Protective Effect And Mechanism Of Compound Yizhi Granule On Aβ-induced PC12 Cell Injury

Objective:In this study,MTS,high content and other detection methods were used to target Aβ.By observing the protective effect of compound Yizhi granule and Yizhi granule fraction on nerve cell damage caused by Aβ,the effect of compound Yizhi granule on Aβ-induced PC12cell injury was elucidated.The research on protection and preliminary mechanism is expected to provide a favorable experimental basis for the further development and utilization of compound Yizhi granule.Methods:1.The compound Yizhi granule are divided into 6fractions（F1,F2,F3,F4,F5,F6）by semi-preparative high performance liquid chromatography.The PC-12 cell injury induced by Aβ_25-35was used as the AD cell model,and the cells were divided into control group,model group,berberine hydrochloride group and each fraction group（6.25,12.5,25,50,100,250,500,750μg/ml）,three replicate wells per group.MTS experiments screened the effects of six fractions on the proliferation of model cells by modeling 24h and 48h.The effects of different fractions on the proliferation of AD model cells were compared by modeling for 48h.The SH-SY5Y cell injury was induced by Aβ_25-35as an AD cell model,and the selected fraction F4 and fraction F5 were verified.2.The PC-12 cell injury induced by Aβ_25-35was used as the AD cell model,the cells were divided into control group,model group,the whole side group,berberine group,fraction F4（12.5,25,50,100,250μg/ml）,fraction F5（12.5,25,50,100,250μg/ml）.After 48 hours of administration,the cell cycle kit was used for staining according to the instructions,and the high-content detection of the fractions of F4 and F5on the cell cycle of the AD cell model was examined.3.The PC-12 cell injury induced by Aβ_25-35was used as the AD cell model,the cells are divided into control group,model group,whole group,berberine group,fraction F4 group,fraction F5 Group（12.5,25,50,100,250μg/ml）.After 48 hours of administration,immunocytochemical（ICC）staining was performed.High-intensity detection of effective sites F4 and F5 on Aβresulted in PC12 cell damage.Apoptosis proteins Caspase-3,Caspase-9,Caspase-12 fluorescence intensity,Bax/Bcl fluorescence intensity,Glucose Regulators GRP78,GRP94 and Transcription Factor DDIT3 Expression EffectsResults:1.Compound Yizhi granule extract and fractions F1,F2,F3,F4,F5,AD models have increased cell viability.The results of modeling for 24 hours were consistent with the administration of 48 hours,and the subsequent experiments were carried out for 48 hours.F4 and F5 have the best protective effect on AD model cells and are used for subsequent mechanism research.2.The proportion of S phase cells in the control group was 27.6%,the proportion of cells in M phase was 42.6%,the proportion of cells in the S phase of the model group was 24.4%,and the proportion of cells in the M phase was 63.8%.Fractions F4 and F5 all increased the proportion of cells in S phase,decreased the proportion of cells in M phase,and countered the cell cycle arrest and division inhibition of Aβ_25-35.3.Fraction F4,F5 reduced apoptotic protein Caspase-3,Caspase-9expression,and decrease the ratio of the apoptotic protein Bax to the anti-apoptotic protein Bcl-2,reduced apoptotic protein Caspase-12expression.Inhibits the expression of glucose regulatory proteins GRP78and GRP94,down-regulates DDIT3 expression factor and reaches anti-apoptotic effect.Conclusions:1.Compound Yizhi granule extract and fractions F1,F2,F3,F4,F5 are protected Aβ_25-35induced PC-12 cell damage and protect SH-SY5Y cells induced injury Aβ.2.Fractions F4 and F5 may play a role in protecting AD model cells by counteracting cell cycle arrest and division inhibition of Aβ_25-35.3.Fraction F4,F5 reduced apoptotic protein Caspase-3,Caspase-9expression,and decrease the ratio of the apoptotic protein Bax to the anti-apoptotic protein Bcl-2,reduced apoptotic protein Caspase-12expression.Inhibits the expression of glucose regulatory proteins GRP78and GRP94,down-regulates DDIT3 expression factor and reaches anti-apoptotic effect.