Study On Ferroptosis In The Auditory Hair Cell Of Mice

In 2012,Stockwell reported a new cell death way named ferroptosis,which relies on intracellular ions and is different from known apoptosis,necrosis,and autophagy in terms of morphology and biochemistry.With the in-depth research on ferroptosis,it is generally believed that cells inhibit ferroptosis mainly through two major pathways,which are glutathione peroxidase 4(GPX4)and ferroptosis suppressor protein 1(FSP1)antioxidant system.When these two antioxidant systems are destroyed,redox imbalance occurs and causing intracellular lipid peroxidation,ultimately leading to cell ferroptosis.Ferroptosis was first proposed in some engineered cells that overexpress oncogenic RAS,so many scholars began to study how to activate ferroptosis rationally,so as to achieve the purpose of killing cancer cells.However,more and more studies have found that ferroptosis also affects the cell activities of normal tissue.Many diseases such as stroke or acute kidney injury are related to the activation of ferroptosis.Some studies have found that knockout of Gpx4 is in normal tissues(such as kidney,retina,liver,forebrain,etc.)can lead to various diseases.Therefore,although the activation of ferroptosis has great application prospects in the elimination of tumors,the prerequisite is whether it can affect other normal tissues.This study aims to explore the ferroptosis sensitivity of auditory hair cells.The production of hearing involves a series of complex biochemical changes.Sound waves are mechanical vibrations that are transmitted into the tympanic membrane through the external auditory canal,causing the vibration of auditory ossicles,which then drives the flow of endolymph fluid in the cochlea.The auditory stereocilia located on the surface of the hair cells can convert mechanical signal(lymph fluid vibration)into an electrical signal,finally the electrical signal is transmitted to the auditory cortex to produce hearing.Therefore,cochlear hair cells are the sensors of auditory production and play a very important role in auditory production.Hair cells are very fragile.Many stresses such as noise,trauma,ototoxic drugs,gene mutations,etc.can cause irreversible damage.Therefore,many researchers in the peripheral auditory system focus their research on the cochlear hair cells.In this study,we aimed to explore whether ferroptosis occurs in cochlear hair cells,and if it does,which pathway plays a major role.In recent years,the research on ferroptosis has developed rapidly,and its signaling molecular pathways have been continuously expanded and improved.In order to make the research more representative,we chose to study the function of the ferroptosis-regulated antioxidant gene Gpx4 in the mouse cochlea.At the same time,we have also conducted a series of analyses on whether Fsp1 will affect the hearing of mice.First,we used an common cell lines(HEI-OC1 cell line),which usually used to drugs screen,to conduct preliminary in vitro experiments.The cells were treated with the common inhibitor of Gpx4 RSL3 and common ferroptosis inhibitors,and the cytotoxicity was determined.This experiment found that 1μM RSL3 caused a large number of cell deaths,and after 2h of pretreatment with ferroptosis inhibitors Fer-1 and DFO,the cell death was significantly alleviated.The immunofluorescence staining of 4-Hydroxynonenal(4-HNE)and Transferrin receptor 1(Tfrl)also proved that the way of cell death was ferroptosis.Therefore,we preliminarily concluded that cochlear hair cells are regulated by the Gpx4 ferroptosis pathway from an in vitro perspective.Subsequently,we used the knockout mice to further verify,Gpx4 flox mice were mated with cochlear hair cell-specific Cre(Atoh1-Cre)mice to obtain Gpx4fl/fl/Atoh1-Cre+mice(Gpx4-cKO mice),and check its audiological phenotype in some different time points.We found that Gpx4-cKO mice had severe hearing loss at P30,and the basilar membrane immunofluorescence staining showed that all outer hair cells had been lost.Therefore,Gpx4 is necessary for the survival of mouse hair cells,and the ferroptosis Marker Tfr1 verifies that the cell death is ferroptosis.Not only that,we also found that ferroptosis induced by knockout of Gpx4 only exists in outer hair cells.In addition,we used the Fsp1 systemic knockout mouse model to explore whether mouse cochlear hair cells are sensitive to ferroptosis regulated by the Fsp1 pathway.The results showed that the hearing threshold of Fsp1 knockout mice was still normal at the age of 3 months,and cochlear basilar membrane immunofluorescence staining also proved that there was no obvious death of hair cells.Therefore,we concluded that in the mouse cochlea,the Fsp1 pathway is not essential.Finally,we explored the relationship between the ototoxicity of cisplatin and ferroptosis,and found that cisplatin-induced hair cell death is not only related to apoptosis,but ferroptosis also plays an important role in it.In summary,we have studied and found that ferroptosis pathway is existed in mouse cochlear hair cells,and Gpx4 is the main protein in hair cells that resists lipid peroxidation and inhibits ferroptosis,as well as the ototoxicity of cisplatin is related to the activation of ferroptosis of hair cells.