Asiaticoside Attenuates Cell Growth Inhibition And Apoptosis Induced By A?_1-42 Via Inhibiting The TLR4/NF-?B 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-?1-42(A?_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 A?and the molecule mechanism remains unclear that whether AS can inhibit the activation of TLR4/NF-?B signaling pathway.Therefore,we investigate the protective effect of asiaticoside on A?_1-42-induced apoptosis as well as associated mechanism in hBMECs with commonly used in vitro model that A?induced 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?3.125?mol/L,6.25?mol/L,12.5?mol/L,25?mol/L,50?mol/L,100?mol/L,200?mol/L?for 6h,12h,24h and 48h,as detected by CCK-8 assay.?2?The excellent concentration and suitable time stimulated by A?_1-42-42 in hBMECs,the cells were treated by A?_1-42-42 with various concentration such as12.5,25,50,100 and 200?mol/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 A?_1-42,as detected by CCK-8 assay,the cells were divided several groups including negative control group,model group,asiaticoside groups?25?mol/L,50?mol/L and 100?mol/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 A?_1-42?50?mol/L?for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25?mol/L,50?mol/L,100?mol/L for 12h,and then treated by 50?mol/L A?_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242?1?mol/L?for 12 h,and then treated by A?_1-42-42 for 24 h in TAK-242 group.?4?The effects of asiaticoside on cells apoptosis induced by A?_1-42,as detected by Hoechst 33258 staining assay.The cells were divided several groups including negative control group,model group,asiaticoside groups?25?mol/L,50?mol/L and 100?mol/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 A?_1-42?50?mol/L?for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25?mol/L,50?mol/L,100?mol/L for 12h,and then treated by 50?mol/L A?_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242?1?mol/L?for 12 h,and then treated by A?_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 A?_1-42,as detected by Annexin V-FITC.The cells were divided several groups including negative control group,model group,asiaticoside groups?25?mol/L,50?mol/L and 100?mol/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 A?_1-42?50?mol/L?for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25?mol/L,50?mol/L,100?mol/L for 12h,and then treated by 50?mol/L A?_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242?1?mol/L?for 12 h,and then treated by A?_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 A?_1-42,as detected by fluorescence microscope.The cells were divided several groups including negative control group,model group,asiaticoside groups?25?mol/L,50?mol/L and 100?mol/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 A?_1-42?50?mol/L?for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25?mol/L,50?mol/L,100?mol/L for 12h,and then treated by 50?mol/L A?_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242?1?mol/L?for 12 h,and then treated by A?_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 A?_1-42,as detected by Western blotting.The cells were divided several groups including negative control group,model group,asiaticoside groups?25?mol/L,50?mol/L and100?mol/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 A?_1-42?50?mol/L?for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25?mol/L,50?mol/L,100?mol/L for12h,and then treated by 50?mol/L A?_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242?1?mol/L?for 12 h,and then treated by A?_1-42-42 for 24 h in TAK-242 group.The expressions of protein involving TLR4,MyD88,TRAF6,p-NF-?B p65,total NF-?B p65 were determined by Western blotting.?8?The effects of asiaticoside on NF-?B p65 translocation in cell nucleus induced by A?_1-42,as detected by the immunofluorescence.The cells were divided several groups including negative control group,model group,asiaticoside groups?25?mol/L,50?mol/L and 100?mol/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 A?_1-42?50?mol/L?for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25?mol/L,50?mol/L,100?mol/L for 12h,and then treated by 50?mol/L A?_1-42-42 for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242?1?mol/L?for 12 h,and then treated by A?_1-42-42 for 24 h in TAK-242group.The NF-?B p65 protein translocation from cytoplasm to nucleus was observed by the immunofluorescence analysis.?9?The effects of asiaticoside on inflammation factors including TNF-?and IL-6 in supernate of cells medium induced by A?_1-42,as detected by the ELISA.The cells were divided several groups including negative control group,model group,asiaticoside groups?25?mol/L,50?mol/L and 100?mol/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 A?_1-42?50?mol/L?for 24 h,the cells were pre-treated by asiaticoside with various concentration including 25?mol/L,50?mol/L,100?mol/L for 12h,and then treated by 50?mol/L A?_1-42for 24 h in asiaticoside groups.The cells were pre-treated by TAK-242?1?mol/L?for 12 h,and then treated by A?_1-42-42 for 24 h in TAK-242 group.The level of inflammation cytokines such as TNF-?and 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 3.125?mol/L,6.25?mol/L,12.5?mol/L,25?mol/L,50?mol/L,100?mol/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 200?mol/L asiaticoside for 24h and 48h,which have no significant difference.?2?The result for the concentration and time of A?_1-42-42 have shown that the cells viability appear no difference after treatment with A?_1-42-42 in 12.5?mol/L and 25?mol/L for 6h,12h,24h and 48h,A?_1-42-42 in50?mol/L,100?mol/L for 6h and 12h,A?_1-42-42 in 200?mol/L for 6h when compared with negative control group,but the cells viability was decreased by A?_1-42-42 in 50?mol/L and 100?mol/L for 24h and 48h,A?_1-42-42 in 200?mol/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 A?_1-42,which have shown that cells viability was decreased by A?_1-42-42 in model group when compared with negative control group,but the cells viability was elevated by asiaticoside in the A?1-42-induced decline,compared with model group,the difference was statistically significant?p<0.05?.?4?The effects of asiatisoside on cells apoptosis induced by A?_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?25?mol/L,50?mol/L,100?mol/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 A?_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?25?mol/L,50?mol/L,100?mol/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 A?1-42,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?25?mol/L,50?mol/L,100?mol/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 A?1-42,which have shown that the level of various proteins including TLR4,MyD88,TRAF6 and p-NF-?B 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-?B p65 in hBMECs was obviously decreased in several asiaticoside groups?25?mol/L,50?mol/L,100?mol/L?and TAK-242 group compared with model group,the difference have statistical significance?p<0.01?.?8?The effects of asiaticoside on NF-?B p65 translocation in cell nucleus induced by A?_1-42,which have shown that the cells with NF-?B 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-?B p65 translocation were decreased in several asiaticoside groups?25?mol/L,50?mol/L,100?mol/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-?and IL-6 in supernate of cells medium induced by A?1-42,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?25?mol/L,50?mol/L,100?mol/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 A?_1-42-42 in hBMECs.?2?Asiaticoside can decrease the expression of proteins including TLR4,MyD88,TRAF6 and p-NF-?B p65 in TLR4/NF-?B signaling pathway,inhibiting the NF-?B p65 translocation from cytoplasm into nuclear and the release of inflammation factors such as TNF-?and IL-6.