| Alzheimer’s disease(AD)is a common neurodegenerative disease.The most obvious performance of the patient is that the irreversible decline of memory for people and things and cognitive function in daily life.Old age is the main cause of the disease,so this disease is also used to called Senile Dementia.Existing studies have shown that the pathological changes of AD are due to the formation of extracellular senile plagues and intracellular neurofibrillary tangles.The formation of senile plaques caused by abnormal aggregation of amyloid β-protein and pathological hyperphosphorylation of tau protein in cells are the basis for forming neurofibrillary tangles.Aβ1-42 has a significant injury effect on neuronal microglia,which can induce the occurrence and development of neuroinflammation.Tau is widely present in human neurons,its single gene expression and the only effective coding region are located on autosomal chromosome 17.As one of the microtubule related proteins,Tau protein can effectively polymerize and stabilize microtubules in the cycle of cell division and maturation,which plays an important role in the growth and movement of nerves,and plays a more obvious role in the axons of nerve cells.Pathological Aβ can induce protein phosphokinase(such as PKA,GSK3β)and protein phosphatase(such as PP-1,PP2A)to fold correctly,and the induced enzyme after this misfolding can cause hyperphosphorylation of Tau protein.During the occurrence and development of AD,Aβ was deposited continuously,and then the pathological changes of cells such as autophagy and apoptosis were induced.Because the aggregation of Aβ is the upstream mechanism of Tau protein lesion,and the lesion of Tau protein promotes the aggregation of Aβ through feedback mechanism,the production of Aβ and the phosphorylation of Tau protein complement each other,and they are closely related to the occurrence and development of AD.Mouse neuroblastoma N2 a cells(N2a cells)can gradually differentiate into cell lines and no longer have the characteristics of tumor cells.This cell can produce a large number of tubulin and act as a contractile system in nerve cells.Our laboratory has previously found that Aβ25-35 has toxic effect on N2 a cells,and its toxic fragment is one of the main mechanisms of AD,which will lead to hyperphosphorylation of Tau protein in the brain of AD patients.Therefore,N2 a cells were selected in this study to explore the pathogenesis of AD and to provide a new contract point for clinical treatment of AD.Scutellaria barbata flavonoids(SBF)was isolated extracts from the aerial parts of Scutellaria barbata D.Don.Through the previous study in our laboratory,it was found that the drug concentration gradient was more effective in the range of 1.125 mg/L,2.25 mg/L and 4.5 mg/L.With the deepening of the pharmacological mechanism of SBF,its anti-tumor,anti-Alzheimer’s,anti-body oxidation and lipid-lowering effects have been gradually applied to the treatment of clinical diseases.A large number of literature studies have shown that the phosphorylation level of Tau protein is closely related to the attack rate of AD.In our previous study,it was found that Aβ25-35 could effectively induce the injury of N2 a cells,and the most critical role of SBF in the process of Aβ25-35 induced injury of N2 a cells was the obvious inhibition of Tau protein phosphorylation.In this study,Aβ25-35 was used to establish a quasi-AD model.The phosphorylation of Tau proteins at Ser199,Ser214,Ser404,Thr231 and Thr205 sites in N2 a cells and the activities of PKA and PP-1 were detected and regulated to explore the mechanism of SBF on Tau protein phosphorylation induced by Aβ25-35 in N2 A cells.Objective:Protein phosphokinase(PKA)inhibitor H-89 and protein phosphatase(PP-1)inhibitor OA were used to investigate the damage effects of Aβ25-35 on N2 a cell,and the abnormal phosphorylation of Tau protein,and the intervention mechanism of SBF.Methods:1 N2 a Cell Culture: N2 a cells were cultured in high glucose DMEM complete culture medium,which contains 10% FBS and 100μL/m L penicillin and placed in 37 ℃ and 5% CO2 incubator.The cells were adhered growth and the culture medium was