四氢紫堇萨明对AD细胞模型Tau蛋白磷酸化的影响及其可能机制研究
|
摘要
研究四氢紫堇萨明对Aβ_(25-35)诱导的阿尔茨海默病细胞模型Tau蛋白磷酸化的影响及其可能作用机理。以神经细胞PC-12为载体,Aβ_(25-35)诱导48 h建立AD细胞模型,MTS试剂盒检测细胞活力,激光共聚焦显微镜观察细胞核和细胞微管变化情况,Western blot法检测蛋白表达水平。结果显示,四氢紫堇萨明可显著增强模型细胞活力,改善微管形态,降低p-Tau(Ser396)和p-GSK-3β(Tyr216)表达水平,升高p-GSK-3β(Ser9)和p-AKT表达水平(P <0.05);PI3K抑制剂LY294002可部分阻断四氢紫堇萨明对模型细胞的上述改善作用。以上结果表明四氢紫堇萨明可显著改善AD细胞模型Tau蛋白过度磷酸化,从而改善细胞骨架微管形态,增强细胞活力,其机理可能与激活PI3K/Akt信号通路,降低GSK-3β活性,改善蛋白激酶/蛋白磷酸酯酶系统失衡相关。
To investigate the effect of tetrahydropyrimin(SQZJSM) on the phosphorylation of Tau protein in Alzheimer's disease(AD) cell model and its possible mechanisms.AD cell model was established using PC-12 cell induced by Aβ_(25-35) for 48 h.Cell viability was detected by MTS,the changes of nuclei and microtubules were observed by laser confocal microscopy,the expression of protein was detected by Western blot.The results showed that SQZJSM can improve the cell viability and microtubule morphology of model cells,reduce the expression of p-Tau(Ser396) and p-GSK-3β(Tyr216) and up-regulate the expression of p-GSK-3β(Ser9) and p-AKT significantly(P<0.05).PI3 K inhibitor LY294002 can partial block the improvement of SQZJSM on model cells.The above results indicate that tetrahydrocorysamine can significantly reduce the hyperphosphorylation of Tau protein in AD cell model,thereby improving the morphology of cytoskeletal microtubules and enhancing cell viability.The mechanism may be related to activating PI3K/Akt signaling pathway,decreasing GSK-3β activity and improving the imbalance of protein kinase/protein phosphatase system.
To investigate the effect of tetrahydropyrimin(SQZJSM) on the phosphorylation of Tau protein in Alzheimer's disease(AD) cell model and its possible mechanisms.AD cell model was established using PC-12 cell induced by Aβ_(25-35) for 48 h.Cell viability was detected by MTS,the changes of nuclei and microtubules were observed by laser confocal microscopy,the expression of protein was detected by Western blot.The results showed that SQZJSM can improve the cell viability and microtubule morphology of model cells,reduce the expression of p-Tau(Ser396) and p-GSK-3β(Tyr216) and up-regulate the expression of p-GSK-3β(Ser9) and p-AKT significantly(P<0.05).PI3 K inhibitor LY294002 can partial block the improvement of SQZJSM on model cells.The above results indicate that tetrahydrocorysamine can significantly reduce the hyperphosphorylation of Tau protein in AD cell model,thereby improving the morphology of cytoskeletal microtubules and enhancing cell viability.The mechanism may be related to activating PI3K/Akt signaling pathway,decreasing GSK-3β activity and improving the imbalance of protein kinase/protein phosphatase system.
引文
1 Calderon-Garcidue1as AL,Duyckaerts C.Alzheimer disease[J].Handb Clin Neurol,2017,145:325-337.
2 Wang SS,Zhang Z,Zhang X,et al.Advances in immunotherapy of Alzheimer’s disease with traditional Chinese medicine[J].Acta Pharm Sin(药学学报),2018,53:1023-1029.
3 Maccioni RB,Munoz JP,Barbeito L,et al.The molecular bases of Alzheimer’s disease and other neurodegenerative disorders[J].Arch Med Res,2001,32:367-381.
