补肾活血方通过PI3K/AKT/mTOR信号通路对衰老小鼠听功能的保护作用  

Protective effect of Bushen Huoxue Formula on auditory function of aging mice via PI3K/AKT/mTOR signal pathway

作　　者：张梦娴 周颖东 王青玲 康浩然 江利敏 郭向东 王庆林 ZHANG Meng-Xian;ZHOU Ying-Dong;WANG Qing-Ling(Henan University of Chinese Medicine,Zhengzhou 450046,Henan,China)

机构地区：[1]河南中医药大学,河南郑州450046 [2]河南中医药大学第一附属医院 [3]河南医学高等专科学校

出　　处：《中国老年学杂志》2024年第24期6008-6014,共7页Chinese Journal of Gerontology

基　　金：国家自然科学基金(81403439);河南省科技攻关计划(222102310604,232102310430);河南省中医药科学研究专项重点课题(20-21ZY1045);河南省高等学校重点科研项目(23A360028)。

摘　　要：目的探讨补肾活血方(BSHXF)通过磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路调节自噬减轻衰老小鼠听力损伤的作用机制。方法将32只7月龄C57BL/6J雄鼠随机分为衰老组、BSHXF组、BSHXF+PI3K抑制剂组、PI3K抑制剂组,每组8只。另购8只2月龄听力正常的同品系小鼠作为对照组。衰老组、BSHXF组、BSHXF+PI3K抑制剂组、PI3K抑制剂组连续给药12 w后取材。对照组适应性喂养1 w后取材。测定给药前后各组听性脑干反应(ABR)阈值,检测血清丙二醛(MDA)、超氧化物歧化酶(SOD)水平,苏木素-伊红(HE)染色观察耳蜗组织结构,免疫荧光染色耳蜗基底膜铺片观察毛细胞形态和数量的变化,免疫组织化学法检测耳蜗组织中自噬相关蛋白Beclin1、微管相关蛋白轻链(LC)3的表达,Western印迹检测耳蜗组织中P-PI3K/PI3K、P-AKT/AKT、P-mTOR/mTOR的表达。结果与对照组相比,衰老组ABR阈值、MDA含量升高,SOD活性下降,耳蜗组织的形态结构紊乱,耳蜗基底膜铺片毛细胞数量缺失,排列不规则,耳蜗组织中Beclin1、LC3表达均下调,P-PI3K/PI3K、P-AKT/AKT、P-mTOR/mTOR表达上调,差异均有统计学意义(P<0.01,P<0.001)。与衰老组相比,BSHXF组、BSHXF+PI3K抑制剂组、PI3K抑制剂组ABR阈值、血清MDA含量下降,SOD活性升高,耳蜗基底膜毛细胞缺失数量减少,排列有序,耳蜗组织中Beclin1、LC3的表达上调,P-PI3K/PI3K、P-AKT/AKT、P-mTOR/mTOR的表达降低,差异具有统计学意义(P<0.05,P<0.01,P<0.001)。结论BSHXF可上调自噬对衰老小鼠的听力损伤起保护作用,其机制可能是通过抑制PI3K/AKT/mTOR信号通路实现的。Objective To investigate the mechanism of Bushen Huoxue Formula(BSHXF)reducing hearing damage in aging mice by autophagy regulated by phosphatidylinositol-3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signaling pathway.Methods Thirty-two seven-month-old male C57BL/6J rats were randomly divided into aging group,BSHXF group,BSHXF+PI3K inhibitor group and PI3K inhibitor group,with 8 rats in each group.Another eight two-month-old hearing-normal mice of the same strain were purchased as the control group.The samples were collected from aging group,BSHXF group,BSHXF+PI3K inhibitor group and PI3K inhibitor group after continuous administration for 12 w.The control group were selected after adaptive feeding for 1 w.The auditory brainstem response(ABR)threshold of mice in each group before and after dosing was measured,and the serum levels of malondialdehyde(MDA)and superoxide dismutase(SOD)were detected.The structure of the cochlea was observed by hematoxylin-eosin(HE)staining.The morphology and number of hair cells were observed by immunofluorescence with cochlear basement membrane spread.The expressions of autophagy-related proteins Beclin1 and microtubule-associated protein light chain(LC)3 in the cochlea tissue were detected by immunohistochemistry.P-PI3K/PI3K,P-AKT/AKT and P-mTOR/mTOR expressions in the cochlea tissue were detected by Western blotting.Results Compared with the control group,ABR threshold and MDA content were increased,SOD activity was decreased,the morphology and structure of cochlear tissue were disordered,the number and arrangement of basal membrane hair cells were missing,and the expressions of Beclin1 and LC3 in cochlear tissues were down-regulated,the expressions of P-PI3K/PI3K,P-AKT/AKT,P-mTOR/mTOR were up-regulated in aging group,and the differences were statistically significant(P<0.01,P<0.001).Compared with aging group,ABR threshold and serum MDA content were decreased,SOD activity was increased,and the number of missing hair cells in the basement membrane of cochl

关 键 词：补肾活血方 自噬 磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR) 衰老 

分 类 号：R764.35[医药卫生—耳鼻咽喉科]