| Objectives:Alzheimer’s disease(AD)is a complex neurodegenerative disease with main pathological feature of the deposition ofβ-amyloid protein(Aβ)plaques,neurofibrils in the brain and neurons loss.At present,there is no ideal drug for treating AD in clinical practice.Liuwei Dihuang Decoction(LW)is a classic representative traditional Chinese medicine to protect kidney.Clinical and pharmacological studies have shown that LW has a role in preventing AD.LW-AFC is a five new Chinese medicine derived from LW.The results of previous studies in our laboratory showed that LW-AFC can improve the cognitive function of PrP-hAβPPswe/PS1dE9(APP/PS1)transgenic mice and senescence-accelerated mouse prone 8(SAMP8).At the same time,LW-AFC can reduce the deposition of Aβin the brain and the loss of neurons.In order to further clarify the material basis and the mechanism of action of the above mentioned pharmacological action in LW,from the perspective of neurogenesis,we study mechanisms by which LW and its active fractions reduce neuronal loss in the brain by using AD animal models and induced pluripotent stem cell derived-Neural stem cell(iPSC-derived NSC)model.LW was used for the treatment of children with kidney deficiency by a famous doctor in the Song Dynasty.In this study,immunofluorescence technique was used to observe the proliferation and migration of neural stem cells in the juvenile period of SAMP8 and its control SAMR1 mice to find out foundation of the neurogenesis in young AD mice.Secondly,learning and memorybehavioralexperiments,electrophysiologicaltechniquesand immunofluorescence experiments were used to observe the learning and memory function and synaptic plasticity of LW and its active fractions in young SAMP8 mice,as well as their role in proliferation and differentiation of neural stem cells in the brain;then,the Incu Cyte Zoom live cell dynamic imaging analysis system,CCK8 assay,and Caspase 3/7 assay were used to observe the activity,proliferation and apoptosisof blood-brain barrier monomers of LW on iPSC-derived NSC model;Finally,Nissl’s staining,immunofluorescence and brdu incorporation methods were used to study the number of neural stem cells and neurons loss effect on adult SAMP8 and APP/PS1 transgenic mice.Part 1.Change of neurogenesis in the brain of Juvenile SAM miceSAMP8 mice is classical AD model mice showing cognitive impairment at 2 months of age.In order to observe the changes in neurogenesis of SAMP8mice at young age,we observed the proliferation,migration,and differentiation of neural stem cells in mice brains of 5,15,30,and 45 days of age.Results were as follows:1.Cell proliferation change in the brain of juvenile SAM miceThe immunofluorescence technique was used to observe the proliferation of EdU labeled proliferating cells in the brains of young SAMR1 and SAMP8mice.The results showed that for SAMR1 mice,the number of EdU~+cells in the brain continued to decrease as the age increased.In SAMP8,the number of Ed U~+cells in the brains of 15th,30th,and 45th day old mice was significantly decreased compared with 5th day,but there was no significant difference in the number of EdU~+cells in the brains of 15,30,and 45 days old.Compared with SAMR1 mice,the number of EdU~+cells in the brain of SAMP8 mice was significantly reduced at the age of 5 days.This result shows that as the age increases,the proliferation of cells in the brain of SAMR1 and SAMP8 mice is reduced,and compared with the same age SAMR1 mice,the cells in the brain of SAMP8 mice increase less.2.Change of NSC Cell proliferation in the brain of juvenile SAM miceUsing immunofluorescence techniques to observe SOX2/EdU dual labeling of neural stem cells in the brains of young SAMR1 and SAMP8 mice,the results showed that,for SAMR1 mice,SOX2~+/EdU~+in brain of 15,30,and45 days compared with 5 days of age.The number of cells decreased significantly,but there was no significant difference in the number of SOX2~+/Ed U~+cells in the brains of 15,30,and 45 days old mice.In SAMP8,as the age increases,the number of SOX2~+/EdU~+cells in the brain continues to decrease.Compared with SAMR1 mice,the number