Study On The Mechanism Of Protection Of P-15,an Active Component Of Tongluo Xingnao Effervescent Tablets,in Alzheimer' Dissease Through Autophagy-lysosome Pathway

Screening of active components from Tongluo Xingnao effervescent tablets based on AD cell modeObjective:SH-SY5Y cells injured by H₂O₂ or A?_25-355-35 were used as AD cell model and to screen the active components of the Tongluo Xingnao effervescent tablet,which had the protective effect of nerve cells.We determined the target active compound through preliminary results in vitro so that lay the foundation for the follow-up study.Method:Viability of SH-SY5Y cells treated with H₂O₂?50,100,150,200,400,500,600and 800uM?for 2,4,8,12h was determined by MTT assay and the optimum concentration and time of H₂O₂ injuring cells was chosen.Viability of SH-SY5Y cells treated with Oligomer A?_25-35?5,10,30,60 and 100uM?for 24 or 48h was determined with MTT assay and the optimum concentration and time of H₂O₂ injuring cells was chosen.Cells were pretreatedwith components from Tongluo Xingnao effervescent tablets and other sources?12.5,25,50uM?for 24h and then was injured by 20uM A?_25-35(IC₅₀)for 24h.Cell viability was determined by MTT assay.After screening and verification repeatedly,the active components from Tongluo Xingnao effervescent tablet which has protective effects on cells injured by H₂O₂ or A?_25-355-35 determined to be used for follow-up study.Results:H₂O₂ decreased cell viability in a dose-dependent manner and the IC₅₀0 of H₂O₂injuring cells for 2h was 500uM.After 3 times blind screening,it was found that the protective effect of GHX-13,P-15 and GHC-6 on cells injured by H₂O₂ was clear and stable.A?_25-355-35 decreased cell viability in a dose-and time-dependent manner and the IC₅₀0 of A?_25-355-35 injuring cells for 24h was 500uM.After 2 times blind screening,it was found that the protective effect of P-15 and GHC-6 on cells injured by A?_25-355-35 was clear and stable.By comparing the source of these compound and blinded revelation P-15 was from the ingredients of Tongluo Xingnao effervescent tablet,so P-15 is the active component of Tongluo Xingnao effervescent tablet,which is used for follow-up study.Conclusion:Multiple blinded screening found that P-15 derived from the Tongluo Xingnao effervescent tablet has a clear protective effect on the SH-SY5Y cells damaged by H₂O₂ and A?_25-35,so that the follow-up study will be carried out with P-15 as the research object.Regulation and mechanism of P-15 on autophagy lysosomal pathway in SH-SY5Y cells injured by A?_25-35Objective:Verify the neuroprotective effect of P-15 on the autophagy-lysosomal pathway and on the mTOR/TFEB signing pathway in the SH-SY5Y cells injured by A?_25-355-35 and reveal the molecular mechanism of the regulation of the anti-AD.Method:1.autophagy flux arrest block was involved in the abnormal accumulation of autophagic vacuoles mediated by A?_25-351.2 Study on the time dependent changes of autophagic vacuoles in SH-SY5Y cells damaged by A?_25-35SH-SY5Y cells were treated with A?_25-355-35 for 0 h,3 h,6 h,12 h,24 h and 48h.The autophagic vesicles were detected by MDC staining,and the expression level of the protein level was detected by Western Blot.1.3 Correlation between abnormal accumulation of autophagic vacuoles and autophagy overactivation in SH-SY5Y cells injured by A?_25-35After treating cell for 2h with 6.25¹00uM pretreated cells,the cells were given20uM A?_25-355-35 or 24h in culture medium,and cell activity was determined by MTT method,and the effect of the maximum non-toxic concentration of CQ and CQ on the cell activity of A?_25-355-35 damaged cells was determined.The cells were set into 4 groups:?1?normal group?without any treatment?,group CQ?cells pretreated with 25uM CQ for 2h?,25uM CQ group?20uM?,A?_25-35+CQ group?cells pretreated with 25uM CQ for 2h?.2.Protective effect of P-15 in SH-SY5Y cells injured by A?_25-355-35 and regulation of autophagy lysosomal pathway2.1 Protective effect of P-15 in SH-SY5Y cells injured by A?