| ObjectiveTo explore the etiological factor and effect of replenishing kidney-essence,removing phlegm therapy on treating Alzheimer's disease(AD) in theory. And to investigate the effect and mechanism of serum containing-Bushen Huatan Recipe(BHR) on neuronal apoptosis of PC12 cell model of Alzheimer's disease(AD) that was induced by Aβ25-35 segment neurotoxin.MethodsThe Wistar rats were randomly divided into normal groups and BHR groups which were fed on BHR decotions with dosage as ten times as human normal. Then stripple blood from the rats and contract the serum containing-BHR, in terms of serum pharmacology principle . Use PC12 cells induced with 20μmol/L Aβ25-35 as experimental model in vitro. Devide cells into different groups and add the different concentration rats serum containging-BHR(5%,10%, 20%) as culture medium to the cells, compared to the complete medium(RPMI1640) and the matched drug-free rats serum medium. Then , Neuronal survival was assessed by counting MTT assay, cell death rate was checked with Trypan-blue tincture. Apoptosis of PC12 cells was determined with flow cytometric analysis. The protein expression of apoptosis associated protein(caspase-3) was examined by using immunocytochemical SABC method,while the configuration of PC12 cells was observed with microscopy.Results1.Serum containing-BHR effect on proliferation in PC12 cells After 12, 24, 48hours, the serum containing-BHR promoted the PC12 cells' growth, and increased in a time and dose-dependant manner . There was no difference between the complete medium and drug-free serum medium (p>0.05)o However, the proliferation treated by the serum containing-BHR are significantly higher than the complete medium and drug-free serum medium (P<0.05). Among the serum containing-BHR groups; the proliferation rate is statistically increased for 20% serum group compared to the other two groups.2.Setting up Alzheimer's disease cell apoptosis model with PC12 cell induced byAβ25-35Compared to blank medium, Trypan-blue tincture method showed that cell deathrate increased with different concentrations of A625.35 (0.1μM,1μM, 5μM,10μM, 20μM) in a dose-dependant relationship. Therefore, we choose the 20μmol/LAB25-35 for AD model.3.Serum containing-BHR effect on protecting neurotoxic action of Aβ25-35(l)Histological examinationWithout Aβ25-35 treated,the drug-free serum medium and serum containing-BHR could promote the PC12 cells' growth with clear nucleolus, bright extranuclear, probuberance and floccus in the vicinity of cells. However,with the neurotoxic AB25-35 treated, we observed that the cell body became shrunken ,the chromation was compacted and probuberance disappeared, arised macula around the cells. But results of serum containing-BHR are better than drug-free serum medium(2)Serum containing-BHR effect on decreaseing cell death rateCell death rate of the AD model groups were significantly higher than other groups (p<0.01). There were also significant differences between the drug-free serum medium and serum containing-BHR groups (p<0.01). In the range of 5%,10%, 20% concentration of serum containing-BHR, cells death rate decreased with the increase of concentration.(3)Serum containing-BHR effect on neuronal survivalNeuronal survival of the AD model groups are significantly lower than the other groups(p<0.01). There were significant differences between the drug-free serum medium and serum containing-BHR groups(p<0.01 ). Among the serum containing-BHR groups, the neuronal survival of cells was statistically increased for 20% serum group compared to the other tow groups.(4) Serum containing-BHR effect on cell apoptosisIn AG25-35 treated cells, folw cytometric analysis displayed the peak of hypodiploid DNA content . The cell apoptosis rate was lower in Serum containing-BHR groups than that of AD model group. There were significant differences between the drug-free serum medium and serum containing-BHR groups(p<0.01). Among the serum containing-BHR groups, the cell apoptosis is statistically decreased.(5)Serum containing-BHR effect on... |
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