The Effect Of Resveratrol And Presenilin-1 On Phagocytosis Function Of BV-2 Cell And The Underlying Mechanisms

ObjectiveAim 1:by the study on the effect of resveratrol on the phagocytosis of A β,the inflammatory cytokine secretion and the regulation of protein expression of Trem2,we would like to discover the potential of resveratrol to be an anti-AD drug.Aim 2:,by the study on the effect of PS1 overexpression on phagocytosis of A β and the regulation of Trem2 trafficking,we provide a new possible way to the screening of Trem2-targeted traditional herbs.Methods1.The effect of resveratrol on phagocytosis function of BV-2 cell line and the underlying mechanisms1.1 CCK-8 cell viability:Select 5 μ M,10 μ M,20 μ M and 40 μ M as treating concentrations.24 hours or 48 hours later,add CCK-8 reagent and incubated for 2 hours at 37 ℃.Then read the absorbance values at 492nm.1.2 Flow cytometry:Treat BV-2 cell line with different concentrations of resveratrol or sTrem2 for 24 hours.Then incubate with fluorescent labeled Aβ for 2 hours,followed by flow cytometry to detect the phagocytosis of Aβ.1.3 Molecular biology experiments:Treated with different concentrations of resveratrol,Trem2 expression and sTrem2 secretion of BV-2 cells were detected by Westerm Blot.IL-1 β and TNF-α mRNA transcript levels was detected by real-time PCR.2.The effect of PS1 on phagocytosis function of BV-2 cell line and the underlying mechanisms2.1 Molecular biology experiment:Detect the interaction between PS1 and Trem2 by co-immunoprecipitation,and study the effect of PS1 overexpression on Trem2 trafficking by cell surface biotinylation.2.2 Viral vector infection:Screen PS1/Trem2 overexpression stable BV-2 cell lines.2.3 Flow cytometry:Flow cytometry was used to detect the effect of PS1 overexpression on phagocytosis of BV-2 cell line.Results1.The effect of resveratrol on phagocytosis function of BV-2 cell and the underlying mechanisms1.1 With the treatment of 5 u M,10 u M or 20 u M of resveratrol for 24h,there is no effect on cell viability compared with the control groups respectively,while 40 μM resveratrol can damage the cell viability of BV-2 cell line.With the treatment of 5 μ M or 10 μ M of resveratrol for 48h,there is no effect on cell viability compared with the control groups respectively,while 20 μM and 40 μ M resveratrol can damage the cell viability of BV-2 cell line.1.2 5μM resveratrol increased BV-2 phagocytosis slightly.But compared to the control group,the difference was not statistically significance;10 μ M and 20 u M resveratrol significantly increased BV-2 phagocytosis compared with control group.1.3 10μM resveratrol increases Trem2 expression slightly,but there is no statistical difference compared with the control group(P>0.05);20 μ M resveratrol significantly increases the expression of Trem2,the expression of which is about 1.7 times relative to the control group,and there is statistically difference between the two groups(P<0.01).1.4 10 u M resveratrol may reduce sTrem2 secretion,but there is no statistical difference compared with the control group(P>0.05);20 u M resveratrol significantly reduces sTrem2 secretion,the expression of which is about 70%of the control group,(P<0.01).1.5 4nM and 20nM Trem2-Fc reduce phagocytosis of Aβ slightly,but there is no statistical difference compared with the control group(P>0.05).40nM dose group reduces the phagocytosis of A β,and the difference is statistically significant(P<0.01).1.6 With the treatment of different doses of resveratrol,the mRNA levels of IL-1β are reduced in BV-2 cell line,and the differences are statistically significant(P<0.05).5μM dose resveratrol do not affect the TNF-α mRNA transcription,but resveratrol 10 u M and 20 u M group,mRNA expression level of TNF-a are decreased,but the decreases are not statistically significant(P>0.05).2.The effect of Presenilin-1 on phagocytosis function of BV-2 cell and the underlying mechanisms1.Trem2 full-length protein can be co-precipitated with PS1(PS1 full lenth,PS1-FL),PS1-NTF and PS1-CTF,but can not detect the expression of NCT,and the protein band is not detected in the IgG control group.As verified by the reverse experiments,Trem2 expression can be detected in the immunoprecipitates of PS1-NTF antibody(Ab14)and PS1-CTF antibodies(Anti PS1-loop),and the control IgG group does not show a corresponding protein bands band.2.The Term2 overall expression level in BV-2-Vector Group is consistent with that of BV-2-PS1 group,without significant difference.Results from Surface Trem2 and Total Trem2 expression analysis show that the Surface/Total Trem2 ratio in BV-2 overexpressed PS1 group is significantly decreased and the difference is statistically significant(P<0.05).3.The phagocysis effect of BV-2-PS1 cell line on FAM-Aβ is significantly lower than that of BV-2-Vector cell lines,and the difference is statistically significant(P<0.05).Compared to the BV-2-PS1 cell line,the phagocysis effect of BV-2-PS1-Trem2 on FAM-Aβ is significantly increased,and the difference is statistically significant(P<0.05).Conclusion1.The effect of resveratrol on phagocytosis function of BV-2 cell and the underlying mechanisms.1.1 Resveratrol interventions can enhance microglial phagocytosis,and enhancement shows dose-dependent effect.1.2 Resveratrol can upregulate the overall protein expression of Trem2 and downregulate the secretion of sTrem2.1.3 The mechanisms underlying the regulation of resveratrol on the BV-2 microglial phagocytosis possibly involve the upregulation of total Trem2 and downregulation of sTrem2 secretion.1.4 Resveratrol can significantly reduce the secretion level of IL-1β from Aβ-induced BV-2 microglial cells and has downward effect on the secretion of TNF-α.However,the results are not statistically significant,which require further exploration.2.Regulation and mechanism of PS1 within the BV-2 microglia endocytosis 2.1 Protein interactions exist between PS1 and Trem2.2.2 Overexpression of PS1 can reduce the membrane transcportation of Trem2 in BV-2 microglial cells.2.3 Overexpression of PS1 decreases the phagocytosis function of BV-2 microglia.Based on this,overexpression of Trem2 can restore the phagocytosis to the baseline level.This suggests that PS1 reduced microglia phagocytosis may be induced by the reduction of Term2 membrane transportation.