| Genistein (Genistein, Gen), is one of the main components of soy isoflavoneaglycone，widely exists in leguminous plants. Gen is known as phytoestrogen because ofthe similar chemic structure to estradiol, perform dual effects as estrogenic andanti-estrogenic. Gen could protect and treat tumor、cardiovascular disease、osteoporosis andclimacteric symptom, and could enhance the organic immune function, it is applied inpharmaceutical industry, nutritional products and so on. Studies show that Gen has anapparent neuroprotective effect, and it can be used for prophylaxis and cure of senileneurodegenerative diseases such as AD (Alzheimer’s disease). The previous cellular levelstudies showed Gen could activate the PKC (Protein kinase C) signal pathway, throughup-regulating α-secretase activity and down-regulating β-secretase activity, reducing theformation and deposition of β-amyloid and inhibiting the toxicity of Aβ on hippocampalneurons, and play the neuroprotective effect. We will continue to further study the protectiveeffect of Gen and its mechanism in cellular level in this paper. The concrete job are asfollows:1. To set up the AD cell model using PC12cells induced with Aβ25-35. The PC12cellswere pre-incubated with different concentrations Gen for2h, then cultured with Aβ25-35for24h. We detected cell morphological alterations and cell viability by microscopicexamination and MTT assay. Morphological apoptosis and apoptosis ratio were determinedby staining with Hoechst33342/PI. The spectrophotometry was used to detect Caspase-3activity, and the fluorescent probe Fluo/AM was used to determine the intercellular calciumconcentration by fluorospectrophotometer. The results revealed that, the toxic effects of Aβ25-35on PC12cells showed in a dose-dependent manner, PC12cells were cultured with20μM Aβ25-35for24h, cells injury was observed.25μM Gen could lighten themorphological damage, increase cell viability, reduce apoptosis ratio, decrease Caspase-3activity and inhibit the intercellular calcium overload.The results showed Gen could protectPC12cells damage induced by Aβ25-35.2. The PC12cells were pre-incubated with50μM PKC inhibitor (MyristoylatedProtein Kinase C Peptide Inhibitor，Myr),1h latter, treated with Gen for2h, then culturedwith Aβ25-35for24h. We determined the change of PKC activity, then by morphologicalobservation, cell viability and apoptosis assay, Caspase-3activity and intercellular calciumconcentration determination, we discussed the effect of Myr to the cytoprotection of Gen.The method of Real time PCR was used in this study to discuss the relationship betweenGen、Myr and expression of Bcl-2, Bax mRNA. It provided the further discussion for themechanism of Gen against Aβ25-35-induced PC12cells injury. The results showed that, Gencould improve PKC activity of PC12cells by induced Aβ25-35, however the phosphorylationcould be inhibited by Myr. Myr could increase the morphological damage, reduce cellviability, improve apoptosis ratio、 Caspase-3activity and the intercellular calciumconcentration. So pretreatment of cells with Myr significantly bloked the protective effectsof Gen against Aβ25-35-induced PC12cells injury. The results also showed that Gen couldincrease Bcl-2expression, decrease Bax expression, and increase the ratio of Bcl-2/Bax,which indicated that Gen could inhibit the apoptosis induced by Aβ25-35, and Myr couldblock the effect of Gen.It was indicated that Gen protected PC12cells from Aβ25-35-induced cytotoxicity. Itspossible molecular mechanism may be that Gen increased Bcl-2expression, decreased Baxexpression, and increased the ratio of Bcl-2/Bax, thus it reduced the apoptosis ratio; Genregulated PKC signal pathway, inhibited the deposition and toxicity of Aβ, and PKC signalpathway involved in the Bcl-2and Bax expression. The research of this dissertationprovided the theory basis for the prevention and treatment to AD. |
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