Study On The Neuroprotective Mechanism Of Active Ingredients Of Schisandra Chinensis And Glycyrrhiza Glycyrrhiza And Aminomethylindole Derivatives

Alzheimer’s disease（AD）is by far the most common neurodegenerative disease.AD involves a variety of pathogenic mechanisms,including Aβoligomer neurotoxicity,tau protein hyperphosphorylation,inflammation,neurotransmitter deficiency,oxidative stress,accumulation of unfolded/misfolded proteins,impaired mitochondrial transport,etc.Although the histopathological study of AD has made many breakthroughs and improved people’s understanding of AD,unfortunately,there is no effective treatment for AD up to now.In view of the complex pathogenic mechanism of AD and the unsuccessful experience of developing drugs targeting a single target,the exploration of multi-component and multi-target synergistic treatment of AD or the discovery of multifunctional molecules that can interact with multiple targets has become a new mode of anti-AD drug research.In this paper,we first used network pharmacology to explore the multi-component and multi-target synergistic action material basises and molecular mechanisms of Schisandra chinensis and Glycyrrhiza uralensis against AD,and further verified the neuroprotective effects of their representative active ingredients.Then,inspired by the anti-neuroinflammatory activity of the active ingredients from Schisandra chinensis and the neurite outgrowth-promoting activity of the active ingredients from Glycyrrhiza uralensis,we designed three series of 35 new aminomethylindole derivatives,mainly study their biochemical multifunctional profiles.The main achievements of the paper are as follows:16 active components of S.chinensis with good pharmacokinetics and biological activities were obtained by searching literatures and filtering by oral bioavailability（OB）,drug-likeness（DL）and blood-brain barrier（BBB）of TCMSP.The targets of these active substances were predicted by SEA,STITCH and Swisstarget Prediction databases.The relationships between diseases and targets were explored by CTD,TTD,MAS and DAVID databases,and 51 AD related targets were obtained.Among them,GSK3βis the most critical.DAVID database was used for GO analysis and KEGG analysis.It was indicated that the targets were mainly related to Aβgeneration,tau phosphorylation and inflammation,and the Alzheimer’s disease signal pathway was the most significant enrichmentpathway.Interestingly,the molecular docking results showed the target of schisandrin B was GSK3β,and the binding model of schisandrin B in GSK3βwas determined by DOX calculation.Finally,the protective effects of schisandrin and schisantherin A were experimentally verified by LPS-induced microglia BV2 neuritis model.The results of NO content determination and western blot showed that the two active ingredients could significantly inhibit the production of NO,iNOS and COX in LPS-induced Bv2 cells.Moreover,they also regulate GSK3β（Ser 9）phosphorylation,and inhibit the activityof GSK3β.（2）71 active ingredients of Glycyrrhiza uralensis with good pharmacokinetic and biological activities and 48 AD related targets were obtained by using the same screening method and target prediction method.GO and KEGG analysis showed that the targets of these active ingredients were mainly involved in drug combination,aging,NO biosynthesis,response to lipopolysaccharide,response to hypoxia,redox process,TNF signaling pathways,estrogen signaling pathways,arginine and proline metabolism,and alzheimer’s disease pathways.Finally,we tested the neurite growth-promoting activity of Glycyrrhizal chalcone A by PC12 cells.The results showed that it significantly promoted the growth of nerve processes,and had the NGF-like and NGF-enhancing activities.To further investigate the mechanism,the expression of autophagy marker proteins LC3B Ⅱ and P62 was studied by Western blot assay.The results showed that Glycyrrhizal chalcone A increased the expression of LC3B Ⅱ and P62 in PC12 cells,and it may promote the growth of nerve processes by promoting autophagy.（3）Our initial screening showed that most of the aminomethylindole derivatives potently inhibited lipopolysaccharide（LPS）-stimulated production of NO in microglial cells and potentiated the action of NGF to promote neurite outgrowth of PC12 cells.Interestingly,with outstanding NO/TNF-αproduction inhibition and neurite outgrowth-promoting activities,compounds 8c and 8g were capable of rescuing cells after injury by H₂O₂.Their anti-neuroinflammatory effects were associated with the downregulation of the LPS-induced expression of the inflammatory mediators inducible nitric oxide synthase（iNOS）and cyclooxygenase 2（COX2）.Western blotting and immunofluorescence assay results indicated that the mechanism of their anti-neuroinflammatory actions involved suppression of the MAPK/NF-κB signal pathways.Further studies revealed that another important reason for the high comprehensive anti-neuroinflammatory activity was the anti-COX2 capabilities of the compounds.In all,the active ingredients of Schisandra chinensis and Glycyrrhiza uralensis were obtained and their multi-component and multi-target synergistic action material basises and molecular mechanisms were discussed by the network pharmacology method.Furthermore,the neuroprotective effects of the representative active ingredients was verified by wet experiments.The combination of theory and experiment revealed the molecular basis of Schisandra chinensis and Glycyrrhiza uralensis in the prevention and treatment of AD.Importantly,lead molecules represented by compounds 8c and 8g that have both anti-neuroinflammation and pro-neurite growth activitieswere screened out.All these results suggest that the biochemical multifunctional profile of the compounds provide a new sight for the treatment of AD diseases.