of SOX2~+/Ed U~+cells in the brain of SAMP8 mice was significantly reduced at 5 days of age.This result shows that as the age increases,the proliferation of neural stem cells in the brains of SAMR1 and SAMP8 mice is reduced,and compared with SAMR1mice of the same age,the proliferation of neural stem cells in the brain of SAMP8 mice is less.3.Change of NSC Cell migration in the brain of juvenile SAM miceImmunofluorescence technique was used to observe Ed U/DCX dual labeling of migrating neural stem cells in the brains of young SAMR1 and SAMP8 mice.The results showed that there was no significant difference in the number of SOX2~+/DCX~+cells in brains of mice at 15,30,and 45 days of age in SAMR1 mice.For SAMP8 mice,there was no significant difference in the number of SOX2~+/DCX~+cells in the brains of 15,30,and 45 days old mice.Compared with SAMR1 mice of the same age,there was no significant difference in the number of SOX2~+/DCX~+cells in the mouse brain.This result shows that as the age increases,there is no significant change in the number of neural stem cells in the brain of SAMR1 and SAMP8 mice.Part 2.Effect of LW and its active fractions on neurogenesis in juvenile SAMP8miceSAMP8 mice are classical AD model mice and exhibit cognitive impairment at 2 months of age.To observe the effect of LW on cognitive function in SAMP8 mice,the study was performed by intragastric administration of 25-day-old SAMP8.Mice with LW(1.17 g/kg)and LW-AFC(1.6 g/kg)for 47 days were observed for cognitive function changes in mice.The results are as follows:1.Effect on the proliferation and differentiation of NSC in the brainUsing 25-day-old SAMP8 mice,piracetam,LW,and LW-AFC were continuously administered for 5 weeks,and the proliferation and migration of neural stem cells in the brain were observed by immunofluorescence.The proliferation of neural stem cells was observed using EdU/DCX double labeling.It was found that compared with SMAR1 mice,there was no significant change in the migration of neural stem cells in the brain of SAMP8 mice.After administration of piracetam,LW,and LW-AFC,There was no effect on the migration of neural stem cells in the mouse brain.The proliferation of neural stem cells was observed by EdU/SOX2 double labeling.Compared with SMAR1 mice,the proliferation of neural stem cells in the brain of SAMP8 mice was significantly reduced.After administration of piracetam,LW,and LW-AFC,the nerves of the mouse brain were significantly reduced.Stem cell proliferation has no effect.The results showed that the administration of piracetam,LW,and LW-AFC had no effect on the proliferation and migration of neural stem cells in the brain of SAMP8 mice.2.Effect on synaptic plasticityThrough LTP experiments,the effect of LW on synaptic plasticity in SAMP8mice was observed.The results showed that the synaptic plasticity of SAMP8mice was significantly lower than that of SAMR1 of the same age.LW-AFC(1.6 g/kg)and piracetam(0.6 g/kg)could significantly increase the long term potentiaton of SAMP8.3.Effect on learning and memory functionThe effects of LW on the central learning and memory function of SAMP8mice were observed through new object recognition experiments,darkness avoidance experiments,and Morris water maze experiments.The results showed that compared with SAMR1 of the same age,there was no significant difference in the object recognition function,passive avoidance ability,and spatial memory ability of SAMP8 mice.And LW(1.17 g/kg)and LW-AFC(1.6 g/kg)have no effect on mice’s learning and memory function.Part3.Effect of LW monomer which can penetrate the blood-brain barrier(BBB)on Neural Stem CellsPrevious experiments were observed that the monomers(catapol,acteoside,pachymic acid,sweroside,benzonic acid)in the LW can penetrate the blood brain barrier(BBB).In order to further explore the specific components of LW in reducing neuronal cell loss in the brain,we use iPSC-derived NSC which comes from cognitive normal and AD patients to observe the effect of the monomers in viability,proliferation and apoptosis.The results were as follows:1.Effect on the viability of iPSC-derived