The Role And Mechanism Of Endoplasmic Reticulum Stress Regulation In Modified Shuyu Pills To Reduce Neuronal Apoptosis In App/PS1 Double Transgenic Mice

ObjectiveThe propose of this study is to discuss the pathogenesis of Alzheimer's disease from the perspectives of yin and yang,deficiency,phlegm,and stasis,and to clarify the theoretical basis of treating AD with Modified Shuyu pill?MSP?.From the perspectives of neuronal apoptosis induced by excessive endoplasmic reticulum stress,we explored the effect of the learning and memory of APP/PS1 double transgenic mice through behavioral testing after the intervention of MSP,exploiting real-time quantitative PCR,immunofluorescence,transmission electron microscopy,and western blotting to evaluation the effects of MSP protect affection neural cell apoptosis though regulating the endoplasmic reticulum-related degradation and autolysosome pathway induced by excessive endoplasmic reticulum stress,and the possible mechanism of the addition and subtraction of MSP.Methods1.Theoretical exploration:Systematic review and analysis of ancient and modern doctors'cognition of AD from the perspectives of pathological state deficiency by yin and yang,deficiency,sputum,and stasis.From the point of view of group analysis,previous research results of the team and the modern pharmacological mechanism of the drug ingredients in the formula to clarify the theoretical basis of treating AD with MSP.2.Experiment 1 MSP regulates Nrf2 to inhibit excessive endoplasmic reticulum stress and decrease apoptosis in APP/PS1 double transgenic mice through the PI3K/AKT/GSK3?pathwayIn vivo experiments:Twenty APP/PS1 double transgenic mice were randomly divided into model group and MSP group,10 littermate wild-type mice were used as control group.The MSP was given MSP 14g/kg/d.intragastrically daily and the control and the model were intragastrically administered with the same amount of saline for four weeks.After drug intervention,Morris water maze and new object recognition test were used to examination the learning and memory ability of mice.And then,brain sections were obtained by perfusion to observe the level of Nissl bodies in hippocampal neurons;TUNEL was used to observe hippocampal neuron apoptosis;fresh hippocampal tissue was taken for Western Blot and real-time quantitative PCR detection.Western blot was used to detect GRP78,PERK,p-PERK,eIF2?,p-eIF2?,CHOP,Cleaved Caspase-3,and Nrf2 protein expressions,and real-time quantitative PCR was used to detect Nrf2,Hrd1,VCP,and Os9mRNA levels.The level of A?₄₂2 was detected by ELISA.In vitro experiments:Neonatal mice?within 0-24h?delivered by APP/PS1pregnant mice,which tails were subjected to PCR amplification to identify APP/PS1 homozygous background and wild type background mice for primary neuron culture.Immunofluorescence staining was used to identify the purity of primary neurons.The MTT method was used to detect the effects of different concentrations of MSP on cell viability.APP/PS1 homozygous background primary neurons were divided into model group,MSP group,MSP+tunicamycin group,MSP+PI3K/Akt pathway inhibitor group,wild-type mouse primary neurons were used as the control group;control group and model group were given 10%blank serum,MSP group was given 5%MSP-containing serum intervention.Tunicamycin Group and PI3K/Akt inhibitor group were supplemented with 2mg/L tunicamycin and 10?mol/L LY294002 on the basis of drug-containing serum respectively.The protein expressions of GRP78,PERK,p-PERK,eIF2?,p-eIF2?,CHOP,Cleaved Caspase-3,p-Akt,Akt,GSK3?and Nrf2 were detected by Western blot.3.Experiment 2:MSP regulates TFEB-mediated autophagy lysosome pathway to reduce excessive endoplasmic reticulum stress in APP/PS1 neurons.In vivo experiment:the grouping and intervention were the same as experiment 1.fresh hippocampus was fixed with glutaraldehyde and examined by transmission electron microscopy.the expression of p62,LAMP1,CSTB,LC3II/LC3I and TFEB protein inside and outside the nucleus were detected by Western Blot.In vitro experiment:the primary neurons of APP/PS1 homozygous background mice were divided into model group,MSP group,MSP+chloroquine group,wild type mouse primary neurons as control group;MSP group was given 5%MSP containing serum,and MSP+chloroquine group was added 25?mol/L chloroquine on the basis of drug-containing serum.After12-24 hours of drug intervention,Western Blot were used to detected the protein expression levels of p62,LAMP1,CSTB,LC3II/LC3I,GRP78and p-PERK/PERK,CHOP,Cleaved Caspase-3;TFEB for primary neurons were detected by immunofluorescence assay.Results.Experiment one.In vivo:1.Morris water maze test:the escape latency of mice in each group was gradually shortened on the day of navigation,compared with the model group,the escape latency in the MSP group was shorter than the model group on the fourth day?P<0.05?.Compared with the model group,the times of crossing the platform and the proportion of residence time in the original platform quadrant in the MSP group were significantly higher than the model group in the spatial