Active Compounds Of Three Chinese Herbs Through Regulating Iron Metabolism Related Protein Performing The Neuroprotective Effect In The APP/PS1 Transgenic Mouse

Alzheimer’s disease(AD)is a neurodegenerative brain disorder,which is the most common cause of dementia,characterized by amyloid plaque accumulations,intracellular tangles and neuronal loss in selective brain regions.The frontal cortex,important for executive functioning,is one of the regions affected.The present study evaluated the effects of the active compounds of Epimedium,Astragaoside and Puerarin on the six-month-old APPswe/PS1ΔE9(APP/PS1)double transgenic mouse model of AD.Increasing evidence indicates that a disturbance of normal iron metabolism,which may catalyze free radical reactions contributing to the pathology of AD.However,the underlying mechanisms resulting in abnormal iron load in the AD brain is unclear.Deferoxamine(DFO)is an iron chelator that significantly alleviates the symptoms of patients with AD has been reported.However,oral side effects have been exhibited.Chinese herbal medicine compatibility is the predominant form for clinical medication for three thousand years.Epimedium,Astragaoside,Puerarin are the highly valued traditional Chinese medicine and were widely applied in clinics with remarkable effect on neuroregulatory and neuroprotective activities.For example,several studies showed that Epimedium has neuroprotective effects on β amyloid induced neurotoxicity to neuronal cells and significantly attenuated Aβ deposition in amyloid plaques in cortex and hippocampus of transgenic mice.It was demonstrated that Astragaoside membranaceus has an inhibitive effect on oxidative stress induced by metal and against amyloid beta1-42 neurotoxicity as an effective anti-oxidant in Human SK-N-SH neuroblastoma cells.Puerarin,an isoflavone purified from the root of Puerarin lobata,which has been reported to attenuate learning and memory impairments in the transgenicmouse model of AD and could protect neurons from oxidative stress-induced apoptosis.Therefore,these compounds may have the potential effectively active compounds on AD underlying certain pathogenesis.The results showed that the treatment with the active compounds reduced cognitive and memory deficits and damaged of cell ultrastructure in AD model mice and affected the iron metabolism proteins.Our founding suggest that the change in the levels of four major iron uptake proteins including divalent metal transporter with iron response element(DMT1-with IRE),divalent metal transporter without iron response element(DMT1-without IRE),transferrin(TF)and transferring receptor 1(TfR1);three release proteins including the exporter ferroportin 1(FPN1),ceruloplasmin(CP)and hephaestin(HEPH),one increased storage iron protein ferritin and one iron regulating hormone Hepcidin which involved the iron metabolic regulation mechanism in the cortex of the APP/PS1 double transgenic mouse brain may due to the administraion of active compounds of Epimedium,Astragaoside and Puerarin.The Hepcidin is a key regulator hormone of the entry of iron in the circulation of the mammals,which is secreted by the liver and controlled by iron stores within inflammation,macrophages,hypoxia,and erythropoiesis.Hepcidin inhibits iron transport by binding to the iron export channel FPN1.The recent study has demonstrated that Hepcidin could significantly reduced brain iron in the iron overloaded rats and suppressed transport of transferrin-bound iron from the periphery into the brain.Therefore,the present study aimed to investigate the effects of the active compounds of Epimedium,Astragaoside and Puerarin on the above iron metabolism related proteins in the frontal cortex of the AD model mice with 3months administration.Part one The compounds of Epimedium,Astragaoside,Puerarine onbehavioral changes and Pathological changes in APP/PS1transgenic mouse modelObjective: To investigate the effects of Epimedium,Astragaoside,Puerarin on behavioral changes and Pathological changes in the APP/PS1 double transgenic mouse model of AD.Methods: A total of 84 specific-pathogen-free male APP/PS1 double transgenic mice aged 6months were equally and randomly assigned to model,Epimedium,Astragaoside,Puerarin,compound and DFO groups.An additional 12 6-month-old C57BL/6J mice served as negative controls group.Mice in the Epimedium,Astragaoside,Puerarin and compound groups were given120-mg/kg Epimedium herb,80-mg/kg milkvetch root,80-mg/kg kudzuvine root,120-mg/kg Epimedium herb + 80-mg/kg milkvetch root + 80-mg/kg kudzuvine root via intragastric administration,once a day,for 3 consecutive months.Mice in the deferoxamine group were intraperitoneally injected with30-mg/kg deferoxamine,once a day,for 3 consecutive months.Mice in the model and control groups were intragastrically administered 1 mL of normal saline.Using Morris water maze and novel object recognition to investigated effects of Epimedium,Astragaoside,and Puerarin on behavioral changes in the APP/PS1 double transgenic mouse model of AD.Use the immunohistochemistry analysis to analysis the change of Aβ-42 and the transmission electron microscope to explore the ultrestructure change of each group.Results:1.The active compounds of Epimedium,Astragaoside and Puerarin ameliorate episodic memory decline in AD model mice.During the familiarity stage of novel object recognition(NOR),the discrimination indices showed no difference in time spent in exploring two