Small Molecule Drugs Troxerutin Regulate Neuronal Autophagy Through Inhibiting The MTORC1 To Protect Against Cerebral Ischemia-reperfusion Injury

Background: Early or even ultra-early drug thrombolysis or mechanical thrombectomy to achieve vascular recanalization and improve collateral circulation in the acute stage of ischemic stroke are currently the most critical and effective methods.However,while the above-mentioned treatments improve tissue perfusion in the ischemic penumbra area,they will also cause unavoidable secondary nerve injury,namely Cerebral Ischemia-Reperfusion Injury(CIRI).Studies have found that in the early stage of CIRI,autophagy can play a neuroprotective role by removing misfolded proteins and/or aging and damaged organelles and preventing neuronal apoptosis.Mammalian Target of Rapamycin Complex 1(mTORC1)is a key protein that regulates autophagy,and its inhibitor rapamycin has been confirmed to have neuroprotective effects in ultra-early and early CIRI.Therefore,mTORC1 is expected to be an important target for the treatment of CIRI,thereby improving the prognosis of patients with ischemic stroke.Object: 1.To screen drugs targeting mTORC1 Computer Aided Drug Screening Technology.2.To verify by in vitro experiments that the drug plays a neuroprotective role in CIRI by inhibiting mTORC1 and activating neuronal autophagy.Methods: 1.Screening of mTORC1 Targeted Drugs Using Computer-Aided Drug Screening Technology(1)First,through the Deep Screening server,mTOR(Mammalian Target of Rapamycin)-targeted drug data sets were used for deep learning to build a drug screening model for mTOR proteins.The model was used to calculate and score drugs in the US Food and Drug Administration(FDA)database.(2)We downloaded the complex of FRB(FKBP-Rapamycin Binding)sequence of the mTORC1 protein crystal structure and the inhibitor rapamycin from the PDB protein database.Through the Libdock module of Discovery Studio 4.5(DS 4.5)software,the drugs with a score higher than 0.9 in the FDA and the control drug rapamycin were docked with mTORC1,and the Libdock score was calculated.(3)The TOPKAT(Toxicity Prediction by Computer Assisted Technology)and ADME(absorption,distribution,metabolism,excretion,absorption)modules of DS 4.5 were conducted to access the toxicological and pharmacological properties of drugs with higher Libdock scores than rapamycin.Subsequently,two of the drugs were selected for further research.(4)The CDOCKER module of DS 4.5 was used to carry out accurate docking analysis of target proteins and small molecule drugs.The affinity and stability of their complexes were evaluated by calculating the CDOCKER interaction energy and the number,type,and bond length of the interacting chemical bonds.(5)Next,the molecular dynamics simulation analysis module of DS 4.5 was applied to analyze the root mean square deviation(RMSD)and potential energy of the ligand-protein complex to evaluate the stability of the complex in the natural environment.In addition,the 3D-QSAR(3D-Quantitative Structure-Activity Relationship)pharmacophore generation module of DS 4.5 was also used to reveal the pharmacophore of small molecule drugs and rapamycin.Finally,considering all the above results comprehensively,an optimal drug was selected for experimental verification in vitro.2.Verify by in vitro experiments that the selected drug could inhibit the mTORC1 to activate neuronal autophagy and played a neuroprotective role in CIRI(1)HT22 cells were used to construct an oxygen glucose deprivation/reperfusion(OGD/R)model.(2)Experiments were grouped into normal control group,OGD/R model group,OGD/R+ small molecule drug group,OGD/R+ rapamycin group,OGD/R+ mTOR activator group,OGD/R+ drug+ mTOR activator group.(3)Western blotting was used to determine the expression levels of mTOR and pmTOR proteins in HT22 neurons in each group.(4)And the survival rate of HT22 neurons in each group was detected by methylthiazolyldiphenyl-tetrazolium bromide(MTT)method.Results: 1.Screening of mTORC1 Targeted Drugs Troxerutin Using Computer-Aided Drug Screening Technology(1)According to the analysis of the mTOR protein screening model constructed by deep learning,a total of 559 drugs in the FDA database had a score higher than 0.9.(2)Through Libdock docking analysis,it was found that 13 FDA drugs had higher Libdock scores than the control drug rapamycin.(3)The pharmacological and toxicological properties of the ADME and TOPKAT modules showed that the 13 small molecule drugs and rapamycin had potential developmental toxicity.Except for S2292,which was carcinogenic to rodents,and S4523 and rapamycin,which were mutagenic,the pharmacological and toxicological properties of other small molecule drugs were within a reasonable range.Considering the above results comprehensively,we chose S2369(Troxerutin)and S4690(Escin)for further research.(3)By CDOCKER analysis,the CDOCKER interaction energies of these two small molecule drugs were lower than those of rapamycin.Compared with rapamycin,both S2369(Troxerutin)and S4690(Escin)formed multiple chemical bonds with mTORC1.(4)The results of molecular dynamics simulation analysis showed that the RMSD and potential energy curves of S2369(Troxerutin)and S4690(Escin)tended to be stable over time like rapamycin.According to pharmacophore analysis of 3D-QSAR module,both S2369(Troxerutin)and S4690(Escin)had more pharmacophores than rapamycin.Since S2369(Troxerutin)had more pharmacophores and more stable RMSD and potential energy curves than S4690(Escin),it was chosen for experimental verification in vitro.2.Verify by in vitro experiments that the selected drug could inhibit the mTORC1 to activate neuronal autophagy and played a neuroprotective role in CIRI(1)The results of western blotting showed that compared with the OGD/R group,the OGD/R+ Troxerutin group(P ﹤ 0.05)and the OGD/R+ Troxerutin+ mTOR activator group(P﹤0.05)had significantly higher expression level of mTOR protein.Compared with OGD/R group,OGD/R+ rapamycin group(P ﹤ 0.01),OGD/R+ Troxerutin group(P﹤0.01)and OGD/R+ Troxerutin+ mTOR activator group(P﹤0.01)significantly reduced the ratio of p-mTOR/mTOR,the phosphorylation and activation level of mTOR protein.(2)The results of MTT test showed that compared with the control group,the relative survival rate of cells in the OGD/R group was significantly lower(P﹤0.0001);compared with the OGD/R group,the relative cell survival rate of the OGD/R+ Troxerutin group was significantly higher(P﹤0.05);compared with the OGD/R+ rapamycin group,the relative cell survival rate of the OGD/R+ Troxerutin group was significantly higher(P﹤0.01).Conclusion: The small molecule drug Troxerutin is an ideal targeted mTORC1 inhibitor,and can enhance neuronal cell autophagy by regulating mTORC1 to play a neuroprotective role in CIRI.