| Alzheimer’s disease(AD)is a common neurodegenerative disease in the elderly.Nowadays,the aging process is increasingly intensified,and the incidence of AD is increasing year by year.AD has become the second most important fatal disease after cardiovascular disease and malignant tumor.AD is a disease mediated by multiple causes,and there are many theories on its pathogenesis,including Tau protein abnormality theory,oxidative stress theory,gene mutation theory and metal ion metabolic disorder theory with toxic theory as the core,among which the metal ion metabolic disorder theory is a focus of current research.Modern pharmacology has conducted a large number of studies on the mechanism of icariin,astragaloside IV and radix puerarin in the treatment of AD.The above studies found that icariin,astragaloside IV and radix puerarin had effects on neurotransmitter content,oxidative stress level and inflammatory response in the brain,and insufficient attention was paid to the improvement of iron overload in the brain.At present,relevant studies in this field are rarely reported.Aβ is generated by the catabolism of its precursor protein APP by three different secretases,namely the α,β and γ-secretase responsible for APP cleavageat the site of α,β and γ.Abnormal shear processing of APP,formation of Aβand its transition from soluble to insoluble are key links in the pathogenesis of AD.Studies have shown that intracellular APP has two metabolic pathways: the amyloid metabolic pathway of APP mainly involves the cooperation of α-secretase and γ-secretase.In the first step,APP is cleaved byα-secretase to produce the neuroprotective protein of s APPα,which is released outside the cell,then the remaining fragment C83 is cleaved byγ-secretase to produce P3 and AICD(APP intracellular domain).AICD ramains in the cell,while P3,a toxic substance,is released outside the cell without producing Aβ.The amyloid metabolic pathway is mainly composed of the cooperation ofβ-secretase andγ-secretase,which work together for APP cleavage.In the first step,BACE1 is cleaved byβ-secretase to produce s APPβprotein,which is released outside the cell.Then,the remaining fragment C99 is cleaved byγ-secretase to produce Aβpeptide and AICD,during which Aβis produced.The previous study of our group found that the effective components of icariin,astragaloside IV and radix puerarin compound could effectively reduce the production of Aβ and regulate the disorder of iron metabolism.But its pathophysiological mechanism is not very clear.The purpose of this study was to investigate whether the effective components of epimedium,astragaloside and radix puerarin compound could influence the formation of A by influencing the key proteins in the hydrolysis of APP through the intervention of HAMP gene silenced HT22 cells.The First Part Effects of Active Components of Epimedium,Astragalus and Radix Puerariae on learning and memory and ADAM10 in hippocampus CA3 of APPswe/PS1 d E9 double transgenic Alzheimer’s Disease Model MiceObjective:To investigate the effects of active Components of Epimedium,Astragalus and Radix Puerariae on learning and memory and ADAM10 in hippocampus CA3 of APPswe/PS1 d E9 double transgenic Alzheimer’s Disease Model MiceMethods:Thirty 10-month-old male APPswe/PS1 d E9 double transgenic model mice were randomly divided into compound group,model group and the deferox(DFX)group,and ten 10-month-old C57BL/6J mice were used as normal control group.The effective components of astragalus,epimedium and pueraria were given to mice in the compound group by gavage.The dosage of astragaloside IV,icariin and puerarin were 80mg·kg-1,120mg·kg-1and 80mg·kg-1,respectively.Mice in the normal control group and the model group were gavaged with normal saline,and mice in the DFX group were gavaged with DFX at a dose of 100mg·kg-1 once daily.After the medication was over,the learning and memory ability of mice in each group was measured by Morris water maze,and the brain tissue was taken out after the water maze test.The expression of A disintegrin and metalloproteinase(A disintegrin and metalloproteinase 10,ADAM10)in hippocampal CA3 region of mice in each group was detected by immunofluorescence,Real-time PCR and Western blot.Results:Compared with the normal control group,the escape latency,the swimming distance and the swimming time of the model group mice were significantly prolonged(P<0.05),Crossing-target number,Target-quadrant abidance is significantly decreases(P<0.05),Escape latency for first crossing the platform is significantly prolonged(P<0.05);The escape latency,swimming distance and swimming time of Astragalus,Epimedium and Radix Pueraria effective component compound group and DFX group were significantly shorter than those of APPswe/PS1 d E9 double transgenic AD model mice(P<0.05).Crossing-target number,Target-quadrant abidance is significantly prolonged(P<0.05),Escape latency for first crossing the platform is significantly decreases(P<0.05);There were no significant differences in escape latency,swimming distance,swimming time and cross times between the compound group and the DFX group(P>0.05).Crossing-target number,Target-quadrant abidance