Protective Effects And Its Mechanisms Of Amentoflavone In LPS-induced Microglia Activation And Parkinson’s Disease Models

Part 1:Effect of Amentoflavone on BV-2 cellsObjective In this part,bioinformatics technology was used to study the safe and effective drug concentration,signaling pathway,key factors and conformation of Amentoflavone（AF）on BV-2cells.Methods BV-2 cells were cultured in normal culture.In the logarithmic phase,MTT assay was used to detect the IC₅₀ inhibitory concentration of AF with different concentration gradient on BV-2 cells.SILAC quantitative proteomics assay was used to detect the changes of intracellular whole histone expression and the regulation changes of related signaling pathways and key factors after AF was applied to BV-2 cells.Molecular docking assay was used to detect the degree of conformation of key proteins involved in cell cycle and apoptosis between AF and BV-2 cells.Results The IC₅₀ of MTT test was 21.25μM at 24 h and 13.60μM at 48 h.AF acted on the labeled BV-2 cells for 24 h,SILAC quantitative proteomic analysis:the main disease and functional effects were cell death and survival;the conventional signal pathway was death receptor signal pathway;the molecular and cell function were cell growth and proliferation,cell death and survival,and the toxicity was cell death;a total of 425 protein molecules were regulated,of which 201 were up-regulated and 224 were down regulated.There are 60 kinds of protein molecules that play key roles in cell death receptor signaling pathway,cell cycle G2/M DNA damage checkpoint signaling pathway and apoptosis regulation signaling pathway.Molecular docking experiments showed that when AF was chimeric with Bax,Bcl-2,Bcl-xl,Caspase-3,caspase-9,CDK1,CDK2,cyclin B1,Cytochrome C,p53,p21 and PUMA,the CIE was all low（negative value）,and a complete and stable binding docking structure could be formed.Conclusion AF can effectively apply on BV-2 cells.The concentration of AF on BV-2 cells was 10μμM.AF can play a strong anti-inflammatory,anti-oxidant and anti-apoptotic role in BV-2 cells.Inflammatory protein molecules of TNF-α,IL-1β,IL-6,i NOS,COX-2,IL-4,TGF-β,Arg-1,and CD206 were identified as the research objects to explore the inflammatory effect of AF on BV-2 cells.The mechanism of AF on BV-2 cells was death receptor signaling pathway,which continued to cell cycle and apoptosis regulation of signaling pathways.CDK1,CDK2,Cyclin B1,p53 and p21 were selected as the research objects of cell cycle mechanism,and Bax,Bcl-2,Bcl-xl,Caspase-3,caspase-9,Cytochrome C and PUMA were selected as the research objects of apoptosis mechanism.Part 2:Effect of AF in LPS-induced BV-2 cells activationObjective In this part,molecular biology techniques was used to study the effect and mechanism of AF on the transformation of microglia from neurotoxic M1 phenotype to neuroprotective M2 phenotype after LPS induced BV-2 cell activation.Methods BV-2 cells grouped into did not join any drug treatment for blank control group,AF0 group with LPS treatment and without AF,AF 10μM group with LPS treatment and join the AF10μM,AF 20μM groups with LPS treatment and join AF 20μM.The expression levels of inflammatory factors TNF-α,IL-1β,IL-6,i NOS,COX-2,IL-4,TGF-β,Arg-1 and CD206 in each group were detected by ELISA assay and Western blot assay,respectively,with or without AF.The effects of AF on cell cycle and apoptosis were detected by flow cytometry.Western blotting was used to detect the changes in the expression of cell cycle and apoptosis-related proteins in each group with or without AF.Results ELISA assay and Western blot assay showed that the expressions of pro-inflammatory factors TNF-α,IL-1β,IL-6,i NOS and COX-2 increased and the expressions of anti-inflammatory factors IL-4 and TGF-βdecreased significantly after treatment with different concentrations of AF.Flow cytometry showed that in the absence of AF,the G2/M phase of the cell cycle was significantly blocked,which prevented the cell cycle from normal mitotic circulation.AF could counteract this blocking effect in a concentration-dependent manner.In the absence of AF,the number of apoptotic cells increased significantly,and AF could significantly inhibit the apoptotic effect.CDK1 and Cyclin B1 proteins expression were observably increased by