Study On The Effective Constituents And Mechanism Of Huperzia Serrata Against Alzheimer's Disease

Huperzia serrata(H.serrata)known as Shezushishan,beloging to Huperziaceae,has been used clinically as a folk medicine for improving blood circulation,contusion,strain,swelling,and schizophrenia.Phytochemical studies showed that alkaloids,triterpenes,diterpenes,flavonoids are main secondary metabolites of H.serrata.Among them,huperzine A(Hup.A)has been successfully used to treat Myasthenia Gravis and Alzheimer's disease(AD).In this study,the chemical constituents of H.serrata were systematically investigated.The alleviating effect on AD of Serratinine B is achieved by decreasing the receptor-interacting serine/threonine-protein kinase 1(RIPK1),interfering with the NF-?B signaling pathway,and inhibiting the phosphorylation level of Tau protein.The specific results are as follows:1)Identification of the chemical constituents of H.serrata: 64 compounds have been isolated and identified from the chloroform and ethyl acetate fractions of H.serrata.Among them,10 new compounds and 24 known compounds were isolated from H.serrata for the first time.2)Exploring and mechanism of H.serrata in the treatment of AD and screening the potential active compounds based on system pharmacology: Twenty compounds from H.serrata can transport the blood-brain barrier(BBB)with DL>0.18 were regarded as the potential active components in H.serrata.20 potential compounds with corresponding degree value greater than 3 were selected.16 direct targets such as MAPT,NF-?B,ACHE,BCHE,IL1 B,IMPDH2and TNF were found.Some compounds such as Huperzine M,Serratinine,Lycopodine and Lycobeline C,can regulate the activity of BCHE,ACHE,NOS,NF-?B,IL-1B and TNF,thus indicating that some of the potential active substances can be used to intervene in Alzheimer's disease through these targets.The enrichment of signal pathways showed that these compounds can regulate amino acid metabolism pathway,phosphatidylinositol pathway,and steroid hormone synthesis pathway,inflammatory-mediated TLR pathway and NF-?B signaling pathway.The above results show that some components may slow down the AD process by regulating multiple pathways.3)The in vitro cell experiments showed that the expression levels of IL-1?,IL-6,TNF-?,ROS and NO could be significantly down-regulated by Serratinine B and UHPLC-ESI-HR/MS metabolomics showed that Serratinine B significantly interfered with the biosynthesis and metabolic pathways of sphingolipids,?-aminobutyric acid and hormones in nerve cells.The phosphorylation levels of NF-?B,p38 MAPK,JNK and ERK phosphorylation level were also significantly inhibited,while Serratinine B had no significant effect on the total protein expression level.4)The in vivo behavioral experiments showed that Serratinine B can improve memory,learning and behavioral disorders in mice.Activated astrocytes and microglia were significantly inhibited by Serratinine B,and the expression of inflammatory factors and oxidative stressrelated factors were significantly down-regulated.Serratinine B also significantly interfered with energy metabolism and amino acid metabolism,sphingolipids and other metabolic pathways in mice,which were further supplementary description of Serratinine B anti-AD activity and action mechanism.Serratinine B also significantly interfered with energy metabolism and amino acid metabolism,sphingolipids and other metabolic pathways in mice,which were further supplementary description of Serratinine B anti-AD activity and action mechanism.RIPK3/MLKL-mediated inflammatory cytokines and apoptosis were inhibited by Serratinine B through selectively down-regulating RIPK1 protein.The expression level of I?B-? kinase was up-regulated and NF-?B protein nuclear translocation was reduced in NF-?B signaling pathway.Finally,the symptoms of AD were alleviated by inhibiting the expression of Apo E4 protein and phosphorylation levels of Tau protein.5)Based on the above results and molecular docking,Serratinine B was proposed to inhibit the RIPK1 protein-mediated downstream NF-?B signaling pathway.The phosphorylation level of Tau protein was further down-regulated to alleviate AD and improve learning,memory and behavior disorders.Conclusion:In this study,64 compounds including ten new compounds were isolated and identified from H.Serrata.In vitro and in vivo experiments revealed that the downstream inflammation-related NF-?B signaling pathway and neocroptosis mediated by RIPK1 protein was inhibited by Serratinine B through down-regulating the expression level of RIPK1 protein,the then phosphorylation level of Tau protein was down-regulated,and the learning,memory and disordered behavior on mice were improved.This study enriched the chemical composition of H.Serrata.and provided a scientific basis for the development of new anti-AD drugs.