(-)-Epicatechin Ameliorats Learning And Memory Deficits By Activation Of Autophagy Via Raptor/MTORC1 Signaling In The P301S Mouse Model

Objective: Exploring new drugs that promote tau protein degradation can help to provide new ideas for the treatment of tauopathies.Methods: HEK293 cells were transfected with p EGFP-C1-P301S-tau plasmid and were treated with(-)-Epicatechin(100 μM)for 24 h.The expression levels of tau protein were detected by western blotting.In 7-month-old P301 S transgenic mice and littermate control mice,the expression levels of tau protein were detected by western blotting,immunohistochemistry and immunofluorescence after continuous intragastric administration of(-)-Epicatechin or the same volume of normal saline for 2 months.Tau m RNA were detected by q RT-PCR.Co-treated Chloroquine or Bafilomycin A1 with(-)-Epicatechin and detected the expression of tau protein in vitro.After the p EGFP-C1-P301 Stau plasmid were transfected in HEK293 cells,the expression levels of LC3-II and P62 proteins were detected.The effect of(-)-Epicatechin on autophagosome formation and autophagic flux were detected after m Cherry-LC3 or GFP-RFP-LC3 plasmid were transfected respectively.The expression levels of LC3-II and P62 proteins in P301 S mice treated with(-)-Epicatechin were detected.The number of autophagosomes were shown by transmission electron microscopy.Proteomics technology was applied to expore possible targets of(-)-Epicatechin in P301 S mice.Western blotting was used to detect the effects of(-)-Epicatechin on autophagy-related target proteins and autophagy pathways at the overall level.The mechanism of(-)-Epicatechin degrading tau protein by regulating autophagy was further explored in vitro.Proteomics technology was applied to expore possible targets of(-)-Epicatechin in P301 S mice.Western blotting was used to detect the effects of(-)-Epicatechin on autophagy-related target proteins and autophagy pathways at the overall level.The mechanism of(-)-Epicatechin degrading tau protein by regulating autophagy was further explored in vitro.Synaptic plasticity,neuroinflammation and spatial memory deficits were detected in P301 S transgenic mice.Results:(1)After overexpression p EGFP-C1-P301S-tau plasmid in HEK293 cells and treatment with(-)-Epicatechin(100 μM,24h),the expression levels of tau protein were decereased(p<0.05).After continuously giving(-)-Epicatechin by gavage in 7-month-old P301 S transgenic mice for 2 months,the expression levels of tau protein in the hippocampus and cortex of P301 S mice were decreased(p<0.05).q RT-PCR showed that(-)-Epicatechin did not affect the expression level of tau m RNA in vitro and in vivo(p>0.05).After using Cycloheximide to inhibit the synthesis of proteins,it was found that(-)-Epicatechin could promote the degradation of tau(p<0.05).It was found that Chloroquine or Bafilomycin A1 could inhibit the degradation of tau protein induced by(-)-Epicatechin(p>0.05).P62 protein expression level was increased after overexpression of p EGFP-C1-P301S-tau plasmid(p<0.01),however,P62 decreased(p<0.05)and LC3-II increased(p<0.01)after(-)-Epicatechin treatment.After transfection of m Cherry-LC3 and GFP-RFP-LC3 plasmids,respectively,it was found that(-)-Epicatechin promoted autophagosome formation(p<0.05)and activated autophagic flux(p<0.01).Western blotting and transmission electron microscopy showed that(-)-Epicatechin promoted the formation of autophagosomes in vivo(p<0.05).(2)Proteomics suggested that(-)-Epicatechin down-regulated Raptor and inhibited mTORC1.Western blotting showed that the expression of Raptor was increased in the P301 S models(p<0.05,p<0.0001),mTORC1 was activated(p<0.0001,p<0.0001),and the autophagy pathway was inhibited.(-)-Epicatechin decreased the expression level of Raptor in P301 S model in vitro and in vivo(p<0.001,p<0.001),inhibited mTORC1(p<0.05,p<0.0001),and activated autophagy.After transfection of p LKO.1-sh RNA-Raptor plasmid in HEK293/P301 S cells,autophagy was activated(p<0.001)and the degradation of tau protein was promoted.After overexpression of p RK5-HA-Raptor in HEK293/P301 S cells,autophagosome formation was inhibited(p<0.001).(3)(-)-Epicatechin improved synaptic plasticity and reduced neuron loss(p<0.05).(-)-Epicatechin reduced neuroinflammation in vivo(p<0.05).Behavioral tests shown that(-)-Epicatechin ameliorats learning and memory deficits in P301 S transgenic mice(p<0.05).Conclusion:(-)-Epicatechin can activate autophagy and clear aggregated tau protein.(-)-Epicatechin may be a potential candidate drug for inducing autophagy.Conclusion:(-)-Epicatechin could activate autophagy and clear aggregated tau protein by targeting the Raptor/mTORC1 pathway.(-)-Epicatechin improves learning and memory impairment in P301 S transgenic mice.(-)-Epicatechin may be a potential candidate drug for adjuvant treatment of tauopathies.