Mechanism Of Microglial Activation Induced By OAβ Combined With IFN-γ And Inhibitory Effect Of PF11

Extracellular senile plaque formed by the deposition of amyloid β-protein(Ap)in the brain is a pathological hallmark of Alzheimer’s disease(AD).Aβ is in monomer form in the initial formation stage,then numbers of Aβ monomers quickly agglomerate into oligomeric form and finally form fibrillar AP(fAβ),which is the main component of senile plaques.A large number of activated microglia were found surrounding the Aβdeposits in AD patient brain.Although many literatures reported that Aβ could activate microglia which led to nerve inflammation,specific Aβ form which activate microglia as well as the signaling cascade associated are still little studied.In our previous study,pseudoginsenoside F11(PF11),a component of Panax quinquefolium(American ginseng),was demonstrated to significantly mitigated learning and memory impairment induced by scopolamine,sodium nitrite,ethanol or cerebral ischemia-reperfusion.In addition,PF11 could mitigate cognitive function impairment in APP/PS1 mice and Aβ1-42-treated mice.These findings suggested that some beneficial effects on neuronal and the inhibitory effect on amyloidogenesis,and oxidative stress might contribute to the recognition improvement effect of PF11.On this basis,the following aspects were studied:1.Based on the successful production of three forms of A,fibrillar,oligomeric and aggregated,which were confirmed by AFM,the effects of these three forms of Aβ on N9 microglial cells stimulation were compared.It was found that oligomeric Aβ(oAβ)induced the strongest N9 microglial activation.2.N9 microglial activation model induced by combination of interferon-γ(IFN-γ)and oAP was established and the associated signal cascade pathways in this model were investigated.(1)The role of FPR2 in N9 microglial activation induced by oAβ combined with IFN-y and the relationship between FPR2 and PKC protein family were studied.Results revealed that oAβ combined with IFN-y could significantly induce FPR2 mRNA and protein expression on N9 microglial cell membrane;FPR2 specific inhibitor WRW4,CSH and FPR2 antibody could significantly inhibit release of inflammatory cytokines NO,TNF-a and ROS in N9 microglia induced by oAβ combined with IFN-y;FPR2 specific agonist MMK-1 and WKYMVM could significantly increase release of inflammatory cytokines NO and ROS in N9 microglia.These results suggest that N9 microglial activation induced by oAβ combined with IFN-y is mediated by FPR2.On this basis,we studied the relationship between FPR2 and PKC protein family and found that FPR2 agonist WKYMVM(100 nM)could significantly induce phosphorylation of PKCβ,suggesting that PKCβ may be a downstream protein in FPR2 signaling pathway.(2)The role of NADPH oxidase in oAβ combined with IFN-y induced N9 microglial activation and the relationship between NADPH oxidase,FPR2 and PKC protein family were investigated.Results showed that oAβ combined with IFN-y could significantly increase mRNA and protein expressions of membrane binding subunit gp91phox and cytosolic subunit p47phox of NADPH oxidase in N9 microglia;NADPH oxidase inhibitors,DPI and apocynin,could significantly inhibit release of inflammatory cytokines NO,TNF-a and ROS in N9 microglia induced by oAβ combined with IFN-y;NADPH oxidase activator PMA could significantly induce NO and ROS release in N9 microglia.These results suggest that N9 microglial activation induced by oAp combined with IFN-y is mediated by NADPH oxidase.In relationship study we found FPR2 specific agonist MMK-1 or WKYMVM could remarkably increase protein expressions of gp91phox and p47phox of NADPH oxidase and release of reactive oxygen species(ROS)in N9 microglia;PKC inhibitor Che could remarkably inhibit protein expressions of NADPH oxidase subunit gp91phox and p47phox induced by MMK-1 or WKYMVM in N9 cells;FPR2 specific inhibitors WRW4 or CsH could remarkably inhibit protein expressions of NADPH oxidase subunit gp91phox and p47phoxnduced by oA(3 combined with IFN-γ in N9 cells.The results indicated that the activation of NADPH oxidase is a downstream event after FPR2 activating PKCs in the process of oAβ combined with IFN-γ induced N9 microglia activation.3.The inhibitory effect of PF11 on N9 microglial activation induced by oAβ combined with IFN-γ and the mechanisms of this effect were examined.It was found that the PF11(3-100 μM)could significantly inhibit release of NO,TNF-a and ROS induced by oAβ combined with IFN-γ in N9 cells;PF11(3-100 μM)could significantly inhibit ’NO and ROS release induced by FPR2 specific agonist MMK-1 or WKYMVM in N9 microglia;PF11(10-100 μM)could remarkably inhibit mRNA and protein expressions of FPR2 induced by oAβ combined with IFN-y.These results indicating that FPR2 is a target in the inhibitory effect.of PF11 on N9 microglial activation induced by oAβ combined with IFN-y.Meanwhile,PF11(10-100 μM)could significantly decrease NO and ROS release induced by NADPH oxidase agonist PMA in N9 cells,and PF11 could significantly inhibit mRNA and protein expressions of NADPH oxidase subunits gp91phox and p47phox induced by oAβ combined with IFN-y,suggesting that the PF11 may inhibit oAβ combined with IFN-y induced N9 microglial activation by inhibiting FPR2 and downstream NADPH oxidase.In summary,the effects of three forms of Aβ,fibrillar,oligomeric and aggregated,on N9 microglial cells stimulation were compared and found that oligomeric Aβ(oAβ)induced the strongest N9 microglial activation.On this basis,this study established oAβ combined with IFN-y induced N9 microglial activation model for the first time,and firstly discovered that the activation of NADPH oxidase is a downstream event after FPR2 activating PKCs in the microglial activation process,and FPR2/PKC/NADPH oxidase signaling cascade pathway is involved in microglial activation.On this basis,we found that PF11 may inhibit oAβ combined with IFN-y induced N9 microglial activation by inhibiting FPR2 and downstream NADPH oxidase.This study provides experimental basis for clarifying the relationship between the FPR2 and microglial activation induced AD,and further elucidates that the anti-AD effect of the PF11 may be attributed to the inhibition of Aβ-induced neuronal inflammation,which provide theoretical support for the development of PF11 into new drugs for AD treatment.