| Alzheimer's disease?AD?is a common neurodegenerative disease.The pathological features of AD mainly include senile plaques?SP?caused by accumulation of extracellular?-amyloid?A??in the brain,neurofibrillary tangles?NFTs?and neuronal loss caused by intracellular Tau hyperphosphorylation,the obvious synaptic loss and so on.This study investigated the protective effects of alkaloids of Plumula Nelumbinis?Neferine,Liensinine,Isoliensinine,and total alkaloids from the seed embryo of Nelumbo nucifera?on PC12 cells injury induced by A?25-355-35 and their anti-Alzheimer's disease effect.In the first part of the in vitro activity study,the effects of Neferine,Liensinine,Isoliensinine?0.1-10?M?and total alkaloids from the seed embryo of Nelumbo nucifera?TASENN,0.06-6?g/mL?alone or in combination with A?25-355-35 on the PC12cell viability were investigated by MTT assay.The results showed that the decrease of PC12 cell survival induced by A?25-355-35 was inhibited by the alkaloids of Plumula Nelumbinis significantly.However,the cell viability was not affected by them alone.The morphology of PC12 cells was observed under the inverted microscope by HE staining.The results showed that the number of cells damaged by A?25-355-35 was decreased.The cells showed irregular fusiform shape,pyknosis,and the gap became larger.In addition,some of the nucleus were pyknosis and deep colored.Thus,the alkaloids of Plumula Nelumbinis can inhibit the morphological changes of PC12cells damaged by A?25-35.Furthermore,the apoptosis of PC12 cells was detected by AO-EB and Annexin V-FITC/PI fluorescent staining.The results showed that Neferine,Liensinine,Isoliensinine?10?M?and TASENN?6?g/mL?were able to inhibition of apoptosis of PC12 cells induced by A?25-35.The second part of the in vitro mechanism study firstly used fluorescence spectrophotometer and Fura-2/AM as fluorescent probe to investigate the effects of alkaloids of Plumula Nelumbinis on the[Ca2+]i level in PC12 cells injured by A?25-35.The results showed that the[Ca2+]i level in PC12 cells injured by A?25-35was significantly increased,and the intracellular[Ca2+]i concentration was significantly decreased by treatment the cells with the alkaloids of Plumula Nelumbinis for 24 hours.It was further verified that alkaloids of Plumula Nelumbinis could significantly inhibit the increase of[Ca2+]i concentration in A?25-35-damaged PC12 cells by flow cytometry analysis and Fluo-3/AM staining.The concentration of Neferine,Liensinine and Isoliensinine was 1,3,10?M,and TASENN was 0.6,2,6?g/mL.The effects of drugs on the level of reactive oxygen species?ROS?in PC12 cells injured by A?25-355-35 were examined by fluorescence spectrophotometer and DCFH-DA as fluorescent probe.The results showed that the intracellular ROS levels decreased significantly with the increase of drug concentration.The effects of alkaloids of Plumula Nelumbinis on Tau phosphorylation,total Tau and calmodulin?CaM?expression in PC12 cells induced by A?25-355-35 was measured by western blot analysis.The results showed that alkaloids of Plumula Nelumbinis could significantly inhibit the phosphorylation of Tau and the expression of CaM in A?25-35-damaged PC12 cells.The third part of this paper is the in vivo experiment,in which the anti-Alzheimer's disease effect of TASENN was mainly investigated.The APP/PS1double transgenic AD mice were continuously intragastrically administered to TASENN?50 mg/kg·d?for 28 days.Then the brain tissue was taken for paraffin section and stained.Western blot assay was performed after extracting the total protein of hippocampus and cortex.The experimental animals were divided into three groups:wild-type group,APP/PS1 group and APP/PS1 administration group.The morphology of hippocampal pyramidal cells in paraffin sections of mouse brain tissue was firstly observed by HE staining.The results showed that the nuclei of pyramidal cells of APP/PS1 transgenic AD mice were deeply stained and condensed,and the cells were in a triangular or irregular shape.The hippocampal pyramidal cells of drug-administered group were regular in shape,densely arranged,and the nuclei were large and round,showing uniform and light staining.The apoptosis of mouse brain tissue was observed by TUNEL staining.The results showed that the number of apoptosis in the brain of mice decreased after administration,indicating that the drug can inhibit the apoptosis of mouse neuronal cells.Western blot analysis was used to detect the expression of Tau protein phosphorylation,total Tau protein and CaM in hippocampus and cortex of mice.The results showed that TASENN could significantly inhibit the abnormal phosphorylation of Tau protein in hippocampus and cortex of AD mice and reduce the expression of CaM.In summary,these findings explain that the alkaloids of Plumula Nelumbinis?Neferine,Liensinine,Isoliensinine and TASENN?can inhibit intracellular hyperphosphorylation of Tau protein by inhibiting Ca2+,CaM and ROS levels in PC12 cells injured by A?25-35,thereby reducing apoptosis and protecting PC12 cells.In addition,TASENN can inhibit the expression of CaM in hippocampus and cortex of APP/PS1 transgenic AD mice,thereby inhibiting the phosphorylation of Tau protein and neuronal apoptosis.All these results demonstrated that Neferine,Liensinine,Isoliensinine and TASENN may have potential preventive and therapeutic effects on Alzheimer's disease. |
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