| Alzheimer's disease?AD?,also known as Alzheimer's disease,is a chronic and progressive neurodegenerative disease involving many causes.Clinical symptoms such as cognitive impairment and memory loss are the main criteria for the diagnosis of AD.The study found that AD is closely related to?-amyloid?A??deposition,Tau hyperphosphorylation,glial hyperplasia,brain metabolic disorders and other factors.Neurofibrillary tangles?NFT?induced by Tau hyperphosphorylation in hippocampal,cortical and subcortical tissue cells are recognized as one of the most important mechanism in the academic community.A large number of literatures have shown that A?25-355-35 in condensed state can significantly promote Tau protein hyperphosphorylation to form NFT.So,any method of antagonizing nerve damage may be beneficial to the pathophysiology of AD and the screening of drugs for the treatment of AD.Mouse neuroblastoma cells?N2a?are neural crest-derived cells in mice,which are mainly derived from mouse neuroblastoma.Their biological activity is close to that of neural cells,and they have the morphological and biological characteristics of neural stem cells.They are easy to culture,easy to operate and grow rapidly.N2a cells are often used as a substitute cell for neural cells to carry out basic research of drugs.Therefore,we take the neuron-like N2a cells as the research object and use A?25-35 to construct the model of AD pseudo cells in vitro.In recent years,scientific studies have found that flavonoids have a good preventive or therapeutic effect on neurodegenerative diseases,most of which are multi-target,multi-pathway and multi-system,and have little side effects.Scutellaria barbata flavonoids?SBF?are flavonoid mixture isolated from the aerial parts of Scutellaria barbata,which have many functions such as anti-inflammatory,anti-tumor,anti-oxidation and improving memory disorder.In this study,N2a cells were cultured in vitro,A?25-355-35 toxic fragment injury model was established and SBF intervention was used to observe the effect of SBF on the damage of model cells.By inhibiting the activity of GSK-3?,the regulation mechanism of SBF on the phosphorylation of Tau protein and the antagonistic effect of SBF on AD were discussed,which provided the experimental basis for the clinical application of SBF in the prevention and treatment of AD.Objective:To investigate the protective effect of SBF on N2a cell injury induced by A?25-35 and the regulation mechanism of Tau protein phosphorylation,and to provide the necessary experimental theoretical basis for the application of traditional Chinese medicine monomers in AD prevention and treatment.Methods:1 N2a cells were cultured in vitro and A?25-35 model of N2a cells was established.N2a cells with good growth were randomly divided into control group and A?25-35 treatment group with 25?mol·L-1,30?mol L-1,35?mol L-1 and40?mol L-1,and 45?mol·L-1.The damage degree of the cells was observed under inverted microscope for 12h.The release of lactate dehydrogenase?LDH?in culture medium was measured and the cell cell avibility was determined by MTT colorimetry.2 Screening of the concentration of SBF on N2a cells induced by A?25-35N2a cells with good growth were randomly divided into control group,model group,SBF treatment group(1?g·mL-1,2.75?g·mL-1,4.5?g·mL-1,6.25?g·mL-1,8?g·mL-1).After 12 hours of treatment with different concentrations of SBF,the final concentration of A?25-35 was 35?mol L-1 for12 h.The cell status was observed under microscope.The release of lactate dehydrogenase?LDH?in culture medium was determined by enzyme labeling method and the cell cell avibility was determined by MTT colorimetry.3 Protective effect of SBF on N2a cells damage induced by A?25-35N2a cells with good growth were randomly divided into control group,model group,different concentration of SBF treatment group(1.125?g·mL-1,2.25?g·mL-1 and 4.5?g·mL-1).After 12h of different concentration of SBF treatment,added 35?mol·L-1 A?25-35 for 12 h,and observed cell morphology under microscope.The release of LDH in the culture medium was determined by enzyme labeling method and the cell cell avibility was determined by MTT colorimetry.4 The protein expression level of GSK-3?phosphorylation site Ser9,Tyr216 and Tau protein in the site at Ser202,Ser199,Ser404,Ser214 and Thr231 induced by A?25-35 in N2a cells.N2a cells with good growth were randomly divided into control group,model group,GSK-3?inhibitor 4,6-disubstituted pyrrolopyrimidine?TWS119?group,A?25-35+TWS119 group and three dose SBF treatment group.The control group does not do any processing;The model group was treated with A?25-35 at the final concentration of 35?mol·L-1 for 12 h;The TWS119 group was treated with TWS119 on final concentration of 45nmol·L-1 for 12 hours;The three-dose SBF group was treated with the SBF on the potency of 1.125?g·mL-1,2.25?g·mL-1 and 4.5?g·mL-1,At the same time,SBF group were treated with TWS119,an inhibitor at the final concentration of 45 nmol·L-1 for 12 h and then adding A?25-35 with 35?molL-1 for 12 h;Collecting cells.To detecte the protein expression level of phosphorylated site of