Study On The Effect Of Tongluoxingnao Effervescent Tablet On Aβ Metabolism In Transgenic AD Models

Objective:Based on the transgenic animals and cell models,the study was aimed at the effect of Tongluo Xingnao effervescent tablets（TXET）on learning and memory ability of APPswe/PS1dE9 mice,the production of Aβ-related pathways,the key enzymes and Aβdegrading enzymes in vivo and in vitro.This study also observed the relationship between the anti-AD effect of TXET and the regulation of Aβmetabolism to further elaborate the mechanism of anti-AD in TXET.Methods:1.The study for effectivenessThe spatial learning and memory performance were evaluated using Morris water maze method,to investigate the effect of TEXT on cognitive memory in APPswe/PS1dE9 mice.2.The effect of TXET on AβThe senile plaque formation in hippocampus of APPswe/PS1dE9 mice was evaluated using immunohistochemistry.The secretion of Aβ_1-40-40 and Aβ_1-42-42 were detected by ELISA,which was investigated the effect of TEXT on the secretion of Aβin SH-SY5Y-APP and SH-SY5Y-C99 cells and APPswe/PS1dE9 mice.3.The effect of TXET on Aβgeneration pathwayThe effect of TEXT on the expression of APP,CTF-α,CTF-βand PS1 protein in mouse brain was detected by Western blot,and the activity ofγ-secretase was determined by the fluorescence detection kit.In vitro,the gene and protein expression of APP was detected by RT-PCR and Western blot in SH-SY5Y-APP cells,and the protein expression of sAPPαand sAPPβwas detected by Western blot.The gene,protein level and enzyme activity of BACE1 were determined by qRT-PCR,Western blot and the fluorescence detection kit.4.The effect of TXET on Aβdegradation pathwayWe were detected the protein expression of Aβdegrading enzyme（IDE and NEP）by Western blot in cell and animal experiments,evaluated the effect of TEXT on Aβdegradation.Results:1.Effect of Tongluo Xingnao effervescent tablets on cognitive memory functionof APPswe/PS1dE9 miceThe result of directional navigation experiments show that escape latency of APPswe/PS1dE9 mice,compared with C57BL/6 mice,was significantly prolonged on the third day of training（P<0.05）;compared with the model group,the escape latency of TEXT group was significantly shortened（P<0.05）;compared with the control group,the times of entrance into target quadrants was significantly reduced in APPswe/PS1dE9 mice（P<0.01）and the swimming distance in the target quadrant was significantly shortened（P<0.05）;the swimming distance and the times of entrance into target of in APPswe/PS1dE9 mice was significantly increased（P<0.05）by TEXT administration.2.Effect of Tongluo Xingnao effervescent tablets on AβImmunohistochemistry and ELISA results showed that,the number of senile plaques in the hippocampus of APPswe/PS1dE9 mice was significantly higher than control group（P<0.05）,and the contents of Aβ1-40 and Aβ1-42 in brain were also significantly increased（P<0.01）,but after TEXT administration,the number of senile plaques and the levels of Aβ1-40 and Aβ1-42 in the hippocampus of APPswe/PS1dE9mice were significantly decreased（P<0.01）;The levels of Aβ_1-40-40 and Aβ_1-42-42 in SH-SY5Y-APP cells were significantly inhibited by the serum of TEXT（P<0.05）,but had no effect in SY-SY5Y-C99 cells.3.Effects of Tongluo Xingnao effervescent tablets on AβgenerationIn animal experiments,compared with the control group,the protein expression of CTF-βand PS1 in APPswe/PS1dE9 mice was significantly higher,the expression of CTF-αprotein was significantly decreased（P<0.01）.After TEXT administration,the expression protein of CTF-βwas significantly decreased（P<0.01）,but had no effect on the expression of APP,CTF-αand PS1 protein;The results of theγ-secretase activity test showed that the enzyme activity of APPswe/PS1dE9 mice was significantly higher than the control group,but there was no significant change in TEXT group.In cell experiments,the serum of TEXT（0%⁴0%）had no toxic effects on SH-SY5Y-APP and SY-SY5Y-C99 cells（P>0.05）;The levels of Aβ_1-40-40 and Aβ_1-42in SH-SY5Y-APP cells were significantly inhibited by the serum of TEXT（P<0.05）,but TEXT had no effect on the secretion of Aβin SY-SY5Y-C99 cells;The gene and protein expression of APP and the protein level of sAPPαin SH-SY5Y-APP cells had no significant effect by the serum of TEXT（P<0.05）,but had significantly inhibite sAPPβ（P<0.05）,and the gene,protein level and activity of BACE1was significantly inhibited（P<0.01）.4.Effects of Tongluo Xingnao effervescent Tablets on AβDegradationWestern blot results showed that TEXT had no effect on the protein expression of IDE and NEP in cell and animal experiments.Conclusion:1.TEXT can significantly improve the learning and memory function of APPswe/PS1dE9 mice and has the effect of anti-dementia.2.TEXT can significantly inhibit the secretion of Aβand the formation of senile plaques.3.The effect of TEXT on the inhibition of Aβwas not related to the regulation ofα-secretase andγ-secretase,but also had no effect on IDE and NEP.4.The anti-dementia mechanism of TEXT is down-regulated the BACE1 activity and inhibite the cleavage ofβ-secretase to inhibit Aβproduction,Thereby inhibiting the formation of senile plaques.