replaced in each day.The cells were performed to passage when their density reaches the confluence degree of 80%-90%.2 Determination of Lactate Dehydrogenase(LDH)Release amount in N2 a Cells: LDH kit was used to detect each group of N2 a cell fluid.3 MTT assay for survival rate of N2 a cells: Add 100μL of MTT solution with a final concentration of 0.5mg/L per well.After the incubation time,discard the MTT solution,100μL DMSO per well,and finally,detect it with a microplate reader.4 Cell Grouping: Cells with better growth status were randomly divided into 7 groups: control group,H-89 group,model group,the Aβ25-35+H-89 group,the SBF low dose(1.125 mg/L)treatment group,the SBF medium dose(2.25 mg/L)treatment group and the SBF high dose(4.5 mg/L)treatment group.5 Cell Collection: After each group of cells by centrifugal,the supernatant discarded,to add the appropriate amount of PBS,centrifugal again,will the supernatant discarded again,finally the cells were stored in a-80 °C refrigerator.6 Extraction Protein: 150μL of cell lysate was added to each group of cells,and lysed on ice for 20 min before they were centrifuged.The supernatant was taken as the protein.7 BCA Method Protein Quantification: Corresponding working solution was added to each group of samples as required,after the incubation at 37 °C,for cooling,the detection was carried out with a microplate reader.8 Protein Denaturation: The remaining sample was added to 5× protein loading buffer at 4:1,denatured with 100 °C boiling water for 5 min,cooling,and stored in a-20 °C refrigerator.9 The expression of phosphorylated protein and the protein expression levels of PKA and PP-1 in Ser199,Ser214,Ser404,Thr231 and Thr205 of each group of N2 a cells were detected by Western blot.The regulation of SBF on Tau hyperphosphorylation was studied by Western blot.Results:1 Study by H-89 and OA on the injury of N2 a cells induced by Aβ25-35 and the intervention of SBFAs shown in Figure 1,Compared with the control group,the cell membrane of the model group was incomplete,the cells appeared agglomerated,and the cell growth was slow.As shown in Figure 2,Compared with the model group,the three-dose SBF drug group had better cell state,faster cell growth rate and enhanced refractive index.(2)Compared with the control group,the cell group of the model group was irregular in shape and the cell membrane was incomplete.Compared with the model group,the cell morphology of the three-dose SBF drug group was fusiform,and the cells were full of bottle bottom and enhanced refractive index.2 Study by H-89 and OA on LDH release and survival rate of N2 a cells induced by Aβ25-35 and the intervention of SBF2.1 Study by H-89 and OA on LDH release of N2 a cells induced by Aβ25-35 and the intervention of SBFAs shown in Figure 3,Compared with the control group,the release of LDH in the N2 a cells of the model group was significantly increased(p<0.01).Compared with the model group,the release of LDH in the N2 a cells of the Aβ25-35+H-89 group was decreased(p<0.01);Compared with the Aβ25-35+H-89 group,the LDH release of N2 a cells in the SBF 1.125 mg/m L group and the SBF 2.25 mg/m L group decreased(p<0.01),and the LDH release of N2 a cells in the SBF 4.5 mg/m L group increased(p<0.01).As shown in Figure 4,Compared with the control group,the release of LDH in the N2 a cells of the model group was increased(p<0.01).Compared with the model group,the release of LDH in the N2 a cells of the Aβ25-35+OA group was significantly lower(p<0.01);and compared with the Aβ25-35+OA group,The LDH release of N2 a cells in SBF 1.125 mg/m L group,SBF 2.25 mg/m L group and SBF 4.5 mg/m L group were significantly decreased(p<0.01).2.2 Study by H-89 and OA on the survival rate of N2 a cells induced by Aβ25-35 and the intervention of SBFAs shown in Figure 5,Compared with the control group,the survival rate of N2 a cells in the model group was significantly decreased(p<0.01).Compared with the model group,the survival rate of N2 a cells in the Aβ25-35+H-89 group was significantly increased(p<0.01);and compared with the Aβ25-35+H-89 group,the survival rate of N2 a cells in the SBF 1.125 mg/m L group and the SBF 2.25 