4 Blennow K,De Leon MJ,Zetterberg H.Alzheimers disease[J].Lancet,2006,368:387-403.
5 Khoury R,Patel K,Gold J,et al.Recent progress in the pharmacotherapy of Alzheimer’s disease[J].Drugs&Aging,2017,34:811-820.
6 Zhang HB,Sun Y.Effect of huperzine A on the expression of amyloidβ-protein in transgenic mice brain of Alzheimer’s disease[J].Nat Prod Res Dev(天然产物研究与开发),2017,29:290-293.
7 Wang H,Bi FJ,Lin T,et al.RP-HPLC fingerprint of corydalis yanhusuo and content determination of nine alkaloids[J].J Chin Med Materls(中药材),2017,40:624-629.
8 Yao B,Hou B,Li WY,et al.Effects and mechanism of tetrahydrocorysamine on apoptosis of AD cell model iduced by Aβ25-35[J].Nat Prod Res Dev(天然产物研究与开发),2019,31(1):49-54.
9 Liang JQ,Lyu ZM,Tian SY,et al.Application of tetrahydrocyanin in the preparation of drugs for the treatment of senile dementia(四氢紫堇萨明在制备治疗老年性痴呆药物中的应用):ZL201210098959.4[P].2012-04-06.
10 Weissmann C,Reyher HJ,Gauthier A,et al.Microtubule binding and trapping at the tip of neurites regulate tau motion in living neurons[J].Traffic,2009,10:1655-1668.
11 Bakota L,Brandt R.Tau biology and Tau-directed therapies for Alzheimer’s disease[J].Drugs,2016,76:301-313.
12 Barage SH,Sonawane KD.Amyloid cascade hypothesis:pathogenesis and therapeutic strategies in Alzheimer’s disease[J].Neuropeptides,2015,52:1-18.
13 Wang J,Jing Y,Song L,et al.Neuroprotective effects of Wnt/β-catenin signaling pathway against Aβ-induced tau protein over-phosphorylation in PC12 cells[J].Biochem Biophys Res Commun,2016,471:628-632.
14 Oliveira JM,Henriques AG,Martins F,et al.Amyloid-βmodulates both AβPP and Tau phosphorylation[J].J Alzheimers Dis,2015,45:495-507.
15 Jayapalan S,Natarajan J.The role of CDK5 and GSK-3βkinases in hyperphosphorylation of microtubule associated protein tau(MAPT)in Alzheimer’s disease[J].Bioinformation,2013,9:1023-1030.
16 Li XH,Lyu BL,Xie JZ,et al.AGEs induce Alzheimer-like tau pathology and memory deficit via RAGE-mediated GSK-3activation[J].2012,7:1400-1410.
17 Qin JQ,Shen XY.Functions of protein phosphatase 2A subunits[J].Prog Phys Sci,2011,3:229-233.
18 Liu GP,Wei W,Zhou X,et al.Silencing PP2A inhibitor by Lenti-shRNA interference ameliorates neuropathologies and memory deficits in tg2576 mice[J].Mol Ther,2013,12:2247-2257.
19 Liu R,Zhou XW,Tanila H,et al.Phosphorylated PP2A(tyrosine 307)is associated with Alzheimer neurofibrillary pathology[J].J Cell Mol Med,2008,1:241-257.
20 Xu ZP.Role of glycogen synthase kinase-3βin Alzheimer’s disease and type 2 diabetes mellitus[D].Wuhan:Huazhong University of Science and Technology(华中科技大学),2015.
21 Wang CH,Chen T,Li GX,et al.Simvastatin preventsβ-amyloid 25-35-impaired neurogenesis in hippocampal dentate gyrus throughα7nAChR-dependent cascading PI3K-Akt and increasing BDNF via reduction of farnesyl pyrophosphate[J].Neuropharmacology,2015,97:122-132.
2 Wang SS,Zhang Z,Zhang X,et al.Advances in immunotherapy of Alzheimer’s disease with traditional Chinese medicine[J].Acta Pharm Sin(药学学报),2018,53:1023-1029.
