Effect Of Acetylpuerarin On The Expression Of Caspase-3,NF-κBp65 And Inos In BV-2 Microglia Cells Induced By Aβ

Objective:In this study,the mouse BV-2 microglia cells were activated by Aβ_25-355-35 in condensed state as Alzheimer’s disease inflammatory cell model.We was performed to explore the anti-inflammatory mechanism of acetylpuerarin from different levels（cell,molecular,protein and gene）by investigating the morphological changes of microglia,the concentration of NO in cell culture medium and the expressions of cysteinyl aspartate specific proteinase-3（caspase-3,cleaved）,nuclear ranscription factor-kappa Bp65（NF-κBp65）and inducible nitric oxide synthase（iNOS）in cells.The purpose was to provide new target and experimental basis for the treatment of Alzheimer’s disease and to provide theoretical support for the nursing staff to carry out drug education and propaganda for AD patients.Methods:The mouse BV-2 microglia cells were cultured and activated by Aβ_25-355-35 in condensed state as Alzheimer’s disease inflammatory cell model in vitro and were randomly divided into six groups:control group,Aβ_25-355-35 group（model group）,Aβ_25-35+acetylpuerarin（0.1,0.4,1.6μmol/L）groups and caspase-3 inhibitor（Z-DEVD-fmk）group.The changes of BV-2 microglia cells morphology were observed under inverted phase contrast microscope;the effect of acetylpuerarin on nitric oxide（NO）was detected by Nitrate Reductase assay;the mRNA and protein expressions of iNOS were measured by quantitative Real-time PCR,Western Blot and Immunofluorescence assay;the mRNA and protein expressions of NF-κBp65,caspase-3（cleaved）were examined by quantitative Real-time PCR,Western Blot and Immunofluorescence assay.Results:（1）It was observed that after stimulated by Aβ_25-35,there was obvious expansion of cytoplasmic space with thickening and retraction filopodia,resembling the activated amoeboid phenotype and the cells assembled into groups;the status of cells pretreated with acetylpuerarin at different concentrations were between unstimulated ramified microglia in the control group and activated amoeboid microglia in Aβ_25-355-35 group;the cells in caspase-3 inhibitor D-ZEVD-fmk group were close to the undisturbed microglia cells in the control group;Undisturbed microglia cells in the control group were uniform in size and displayed small round cytoplasm with an off-centered nucleus,resembling the resting ramified phenotype.（2）The effect of acetylpuerarin on inflammatory mediator NO expression in Aβ_25-35-induced BV-2 microglia cells:（1）Nitrate Reductase assay showed that compared with the control group,the expression of NO in the Aβ_25-355-35 group was significantly up-regulated（P<0.001）;compared with the Aβ_25-355-35 group,in low-dose,moderate-dose and high-dose groups,acetylpuerarin could markedly inhibit the expression of NO in a dose-dependent manner（P<0.05,P<0.01,P<0.001）,the expression of NO in the caspase-3 inhibitor group was significantly decreased（P<0.001）;the expression of nitric oxide（NO）in control group was the least.（2）qRT-PCR,Western Blot and Immunofluorescence detection showed that,compared with the control group,the mRNA and protein expressions of iNOS in the Aβ_25-35group were significantly up-regulated（P<0.001）;compared with the Aβ_25-355-35 group,in low-dose,moderate-dose and high-dose groups,acetylpuerarin could markedly inhibit the expressions of iNOS in a dose-dependent manner（P<0.05,P<0.01,P<0.001）,the mRNA and protein expressions of iNOS in the caspase-3 inhibitor group was significantly decreased（P<0.001）.（3）The effect of acetylpuerarin on NF-κBp65 and caspase-3（cleaved）expression in Aβ_25-35-induced BV-2 microglia cells:（1）qRT-PCR and Western Blot detection showed that,compared with the control group,the mRNA and protein expressions of NF-κBp65 in the Aβ_25-355-35 group were significantly up-regulated（P<0.001）;compared with the Aβ_25-355-35 group,in low-dose,moderate-dose and high-dose groups,acetylpuerarin could markedly inhibit the expressions of NF-κBp65 in a dose-dependent manner（P<0.05,P<0.01,P<0.001）,the mRNA and protein expressions of NF-κBp65 in the caspase-3inhibitor group were significantly decreased（P<0.001）;the mRNA and protein expressions of NF-κBp65 in control group were the least.（2）Immunofluorescence assay detection showed that,the positive material of cleaved caspase-3immunoreactive were red,localized in the cytoplasm,and the intensity of expression was different:The most cleaved caspase-3 positive cells were found in the Aβ_25-35group,with the deepest coloring and the integrated optical density of caspase-3positive reactive cells were the highest（P<0.01）;the cells pretreated with acetylpuerarin at different concentrations were between the control and the Aβ_25-35group;and the positive cells in caspase-3 inhibitor D-ZEVD-fmk group were close to the control group;the positive cells in control group were the least,with the lightest coloring and the integrated optical density of caspase-3 positive reactive cells were the lowest.Conclusion:（1）The microglia cells were successfully activated by Aβ_25-355-35 to establish the Alzheimer’s disease inflammatory cell model.（2）Acetylpuerarin can improve the morphological changes of BV-2 microglia cells and inhibit the inflammatory response in BV-2 microglia cells.The mechanism may be related to its suppressing the activation of caspase-3/NF-κB signal pathway in a dose-dependent manner.