial membrane potential,which can be alleviated by co-treatment of NAC.(3)Treatment of HEI-OC1 cells with 400 ?M GM can cause fluctuations in the expression of PINK1,aggregation of parkin molecules,formation of mitophagy and increase of p53 in cells.Inhibition of ROS levels can reduce the number of parkin particles,mitophagy,and the expression level of p53.(4)Interfering the expression of PINK1 in HEI-OC1 cells with PINK1-specific siRNA can attenuate the activation of PINK1 signaling pathway and autophagy induced by GM,but increase the p53 related apoptosis.(5)Inhibition of p53 signaling pathway by use of PFTa leaded to the increase of cell viability,however inhibition autophagy by 3-MA caused the decrease of cell viability.(6)GM could cause the increase of ROS,mitochondrial membrane potential damage,activation of PINK1/parkin pathway,autophagy and apoptosis in C57BL/6 murine cochlear HCs in vitro,which can be diminished by NAC co-treatment.Conclusions:This study demonstrates that the PINK1 signaling pathway might act against GM-induced HC damage by activating mitochondrial autophagy and inhibiting the p53 signaling pathway,and the activation of the PINK1 signaling pathway is affected by the levels of GM-induced ROS.It is concluded that PINK1 might have universal resistance to damage caused by common ototoxic drugs,such as cisplatin and GM,providing a new regulatory target for the treatment and prevention of drug-induced deafness.