| Genistein(genistein, Gen) is the main active component of soybean isoflavone. Gen, as a phytoestrogen, plays a role in nerve protective effect, but no carcinogenic and other side effects in non-neuronal cells compared with estrogen. Gen’s nerve protective effect arouses the continuing interest. Our previous studies found that, Gen can enhance the activity of PKC Gen, regulate the α-/β secreted enzyme activity, reduce insoluble Aβ deposition, reverse Aβ-induced the Bcl-2 downregulation and Bax upregulation, and reduce the degree of cell apoptosis, eventually improve the vitality of PC12 cells stimulated by Aβ. Currently, Gen nerve protective effects and its related mechanism gradually get deeper study, but its molecular protection mechanism and involved signal remains need to be further elucidated.This paper continue to explore the in vitro neural protection mechanism and molecular mechanism of Gen in protection against Aβ. Specific content is as follows:1. The study of Gen on Aβ-induced PC12 cell vitality, apoptotic staining, apoptosis rate: MTT cell activity assay results showed that 0-25 μM Gen have little impact on cell vitality, but 50 and 100 μM Gen significantly reduced cell survival rate decreased, Aβ significantly reduced the smallest dose of PC12 cell survival rate is 20 μM, 0-100 μM Gen significantly improve the survival rate of Aβ-incubated PC12 cells, and 25 μM Gen have the maximum protection effect. Hoechst 33342 apoptosis staining results show that, Aβ-induced PC12 cells were obsersved obvious apoptotic characteristics, and the percentage of apoptotic cells significantly higher compared with the control group, while Gen treatment group(12.5, 25, 50 and 100 μM) markedly reduced the percentage of apoptotic cells. The results of Annexin V- FITC double staining to detect cell apoptosis rate showed that, Aβ group was significantly higher than the control group, and Gen treatment group(12.5, 25, 50 and 100 μM) significantly inhibited the cell apoptosis induced by Aβ, but 25 μM doses of Gen acted maximum inhibition effect.2. q PCR and Western blot to detect the effect of Gen on m RNA and protein expression of Fas and Fas L in Aβ-induced PC12 cells: q PCR results showed that, Aβ group significantly induced the the m RNA expression of Fas L, moderately increased the m RNA expression of Fas, and Gen dramaticlly inhibited Aβ-induced m RNA expression of Fas and Fas L. Westernblot results showed that Aβ group significantly increased the protein expression of Fas and Fas L, however, Gen obviously inhibited Fas and Fas L protein levels induced by Aβ.3. q PCR and Western blot to detect the effect of Gen on m RNA and protein expression of Bcl-w and Bim in Aβ-induced PC12 cells: q PCR results showed that, Aβ group significantly reduced the Bcl-w and increases Bim m RNA expression, and Gen can reversed Aβ-induced the change of Bcl-w and Bim. Western blot results are in consistent with q PCR results.4. Western blot to detect the effect of Gen on cytoplasmic protein expression of Cyt C and Smac in Aβ-induced PC12 cells: the results showed that Gen reversible excite A beta Cyt C and Smac from mitochondria to the release of the cytoplasm.5. q PCR and spectrophotometry to detect Gen on the m RNA and enzymatic activity of Caspase-3 and Caspase-8 in Aβ-induced PC12 cells: the results showed that, Gen inhibited the increase of Caspase-3 and Caspase-8 m RNA induced by Aβ. Enzyme activity determination of Caspase-3 and Caspase-8 further confirmed the effect of Gen against Caspase-3 and Caspase-8 induced by Aβ.6. Using JNK-si RNA and JNK phosphorylation inhibitors SP600125 for interfering JNK to detect m RNA and protein expression of Fas and Fas L in Aβ-induced PC12 cells: the results showed that, Gen, JNK-si RNA and SP600125 inhibited Fas and Fas L m RNA and protein expression induced by Aβ, and Gen+JNK-si RNA or Gen+SP600125 group exerted strongest effect.7. Using JNK-si RNA and JNK phosphorylation inhibitors SP600125 for interfering JNK to detect m RNA and protein expression of Bcl-w and Bim in Aβ-induced PC12 cells: the results showed that, Gen, JNK-si RNA and SP600125 inhibited Bcl-w and Bim m RNA and protein expression induced by Aβ, and Gen+JNK si RNA or Gen+SP600125 group exerted strongest effect.8. Using JNK-si RNA and JNK phosphorylation inhibitors SP600125 for interfering JNK to detect protein expression of Cyt C and Smac in Aβ-induced PC12 cells: the results showed that, Gen, JNK-si RNA and SP600125 inhibited Cyt Cand Smac protein expression induced by Aβ, and Gen+JNK si RNA or Gen+SP600125 group exerted strongest effect.9. Using JNK-si RNA and JNK phosphorylation inhibitors SP600125 for interfering JNK to detect m RNA and enzymatic activity of Caspase-3 and Caspase-8 in Aβ-induced PC12 cells: the results showed that, Gen, JNK-si RNA and SP600125 inhibited m RNA and enzymatic activity of Caspase-3 and Caspase-8 induced by Aβ, and Gen+JNK si RNA or Gen+SP600125 group exerted strongest effect.10. Westernblot to detect the effect of Gen on protein expression of p-JNK and JNK in Aβ-induced PC12 cell: the results showed that, Aβ significantly induced and 46 and 54 k Da bands of p-JNK in PC12 cells, and Gen can significantly blocked p-JNK 54 and 46 k Da bands, however, Gen+SP600125 showed the strongest effect.This study showed that, both JNK-si RNA and SP600125 suppressed Fas-mediated and mitochondrial-initiative apoptotic pathway, suggestting that JNK involved in regulating Fas-mediated and mitochondrial-initiative apoptotic pathway in PC12 cells induced by Aβ. But the results of inhibition of Gen on phosphorylation of JNK induced by Aβ, indicating that Gen may protection against Aβ-induced apoptosis via reduces Aβ-induced phosphorylation of JNK activation, and then inhibits the JNK dependent Fas and mitochondrial apoptosis pathway. However, the results of Gen+JNK-si RNA or Gen+ SP600125 group suggested that the the effect of Gen on Aβ-induced Fas and mitochondrial apoptotic pathways might be one of the Multi-target antiapoptotic effect of Gen, its neuroprotective mechanism remains to be elucidated. |
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