3 Maccioni RB,Munoz JP,Barbeito L,et al.The molecular bases of Alzheimer’s disease and other neurodegenerative disorders[J].Arch Med Res,2001,32:367-381.
4 Blennow K,De Leon MJ,Zetterberg H.Alzheimers disease[J].Lancet,2006,368:387-403.
5 Khoury R,Patel K,Gold J,et al.Recent progress in the pharmacotherapy of Alzheimer’s disease[J].Drugs&Aging,2017,34:811-820.
6 Zhang HB,Sun Y.Effect of huperzine A on the expression of amyloidβ-protein in transgenic mice brain of Alzheimer’s disease[J].Nat Prod Res Dev(天然产物研究与开发),2017,29:290-293.
7 Wang H,Bi FJ,Lin T,et al.RP-HPLC fingerprint of corydalis yanhusuo and content determination of nine alkaloids[J].J Chin Med Materls(中药材),2017,40:624-629.
8 Yao B,Hou B,Li WY,et al.Effects and mechanism of tetrahydrocorysamine on apoptosis of AD cell model iduced by Aβ25-35[J].Nat Prod Res Dev(天然产物研究与开发),2019,31(1):49-54.
9 Liang JQ,Lyu ZM,Tian SY,et al.Application of tetrahydrocyanin in the preparation of drugs for the treatment of senile dementia(四氢紫堇萨明在制备治疗老年性痴呆药物中的应用):ZL201210098959.4[P].2012-04-06.
10 Weissmann C,Reyher HJ,Gauthier A,et al.Microtubule binding and trapping at the tip of neurites regulate tau motion in living neurons[J].Traffic,2009,10:1655-1668.
11 Bakota L,Brandt R.Tau biology and Tau-directed therapies for Alzheimer’s disease[J].Drugs,2016,76:301-313.
12 Barage SH,Sonawane KD.Amyloid cascade hypothesis:pathogenesis and therapeutic strategies in Alzheimer’s disease[J].Neuropeptides,2015,52:1-18.
13 Wang J,Jing Y,Song L,et al.Neuroprotective effects of Wnt/β-catenin signaling pathway against Aβ-induced tau protein over-phosphorylation in PC12 cells[J].Biochem Biophys Res Commun,2016,471:628-632.
14 Oliveira JM,Henriques AG,Martins F,et al.Amyloid-βmodulates both AβPP and Tau phosphorylation[J].J Alzheimers Dis,2015,45:495-507.
15 Jayapalan S,Natarajan J.The role of CDK5 and GSK-3βkinases in hyperphosphorylation of microtubule associated protein tau(MAPT)in Alzheimer’s disease[J].Bioinformation,2013,9:1023-1030.
16 Li XH,Lyu BL,Xie JZ,et al.AGEs induce Alzheimer-like tau pathology and memory deficit via RAGE-mediated GSK-3activation[J].2012,7:1400-1410.
17 Qin JQ,Shen XY.Functions of protein phosphatase 2A subunits[J].Prog Phys Sci,2011,3:229-233.
18 Liu GP,Wei W,Zhou X,et al.Silencing PP2A inhibitor by Lenti-shRNA interference ameliorates neuropathologies and memory deficits in tg2576 mice[J].Mol Ther,2013,12:2247-2257.
19 Liu R,Zhou XW,Tanila H,et al.Phosphorylated PP2A(tyrosine 307)is associated with Alzheimer neurofibrillary pathology[J].J Cell Mol Med,2008,1:241-257.
20 Xu ZP.Role of glycogen synthase kinase-3βin Alzheimer’s disease and type 2 diabetes mellitus[D].Wuhan:Huazhong University of Science and Technology(华中科技大学),2015.
21 Wang CH,Chen T,Li GX,et al.Simvastatin preventsβ-amyloid 25-35-impaired neurogenesis in hippocampal dentate gyrus throughα7nAChR-dependent cascading PI3K-Akt and increasing BDNF via reduction of farnesyl pyrophosphate[J].Neuropharmacology,2015,97:122-132.