_25-353.125⁴00uM P-15 was used to treat 24h and 48h respectively.The cell viability was determined by MTT method and the maximum non-toxic concentration of P-15was determined.The cells were set up as normal group?without treatment?,A?_25-35(20uMA?_25-35),groupP-15(20uMA?_25-35+3.125⁵0uMP-15),(20uM A?_25-35+3.125⁵0uM P-15),P-15 pretreated cells 24h and 48h.The best time and concentration range of P-15 in the protective effect of damaged cells was determined;The morphology of cells was observed by inverted microscope,and the effect of P-15 on the cell morphology of A?_25-355-35 damaged cells was observed.2.2Effects of P-15 on autophagic vacuoles accumulation in SH-SY5Y cells injured by A?_25-35The cells were set up as normal group?without treatment?,A?_25-355-35 group(20uM A?_25-35),and P-15 group(20uM A?_25-35+6.25⁵0uM P-15),.The cells of each group were treated with 3,6,12,24,48h,and MDC staining method was used to detect the average fluorescence intensity of MDC.2.3 Effect of P-15 on lysosomal function in SH-SY5Y cells injured by A?_25-35The cells were set up as normal group?no treatment?,A?_25-35(20uM A?_25-35),group P-15(20uM A?_25-35+50uM P-15),.EBSS control Group?EBSS,30min?,CQ control group?25uM CQ,2h?,P-15 pretreated cell 24h,and 20uM A?_25-355-35 injury was given.Lyso-Tracker fluorescence probe was used to detect lysosome number and Lyso-Tracker average fluorescence intensity.2.4 Effects of P-15 on autophagy flux in SH-SY5Y cells injured by A?_25-35The cells were set as normal group?no treatment?,A?_25-35(20uM A?_25-35),P-15group(A?_25-35+6.25⁵0uM P-15)and P-15 pretreated cell for 24h and 20uM A?_25-35damage was given after P-15 pretreatment,Western Blot was used to detect LC3-II/I ratio and P62/SQSTM1 protein level.SH-SY5Y cells were transfected with mCherry-GFP-LC3 adenovirus?MOI=2?,and the transfected cells were divided into 1normal group?without any treatment?,(20uM A?_25-35),and group P-15(50uM P-15+20uM A?_25-35).After the cells were treated for 24h,the effect of P-15 on autophagic flux was observed under laser confocal microscopy.3.Effects of P-15 on cell viability of SH-SY5Y cells injured by A?_25-355-35 with CQ pretreatment.The cells were set up as normal group?without any treatment?,group CQ?25uM CQ pretreated for 2h?,group A?_25-35,P-15+A?_25-355-35 group+CQ group?25uM CQ preprocessing for 2h?.Cell viability was detected by MTT assay.4.The effect of P-15 on mTOR/TFEB pathway in SH-SY5Y cells injured by A?25-35Cells were set up as normal group?without treatment?,A?_25-35(20uM A?_25-35),P-15 group(20uM A?_25-35+6.25⁵0uM P-15),and P-15 pretreated cells for 24h were set up and 20uM A?_25-355-35 given after P-15 pretreatment,and the protein level of TFEB in nucleus or total protein,the p-m TOR/mTOR was detected by Western Blot.Resuts:1.autophagy flux arrest block was involved in the abnormal accumulation of autophagic vacuoles mediated by A?_25-351.1 Study on the time dependent changes of autophagic vacuoles in SH-SY5Y cells damaged by A?_25-3520uM A?_25-355-35 enhanced the average fluorescence intensity of MDC in time-dependent?p<0.05 or p<0.01?,increased LC3-II/I ratio?p<0.05 or p<0.01? and P62/SQSTM1 protein level?p<0.05 or p<0.01?,suggesting that A?_25-355-35 increased time-dependent autophagic vesicles in cell cytoplasm.1.2 Correlation between abnormal accumulation of autophagic vacuoles and autophagy overactivation in SH-SY5Y cells injured by A?_25-35 6.25²5uM CQ had no significant effect on the cell activity of the normal group?p>0.05?,but in the dose range,CQ was dose-dependent and A?_25-355-35 induced the decrease of cell activity?p<0.01?.Compared with the normal group,25uM CQ pretreated 2H or A?_25-35cell 24h,which could significantly increase the LC3-II/I ratio?p<0.01?.Compared with the A?_25-355-35 group,the LC3-II/I ratio of A?_25-35+CQ group was not significantly different?p>0.05?,suggesting that the abnormal aggregation of autophagic mediated by A?