Investigate The Ameliorate Effect Of Icariin On Neuron Autophagy Level Mediated By AMPK-mTOR-ULK1 Pathway In Natural Aging Rats

Objective In this study,we investigated the effect and mechanism of Icariin(ICA)on autophagy of cortical and hippocampal neurons in nutural aging rats,so as to provide experimental basis for Icariin in the prevention and treatment of central neurodegenerative diseases.Methods Twenty-four 16-month-old SD rats were randomly divided into the aging model group(20M),low-dose ICA group(ICAL)and high-dose ICA group(ICAH),each group of 8 rats.The aging model group was treated with normal feed,ICAL and ICAH were administrated via food at a dosage of 2 mg/kg and 6 mg/kg for 4 months until 20 months old.In addition,8 rats aged 6 month were used as the youth control group(6M).After continuous administration for 4 months,rat were sacrificed after urethane anesthesia,blood sample was collected from the abdominal aorta,and then the cortex and hippocampus were separate and storage at-80℃.After that,HE staining were used to observe the pathological changes of cortex and hippocampus.Nissl staining was used to measure the nissite changes in the cortex and hippocampus of rats.The expressions of LC3 B,p62,Atg5,Beclin1,p-ULK1(ser757),p-m TOR,p-AMPK and AMPK were detected by Western Blot.The co-localization of LC3 and neurons were detected by immunofluorescence.Results 1)HE staining results showed that compared with 6M,a large number of neurons in the 20 M cortical tissue were denatuated,which were featured with the prominent manifestations of shrinkaged nuclear and decreased cytoplasm,and all the damages could be alleviated by ICA intervention.The number of neuronal layers in the hippocampus were significantly decreased in 20 M rats,meanwhile,neurons were loosely arranged,and nuclear were dark dye and shinkage.After icariin intervention,the number of neuronal layers were increased in the hippocampus and in a dose-dependent manner.2)Nissl staining results showed that compared with 6M,nissl bodies stain shallow in the 20 M cortex and hippocampus,but icariin could significantly improve the Nissl bodies.3)Western Blot results showed that p62 protein in the cortex and hippocampus tissues of 20 M rats was significantly increased compared to 6M,while the LC3 B,Beclin1 protein level was significantly decreased.p-AMPK protein in hippocampus tissues increased significantly in 20 M rats,meanwhile,the downstream protein p-m TOR and p-ULK1(ser757)level increased obviously.Low and high dose of ICA could promote the expression of autophagy associated protein LC3 B,Beclin1 and atg5 in aging brain,while reduce the expression of p62 p-m TOR and p-ULK1(ser757).4)Immunofluorescence results confirmed that LC3 was co-localization with neurons in the cortex and hippocampus,and the intensity of LC3 was notebaly weakened which was rescured by ICA intervition.Conclusion ICA can effectively improve the morphology of neurons in cortex and hippocampal of aging rats.The underlying mechanism is that ICA could activate AMPK,and inhibit the activation of m TOR in the cortex and hippocampal of aging rats,thereby inhibiting the phosphorylation of serine at ULK1(Ser757)site.As a result,neuronal autophagy is enhanced and cell function is improved.