Study On The Mechanism Of Anti-AD Model Inflammation And Apoptosis Of Forsythiaside

Alzheimer's disease is a neurodegenerative disease that causes learning and memory disorders.Due to the increasing aging of the population in our country,it has become one of the most serious diseases that threaten the elder and has brought a very serious burden on the country.Therefore,the treatment or prevention of AD can not be delayed.Forsythiaside has been widely known for its antibacterial,anti-inflammatory,and antioxidative properties.It can improve learning and memory impairment of AD in animal behavior.However,the mechanism is not yet known.Therefore,the molecular mechanism of forsythiaside on A?_25-35-induced hippocampal slice induced neuroinflammation and apoptosis had been investigated.The specific research methods and results are as follows:1.Effects of forsythiaside on hippocampal slice injury induced by A?_25-35aggregates:A?_25-35 aggregation time,concentration and forsythiaside concentration determination and the effect of forsythiaside on A?_25-35aggregation.The A?_25-35 aggregate was obtained by placing A?_25-35 in a 37°C incubator for 96 h.The optimum concentration of forsythiaside was 50?g·mL^-1 as measured by the lactate dehydrogenase kit and NO kit,and the optimal concentration of A?_25-35 aggregate was 5?M,and forsythiaside improved A?_25-35 aggregates induced hippocampal slice damage.The concentration of forsythiaside and A?_25-35 aggregates in hippocampal slices and the conditions required for the accumulation of A?_25-35 were determined to establish the basis for successful follow-up experiments.2.The molecular mechanism of forsythiaside on hippocampal neuronal inflammation and apoptosis induced by A?_25-35 aggregates:?1?Forsythiaside upregulated 2-AG signaling;?2?Forsythiaside up-regulated endogenous2-AG by inhibiting the metabolism of 2-AG:Forsythiaside decreased the expression of 2-AG-specific hydrolase MAGL to increase 2-AG;?3?2-AG acts as a substrate for the production of the inflammatory factor PGE2 by COX-2,while forsythiaside decreased the expression of COX-2 and decreased the release of inflammatory factor PGE₂.And by inhibiting of enzyme experiments,the IC₅₀ value of forsythiaside against COX-2 was 4.89?M,indicating that forsythiaside had a direct inhibitory effect on COX-2.Residues of COX-2 and forsythiaside formed six hydrogen bonds by molecular docking simulations;?4?Detection of inflammatory cytokines?TNF-?,PGE2?by Elisa kit and detection of inflammation?TNF-??and apoptosis proteins?Bax/Bcl-2,Active caspase-3?expression levels by Western blot,forsythiaside improved hippocampal slice inflammation and apoptosis induced by A?_25-35,additionally,this effect was reversed by the CB1 antagonist,found that forsythiaside improved A?_25-35 induced hippocampal slice inflammation and apoptosis through CB1R;?5?Forsythiaside decreased expression of phosphorylation NF-?B protein induced by A?_25-35 indicating that forsythiaside protected A?_25-35-induced neuroinflammation and apoptosis through NF-?B signaling pathway?6?Long-term potentiation?LTP?is a typical learning and memory type,recording field potential showed that forsythiaside improved the learning and memory impairment induced by A?_25-35;?7?The change of LTP was due to change in the content of neurotransmitters such as GABA and Glu after synapses,while forsythiaside changed the content of neurotransmitters related of AD.The above mechanism provides experimental basis for the anti-inflammatory and other effects of Forsythiaside,and lays a solid foundation for forsythiaside as a drug for the treatment or prevention of AD.3.Effects of extract of Forsythia suspensa leaf and forsythiaside on LPS-induced injury of hippocampal slices in mice:5 and 50?g/mL Forsythia suspensa leaf extract and forsythiaside significantly inhibited leakage of LDH on hippocampal slices,so extract of Forsythia suspensa leaf and forsythiaside had a protective effect on the survival of hippocampal slices.We used 1?g/mL LPS-induced hippocampal slice for 24 h to establish damage model in vitro,the effect of 5 and 50?g/mL forsythia leaf extract and forsythiaside had been investigated through lactate dehydrogenase kits on damage model;Determination of inflammatory factors NO,IL-1?and TNF-?;Determination of protein expression levels of apoptosis protein caspase-3 and cleaved caspase-3.Forsythiaside and the forsythia leaf extract had improved hippocampal injury induced by LPS.In conclusion,we demonstrated that extract of Forsythia suspensa leaf more effectively inhibited LPS-induced inflammation and decreased apoptosis on hippocampal slice than an equivalent forsythiaside.This laid the foundation for the further development of Forsythia suspensa leaf.