Asiaticoside Attenuates Cell Growth Inhibition And Apoptosis Induced By Aβ_1-42 Via Inhibiting The TLR4/NF-κB Signaling Pathway In Human Brain Microvascular Endothelial Cells

Objective:Human brain microvascular endothelial cells（hBMECs）is a main component of blood brain barrier（BBB）,amyloid-β1-42(Aβ_1-42)can increase BBB permeability through inducing the apoptosis in hBMECs and it is deposited in neuronal cells that resulted in degeneration and necrosis of neurons or impaired function,finally,which promote the development of AD.Asiaticoside（AS）has an anti-inflammation,anti-oxidation and anti-apoptotic effect,but it still remains unclear whether AS have a protection effects for the apoptosis on hBMECs induced by Aβand the molecule mechanism remains unclear that whether AS can inhibit the activation of TLR4/NF-κB signaling pathway.Therefore,we investigate the protective effect of asiaticoside on Aβ_1-42-induced apoptosis as well as associated mechanism in hBMECs with commonly used in vitro model that Aβinduced the apoptosis incidence in hBMECs,which will provide a theoretical foundation that asiaticoside as a novel agent for the therapy of AD in clinic.Methods:（1）The choice of treatment concentration of AS that hBMECs were pre-treated with various asiaticoside（3.125μmol/L,6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L,100μmol/L,200μmol/L）for 6h,12h,24h and 48h,as detected by CCK-8 assay.（2）The excellent concentration and suitable time stimulated by Aβ_1-42-42 in hBMECs,the cells were treated by Aβ_1-42-42 with various concentration such as12.5,25,50,100 and 200μmol/L for several time including 6h,12h,24h and48h,as detected by CCK-8 assay.（3）The effect of AS on cells viability induced by Aβ_1-42,as detected by CCK-8 assay,the cells were divided several groups including negative control group,model group,asiaticoside groups（25μmol/L,50μmol/L and 100μmol/L）,TAK-242 group.The cells were cultured in the common condition,the cells was treated with nothing in negative control group,the cells were treated by Aβ_1-42（50μmol/L）for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25μmol/L,50μmol/L,100μmol/L for 12h,and then treated by 50μmol/L Aβ_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242（1μmol/L）for 12 h,and then treated by Aβ_1-42-42 for 24 h in TAK-242 group.（4）The effects of asiaticoside on cells apoptosis induced by Aβ_1-42,as detected by Hoechst 33258 staining assay.The cells were divided several groups including negative control group,model group,asiaticoside groups（25μmol/L,50μmol/L and 100μmol/L）,TAK-242group.The cells were cultured in the common condition,the cells was treated with nothing in negative control group,the cells were treated by Aβ_1-42（50μmol/L）for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25μmol/L,50μmol/L,100μmol/L for 12h,and then treated by 50μmol/L Aβ_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242（1μmol/L）for 12 h,and then treated by Aβ_1-42-42 for 24 h in TAK-242 group.The cells in all groups were detected by Hoechst 33258staining.（5）The effects of asiaticoside on cells apoptosis ratio induced by Aβ_1-42,as detected by Annexin V-FITC.The cells were divided several groups including negative control group,model group,asiaticoside groups（25μmol/L,50μmol/L and 100μmol/L）,TAK-242 group.The cells were cultured in the common condition,the cells was treated with nothing in negative control group,the cells were treated by Aβ_1-42（50μmol/L）for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25μmol/L,50μmol/L,100μmol/L for 12h,and then treated by 50μmol/L Aβ_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242（1μmol/L）for 12 h,and then treated by Aβ_1-42-42 for 24 h in TAK-242 group.The apoptosis ratio of cells in all groups were detected by FCM flow cytometry.（6）The effects of asiaticoside on cells mitochondrial membrane potential induced by Aβ_1-42,as detected by fluorescence microscope.The cells were divided several groups including negative control group,model group,asiaticoside groups（25μmol/L,50μmol/L and 100μmol/L）,TAK-242 group.The cells were cultured in the common condition,the cells was treated with nothing in negative control group,the cells were treated by Aβ_1-42（50μmol/L）for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25μmol/L,50μmol/L,100μmol/L for 12h,and then treated by 50μmol/L Aβ_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242（1μmol/L）for 12 h,and then treated by Aβ_1-42-42 for 24 h in TAK-242 group.The mitochondrial membrane potential was detected by fluorescence microscope after treating cells with JC-1staining.（7）The effects of asiaticoside on main proteins expression in MyD88-dependent TLR4 signaling pathway induced by Aβ_1-42,as detected by Western blotting.The cells were divided several groups including negative control group,model group,asiaticoside groups（25μmol/L,50μmol/L and100μmol/L）,TAK-242 group.The cells were cultured in the common condition,the cells was treated with nothing in negative control group,the cells were treated by Aβ_1-42（50μmol/L）for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25μmol/L,50μmol/L,100μmol/L for12h,and then treated by 50μmol/L Aβ_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242（1μmol/L）for 12 h,and then treated by Aβ_1-42-42 for 24 h in TAK-242 group.The expressions of protein involving TLR4,MyD88,TRAF6,p-NF-κB p65,total NF-κB p65 were determined by Western blotting.（8）The effects of asiaticoside on NF-κB p65 translocation in cell nucleus induced by Aβ_1-42,as detected by the immunofluorescence.The cells were divided several groups including negative control group,model group,asiaticoside groups（25μmol/L,50μmol/L and 100μmol/L）,TAK-242 group.The cells were cultured in the common condition,the cells was treated with nothing in negative control group,the cells were treated by Aβ_1-42（50μmol/L）for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25μmol/L,50μmol/L,100μmol/L for 12h,and then treated by 50μmol/L Aβ_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242（1μmol/L）for 12 h,and then treated by Aβ_1-42-42 for 24 h in TAK-242group.The NF-κB p65 protein translocation from cytoplasm to nucleus was observed by the immunofluorescence analysis.