GSK-3?at Ser9 and Tyr216 in N2a cells and Tau protein site at Ser202,Ser199,Ser404,Ser214,Thr231 in each group by Western blot.All the data were processed by SPSS19.0 software.The data were expressed as mean±standard deviation?P<0.05?.Results:1 Determination of the damage concentration of A?25-35 to N2a cellsN2a cells were treated by A?25-35 treatment groups with different concentrations(25?mol·L-1,30?mol·L-1,35?mol·L-1,40?mol·L-11 and 45?mol·L-1)for 12 h.Compared with the control group,with the increase of the concentration of A?25-35 cell morphological structure was damaged,the cells became round,cell-to-cell links destroyed,part of the cell aggregation,refractive index decreased,the release amount of LDH gradually increased and then decreased,the cell avibility of cells was gradually decreased.When the concentration of A?25-35 was 35?mol·L-1,the cell membrane folds,atrophy,abscission and even rupture,cell death,cell cell avibility decreased significantly?P<0.01?,the release amount of LDH reached the highest level?P<0.01?,so the final choice of the concentration of A?25-35 is 35?mol·L-1.2 Establishment of the concentration of SBF on N2a cells damaged by A?25-35Compared with the control group,cell membrane damagethe,LDH release increased by 25.02%and cell viability of the model group was decreased by 13.75%.Compared with the model group,the degree of cell damage in different concentrations(1?g·mL-1,2.75?g·mL-1,4.5?g·mL-1,6.25?g·mL-1,8?g·mL-1)of SBF treatment group was significantly reduced.With the increase of the concentration,the refractive index of cells was increased and the aggregation decreased.The release of LDH decreased4.81%,8.53%,11.06%,8.30%and 5.46%respectively.The cell avibility of the cells increased 5.12%,8.14%,8.56%,4.9%and 1.87%respectively.It can be seen from the data that when the concentration of SBF exceeds 4.5?g·mL-1,the release of SBF increases and the cell avibility decreases.The release of SBF increases and the cell survival rate decreases.It may be caused by the toxicity of the impurity in the drug SBF.So,the concentration of SBF should be in the range of 1?g·mL-14.5?g·mL-1.3 Protective effect of SBF on N2a cells injury induced by A?25-35After 12h of A?25-35 treatment,compared with the control group,the model group showed that the cell process disappeared,the cell contour was unclear,the cell membrane was damaged incompletely,even the cells rupture.The LDH released by the cells increased by 17.45%?p<0.01?,and the cell avibility of the cells decreased by 20%?p<0.01?.Compared with the model group,three doses of SBF,reduce the A?25-355-35 damage induced by N2a cell structure,cell processes increased,cell structure is clear,and the medium LDH emissions were reduced by 10.03%?p<0.05?,10.23%?p<0.05?and 11.37%?p<0.05?respectively.Cell cell avibility were increased5.30%?p<0.01?,6%?p<0.01?and 7.43%?p<0.01?respectively.The effects of SBF were dose dependent.4.SBF phosphorylation site of GSK-3?of N2a cells induced by A?25-35,Ser9,Tyr216 and Tau protein expression levels in Ser202,Ser199,Ser404,Ser214,Thr231 sites.Compared with the control group,the protein content of GSK-3??Ser9?in the model group was decreased?p<0.01?,but the protein content of the P-Tau?Thr231?in the model group was not significantly changed and the protein content at the other sites was increased in the model group.The protein content of P-Tau?Ser404?and P-Tau?Ser214?in TWS119 group was decreased?p<0.01?,while the other protein levels were increased or unchanged.Compared with the model group,the expression level of P-Tau?Ser202?,P-Tau?Ser404?,P-Tau?Ser214?and GSK-3??Tyr216?decreased significantly in A?25-35+TWS119 group?p<0.01?.The expression level of histone in A?25-35+TWS119 group of GSK-3??Ser9?,P-Tau?Ser199?and P-Tau?Thr231?was increased or had no obvious change.Compared with the A?25-35+TWS119 group,the protein content of the SBF treatment group of GSK-3??Ser9?increased,while the histone level of SBF treatment at other sites decreased?p<0.01 or p<0.05?.Conclusion:1 The concentration of A?25-35 induced N2a cell damage was established,the concentration of A?25-35 at 35?mol·L-1 could cause obvious damage to N2a cells.2 The concentration range of SBF on N2a cells induced by A?25-35 was established.The concentration of SBF in the range of 1?g·mL-14.5?g·mL-1could improve the damaged N2a cells.3 The protective concentration of SBF on N2a cells induced by A?25-35was 1.125?g·mL-1,2.25?g·mL-1and 4.5?g·mL-14 SBF inhibits the phosphorylation of Tau protein at the sites of Ser202,Ser404 and Ser214,by regulating the activity of GSK-3?.The reduction of Tau protein hyperphosphorylation at the site of Ser202,Ser199 and Thr23 by SBF may be caused by affecting other protein kinases.TWS119,an inhibitor of GSK-3?,had inhibitory effect on normal GSK-3??Ser9?,but not on normal GSK-3??Tyr216?.It inhibited the abnormal GSK-3??Ser9?and GSK-3??Tyr216?.According to the phosphorylation of Tau protein,SBF inhibits the Tau protein phosphorylation at the site of Ser404 and Ser214 is accomplished by the regulation of GSK-3??Ser9?and GSK-3??Tyr216?. |
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