| Alzheimer's disease?AD?is one kind of neurodegenerative diseases,it is the most commen type of dementias and manifestied as memory loss and other cognitive impairments in clinic,often accompanied with mental and behavior changes.Senile plaques deposited with?amyloid?A??and neurofibrillary tangles deposited with hyperphosphorylated tau protein are the major pathological changes of AD,but the therapeutic strategies against A?and tau protein have not get any success until now.Autophagy is widely existed in eukaryotic cells and plays an important role in cytosolic material degradation,it can degrade misfolded or damaged long-lived proteins,macromolecular protein and cell organelles,and keep material and energy balance in cells.Mammalian target of Rapamycin?mTOR?is the most important signaling pathway in the regulation of autophagy,hightly activation of mTOR signaling pathway and lysosomal dysfunction that caused the inhibition of autophagy function and the deposition of misfolded proteins may be the important pathological basis and contribute to the occurrence and development of AD.In this study,we transfected APP and PS1 genes to the HEK293 cells to eatablish an AD cell model,and further observed the regulating effects and mechanisms of Fuzheng Quxie Fang?FQF?-an small Chinese herbal medicine formular,on the autophagy function of AD model cells.This may contribute to the understanding of the mechanisms of FQF on the treatment of AD.Objectives:To investgate the effectiveness of FQF on regulation of autophagy in AD model cells,and further detect the mechanisms of this function.Methods:Liposome transfection method and lipofectamine 2000 transfection reagent were used for gene transfection,puromycin and G418 were used as the screening antibiotics for the obtaining of HEK293 cells that expressing APP695 and PSEN1dE9genes.Amyloid precursor protein?APP?and presenilin 1?PS1?were detected by western blot and A?1-40 and A?1-42 were detected by enzyme linked immunosorbent assay?ELISA?after the gene transfention.Fourty Sprague Dawley?SD?rats were used to get serum containing FQF,rats were devided in four groops randomly,FQF high does group?18g/kg/d,crude drugs?,FQF medium does group?9g/kg/d,crude drugs?,FQF low does group?1.8g/kg/d,crude drugs?and control group?same volume of distilled water?,ten in each group,drugs were intragastric administrated for 7consecutive days at the does of 1mL/100g/d,blood was drawed through abdominal aorta to obtain serum,brain homogenate of the rats of FQF medium group and FQF high dose group were also made and freezed in-80?.Liquid chromatography coupled with tandem mass spectrometry?LC-MS/MS?were used to detecte the active components in the serum containing FQF and the brain homogenate of rats.Rapamycin were used as the positive control drug,the experimental cells were divided into 6 groups:control group,model group,rapamycin group,FQF low does group?FQF-L?,FQF medium does group?FQF-M?,FQF high does group?FQF-H?.Cell growth states of each group were observed under inverted microscope,transmission electron microscopy was used to observe the autophagic vacuoles and mitochondria in each group,cell counting kit-8?CCK8?were used to detect the cell viability,alteration of cell cycle was defected by flow cytometry,A?levels of each group were detected by ELISA,the protein and mRNA expression in each group were detected by western blot and real time PCR.Results:1 4?g/m L of puromycin and 1000?g/m L of G418 were used to screen positive cells after the APP and PSEN1 gene transfection,cell morphology changes were not obvious after the gene transfection.Compared with the HEK293 cells,the APP and PS1 protein levels in the APP and APP-PS1 HEk293 cells were increased significantly?P<0.05?,A?1-40 and A?1-42 levels in the cell culture supernatant also increased significantly?P<0.01?.2 Many active components have been detected from serum containing FQF,among them,ginsenoside Rg1?Re?Rb1?Rd?Rc and Rb2/3 were originated from Ginseng?Panax ginseng C.A.Mey?,berberine,palmatine,worenine,protopine,epiberberine,coptisine and jatrorrhizine were originated from Rhizoma Coptidis?Coptis chinensis Franch?,ferulic acid was originated from Rhizoma Chuanxiong?Ligusticum chuanxiong Hort?.Active components detected from brain homogenate include