_25-355-35 was not related to autophagic activation.2.Protective effect of P-15 in SH-SY5Y cells injured by A?_25-355-35 and regulation of autophagy lysosomal pathway2.1 Protective effect of P-15 in SH-SY5Y cells injured by A?_25-35The drug concentration of P-15 acted on the cell 24h and 48h in the 3.125⁵0uM interval,and had no effect on the activity of SH-SY5Y cells?p>0.05?.When the dosage was 100⁴00uM,the cell activity decreased significantly?p<0.01?,and the concentration of P-15 was within the range of 6.25⁵0uM,and there was a significant protective effect on the viability damage induced by A?_25-35?p<0.05 or p<0.01?can significantly antagonize cell morphologic changes such as cell body crinkle,cell membrane rupture,large cell space,particulate matter deposition and increased vacuolated structure in the cytoplasm.2.2Effects of P-15 on autophagic vacuoles accumulation in SH-SY5Y cells injured by A?_25-35Compared with the normal group,A?_25-355-35 increased the average fluorescence intensity of MDC in time dependence?p<0.05 or p<0.01?.Compared with the A?_25-35group,P-15 action 12h and 24h significantly lowered the MDC average fluorescence intensity?p<0.05 or p<0.01?.2.3 Effect of P-15 on lysosomal function in SH-SY5Y cells injured by A?_25-35Compared with the A?_25-355-35 group,P-15 could obviously increase the level of Cathepsin D?p<0.05 or p<0.01?.Compared with the normal group,EBSS could increase the average fluorescence intensity of LysoTracker?p<0.01?by starvation,and CQ and A?_25-355-35 reduced the average fluorescence intensity of LysoTracker?or0.01?.The average fluorescence intensity of LysoTracker decreased?p<0.05?.2.4 Effects of P-15 on autophagy flux in SH-SY5Y cells injured by A?_25-35Compared with the A?_25-355-35 group,P-15 could obviously reduce the LC3-II/I ratio and the expression level of P62/SQSTM1 protein?p<0.05 or p<0.01?.mCherry-GFP-LC3 adenovirus was successfully transfected to SH-SY5Y cells.Compared with the blank group,the yellow spot ratio of the cells in the A?_25-355-35 group could be observed to decrease,and P-15 could obviously reduce the number of yellow spots and increase the red number.The number of spots increased and the ratio of red/red yellow spots increased,suggesting that P-15 can promote autophagy flux.3.Effects of P-15 on cell viability of SH-SY5Y cells injured by A?_25-355-35 with CQ pretreatmentAfter CQ pretreatment for 2h blocked the lysosomes,compared with the P-15+A?_25-355-35 group,P-15 increased cell activity?p<0.05?,suggesting that P-15 increased the cell activity induced by A?_25-355-35 and increased the function of lysosome.4.The effect of P-15 on mTOR/TFEB pathway in SH-SY5Y cells injured by A?25-35Compared with blank group,A?_25-355-35 had no significant effect on the level of TFEB in the total cells?p>0.05?,but could significantly reduce the TFEB protein level?p<0.05 or p<0.01?and increase the p-mTOR/mTOR ratio?p<0.01?.Compared with the A?_25-355-35 group,P-15 had no significant effect on the TFEB level in the total cells?p>0.05?,but could not significantly affect the level of TFEB in the total cells?p>0.05?.Significantly increased the level of TFEB protein?p<0.05 or p<0.01?and decreased the p-mTOR/mTOR ratio?p<0.05 or p<0.01?.Conclusion:20uM A?_25-355-35 induced abnormal accumulation of autophagic vacuoles in the cytoplasm of SH-SY5Y cells in time dependent manner.The abnormal accumulation of autophagic vacuole mediated by A?_25-355-35 is independent of autophagy and is related to autophagic flow arrest.P-15 has an obvious protective effect on A?_25-355-35 induced cell damage,and its effective concentration is 6.25⁵0uM.It can partially restore lysosome function through the activation of mTOR/TFEB signaling pathway,promote autophagic flow to mediate the removal of autophagic vesicles,and play a protective role in the SH-SY5Y cell model of A?