and Escape latency for first crossing the platform was no significant difference.Immunofluorescence,Real-time PCR and Western blot all showed:Compared with normal control group mice,model group increased the expression of hippocampal CA3 area ADAM10(P<0.05),and the expression of ADAM10 in the hippocampal CA3 region of the compound group and DFX group was lower than that of the model group(P<0.05),but there was no significant difference in the expression of ADAM10 between the compound group and the DFX group.Summary:Epimedium,Astragalus,Radix Puerariae and effective components of compound can shorten APPswe/PS1 d E9 double transgenic escape latency,swimming distance and AD model mice swimming time,increased number of crossing platform area,platform area residence time,shorten the escape latency period through the platform for the first time,effectively improve APPswe/PS1 d E9 double transgenic AD mice learning memory ability,its mechanism may be related to lower hippocampal CA3 area ADAM10 m RNA and protein expression.The Second Part Effects of Active Components of Epimedium,Astragalus and Radix Puerariae on the Expression of HAMP in hippocampus CA3 in APPswe/PS1 d E9 double transgenic Alzheimer’s Disease Model MiceObjective:To explore the effect of a combination of Epimedium,Astragalus and Radix Pueraria on the expression of HAMP in hippocampal CA3 region of APPswe/PS1 d E9 double transgenic AD model mice.Methods:Thirty 10-month-old APPswe/PS1 d E9 double transgenic model mice were randomly divided into three groups: the compound group,the model group and the deferox(DFX)group,and ten 10-month-old C57BL/6J mice.As a normal control group.In the study,mice in the normal group and the model group were intragastrically administered with normal saline.Subsequently,the brain tissue of the mice was taken out,and the expression of HAMP in the hippocampal CA3 region of each group of mice was detected by immunofluorescence combined with Real-time PCR and Western blot detection.Results:Compared with the normal group,the expression of HAMP was decreased in the model group.Active Components of Epimedium,Astragalus and Radix Puerariae and DFX were given intragastric administration,HAMP expression levels are APPswe/PS1 d E9 double transgenic AD model mice hippocampal CA3 area increased significantly.Compared with the DFX group,the compound group There were no significant differences in m RNA expression and HAMP protein indices.Each group met P>0.05.Summary:The effective components of Epimedium,Astragalus and Radix puerariae could up-regulate the expression levels of HAMP m RNA and protein in the hippocampal CA3 of APPswe/PS1 d E9 transgenic AD model mice,which may be one of the mechanisms of improving the learning and memory of AD model mice.The Third Part Effect of effective components of Epimedium,Astragalus and Radix Puerariae on the hydrolysis of key enzymes of APP in HT22 cells by HAMPObjective:To study the effect of effective components of Epimedium,Astragalus and Radix Puerariae on the hydrolysis of key enzymes of APP in HT22 cells by HAMPMethods:HT22 cells were divided into Control group(cell culture of equal volume without adding any drugs),Aβ group(treated by Aβ25-35 for 24h),RNAi group(transfection of RNAi-3),Aβ+RNAi group(transfection of RNAi-3 after being treated by Aβ25-35 for 24h),Aβ+TCM(treated by TCM for 24 h after adding Aβ25-35 for 24h),RNAi+ TCM(treated by TCM for 24 h after transfection of RNAi-3 for 24h),Aβ+ RNAi+ TCM(treated by Aβ25-35 for 24h before transfection of RNAi-3 for 24 h,and then treated with TCM for 24h).The expressions of ADAM10,ADAM17 and BACE1 were detected by immunofluorescence,Real-time PCR and Western blot.Results:Compared with the Control group,the Aβ group,the RNAi group and the Aβ+RNAi group have a higher level of BACE1 expression(P<0.05)and a lower level of ADAM10 and ADAM17 expression(P<0.05);Compared with the Aβ group,the Aβ+RNAi group has a higher level of BACE1expression(P<0.05)and a lower level of ADAM10 and ADAM17expression(P<0.05)while the Aβ+TCM group has a lower level of BACE1 expression and a higher level of ADAM10 and ADAM17 expression(P<0.05);Compared with the RNAi group,the Aβ+RNAi group has a higher level of BACE1 expression(P<0.05)and a lower level of ADAM10 and ADAM17 expression(P<0.05)while the RNAi+TCM group has a lower level of BACE1 expression(P<0.05)and a higher level of ADAM10 and ADAM17 expression(P<0.05);Compared with the Aβ+RNAi group,the Aβ+RNAi+TCM group has a lower level of BACE1 expression(P<0.05)and a higher level of ADAM10 and ADAM17 expression(P<0.05).Summary:Traditional Chinese medicine may alleviate AD by up-regulating the expression of HAMP,thus reducing brain iron overload,promoting the expression of ADAM10 and ADAM17,inhibiting the expression of BACE1,and reducing the deposition of Aβ.Conclusion:The effective components of Epimedium,Astragalus and Radix Puerariae could improve the learning and memory ability of mice,and the mechanism was mainly achieved by up-regulating the expression of HAMP,thus promoting the expression of ADAM10 and ADAM17,and inhibiting the expression of BACE1. |
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