The Study Of The Effect And Mechanism Of Total Saponins From Rhizoma Anemarrhenae On Aβ-induced PC12Cell Apoptosis

PurposeThis study is to observe the effects of Anemarrhena total saponins on beta-amyloid (Aβ)-induced PC12cell apoptosis and the mRNA and protein expression of BAX and BCL-2, the apoptosis-related gene, exploring the mechanism and to gain further insight into the development of Alzheimer’s desease in order to provide a scientific basis for clinical treatment of Alzheimer’s desease (AD).Material and methodIn vitro PC12cells was stimulated by β-amyloid (25-35)(10,20,30and40μM)for24,36,48and72h, an MTT tetrazolium assay was used to determine the optimal stimulus concentration and the time of β-amvloid to be20μM and48h. MTT assay was used to determine the appropriate concentration of Anemarrhena total saponins and donepezil HCl to be5,10,20mg/Land1μM repectively. The divided experimental groups:normal group, model group, Anemarrhena total saponins groups(5mg/L,10mg/L and20mg/L), the donepezil HCl group. The cell morphology and proliferation in each group was abserved by inverted phase contrast microscope. The vitality of the cells in each group was measured by MTT assay; the expression of BAX and BCL-2protein was examined by immunohi stochemistry; The apoptosis induction was analyzed and photographed by fluorescence microscopy after Annexin-V/PI double staining; The expression level of the BAX and BCL-2gene was detected using semi-quantitative reverse transcriptase PCR (RT-PCR); The expression levels of BCL-2and BAX protein were assaved by western blot.Results 1. The viability of PC12cells was reduced by Aβ25-35in a dose and time dependent manner. The inhibition ratio was highest when the cells were exposed to Aβ25-35for48h. The inhibition ratio of10μ M,20μ M,30μ M and40μ M Aβ25-35was respectively, obviously higher than that of the normal group and there were no significantly differences between20μ M and30μM Aβ25-35.Therefore, the cell model of AD was ideally established after stimulation for48h by20μ MAPβ25-352. After PC12cells were treated for48h both with Anemarrhena total saponins (0.5,5,10,20,40mg/L)/donepezil HC1(0.1,0.5,1,5,10,20,40and60μM)and with Aβ25-35of20μM, MTT assay show that there were statistically significant differences in cell viability between Anemarrhena total saponins (5,10,20mg/L)/donepezil HCl(1μM)group and normal group (p＜0.01), but was reduced at concentration of40mg/L.3. Anemarrhena total saponins (5,10,20mg/L) and donepezil HCl1μM treatment markedly attenuate Aβ-induced injury in PC12cells as shown by MTT, flow cytometry and fluorescent staining, compared with model group (p＜0.05or p＜0.01)4. Anemarrhena total saponins(5,10mg/L) and donepezil HCl1μM treatment dramatically reduced the expression levels of pro-apoptotic gene BAX mRNA and protein as shown by RT-PCR, immunohistochemistry and western blot, compared with those of model group (p＜0.05or p＜0.01)5. As revealed by MTT assay, flow cytometry and fluorescent staining, Anemarrhena total saponins(5,10and20mg/L) and donepezil HCl1μM treatment significantly reduced the expresson level of anti-apoptotic gene BCL-2mRNA and protein compared with those of model group (p＜0.05or p＜0.01), At concentrations of5,10mg/L, Anemarrhena total saponins works best as did the donepezil HCl.Conclusion1. Anemarrhena total saponins treatment improved the cytotoxic effects of A β25-35on the viability of PC12cells, and decreased Aβ25-35-induced the apoptosis rate of PC12cells. 2. Anemarrhena total saponins treatment decreased the expression levels of BA mRNA and protein in PC12celll induced by Aβ25-35but increased the expression of BCL-2mRNA and protein.