ginsenoside Rb1,berberine,palmatine,worenine,protopine,epiberberine,coptisine,jatrorrhizine,dehydrocorydaline and tetrahydroJatrorrhizine.3 The cell viability were not affected significantly as the concentrations of rapamycin was 0-100nM,when the concentrations of rapamycin passed 100nM,the cell viability decreased significantly compared with the 0nM group?P<0.01?.The blank serum can impair the cell viability as the concentration of it were higher than 10%?P<0.05,P<0.01?.4 The cell viability and the S phase ratio of the model group were decreased significantly compared with the control group?P<0.05,P<0.01?,10%serum containing FQF could increase the cell viability and the S phase ratio compared with the model group?P<0.05,P<0.01?,along with the increase of FQF dosage,the cell viability and the S phase ratio increased.The amount of autophagic vacuoles increased and the mitochondria impairment aggravated in the model group cells compared with the control group cells,rapamycin and FQF could alleviate these changes.5 In APP HEK293 cells,the levels of LC3II decreased and P62 increased at the 24h phase and 48h phase in model group compaired with the control group?P<0.01?,the levels of LC3II increased and P62 decreased in rapamycin,FQF-M and FQF-H group?P<0.05,P<0.01?,there is no significant difference compared with the rapamycin group and FQF groups in the expression of LC3II protein?P>0.05?.Rapamycin,FQF-M and FQF-H dosage could decrease the levels of A?1-42 in the cell culture supernatant compaired with the model group?P<0.01?,there is no significant difference compared with the rapamycin group and FQF groups?P>0.05?,with the increase of the FQF dosage,the expression of A?1-42 decreased gradually.6 In APP-PS1 HEK293 cells,the levels of LC3II and P62 increased,rapamycin and FQF could decrease the expression of them?P<0.05,P<0.01?,FQF decrease the levels of LC3II and P62 with dose-effect relationship.The mRNA levels of LC3 and P62decreased in APP-PS1 HEK293 cells?P<0.01?,rapamycin and FQF could increase the expression of them?P<0.05?.The protein and m RNA levels of Beclin 1 in APP-PS1HEK293 cells decreased compared with the control cells,but the levels of Bcl-2protein and mRNA increased?P<0.01?,FQF could increase the expression of the protein and mRNA levels of Beclin 1,decrease the expression of the protein and mRNA levels of Bcl-2?P<0.05,P<0.01?.7 In APP-PS1 HEK293 cells,the levels of mTOR and p-mTOR protein increased,so as the mRNA level of mTOR?P<0.05,P<0.01?,rapamycin and FQF could inhibite the expression of mTOR,p-mTOR protein and m TOR mRNA?P<0.05,P<0.01?,there is no significant difference compared with the rapamycin group and FQF groups?P>0.05?,and there got a dose-effect relationship in the influence of FQF in the levels of mTOR,p-mTOR protein and mTOR mRNA.Rapamycin and FQF could not influence the levels of p70S6K protein and mRNA levels in APP-PS1 HEK293 cells.The 4EBP1 protein levels in the rapamycin group and FQF-H group increased?P<0.05?,but no significant changes of the levels of 4EBP1 mRNA were found in different treating groups?P>0.05?.8 In APP-PS1 HEK293 cells,LAMP1 protein level was not changed,there is a rising trend of the levels of LAMP1 in FQF groups,but no significant difference were found between between the model group and the FQF groups?P>0.05?.Cathepsin D and V-ATPase levels were decreased significantly in APP-PS1 HEK293 cells?P<0.05?,FQF could increase the levels of Cathepsin D and V-ATPase?P<0.05,P<0.01?,and with the increase of the dosage of FQF,the levels of Cathepsin D and V-ATPase increased gradually.Cathepsin D and V-ATPase levels got a increased trends in rapamycin group compared with the model group.9 In APP-PS1 HEK293 cells,the levels of A?1-42 in rapamycin group and FQF groups decreased?P<0.01?,there is no significant difference compared with the rapamycin group and FQF groups?P>0.05?,and there got a dose-effect relationship in the influence of A?1-42-42 levels of FQF.Conclutions:FQF can upregulate the autophagy function and the cell viabilities of the AD model cell lines,the mechanisms may related to the inhibition of m TOR signaling pathway and the ameliorate of the lysosomal functions,this may contribute to the therapeutic mechanisms of FQF in the treatment of AD. |
PDF Full Text Request