Study On The Mechanism Of 4-octyl Itaconate Affecting The Development Of Alzheimer’s Disease By Regulating Neuroinflammation And Oxidative Stress

Objective To study the relationship between 4-octyl itaconate(4OI)and Alzheimer’s disease(AD)by means of network pharmacology.The effects of 4OI on AD were observed in vivo and in vitro.It provides new scientific basis or probability for the study of 4OI in treatment of AD.Methods The effective components of 4OI were obtained based on Pub Chem and the target genes were obtained through Pharmmapper.All AD genes were obtained through Gene Cards,OMIM and Uniprot databases.The effective components and disease targets were intersected by Venny diagram.Using STRING online,the4OI-AD intersection target was calculated and the core target map of protein interaction network was constructed.Then,the active component-target network of4OI-AD is built by Cytoscape software.GO enrichment analysis was performed by David’s online website.In the vitro experiment,BV2 and SH-SY5 Y cells were cultured.The morphology of BV2 cells was observed,and the expression of inflammatory factors was evaluated by immunofluorescence and Elisa.The ROS of BV2 cells and apoptosis of SH-SY5 Y cells were analyzed.The expression of inflammatory factors,Nrf2,HO-1 and apoptosis factors were evaluated by PCR and Western blot.Based on cell research,AD animal model was obtained by injecting with Aβ 1-42 in the lateral ventricle,and tested by WMW.To observe the effect of4 OI on cognitive function and the structural changes of neurons in the hippocampus.The expression of inflammatory factors was evaluated through Elisa.The expression levels of TNF-α,IL-1β,Nrf2,HO-1,Caspase3,Bax and Bcl-2 in hippocampus were detected by PCR and Western blot.Results There are 17 targets for 4OI-AD’s intersection.And there are 9 targets intersected between neuroinflammation/oxidative stress and 4OI.The common targets are HDAC6,VCP and ERN1.12 biological processes that 4OI-AD targets involved was found through GO functional enrichment analysis,including DNA binding,ubiquitin protein ligase binding,RNA binding,ribonucleoprotein complex,etc.Based on CCK8 experimental results,4OI(62.5μM、125μM)were used in the following course.It was divided into normal group,model group,solvent group,treatment 1 and 2 group.Compared with the model group,M1 microglia decreased in the treatment group.Results showed that BV2 cells in the model group released more inflammatory factors by Elisa,and the effect of treatment group 2 was stronger than that of treatment group 1 in inhibiting inflammation.4OI makes ROS of BV2 cells decreased.And it showed 4OI may reduce the apoptosis of neurons.And other results showed that there are higher expressions of IL-1β,TNF-α m RNA in model group.On the contrary,there are lower expressions of IL-1β,TNF-αm RNA,a higher expression of TGF-β in treatment group.There was also a statistical difference between the two treatment groups.Compared with the model group,the expressions of Nrf2 and HO-1 m RNA in the treatment group increased significantly.There were also statistical differences in Nrf2 and HO-1 m RNA between the two treatment groups.The expressions of Caspase3 and Bax m RNA increased in the model group.The expressions of Caspase3 and Bax m RNA decreased and the expression of Bcl-2 m RNA highly increased in the treatment group.There were also statistical differences in the expression of apoptotic genes between treatment groups.The expressions of IL-1β,TNF-α protein in model group increased,while IL-1β,TNF-α protein expressions in treatment group decreased,and the TGF-β protein highly increased.The protein results showed that the protein of Nrf2 and HO-1 increased in the treatment group.In addition,the protein expressions of Caspase3 and Bax in the model group increased significantly.Compared with the model group,the protein expressions of Caspase3 and Bax of the treatment group decreased,and the protein expression of Bcl-2 highly increased.There was no significant difference in body weight of rats in each group.In the WMW,the average escape latency of the model group mice was significantly longer than those of the treatment group.The hippocampal neurons in the model group were irregularly arranged and the perinuclear space around nucleus was significantly widened.In treatment group,the damage of hippocampal neurons was alleviated and the perinuclear space was narrowed.The m RNA expressions of TNF-a,IL-1β,Caspase3 and Bax of hippocampus in the model group mice increased,while in the treatment group decreased.And the m RNA expressions of Nrf2,HO-1,Bcl-2,TGF-β in the treatment group highly increased.The relative protein results are the same as the expressions in PCR experiment.Conclusion As an intermediate metabolite of tricarboxylic acid cycle,4OI may play an anti-inflammatory and anti-oxidative stress role by participating in various metabolic processes,such as protein-DNA,protein-RNA,and tricarboxylic acid cycle.According to this study,in the progress of AD,4OI may improve cognitive function through anti-neuroinflammation and anti-oxidative stress course.It was also proved that 4OI may participate in AD disease by regulating Nrf2 pathway.4OI may have significant protective effects on AD.