Neuroprotective Effect Of Huangpu Tongqiao Capsule On AD Cell Modeland The Effect Of CaM-CaMKIV Signaling Pathway

ObjectiveTo observe the neuroprotective effect and molecular mechanism of Huangpu Tongqiao Capsule medicated serum on AD（Alzheimer’s disease）cell model via cultured hippocampal neurons in vitro,for providing the basis for modern experimental clinical Huangputongqiao Capsule in the treatment of the use of AD,meanwhile,providing new ideas and methods for the treatment of AD.MethodsThe SD rats in the newborn 24h were isolated from the hippocampus,and the primary hippocampal neurons were cultured,the morphological changes were observed by microscope and the identification of hippocampal neurons by immunofluorescence;The MTT method was used to screen the optimal concentration range of Huangpu Tongqiao Capsule medicated serum,the optimum concentration of Aβ_25-35,and detection effect of cell viability of Huangpu Tongqiao Capsule medicated serum on cell model;Inverted microscope was used to observe the morphological changes of neuron injury of Huangpu Tongqiao Capsule medicated serum on cell model;Detection effect of apoptosis rate of Huangpu Tongqiao Capsule medicated serum on cell model by double staining flow cytometry of Annexin V-FITC/propidium iodide（PI）;Observed the ultrastructural changes of hippocampal neurons injury of Huangpu Tongqiao Capsule medicated serum on cell model by transmission electron microscopy.After that the experiment was divided into normal control group,model group(AD 30μM Aβ_25-35induced 24h),KN62 group（pretreatment with 10μM KN62 before modeling）,Huangpu Tongqiao Capsule medicated serum group（treat with 15%Huangpu Tongqiao Capsule medicated serum after 24h modeling）,KN62+Huangpu Tongqiao Capsule medicated serum group and Nimodipine group.The expression levels of Ca M,Ca MKK,Ca MKIV and tau m RNA in hippocampal neurons detected by RT-PCR;The expressions of Ca M,Ca MKK,Ca MKIV,p-Ca MKIV,tau and p-tau protein in hippocampal neurons of each group was detected by Western Blot.Results1、When cultured 7^thday,the neurons aggregation,cell body rounded,densely distributed neurite growth,mature in good condition,which be good for the subsequent test;Map-2 immunofluorescence staining showed that Cy3 labeled Map-2 was expressed in the cytoplasm of hippocampal neurons,and it was stained with orange red fluorescence.Hochest33258 positive staining showed blue fluorescence.After image synthesis,the purity of hippocampal neurons was over 90%.2、MTT results showed that Huangpu Tongqiao Capsule medicated serum has a good protective effect on hippocampal neurons at concentration of 5%,10%,15%,the concentration of 30μM of Aβ_25-35on neuronal inhibition rate reached 50%,which was selected to establishment of AD cell model.MTT and flow cytometry results showed that,compared with the model group,different concentration of Huangpu Tongqiao Capsule medicated serum group,Donepezil group,Nimodipine group can significantly improve the survival rate of hippocampal neurons.The effect is best when its concentration is 15%,and this concentration was selected as a follow-up test.Under the inverted microscope,hippocampal neurons were shrunken,slightly shedding,protrusion,retraction and breakage.The cell morphology of each drug treatment group was different.The neurons in the treated group recovered,neurite recovered and neurons adhered well.Transmission electron microscopy showed that compared with the normal group,the ultrastructure of neurons in the model group was severely damaged,the number of ribosomes decreased,the endoplasmic reticulum dilated and swollen,mitochondria swelled and vacuolar degeneration.Compared with the model group,Huangpu Tongqiao Capsule medicated serum group significantly improved cell morphology damage.3、RT-PCR results showed that compared with the normal group,the m RNA expressions of Ca M,Ca MKK,Ca MKIV and tau were significantly increased in model group（P<0.01）;compared with the model group,the m RNA expressions of Ca M and Ca MKK were not changed significantly,and the m RNA expressions of Ca MKIV and tau were significantly reduced in inhibitor group（P<0.01）,the m RNA expressions of Ca M,Ca MKK,Ca MKIV and tau were significantly reduced in Huangpu Tongqiao Capsule medicated serum group,the inhibitor+Huangpu Tongqiao Capsule medicated serum group and Nimodipine group（P<0.01）.4、Western blot results showed that compared with the normal group,the protein expressions of Ca M,Ca MKK,Ca MKIV,p-Ca MKIV,tau and p-tau were significantly increased in model group（P<0.01）;compared with the model group,the protein expressions of Ca M and Ca MKK were not change significantly and the protein expressions of Ca MKIV,p-Ca MKIV,tau and p-tau were significantly reduced in inhibitor group（P<0.01）,the protein expressions of Ca M,Ca MKK,Ca MKIV,p-Ca MKIV,tau and p-tau were significantly reduced in Huangpu Tongqiao Capsule medicated serum group,the inhibitor+Huangpu Tongqiao Capsule medicated serum group and Nimodipine group（P<0.01）.Conclusions1.Huangpu Tongqiao Capsule medicated serum could improve neuronal damage which induced by Aβ_25-35and has a protective effect on hippocampal neurons.2.The mechanism of Huangpu Tongqiao Capsule medicated serum protected hippocampal neurons in AD cell model,may via regulating calcium/calmodulin dependent protein kinase IV signal pathway,and relating to the reduction of overphosphorylation of tau protein.