The Study Of The Mechanism Underlying The Effect Of Calcium Homeostasis And Its Responding Channels On Cisplatin-Induced Ototoxicity

ObjectiveSensorineural hearing loss(SNHL),as a common sensory defect in humans,is a global health problem.Cisplatin,as a commonly used chemotherapeutic drug,can cause bilateral and progressive hearing loss,which limits its clinical application.At present,SNHL-induced by cisplatin is mostly attributed to the death of auditory cells caused by endoplasmic reticulum(ER)stress,inflammation,apoptosis,and autophagy.As a second messenger widely present in eukaryotic cells,calcium participates in many physiological processes,and the imbalance of calcium homeostasis can lead to many diseases.The ER and mitochondria,as important calcium reservoirs,play a role in the maintenance of intracellular calcium homeostasis.Inositol 1,4,5-triphosphate receptor(IP3R)/Glucose-regulated protein 75(Grp75)/Voltage-dependent anion channel 1(Voltage-The dependent anion channel 1,VDAC1)complex,as a vital structure between the ER and mitochondria,participates in calcium regulation between them.Studies have shown that the imbalance of calcium homeostasis caused hair cells apoptosis.However,its roles in hair cells damage caused by cisplatin are still unclear.This study aims to discuss the new mechanism of cisplatin-induced hair cells damage,focusing on the changes in calcium homeostasis and responding channels in the subcellular structure.Methods(1)Flow cytometry and TUNEL staining were used to verify the damage of 30 μM cisplatin to HEI-OC1 cells and neonatal mouse hair cells.(2)Immunofluorescence was used to observe the activated caspase-3 in HEI-OC1 cells and neonatal mouse hair cells after 30 μM cisplatin was treated for 24 h.(3)The change of p-PERK,ATF6,Caspase-12,and other endoplasmic reticulum stress indicators was exhibited by WB,after 30 μM cisplatin treatment for 0,6,12,24 h;the variation of Caspase-12 in hair cells of suckling mice after 30 μM cisplatin treatment for 24 h was shown by IF.(4)WB was used to observe the mitochondrial apoptosis indicators such as Bcl-2,Bax,cleaved-caspase 9 at 30 μM cisplatin for 0,6,12,24 h;IF was used to observe the activated caspase-9 in hair cells of suckling mice,after 30 μM cisplatin stimulus for 24 h.(5)The calcium ions of the ER and mitochondria were labeled by Mag-fluo-4 AM and Rhod2 AM respectively,to observe their variation in HEI-OC1 cells and hair cells of suckling mice after 30 μM cisplatin addition for 0,6,12,24 h.(6)WB and IF were used to observe the changes of ER calcium channel IP3R and mitochondrial calcium channel VDAC1 in HEI-OC1 cells and suckling mice’s hair cells when 30 μM cisplatin was treated for 0,6,12,24 h.(7)The interaction between IP3R and beclin-1 in HEI-OC1 cells was exhibited by IF at 30 μM cisplatin exposure for 0,6,12,24 h;The expression changes of autophagy indicators Beclin-1 and LC3B in HEI-OC1 cells after 30 μM cisplatin treatment for 0,6,12,24 h were shown by WB;IF was chosen to observe of the expression changes in neonatal mice’s hair cells after 30 μM cisplatin addition for 24 h.Results(1)After 30 μM cisplatin treatment for 24 h,the proportion of HEI-OC1 cell apoptosis increased significantly,and the hair cells of suckling mice showed an obvious loss,disorder,and apoptosis.(2)After addition with 30 μM cisplatin for 24 h,caspase-3 in HEI-OC1 cells and hair cells of suckling mice was significantly activated.(3)After 30 μM cisplatin was added,the ER stress indicators p-PERK and ATF6 in HEI-OC1 cells first increased and then decreased,and caspase-12 increased in a time-dependent way;the expression of caspase-12 in hair cells of suckling mice enhanced significantly,compared with the control group.(4)Under 30 μM cisplatin exposure,the mitochondrial apoptosis indicators Bcl-2 in HEI-OC1 cells decreased,Bax increased,and caspase-9 was significantly activated;similarly,caspase-9 in hair cells of suckling mice was activated obviously,compared with the control group.(5)After 30 μM cisplatin addition,the calcium level in the ER decreased firstly followed by increment in HEI-OC1 cells and suckling mice hair cells,while the calcium level in mitochondria accumulated with time.(6)Correspondingly,under 30 μM cisplatin elicitation,the level of IP3R protein in HEI-OC1 cells and suckling mice hair cells increased first and then decreased,while the level of VDAC1 protein increased in a time-dependent way.(7)Under 30 μM cisplatin exposure,in HEI-OC1 cells,IP3R and beclin-1 gradually dissociated,the expression of beclin-1 and LC3B increased,LC3B in suckling mice hair cells gradually increased,and more punctate aggregation appeared.Conclusions(1)Cisplatin may influence the fate of HEI-OC1 cells and neonatal mouse hair cells,via affecting the expression of IP3R and VDAC1,resulting in changes of calcium levels in the ER and mitochondria,which activates ER stress and mitochondrial pathways.(2)Cisplatin may participate in the damage of HEI-OC1 cells and neonatal mouse hair cells by promoting the dissociation of IP3R and beclin-1 to induce autophagy.