NSCCCK-8 results showed that catapol(1μM,10μM,100μM)had inhibitory effects on the activity of iPSC-derived NSC derived from normal people.High concentration of acteoside(100μM)and pachymic acid(100μM)inhibited the activity of iPSC-derived NSC derived from AD.2.Effect on iPSC-derived NSC proliferationThe Incucyte Zoom live cell dynamic imaging analysis system showed that catapol(100μM)and pachymic acid(100μM)could inhibit the proliferation of AD-derived iPSC-derived NSC cells.3.Effect on apoptosis of i PSC-derived NSCApoptosis was detected using Caspase 3/7 reagent.It was found that catapol(1μM,10μM,100μM)and high concentration of acteoside(100μM)inhibited the apoptosis of iPSC-derived NSC from AD,while the lower concentration of acteoside(1μM,10μM),pachymic acid(1μM,10μM),sweroside(1μM,10μM,100μM)and benzoic acid(100μM)promoted the apoptosis of i PSC-derived NSC from AD.Part4.The effect of LW and its active fraction on the cells number in the brain of AD model miceBrain atrophy is an important marker in patients with AD.Structural nuclear magnetic resonance(NMR)show that patients with AD progressively with the volume of ventricles expanding,and the hippocampus and the cortex of the entorhinal area shrinking.In order to observe the effect of LW and its active fractions on reducing neuronal loss and preventing brain atrophy,this study was performed by intragastric administration of LW(1.17 g/kg)and LW-AFC(1.6g/kg)respectively to 7-month-old male SAMP8 mice and CA-30(1.224 g/kg)for 198 days,and by intragastric administration of LW(10 g/kg),LW-AFC(1.6g/kg)and CA-30(0.96 g/kg)respectivelyto 10 months old male APP/PS1transgenic mice for 117 days.The number of neurons,astrocytes,microglia,and proliferation and differentiation of neural stem cells in the brain were observed.The results are as follows:1.Effect on the number of neuronsThe results of Nissl staining showed that the number of Nissl bodies in the hippocampus and CA3 area of??APP/PS1 transgenic mice were reduced compared with the wild type control,and CA-30 could increase the number of hippocampal Nissl bodies in APP/PS1 transgenic mice.LW can increase the number of Nissl bodies in CA3 area of hippocampal.2.Effect on the number of astrocyteThe results of immunofluorescence showed that the number of GFAP~+cells in hippocampus of APP/PS1 transgenic mice was increased compared with WT mice.Both LW-AFC and CA-30 could increase the number of hippocampal GFAP~+cells in APP/PS1 transgenic mice.Compared with SAMR1 mice,the number of GFAP~+cells in the hippocampus of SAMP8 mice was significantly increased,and they were not affected by LW,LW-AFC,and CA-30 respectively.3.Effect on the number of microgliaThe results of immunofluorescence showed that the number of Iba1~+cells in hippocampus of APP/PS1 transgenic mice was increased compared with WT mice,and the number of Iba1~+cells could be decreased by LW,LW-AFC,and CA-30 respectively.Compared with SAMR1 mice,the number of Iba1~+cells in the hippocampus of SAMP8 mice was increased.LW and LW-AFC can decreased Iba1~+cells respectively.4.Effect on proliferation and differentiation of NSCBrdu~+,Brdu~+/SOX2~+,and Brdu~+/NenN~+were not detected in brains of WT mice and APP/PS1 transgenic mice by Brdu incorporation assay and immunofluorescence assay.LW,LW-AFC,and CA-30 can not change the results.Conclusions:1.The proliferation of neural cells and neural stem cells in the brain of SAM mice decreased with increasing age,and the proliferation of cells and neural stem cells in the brain of SAMP8 mice was significantly less than that of SAMR1 mice at the age of 5 days.2.Synaptic plasticity in SAMP8 mice was significantly reduced compared to juvenile SAMR1 mice,and LW-AFC can significantly increased synaptic plasticity in SAMP8 mice.3.Catapol and acteoside which penetrate BBB can inhibit AD-derived iPSC-derived NSC cell apoptosis.4..For AD model mice,LW,LW-AFC,CA-30 have the effect of reducing the activation of microglia in the brain and increasing the number of neurons,and LW-AFC,CA-30 can increase the number of astrocytes in the brain. |
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