温阳解郁方通过调控PI3K/Akt/mTOR信号通路对抑郁小鼠前额皮质自噬的影响  

Effect of Wenyang Jieyu Formula on Autophagy in Prefrontal Cortex of Depressed Mice via Regulating PI3K/Akt/m TOR Signaling Pathway

作　　者：佘楷杰 巩子汉 孟丹华 梁文青 杨婧雯 岳广欣[1] SHE Kaijie;GONG Zihan;MENG Danhua;LIANG Wenqing;YANG Jingwen;YUE Guangxin(Institute of Basic Theroy for Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China;School of Basic Medicine,Henan University of Chinese Medicine,Zhengzhou 450046 Henan,China)

机构地区：[1]中国中医科学院中医基础理论研究所,北京100700 [2]河南中医药大学基础医学院,河南郑州450046

出　　处：《中药新药与临床药理》2022年第11期1478-1486,共9页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金面上项目(82174251,81573846);中国中医科学院科技创新工程项目资助(CI2021A00607);中央级公益性科研院所基本科研业务费专项资金资助(YZ-202107,YZ-202153)。

摘　　要：目的探讨温阳解郁方对抑郁小鼠前额皮质自噬水平的影响及磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/m TOR)信号通路的调控作用。方法仔鼠出生后随机分为空白对照组12只、模型组60只,模型组采用母婴分离联合慢性束缚应激制备抑郁小鼠模型,离乳后随机分为模型组,温阳解郁方低、中、高剂量(8.35、16.70、33.40 g·kg^(-1))组和氟西汀(2.6 mg·kg^(-1))组,于离乳第21天至束缚完成第111天给予药混饲料干预。采用糖水偏好、悬尾实验和旷场实验评价小鼠焦虑抑郁行为;免疫组化法检测小鼠前额皮质泛素结合蛋白(p62)的表达;免疫荧光组织化学法检测自噬效应蛋白(Beclin-1)、自噬微管相关蛋白1轻链3(LC3)蛋白的表达;Western Blot和Real-time PCR法检测PI3K/Akt/m TOR信号通路及自噬相关指标的蛋白和m RNA表达水平。结果行为学实验中,与正常对照组比较,模型组小鼠糖水偏好度、中央区活动时间及中央穿越次数明显降低,悬尾不动时间明显升高(P<0.01);与模型组比较,温阳解郁方中、高剂量组和氟西汀组小鼠糖水偏好度、中央区活动时间及中央穿越次数明显增加,悬尾不动时间明显缩短(P<0.05,P<0.01)。免疫组化、免疫荧光、Western Blot和Real-time PCR实验中,与正常对照组比较,模型组小鼠PI3K/Akt/m TOR信号通路和p62的蛋白及m RNA表达明显降低(P<0.05,P<0.01)、Beclin-1和LC3的蛋白及m RNA表达水平明显升高(P<0.05,P<0.01);与模型组比较,温阳解郁方中、高剂量组和氟西汀组小鼠PI3K/Akt/m TOR信号通路和p62的蛋白及m RNA表达明显升高(P<0.05,P<0.01),Beclin-1和LC3的蛋白及m RNA表达水平明显降低(P<0.05,P<0.01)。结论温阳解郁方可能通过激活PI3K/Akt/m TOR信号通路,降低前额皮质神经元和胶质细胞自噬水平,进而发挥抗抑郁疗效。Objective To explore the effect of Wenyang Jieyu Formula on the autophagic activity in prefrontal cortex of depressed mice and the regulation of PI3K/Akt/m TOR signal pathway.Methods The new born offspring of mice were randomly divided into a normal control group(n=12)and an experimental group(n=60).The depressed model mice were prepared by maternal separation combined with chronic restraint stress.The depressed model mice were randomly divided into model group,Wenyang Jieyu group(8.35,16.70,33.40 g·kg^(-1))and fluoxetine group(2.6 mg·kg^(-1)).The treatment groups of mice were fed with corresponding medicine-admixed diet from the 21st day of weaning to the end of 111st day of restraint.Sucrose preference,tail suspension and open field tests were used to evaluate the anxiety-and depression-like behavior in mice.The expressions of ubiquitin binding protein(p62)in prefrontal cortex was detected by immunohistochemistry,while the expression of autophagy effect protein(Beclin-1)and autophagic microtubule-associated protein 1 light-chain 3(LC3)were detected by immunofluorescence histochemistry.The protein and m RNA expression levels of phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)/mammalian rapamycin target protein(m TOR)and autophagy related indexes were detected by Western blot and real-time PCR.Results In the behavioral experiment,compared with the normal control group,the sucrose preference index,time spent in central zone and central crossing times of the model mice decreased significantly,while immobility time in the tail suspension test increased significantly(P<0.01).Compared with the model control group,the sucrose preference index,time spent in central zone and central crossing times in medium-,highdoses of Wenyang Jieyu groups(16.70,33.40 g·kg^(-1))and fluoxetine group significantly increased,while the immobility time in tail suspension test decreased significantly(P<0.05,P<0.01).In the experiments of immunohistochemistry,immunofluorescence,Western Blot and Real-time PCR,compared with the norma

关 键 词：温阳解郁方 抑郁 自噬 磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路 小鼠 

分 类 号：R285.5[医药卫生—中药学]