黄芪甲苷介导Akt/mTOR/HIF-1α通路调控氧糖剥夺PC12细胞自噬的机制研究  

Mechanism of astragaloside Ⅳ in regulating autophagy of PC12 cells under oxygen-glucose deprivation by medicating Akt/mTOR/HIF-1α pathway

作　　者：龙佳欣 田梦芝 陈笑一 熊雨 余黄合[1,2] 龚勇珍[1,2] 丁煌[1,2] 谢明霞[1,2] 杜可[1,2] LONG Jia-xin;TIAN Meng-zhi;CHEN Xiao-yi;XIONG Yu;YU Huang-he;GONG Yong-zhen;DING Huang;XIE Ming-xia;DU Ke(Hunan University of Chinese Medicine,Changsha 410208,China;Laboratory of Pharmacology,Hunan University of Chinese Medicine,Changsha 410208,China;Hunan Provincial Key Laboratory for the Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Traditional Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]湖南中医药大学药理学实验室,湖南长沙410208 [3]中医药防治眼耳鼻咽喉疾病湖南省重点实验室,湖南长沙410208

出　　处：《中国中药杂志》2023年第19期5271-5277,共7页China Journal of Chinese Materia Medica

基　　金：湖南省自然科学基金项目(2020JJ9050);国家“双一流”学科“药学”开放基金项目(2018YX03);国家重点实验室开放基金项目(GTZK201711);研究生创新创业训练计划项目(湘教通[2022]253号);大学生创新创业训练计划项目(湘教通[2022]106号);2020年湖南省级一流本科课程(非线下)项目(湘教通[2021]28号);2022年度湖南省普通高等学校教学改革研究项目(HNJG-2022-0134重点项)。

摘　　要：探讨黄芪甲苷(astragalosideⅣ,AS-Ⅳ)对氧糖剥夺(oxygen-glucose deprivation,OGD)诱导的PC12细胞自噬损伤的保护作用及可能机制。体外建立OGD PC12细胞自噬损伤模型,PC12细胞分为正常组,OGD组,AS-Ⅳ低、中、高剂量组,阳性药右美托咪定(DEX)组。MTT法检测PC12细胞存活率,透射电子显微镜观察自噬小体及自噬溶酶体,MDC荧光染色法观测自噬小体的荧光强度,Western blot检测LC3-Ⅱ/LC3-Ⅰ、Beclin1、p-Akt/Akt、p-mTOR/mTOR、HIF-1α自噬相关蛋白表达情况。与正常组比较,OGD组细胞存活率显著降低(P<0.01),自噬小体显著增多(P<0.01),MDC荧光强度显著增强(P<0.01),Beclin1、LC3-Ⅱ/LC3-Ⅰ、HIF-1α显著上调(P<0.05或P<0.01),p-Akt/Akt、p-mTOR/mTOR显著下调(P<0.05或P<0.01)。与OGD组比较,AS-Ⅳ低、中剂量组及DEX组细胞存活率显著提高(P<0.01),自噬小体显著减少(P<0.01),MDC荧光强度显著减弱(P<0.01),Beclin1、LC3-Ⅱ/LC3-Ⅰ、HIF-1α显著下调(P<0.05或P<0.01),p-Akt/Akt、p-mTOR/mTOR显著上调(P<0.01)。AS-Ⅳ低、中剂量可通过激活Akt/mTOR,继而影响HIF-1α,抑制OGD诱导的PC12细胞自噬损伤,发挥抗神经元自噬损伤的保护作用;AS-Ⅳ高剂量组与OGD组相比差异无统计学意义。该研究可为AS-Ⅳ防治OGD诱导的细胞自噬损伤提供一定的靶标参考,为黄芪及AS-Ⅳ的二次开发积累一定实验室数据。This study explored the protective effect of astragaloside Ⅳ(AS-Ⅳ) on oxygen-glucose deprivation(OGD)-induced autophagic injury in PC12 cells and its underlying mechanism.An OGD-induced autophagic injury model in vitro was established in PC12 cells.The cells were divided into a normal group,an OGD group,low-,medium-,and high-dose AS-Ⅳ groups,and a positive drug dexmedetomidine(DEX) group.Cell viability was measured using the MTT assay.Transmission electron microscopy was used to observe autophagosomes and autolysosomes,and the MDC staining method was used to assess the fluorescence intensity of autophagosomes.Western blot was conducted to determine the relative expression levels of functional proteins LC3-Ⅱ/LC3-Ⅰ,Beclin1,p-Akt/Akt,p-mTOR/mTOR,and HIF-1α.Compared with the normal group,the OGD group exhibited a significant decrease in cell viability(P<0.01),an increase in autophagosomes(P<0.01),enhanced fluorescence intensity of autophagosomes(P<0.01),up-regulated Beclin1,LC3-Ⅱ/LC3-Ⅰ,and HIF-1α(P<0.05 or P<0.01),and down-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.05 or P<0.01).Compared with the OGD group,the low-and medium-dose AS-Ⅳ groups and the DEX group showed a significant increase in cell viability(P<0.01),decreased autophagosomes(P<0.01),weakened fluorescence intensity of autophagosomes(P<0.01),down-regulated Beclin1,LC3-Ⅱ/LC3-Ⅰ,and HIF-1α(P<0.05 or P<0.01),and up-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.01).AS-Ⅳ at low and medium doses exerted a protective effect against OGD-induced autophagic injury in PC12 cells by activating the Akt/mTOR pathway,subsequently influencing HIF-1α.The high-dose AS-Ⅳ group did not show a statistically significant difference compared with the OGD group.This study provides a certain target reference for the prevention and treatment of OGD-induced cellular autophagic injury by AS-Ⅳ and accumulates laboratory data for the secondary development of Astragali Radix and AS-Ⅳ.

关 键 词：黄芪甲苷(AS-Ⅳ) Akt/mTOR/HIF-1α 氧糖剥夺(OGD) 自噬损伤 

分 类 号：R285[医药卫生—中药学]