_25-355-35 damage.Effects of P-15 on learning and memory ability of APP/PS1 transgenic mice and the regulation of autophagy lysosome pathway Objective:The effects of P-15 on the learning and memory function of APP/PS1 transgenic mice,the pathological morphology of hippocampal CA1 area,the ultrastructure of hippocampal tissue and the A?_25-355-35 in the neurons were investigated,and the mechanism of promoting the neuroprotective effect of autophagic lysosome pathway through mTOR/TFEB pathway was investigated to verify the results of the fine cell study.Method:3month-old male APP/PS 1 transgenic mice were randomly divided into group APP/PS1 and P-15 group,and 5 of C57 mice of homosexual age were taken as normal control group.Group P-15 was given P-15?40 mg/kg?,normal control group and APP/PS 1 group were given equal volume of normal saline?NS?,once a day for 6months,using darkness for 6 months.Morris and water maze were used to detect learning and memory ability in mice.HE staining was used to detect the pathomorphology of CA1 region in the hippocampus of mice;the ultrastructure of hippocampus was observed by electron microscopy;A?plaque deposition in hippocampus was measured by immunohistochemistry;Western blot method was used to determine the protein level of LC3-II/I,the level of Cathepsin D protein,the level of TFEB protein in the total cell and the nucleus of the total cell and the p-mTOR/mTOR ratio.Results:Compared with the normal control group,the escape latency of the APP/PS1 mice was shortened?p<0.05?and the number of errors was not significantly different ?p>0.05?.The escape latency of the mice in the Morris water maze was obviously prolonged?p<0.05?,the number of passing through the original platform decreased significantly?p<0.05?,and the percentage of the swimming time in the target quadrant was clear.There was a significant decrease?p<0.05?;the vertebra cells in the hippocampal CA1 area were arranged sparsely and the nuclei were deeply dyed and contracted.The ultrastructure showed that the organelle dissolved in the cytoplasm,the postsynaptic membrane expanded significantly,the myelinated nerve fiber axons showed demyelination,the A?deposition area in the neuron increased,and the LC3-II/I and P62/SQSTM1 protein in the hippocampus tissue The expression level increased?p<0.05?,the expression of Cathepsin D decreased?p<0.05?,the total TFEB level in brain tissue was not significantly affected?p>0.05?,but the expression level of TFEB protein in the nucleus was significantly decreased?p<0.05?,and the ratio of p-mTOR/mTOR in the brain tissue was significantly increased?p<0.05?.Compared with the APP/PS1 group,the dark latency of dark avoidance test in group P-15 was significantly shorter?p<0.05?.Although the number of errors had a decreasing trend,there was no statistical significance?p>0.05?;the escape latency of Morris water maze was significantly shortened?p<0.05?,the number of crossing platform increased significantly?p<0.05?,and the target swimming time was 100points.The degree of pathological changes in hippocampal CA1 area and the ultrastructure of hippocampal tissue were reduced,the A?deposition of neurons in the hippocampus decreased,the protein expression level of LC3-II/I and P62/SQSTM1 in the brain tissue decreased significantly?p<0.05?,the expression level of Cathepsin D increased?p<0.05?,and the TFEB expression level of total brain protein in the brain tissue was significantly increased in the hippocampus?p<0.05?.There was no significant change?p>0.05?,but the TFEB protein level of the nucleus was significantly enhanced?p<0.05?,and the p-mTOR/mTOR ratio of brain tissue was significantly decreased?p<0.05?.Conclusion:P-15 improved the learning and memory function of APP/PS1 transgenic mice,the pathological morphology of the hippocampal CA1 and the ultrastructural damage in the hippocampus,and reduced the A?deposition in the neurons,inhibited the phosphorylation of mTOR,improved the nuclear transcriptional level of TFEB to restore the function of the lysosome,and promote the autophagy-lysosome pathway in Alzheimer'disease.The effect is consistent with the results of cell studies.