Western blot,while CDK2,p53 and p21 proteins expression were significantly decreased after AF treatment.In cell apoptosis,the expression levels of pro-apoptotic factors Bax,Caspase-3,Caspase-9 and Cytochrome C were observably decreased after AF treatment,while the expression levels of anti-apoptotic factors Bcl-2,Bcl-xl and PUMA were significantly increased.Conclusion LPS can successfully induce the activation of BV-2 cells and establish the neuroinflammation model of microglia cells.The expressions of TNF-α,IL-1β,IL-6,i NOS and COX-2 in the activated BV-2 cells induced by LPS were significantly increased,while the expressions of IL-4,TGF-β,Arg-1 and CD206 in the M2 phenotype were significantly decreased.AF can directly act on microglia and inhibit the pro-inflammatory cytokines produced by them.LPS can induce G2/M phase level arrest of activated BV-2 cells,while AF can effectively alleviate this effect.LPS-induced activation significantly increased the number of apoptotic cells in BV-2cells,and AF could regulate the apoptotic protein and thus significantly inhibit the apoptotic effect.Part 3:Effect of AF on LPS induced PD animal modelObjective In this part,behavioral pharmacological techniques was used to study the neuroprotective effect and mechanism of AF in LPS-induced PD animal models.Methods Rats were randomly divided into groups:blank control group,sham operation group,PD model group,AF prevention group and AF treatment group.The animal model of PD was built by stereotactic injection of LPS into the pars compacta of substantia nigra.Rotation test,open field test,forced swimming test and elevated cross maze test were carried out at 4 weeks after operation.At the end of the behavioral experiment,the animals were decapitated and brain samples were taken.The protein and m RNA expressions of TNF-α,IL-1β,IL-6,i NOS,COX-2,IL-4,TGF-β,Arg-1 and CD206 were detected by Western blot assay and q RT-PCR assay,respectively.Immunohistochemical staining and confocal laser scanning microscopies were used to detect the expression of TH positive cells,the key enzyme of DA synthesis,andα-syn,the pathological basis of PD.Results LPS-induced PD animal model was successfully established.In the rotation test,the rotation behavior of AF+LPS group and LPS+AF group were observably reduced more than that of LPS group,and the improvement effect of AF+LPS group was better than that of LPS+AF group.In the open field test,the off-field behavior of AF+LPS group and LPS+AF group was significantly less than that of LPS group,and the duration of shuttling into the intermediate area was increased.In the forced swimming test,the immobility of AF+LPS group and LPS+AF group was significantly less than that of LPS group,and the improvement effect of AF+LPS group was better than that of LPS+AF group.In the elevated cross maze experiment,the number of rats in AF+LPS group and LPS+AF group entering the open arm was significantly higher than that in LPS group.Western blot and q RT-PCR showed that after AF treatment,the up-regulated expression of pro-inflammatory factors（TNF-α,IL-1β,IL-6,i NOS and COX-2）and down-regulated expression of anti-inflammatory factors（IL-4,TGF-β,Arg-1 and CD206）were significantly inhibited,and the improvement effect of AF+LPS group was better than that of LPS+AF group.Immunohistochemical staining showed that the quantity of TH positive neurons in AF+LPS group was observably more than that in LPS group,which had increased to 85.5%of that in blank control group.Confocal laser scanning microscopy showed that the protein expression ofα-syn was significantly increased in LPS-induced PD rats.After AF treatment,the protein expression ofα-syn was significantly decreased in AF+LPS group and LPS+AF group,especially the inhibition effect was more obvious in AF+LPS group.Conclusion After the success of LPS induced PD animal model,the functional defect of PD rats is similar to that of human PD.AF can improve the decrease of excitability,motor dysfunction,depression like behavior and anxiety like behavior.AF plays a neuroprotective role on DA neurons in LPS-induced PD animal models,which is achieved by inhibiting microglia-mediated inflammatory response.In effect evaluation,early prophylactic administration of AF is better than late therapeutic administration.