mg/m L group was significantly increased(p<0.01),the survival rate of N2 a cells in the SBF 4.5 mg/m L group was decreased(p<0.01).As shown in Figure 6,Compared with the control group,the survival rate of N2 a cells in the model group was significantly decreased(p<0.01).Compared with the model group,the survival rate of N2 a cells in the Aβ25-35+OA group was significantly increased(p<0.01);and compared with the Aβ25-35+OA group,the survival rate of N2 a cells in SBF 1.125 mg/m L group,SBF 2.25 mg/m L group and SBF 4.5 mg/m L group were significantly increased(p<0.01).3 Study by H-89 and OA on the mechanism of SBF inhibiting the abnormal phosphorylation of Tau protein induced by Aβ25-35 in N2 a cells3.1 Study by H-89 and OA on the effect of p-Tau(Ser199)induced by Aβ25-35 in N2 a cells and the intervention of SBFAs shown in Figure 7,compared with the control group,the expression level of phosphorylated Tau protein at Ser199 site in N2 a cells increased by 95.61%(p<0.01)in the H-89 group,and the model group protein expression level increased by 1.18 times(p<0.01).Compared with the model group,the expression level of Aβ25-35+H-89 group was decreased by 22.31%(p<0.01).Compared with Aβ25-35+H-89 group,the expression levels of SBF low and medium dose groups respectivelydecreased by 22.34%(p<0.01)and 29.63%(p<0.01),while the SBF high dose group expression level increased by 4.55%(p>0.05).As shown in Figure 8,compared with the control group,the expression level of PP-1 protein in N2 a cells increased by 13.98% in the model group(p>0.05),and the expression level increased by 34.28% in the OA group(p<0.05).Compared with the model group,the protein expression levels increased by 13.09% in the Aβ25-35+OA group(p>0.05).Compared with the Aβ25-35+OA group,the expression level of protein increased by 25.80% in the SBF low dose treatment group(p>0.05),and decreased by 43.51% in the SBF medium dose treatment group(p<0.05),and decreased by 13.24% in the SBF high dose treatment group(p>0.05).3.2 Study by H-89 and OA on the effect of p-Tau(Ser214)induced by Aβ25-35 in N2 a cells and the intervention of SBF As shown in Figure 9,compared with the control group,the expression level of phosphorylated Tau protein at Ser214 site in N2 a cells increased by 32.32%(p<0.01)in the H-89 group,and the model group protein expression level increased by 53.81%(p<0.01).Compared with the model group,the expression level of Aβ25-35+H-89 group was decreased by 18.82%(p>0.05).Compared with Aβ25-35+H-89,the expression levels of SBF three dose groups were respectivelydecreased by 28.29%,20.02% and 34.22%,with significant significance(p<0.01).3.3 Study by H-89 and OA on the effect of p-Tau(Ser404)induced by Aβ25-35 in N2 a cells and the intervention of SBFAs shown in Figure 10,compared with the control group,the expression level of phosphorylated Tau protein at Ser404 site in N2 a cells in the H-89 group increased by 20.94%(p<0.01),and the model group increased by 31.66%(p<0.01).Compared with the model group,the expression level of Aβ25-35+H-89 group was decreased by 26.87%(p<0.01).Compared with Aβ25-35+H-89 group,the SBF low dose group expression level increased by 2.15%(p>0.05),while the expression levels of SBF low and medium dose groups decreased by 2.49% and 13.57%(p>0.05),respectively.As shown in Figure 11,compared with the control group,the expression level of PP-1 protein in N2 a cells increased by 22.72% in the model group(p<0.05),and the expression level increased by 25.10% in the OA group(p>0.05).Compared with the model group,the protein expression levels increased by 21.40% in the Aβ25-35+OA group(p<0.05).Compared with the Aβ25-35+OA group,the expression level of protein decreased by 6.53% in the SBF low dose treatment group(p<0.05),and decreased by 28.70% in the SBF medium dose treatment group(p<0.01),and decreased by 50.10% in the SBF high dose treatment group(p<0.01).3.4 Study by H-89 and OA on the effect of p-Tau(Thr231)induced by Aβ25-35 in N2 a cells and the intervention of SBFAs shown in Figure 12,compared with the control group,the expression level of phosphorylated Tau protein at Thr231 site in N2 a cells increased by 32.24%(p<0.01)of