The Process And Signal Pathway Of Eliminating β-amyloid Protein By Polygala Tenuifolia Extract

Background β-amyloid (Aβ) hypothesis occupies an important position in thepathogenesis research of Aleheimer’s disease (AD) at present. Aβ peptide may lead to aseries of pathological change as the formation of amyloid plaques,hyperphosphorylation of tau protein, neurofibrillary tangles (NFT), neuroninflammation, disfunction of neuron and death as well as dementia. Decreasing thegeneration of Aβ and promoting it’s elimination are the target to delay the progress ofpathology in many researching drugs on anti-AD. Autophagy as the main way ofabnormal body to clear away abnormal substance, has aroused researcher’s extensiveattention, which may become a significant therapeutic strategy to AD.Object The purpose of this study to investigate the effects of Polygala TenuifoliaExtract (PTE) on Aβ peptide levels secreated by Chinese hamster ovary(CHO) cellswhich has transfected with APP and BACE1in vitro, and the role of autophagy has alsodiscussed in further researches of this change as well as signal pathway transformation.Methods Cells were cultured in100mm dishes after resuscitated, change the cuturemedium next day and cell subculture when the cell density reached to80%-90%. Cellswere seeded in culture plate when in the logarithmic phase from the third generation.Cells were treated with different concentration of PTE（0,5,10,20,40,100μg/ml）after adherenced,0μg/ml was considered the control group and investigations wasprocessed as follows: (1) MTT was used to test the effects of vability to SH-SY5Y by PTE. In addition, theprotection effects of PTE was also discussed with the toxicity of exogenous Aβ peptide;(2) ELISA was used to investigate the effects of PTE on Aβ peptide levels whichsecreted by CHO-APP/BACE1cells;(3) The change of autophagy vesicles in SH-SY5Y was observed by fluorescencemicroscope after MDC staining;(4) The expression of autophagy marker protein LC3was investigated by Western blot;(5) Autophay related signal pathway kinases mTOR, p70s6k, Akt, AMPK, Raptor aswell as their phosphorylation protein were tested by Western blot.Results (1) MTT result showed that there is no significant variation betwen PTEtreatment group and control group(P>0.05);(2) The viability of Aβ treatment group had significant decreased compared with that ofcontrol group, indicated that Aβ peptide had notably toxicity to neurone, but the neuroneviability had reverted in a certain extent with the help of PTE when the concentration ofPTE achieved to100μg/ml;(3) The levels of Aβ which were secreated by CHO-APP/BACE1cells had significantreduced in10μg/ml treatment group（P<0.01）, and with dose-dependent manner;(4) The rounded lightspot in cytoplasm which was stained by MDC is autophagyvesicles, had significant increased in PTE treatment group, which was observed byfluorescence microscope;(5) Western blot result indicated that LC3had transformed from type I to type II, andautophagy marker LC3II/LC3I had enhanced along with PTE concentration;(6) Western blot result showd that the phosphorylation protein levels of autophagyinhibited kinase mTOR reduced gradually as well as it’s downstream target p70s6k.Phosphorylation protein levels of AMPK and Raptor had increased, which was locatedupstream of mTOR and was able to inhibit mTOR. Otherwise, there was no obvious change of phosphorylation protein levels of Akt, which was capable of activate mTORin different group.Conclusion PTE has no obvious impact on the viability of SH-SY5Y and has protectioneffect in some extent when the concentration under100μg/ml. Western blot result showthat PTE may inhibit mTOR and p70s6k by activate AMPK/Raptor pathway in neuron,result in autophagy enhanced, The autophagy pathway to promote excessive Aβ cellsremoved, then decreased the secreation of Aβ.