D-半乳糖通过p53调节自噬和凋亡损伤小鼠耳蜗毛细胞  

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

作　　者：阎希芮 王艺蓉 楚敏[1] 刘晴[2] 施建蓉[1] 董杨[1] YAN Xirui;WANG Yirong;CHU Min;LIU Qing;SHI Jianrong;DONG Yang(School of Integrative Medicine Sciences,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;不详)

机构地区：[1]上海中医药大学中西医结合学院,上海201203 [2]上海中医药大学中医学院,上海201203

出　　处：《中华耳科学杂志》2025年第1期101-107,共7页Chinese Journal of Otology

基　　金：国家自然科学基金(81774020);上海中医药大学预算内项目(2021LK030);上海市卫健委上海市进一步加快中医药事业发展三年行动计划项目[ZY(2021-2023)-0208]。

摘　　要：目的在耳蜗基底膜离体培养模型上,观察D-半乳糖对小鼠耳蜗基底膜毛细胞的作用,并从自噬和凋亡途径进一步探讨其分子机制。方法选取SPF级3~4 d龄Wistar大鼠132只(264个耳蜗)体外培养3~4 d龄Wistar大鼠耳蜗基底膜,用100 mmol/L、300 mmol/L、500 mmol/L D-半乳糖处理24 h,将大鼠分为对照组、100 mmol/L D-半乳糖组、300 mmol/L D-半乳糖组和500 mmol/L D-半乳糖组,每组24只(48个耳蜗)。P53抑制剂Pifithrin-α(PFT-α)实验分为对照组、300 mmol/L D-半乳糖组、50μmol/L PFT-α组、300 mmol/L D-半乳糖+50μmol/L PFT-α组,每组9只(18个耳蜗)。采用Alexa Fluo 555-鬼笔环肽标记,进行全耳蜗毛细胞计数和定量分析。Western blot分析耳蜗基底膜自噬相关蛋白LC3-Ⅱ、p62、Beclin-1,细胞凋亡相关蛋白Bax、Bcl-2、Cleaved Caspase-3以及p53蛋白的表达。免疫荧光定位检测耳蜗基底膜毛细胞上p53、LC3-Ⅱ、p62和Cleaved Caspase-3的蛋白表达变化。用p53抑制剂PFT-α抑制p53蛋白表达,检测下游蛋白LC3-Ⅱ、p62和Cleaved Caspase-3表达的变化。结果D-半乳糖可导致耳蜗基底膜毛细胞损伤和缺失,并随剂量增加而加重。Western blot结果显示,D-半乳糖可增加耳蜗基底膜p53、LC3-Ⅱ、p62和Cleaved Caspase-3的蛋白表达水平以及Bax/Bcl-2的比值,降低Beclin-1的表达,且呈一定剂量依赖性。免疫荧光结果显示,D-半乳糖可增强耳蜗基底膜毛细胞上p53、LC3-Ⅱ、p62和Cleaved Caspase-3蛋白表达。P53抑制剂PFT-α可以抑制D-半乳糖引起的p53、p62和Cleaved Caspase-3蛋白表达的增高,但可以进一步升高LC3-Ⅱ蛋白表达。结论D-半乳糖可通过p53调节自噬和凋亡诱导体外培养的耳蜗基底膜毛细胞损伤。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(50μmol/L)group and a D-galactose(300 mmol/L)+PFT-α(50μmol/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 colocalization.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 dosedependent 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-αinhibited the expression of p53,p62 and Cleaved Caspase-3,while increased the expression of LC3-Ⅱfurtherly,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.

关 键 词：老年性聋 D-半乳糖 自噬 凋亡 p53 

分 类 号：R73[医药卫生—肿瘤]