Screening On The Key Active Components Of Litchi Seed Saponin(LSS) Against The Activation Of Microglia BV-2 Cells

Objective:Screening the key active components of Litchi seed saponin(LSS)to inhibit the activation of microglia BV-2 cells induced by A?1-42 via isolation and purification of the components of LSS.Method:(1)Isolation and purification the components of LSS by the AKTA purifier protein purification system.(2)The BV-2 cells were activated by AP1-42(5 ?mol/L),total LSS and its components interfered with activated BV-2 cells.(3),The morphological changes of BV-2 cell were observed by Wright's-Giemsa staining under light microscope.(4)The contents of supernatant inflammatory cytokines(TNF-?,IL-1? and IL-6)were measured by ELISA.(5)The mRNA and protein expressions of TNF-?,L-1? and iNOS in activitied BV-2 cells were measured by Quantitative Real-time PCR and western blotting,respectively.Results:(1)We obtained 12 components which were separated from LSS(1#?12#).(2)Screening the key components of BV-2 cell activation induced by LSS:?We observed BV-2 cells appearing around or oval without cell processes,less unity phenomenon and nucleus regular in the control group.But there were some activated signs of A? group,such as reduced cell number,body enlargement,irregular,nucleus broken and so on,there were less activated signs of cells in LSS treated groups,under inverted microscope(×200).The cellular morphological changes of the groups treated with the components of LSS as following:the cell number has some increased,synaptic reduced,and body enlargement in some cells after the treatment of 1#,5#and 6#components of LSS;the cell number was obviously increased,synaptic reduced and basically no body enlargement after treatment of 3#and 9#components of LSS.? TNF-?,IL-1? and IL-6 of BV-2 cells in the cell culture supernatants were increased in A? treated group(P<0.01);compared to the control,but were significantly reduced in LSS treated group(P<0.01)compared to A?group,TNF-? of BV-2 cells was significantly reduced by 3#,5#,6#,9#and 10#components of LSS(P<0.05 or P<0.01),IL-1? of BV-2 cells was reduced by 1#,3#,9#and 10#components of LSS(P<0.05 or P<0.01)and the IL-6 of BV-2 cells was significantly reduced by 1#,3#,5#,8#,and 9#components of LSS(P<0.05 or P<0.01);compared with the LSS group,there were no significant differences of TNF-?,IL-1? andIL-6 in the cell culture medium between the 3#and 9#components of LSS and LSS group.(3)Verification of the effect of key components of LSS on the inhibition of BV-2 cell activation i:? Morphology and the contents of TNF-?,IL-1? and IL-6 in the cell culture medium:The effect of the morphological changes and contents of TNF-a,IL-1? and IL-6 by total LSS,3#and 9#components of LSS are consistent with the previous results of the preliminary identification.? The mRNA expressions of proinflammatory cytokines:The mRNA expressions of TNF-a,IL-1? and iNOS were significantly increased in A? model group(P<0.01)compared to the control group;but they were significantly decreased in total LSS and the 3#and 9#components of LSS groups(P<0.01)compared to A?group;however,there were no significant difference among the groups of total LSS,the 3#and 9#components of LSS(P>0.05).? The expressions of inflammatory cytokines in cells:The protein expressions of TNF-?,IL-1? and iNOS were significantly increased in A? group compared to the control group(P<0.01);however,the protein expressions of TNF-?,IL-1? and iNOS were significantly decreased in the groups of total LSS and the 3#and 9#components of LSS compared to A? group(P<0.01);there were no significant difference among groups of total LSS,the 3#and 9#components of LSS(P>0.05).Conclusion:(1)We obtained 12 components of LSS using current methods.(2)The 3#and 9#components of LSS are the active components for the inhibition the BV-2 cell activation induced by A?.