the H-89 group,and the model group protein expression level increased by 54.19%(p<0.01).Compared with the model group,the expression level of Aβ25-35+H-89 group was decreased by 21.92%(p<0.05).Compared with Aβ25-35+H-89 group,SBF low dose group expression level decreased by 7.10%(p>0.05),and the expression levels of SBF medium and high dose groups increased by 16.86%(p<0.01)and 5.79%(p>0.05),respectively.As shown in Figure 13,compared with the control group,the expression level of PP-1 protein in N2 a cells increased by 14.23% in the model group(p>0.05),and the expression level increased by 11.21% in the OA group(p>0.05).Compared with the model group,the protein expression levels decreased by 5.23% in the Aβ25-35+OA group(p>0.05).Compared with the Aβ25-35+OA group,the expression level of protein increased by 20.60% in the SBF low dose treatment group(p<0.01),and increased by 2.43% in the SBF medium dose treatment group(p>0.051),and decreased by 24.26% in the SBF high dose treatment group(p<0.01).3.5 Study by H-89 and OA on the effect of p-Tau(Thr205)induced by Aβ25-35 in N2 a cells and the intervention of SBFAs shown in Figure 14,compared with the control group,the expression level of PP-1 protein in N2 a cells decreased by 2.29% in the model group(p>0.05),and the expression level increased by 10.39% in the OA group(p<0.05).Compared with the model group,the protein expression levels increased by 2.26% in the Aβ25-35+OA group(p>0.05).Compared with the Aβ25-35+OA group,the expression level of protein increased by 16.14% in the SBF low dose treatment group(p>0.05),and increased by 6.99% in the SBF medium dose treatment group(p>0.05),and decreased by 2.86% in the SBF high dose treatment group(p>0.05).4 Study by H-89 and OA on the protein expression level of PKA and PP-1 in N2 A cells induced by Aβ25-35As shown in Figure 15,compared with the control group,the PKA protein expression level in N2 a cells in the H-89 group was decreased by 23.77%(p>0.05),and the model group protein expression level increased by 1.88 times(p<0.01).Compared with the model group,the protein expression level of Aβ 25-35 +H-89 was decreased by11.64%(p<0.01).Compared with Aβ 25-35 +H-89 group,the low dose group expression level of SBF decreased by 20.91%(p>0.05),and the expression level of SBF medium and high dose groups increased by8.41%(p>0.05)and 5.72%(p>0.05),respectively.As shown in Figure 16,compared with the control group,the expression level of PP-1 protein in N2 a cells decreased by 0.05% in the model group(p>0.05),and the expression level decreased by 21.65% in the OA group(p<0.05).Compared with the model group,the protein expression levels decreased by 39.22% in the Aβ25-35+OA group(p<0.01).Compared with the Aβ25-35+OA group,the expression level of protein increased by 1.95 times in the SBF low dose treatment group(p<0.01),and increased by 89.11% in the SBF medium dose treatment group(p<0.01),and increased by 1.29 times in the SBF high dose treatment group(p<0.01).Conclusion:1 Aβ25-35 could increase the expression level of PKA protein in N2 a cells of AD model,enhance PKA activity,and also can increase the expression level of Tau protein phosphorylation at Ser199,Ser214,Ser404,Thr231 sites in each group of N2 a cells.2 SBF can decrease the phosphorylation expression level of Tau protein induced by Aβ25-35 at Ser199,Ser214,Ser404,Thr231 sites in N2 a cells by down-regulating PKA activity.3 Aβ25-35 can decrease the expression level of PP-1.And the decrease of PP-1 activity could increase the expression level of Tau protein phosphorylation at Ser199,Thr205,Ser404,Thr231 sites in each group of N2 a cells.4 SBF can up-regulate the activity of PP-1 to decrease the expression level of Tau protein phosphorylation induced by Aβ25-35 at Ser199,Thr205,Ser404,Thr231 sites in N2 a cells.5 SBF can reduce the injury of N2 a cells induced by Aβ25-35 and increase its survival rate,and decrease the hyperphosphorylation of Tau protein at Ser199,Ser214,Ser404,Thr231 and Thr205 sites.And the mechanism of action is achieved by decreasing the activity of PKA and increasing the activity of PP-1. |
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