identical objects among groups and indicated a significant effect in the exploratory stage(P<0.05)for exploring a new object.The result showed the episodic memory impairment was obvious in APP/PS1 double transgenic AD model group than the other groups,as the discrimination index was lower than that of C57,DFO and active compounds and three unilateral groups.The result showed that the episodic memory was impaired in the APP/PS1 double transgenic AD model and was improved by treatment with the active compounds and DFO.There was no significant difference between the active compounds and DFO groups in thediscrimination indice.2.The neuroprotective effect of the active compounds of Epimedium,Astragaoside and Puerarin in the AD transgenic model mice cortex was evaluated using transmission electron microscope(TEM)and immunohistochemistry(IHC)of Aβ-42.3.TEM was used to observe the ultrastructure in frontal cortex neurons.In C57 group mice,the neuronal ultrastructure was normal as exhibited by intact membranes,uniform cytoplasm,and complete organelle structure.In APP/PS1 double transgenic AD model group,the nuclear shrinkaged and deformed,the mitochondrial cristae fused with partial membranes,rough endoplasmic reticulum degranulated,cytoplasm vacuolization,mitochondria swelled and membrane ridges disappeared,which were pointing out the damage of ultrastructure in neurons.The ultrastructure of Epimedium,Astragaoside,and Puerarin group were similar,presenting partial neural edema or loss normal morphology,the nuclear shrinkaged and deformed slightly,seldom mitochondria swelled and deformed or endoplasmic reticulumswere dilated slightly,no significant differences between the three groups.The ultrastructure of active compounds and DFO group were similar as C57 group mice,showing that the karyomorphism and the membrane structure relatively normal,and organelles are relatively complete in morphology.The result showed that the ultrastructure of APP/PS1 double transgenic AD model mice neurons were obviously damaged and the compounds of active Epimedium,Astragaoside and Puerarin could play the neuroprotective effect on the ultrastructure of APP/PS1 double transgenic AD model group.Conclusions: The APP/PS1 double transgenic mice were cognitive deficits on learning and memory performance,and the compounds of Epimedium,Astragaoside,and Puerarin can improve the learning and memory and episodic memory ability of AD model mice.The active compounds of Epimedium,Astragaoside and Puerarin have the neuroprotective effect in the AD transgenic model mice cortex were evaluated using transmission electronmicroscope(TEM)and immunohistochemistry(IHC)of Aβ-42.Part two The active compounds of Epimedium,Astragaoside andPuerarin effect on the iron metabolism relative proteinsexpression in the cerebral cortex of AD transgenic miceObjective: To investigate the effects of Epimedium,Astragaoside,Puerarin on molecular level changes in the APP/PS1 double transgenic mouse.Methods: A total of 60 specific-pathogen-free male APP/PS1 double transgenic mice aged 6 months were equally and randomly assigned to model,Epimedium,Astragaoside,Puerarin,compound and DFO groups.An additional 10 9-month-old C57 mice served as negative controls group.Mice in the Epimedium,Astragaoside,Puerarin and compound groups were given120-mg/kg Epimedium herb,80-mg/kg milkvetch root,80-mg/kg kudzuvine root,120-mg/kg Epimedium herb + 80-mg/kg milkvetch root + 80-mg/kg kudzuvine root via intragastric administration,once a day,for 3 consecutive months.Mice in the deferoxamine group were intraperitoneally injected with30-mg/kg deferoxamine,once a day,for 3 consecutive months.Mice in the model and control groups were intragastrically administered 1 mL of normal saline.Using Real-time PCR,Western bolt and immunohistochemistry to investigated the effects of a compound combining the effective components of Epimedium,Astragaoside,Puerarin,on the levels of four major iron uptake proteins including transferrin(TF)and transferring receptor 1(TfR1);three release proteins including ferroxidaseceruloplasmin(CP)and hephaestin(HEPH),one increased storage iron protein ferritin(FN)and one iron regulating hormone Hepcidin expression in the cerebral cortex of APP/PS1 double transgenic mouse.Results:1.Real time-PCR results showed that the mRNA expression of the four iron uptake proteins in the APP/PS1 double transgenic AD model group was higher than in the C57 group,and these proteins were lower in the DFO and active compounds(P<0.05)groups.The TF mRNA expression showed that theDFO and active compounds groups were lower than the APP/PS1 double transgenic AD model group.The mRNA expression of three unilateral groups were higher than the active compounds(P<0.05).The mRNA expression of TfR1 showed that DFO and active compounds groups were lower than the APP/PS1 double transgenic AD model group and the Astragaoside group were also lower than the APP/PS1 double transgenic AD model group(P<0.05).Among the three main release proteins,the CP mRNA expression showed that APP/PS1 double transgenic AD model group was lower than the DFO and active compounds groups(P<0.05).In the HEPH mRNA expression result showed that the active compounds were higher than APP/PS1 double transgenic AD model group and three unilateral groups(P<0.05).The mRNA expression of storage iron protein ferritin included the Ftl1 and Fth.And the results showed that the mRNA expression in the APP/PS1 double transgenic AD