exploration experiment?P<0.01?.2.New object recognition test:the time of exploring new things in the APP/PS1 model group was lower than the control group,while after the intervention of MSP,the time of exploring new objects and the recognition index were increased.3.Nssil staining:The Nissl bodies of hippocampal nerve cells in the control group were blue-purple,the nucleus was blue and stained clearly;the number of Nissl bodies in APP/PS1 mice decreased and the staining was light;compared with the model group,the Nissl bodies in the MSP group was increased and stained clearly.4.TUNEL staining:Compared with the control group,the apoptosis rate was increased in the model group,and the apoptosis rate of hippocampal neurons CA1 and DG in the MSP group was decreased.5.Western Blot detection:compared with the control group,the expression of GRP78,p-PERK/PERK,p-eIF2?/eIF2?,CHOP and Cleaved Caspase-3 in the model group were increased significantly?P<0.05,P<0.01?.Compared with the model group,the expression of GRP78,p-PERK/PERK,p-eIF2?/eIF2?,CHOP and Cleaved Caspase-3 in the MSP group were down-regulated?P<0.05,P<0.01?.The expression of Nrf2 in the model group was significantly lower than the control group?P<0.05?.Compared with the model group,the expression of Nrf2 in MSP group was significantly increased.6.Real-time fluorescence quantitative PCR detection:Compared with the control group,the expressions of Nrf2,Hrd1,VCP and Os9 mRNA in the model group were significantly decreased,while Nrf2,Hrd1,VCP and Os9mRNA were up-regulated in the MSP group.The results of ELISA showed that the level of A?42 in the hippocampus of the model group was higher than the control group,and that was significantly decreased after the intervention of MSP?P<0.01?.In vitro:Western Blot:the expression of p-Akt/Akt and Nrf2 in MSP group was significantly higher than the model group?P<0.05,P<0.01?,and the level of p-Akt/Akt and Nrf2 in PI3K/Akt pathway inhibitor group was lower than the MSP group?P<0.05,P<0.01?.Compared with the model group,the expression of GSK3?in the MSP group was significantly decreased,and the level of GSK3?in the PI3K/Akt pathway inhibitor group was higher than the MSP group.Compared with the model group,the expressions of GRP78,p-PERK/PERK,p-eIF2?/eIF2?,CHOP and Cleaved Caspase-3 in MSP group were decreased in different degree?P<0.05,P<0.01?,while the levels of GRP78,p-PERK/PERK,p-eIF2?/eIF2?,CHOP and Cleaved Caspase-3 were increased in tunicamycin group compared with MSP?P<0.05,P<0.01?.Experiment 2:In vivo:Transmission electron microscopy:compared with the control group,the number of autophagy-lysosome in the model group was increased,and after the intervention of MSP,the number of autophagy-lysosomes in the hippocampus was significantly decreased.Western Blot:Compared with the control group,the levels of LC3II/LC3I and p62 in the hippocampus of the model group were increased?P<0.05,P<0.01?,the levels of CTSB and LAMP1 was decreased(?P<0.05?.Compared with the model group,there was no significant difference in LC3II/LC3I.the expression of LAMP1 and cathepsin B protein was increased while the expression of p62 was down-regulated in the MSP?P<0.05,P<0.01?.Compared with the model group,the level of TFEB in the nucleus of the MSP group was increased significantly?P<0.01?,but the level of total TFEB had no significantly difference?P>0.05?.in vitro:Immunofluorescence:compared with the model group,the expression of TFEB in the nucleus of the model group increased slightly,while the fluorescence intensity in the nucleus of TFEB increased significantly after the intervention of MSP.Western Blot detection:Compared with the control group,the levels of LC3II/LC3I and p62 in the model group was increased?P<0.05,P<0.01?,the levels of CTSB and LAMP1 was decreased;Compared with the model group the expression of LAMP1 and CTSB were increased and the expression of p62 was decreased?P<0.01?,while there was no significantly difference in LC3II/LC3I in the MSP group.Compared with MSP group,the expression of LC3II/LC3I,P62 in MSP plus chloroquine group was significantly increased,while the increasing trend after the intervention of MSP serum of LAMP1 and CTSB protein expression was significantly inhibited by chloroquine.Compared with the MSP group,the expression of GRP78,p-PERK/PERK,CHOP,Cleaved Caspase-3 was increased after adding chloroquine.Conclusion1.Deficiency of spleen and kidney and mutual accumulation of phlegm and blood stasis are the basic pathogenesis of AD.The treatment of invigorating the spleen,tonifying the kidney,resolving phlegm and removing blood stasis with MSP has a rich theoretical basis.2.MSP improved the ability of learning and memory of APP/PS1 double transgenic mice and reduced neuronal apoptosis.The mechanism may be is that upregulates the expression of Nrf2 through the PI3K/Akt/GSK3?pathway,increases the expression of ERAD-related genes,reduced the level of A?₄₂2 in the hippocampus and reduces excessive ERS.3.MSP increased the nuclear translocation of TFEB,promote ALP,and alleviate the neuroprotective effect of ERS.