（9）The effects of asiaticoside on inflammation factors including TNF-αand IL-6 in supernate of cells medium induced by Aβ_1-42,as detected by the ELISA.The cells were divided several groups including negative control group,model group,asiaticoside groups（25μmol/L,50μmol/L and 100μmol/L）,TAK-242 group.The cells were cultured in the common condition,the cells was treated with nothing in negative control group,the cells were treated by Aβ_1-42（50μmol/L）for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25μmol/L,50μmol/L,100μmol/L for 12h,and then treated by 50μmol/L Aβ_1-42for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242（1μmol/L）for 12 h,and then treated by Aβ_1-42-42 for 24 h in TAK-242 group.The level of inflammation cytokines such as TNF-αand IL-6 in supernate of cells medium was detected by the ELISA.Results:（1）The result for the concentration and time of asiaticoside have shown that the cells viability appear no difference after treatment with asiaticoside in 3.125μmol/L,6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L,100μmol/L for 6 h,12 h,24 h and 48 h when compared with negative control group,but cells viability was mildly decreased after treatment with 200μmol/L asiaticoside for 24h and 48h,which have no significant difference.（2）The result for the concentration and time of Aβ_1-42-42 have shown that the cells viability appear no difference after treatment with Aβ_1-42-42 in 12.5μmol/L and 25μmol/L for 6h,12h,24h and 48h,Aβ_1-42-42 in50μmol/L,100μmol/L for 6h and 12h,Aβ_1-42-42 in 200μmol/L for 6h when compared with negative control group,but the cells viability was decreased by Aβ_1-42-42 in 50μmol/L and 100μmol/L for 24h and 48h,Aβ_1-42-42 in 200μmol/L for12h,24h and 48h,which have significant difference when compared with negative control group.（3）The effects of asiatisoside on cells viability induced by Aβ_1-42,which have shown that cells viability was decreased by Aβ_1-42-42 in model group when compared with negative control group,but the cells viability was elevated by asiaticoside in the Aβ1-42-induced decline,compared with model group,the difference was statistically significant（p<0.05）.（4）The effects of asiatisoside on cells apoptosis induced by Aβ_1-42,which have shown that brillant blue was observed in cell nuclear in model group,the apoptosis cells was obviously increase in model group compared with negative control group and the difference have statistical significance（p<0.05）;The apoptosis cells was obviously decreased in several asiaticoside groups（25μmol/L,50μmol/L,100μmol/L）and TAK-242 group compared with model group,the difference have statistical significance（p<0.05）.（5）The effects of asiaticoside on cells apoptosis ratio induced by Aβ_1-42,which have shown that cells apoptosis ratio was increased in model group compared with negative control group,the difference have statistical significance（p<0.05）;The cells apoptosis ratio was obviously decreased in several asiaticoside groups（25μmol/L,50μmol/L,100μmol/L）and TAK-242 group compared with model group,the difference have statistical significance（p<0.05）.（6）The effects of asiaticoside on cells mitochondrial membrane potential induced by Aβ1-42,which have shown that mitochondrial membrane potential on hBMECs was obviously decreased in model group compared with negative control group,the difference have statistical significance（p<0.05）;The mitochondrial membrane potential on hBMECs was obviously increased in several asiaticoside groups（25μmol/L,50μmol/L,100μmol/L）and TAK-242 group compared with model group,the difference have statistical significance（p<0.01）.（7）The effects of asiaticoside on main proteins expression in MyD88-dependent TLR4 signaling pathway induced by Aβ1-42,which have shown that the level of various proteins including TLR4,MyD88,TRAF6 and p-NF-κB p65 were increased in the model group compared with negative control group,the difference have statistical significance（p<0.01）;The level of various proteins TLR4,MyD88,TRAF6 and p-NF-κB p65 in hBMECs was obviously decreased in several asiaticoside groups（25μmol/L,50μmol/L,100μmol/L）and TAK-242 group compared with model group,the difference have statistical significance（p<0.01）.（8）The effects of asiaticoside on NF-κB p65 translocation in cell nucleus induced by Aβ_1-42,which have shown that the cells with NF-κB p65translocation was obviously increased in model group compared with negative control group,the difference have statistical significance（p<0.01）;The positive cells with NF-κB p65 translocation were decreased in several asiaticoside groups（25μmol/L,50μmol/L,100μmol/L）and TAK-242 group compared with model group,the difference have statistical significance（p<0.05）.（9）The effects of asiaticoside on inflammation factors including TNF-αand IL-6 in supernate of cells medium induced by Aβ1-42,which have shown that the level of inflammation factors in supernate of cells medium was increased in model group compared with negative control group,the difference have statistical significance（p<0.05）;The level of inflammation factors in supernate of cells medium was were decreased in several asiaticoside groups（25μmol/L,50μmol/L,100μmol/L）and TAK-242 group compared with model group,the difference have statistical significance（p<0.05）.Conclusion:（1）Asiaticoside can decreased the cells apoptosis induced by Aβ_1-42-42 in hBMECs.（2）Asiaticoside can decrease the expression of proteins including TLR4,MyD88,TRAF6 and p-NF-κB p65 in TLR4/NF-κB signaling pathway,inhibiting the NF-κB p65 translocation from cytoplasm into nuclear and the release of inflammation factors such as TNF-αand IL-6.