D-galactose Impairs Cochlear Hair Cells through P53-Mediated Autophagy and Apoptosis

YAN Xirui; WANG Yirong; CHU Min; LIU Qing; SHI Jianrong; DONG Yang

doi:10.3969/j.issn.1672-2922.2025.01.020

Abstract：Objective To demonstrate in vitro damage of cochlear hair cells on the basilar membrane by D-galactose(D-gal)and its mechanisms based on autophagy and apoptosis.Methods Cochlear basilar membranes from postnatal 3-4 days Wistar rats were cultured and exposed to D-gal for 24 h,and grouped based on D-gal exposure dosage,i.e.,100 mmol/L,300 mmol/L and 500 mmol/L,as well as a control group,with 48 cochleae in each group.P53 inhibition test included a control group,a D-galactose(300 mmol/L)exposure group,a PFT-α(50 μmol/L)group and a D-galactose(300 mmol/L)+PFT-α(50 μmol/L)group,with 18 cochleae in each group.Specimens were labeled with Alexa Fluo 555-phalloidin and whole cochlear hair cells were counted and analyzed quantitatively.Western blot was employed to assess protein expression levels of p53,autophagy-and apoptosis-related proteins.Changes in protein expression of p53,LC3-Ⅱ,p62,and Cleaved Caspase-3 in hair cells were detected by immunofluorescence co-localization.A P53 inhibitor,Pifithrin-α(PFTα),was used to inhibit the protein expression of p53,while measuring change in expression of downstream proteins including LC3-Ⅱ,p62,and Cleaved Caspase-3.Results In vitro exposure to D-gal led to dose-dependent hair cell damage.Western blot showed increased protein expression of p53,LC3-Ⅱ,p62,Cleaved Caspase-3 and the ratio of Bax/Bcl-2 with exposure to D-gal,but decreased Beclin-1 expression in a dose-dependent manner.Immunofluorescence showed increased the protein expression of p53,LC3-Ⅱ,p62 and Cleaved Caspase-3 in cochlear hair cells with D-gal exposure.Compared to untreated D-gal exposure,Pifithrin-α inhibited the expression of p53,p62 and Cleaved Caspase-3,while increased the expression of LC3-Ⅱ furtherly,indicating that D-gal-induced autophagy and apoptosis were p53-dependent.Conclusion In vitro D-gal exposure can induce damage of cochlear hair cells through p53-mediated autophagy and apoptosis.