model group was higher than the DFO and active compounds groups and three unilateral groups were higher than the active compounds(P<0.05).There was no significant difference between the active compounds and DFO groups.However,in the mRNA expression of FPN1,CP and HEPH the DFO group was lower than the C57 group.2.Western blot assay indicated that in the APP/PS1 double transgenic AD model group the protein relative levels of iron uptake proteins TF and TfR1 were higher than the C57 control group.The protein relative levels of TF in the active compounds group were lower compared with the APP/PS1 double transgenic AD model group.However,there was no significant difference between the active compounds and APP/PS1 double transgenic AD model group in the protein relative level of the TfR1.Among the iron release protein CP,HEPH,the results revealed that in the APP/PS1 double transgenic AD model group these proteins were lower than the C57 group.The protein relative levels in the active compounds were higher than APP/PS1 double transgenic AD model group.The storage protein ferritin in the active compounds was lower in the C57 group than the APP/PS1 double transgenic AD model group.There was no significant difference between the activecompounds and DFO groups in the protein relative levels.Western blot assay results were consistent with the results of Real-time PCR except the TFR1.3.Immunohistochemical staining results revealed that the Integral optical density(IOD)of TF,ferritin,Hepcidin expression of APP/PS1 double transgenic AD model group were higher than the C57 group(P<0.05).The IOD of HEPH,CP were lower(P<0.05)in the APP/PS1 double transgenic AD model group compared with the C57 control group.TF,ferritin expression was lower in the Epimedium,Astragaoside,Puerarine,active compounds and DFO groups than in the APP/PS1 double transgenic AD model group(P<0.05).HEPH,CP expression was higher in the Epimedium,Astragaoside,Puerarine,active compounds and DFO groups than in the APP/PS1 double transgenic AD model group(P<0.05).TF,FN expression was higher(P<0.05),but HEPH,CP expression was lower(P<0.05)in the Epimedium,Astragaoside,and Puerarin groups compared with the DFO group and active compounds.There was no significant difference of IOD in each group about TfR1.No significant difference was detected in TF,FN,TfR1 expression in APP/PS1 double transgenic AD model frontal cortex between DFO and active compounds groups(P > 0.05).Conclusions: Downregulated DMT1-with IRE,DMT1-without IRE,transferring,transferring receptor1,expression,upregulated ferroportin1,ceruloplasmin,hephaestin,expression,the Epimedium,Astragaoside regulated the iron metabolism in the cerebral cortex of mice with Alzheimer’s disease.These compounds reduced damagement of the central nervous system,indicating a new strategy for developing novel drugs for the treatment of Alzheimer’s disease.Part three Effects of effective fraction of Epimedium,Astragaoside,Puerarin on the expression of Hepcidin in the APP/PS1transgenic mouse model of Alzheimer’s diseaseObjective: To investigate the effects of Epimedium,Astragaoside,Puerarin on Hepcidin expression in the cerebral cortex of APP/PS1 doubletransgenic mouse model of AD.Methods: A total of 60 specific-pathogen-free male APP/PS1 double transgenic mice aged 6 months were equally and randomly assigned to model,Epimedium,Astragaoside,Puerarin,compound and DFO groups.An additional 10 6-month-old C57 mice served as negative controls group.Mice in the Epimedium,Astragaoside,Puerarin and compound groups were given120-mg/kg Epimedium herb,80-mg/kg milkvetch root,80-mg/kg kudzuvine root,120-mg/kg Epimedium herb + 80-mg/kg milkvetch root + 80-mg/kg kudzuvine root via intragastric administration,once a day,for 3 consecutive months.Mice in the deferoxamine group were intraperitoneally injected with30-mg/kg deferoxamine,once a day,for 3 consecutive months.Mice in the model and control groups were intragastrically administered 1 mL of normal saline.Using immunohistochemistry and molecular biology methods to investigate the effects of a compound combining the effective components of Epimedium,Astragaoside,Puerarin,on Hepcidin expression in the cerebral cortex of APP/PS1 double transgenic mouse model of AD.Results: Hepcidin has been proposed to have a central role in brain iron homeostasis by regulating the expression of brain iron related proteins.Real-time PCR analysis of Hepcidin showed that compared with the AD model group,the mRNA expression was lower in the C57 group(P<0.05).The mRNA expression of DFO and active compounds groups were lower than the AD model group and there was no no significant difference between the DFO group and active compounds group(P<0.05).The mRNA expression of Epimedium,Astragaoside,Puerarin group were higher than the C57 group and the Astragaoside and Puerarin groups were higher than the active compounds groups.The finding from the immunohistochemical analysis of Hepcidin revealed that it was increased in the AD model group compared with the C57 group,and DFO and active compounds groups decreased the IOD level compared with the AD model group(P<0.05).No significant difference was detected in IOD value between DFO and active compounds groups(P > 0.05).Conclusions: Epimedium,Astragaoside and Puerarin compound could lower the expression of Hepcidin in AD mice,which may be related to the alleviation of AD process by iron related proteins in Epimedium,Astragaoside and Puerarin.