| ObjectiveThe main pathological features of PD are the selective degeneration of substantia nigra and striatum dopamine (dopamine, DA) neurons. Decreasing of DA neurons will contribute to dysfunction of basal ganglia neural. Although it was widely reported that PD is associated with genetic, environmental, aging, oxidative stress, inflammation, immune etc, it is elucidated that the different varieties of pathological factors can lead to incidence of PD through a commen path of cell apoptosis. Apoptosis is the final form of pathological factors contributing to degenenration of DA neurons. More than 80% of brain DA neurons are facing to apoptosis in PD patients. So far, there is still not a very effective method for treating PD, therefore, to screen an effective neuroprotective agents has become the focus and difficult of the medical research.According to the theory of zang organ phenomena"kidney produces marrow, through the brain" our preliminary study found that Plastrum Tstudinis Extracted with ethyl acetate (PTE) may have a good neuroprotective effect, while its mechanism remains unclear. In addition, we found that there exist nine compounds in PTE, but which kind of compounds exersts anti-apoptitic activity remains to be demonstrated. Therefore, this experiment is designed to study the anti-apoptotic activtity and its mechanism of PTE, in turn, to determine the active compound is able to inhibit apoptosis in PTE and to explore its molecular mechanism.Methods(一) Observe the anti-apoptosis activaty of PTE and disclose its potential mechanism(1) The PC12 apoptosis model was established by serum starvation for 3 days. The cells were randomly classified into four groups:Control group, Model group, PTE (3μg/ml) and PTE (30μg/ml) group. Control group was treated by nothing, model and drug group was cultured without fetal bovine serum, drug group added with different concentrations of PTE, the model group added with the same amout of DMSO with drug. The effects of PTE on serum starvation induced apoptosis of PC12 cells were determined after treatment. Cell optical density was determined by MTT, cell morphology was observed by optical microscope, ratio of cell apoptosis was examined by Annexin-V/PI double stain flow cytometry (FCM), morphology of cell nuclear was examined by Hoechst staining.(2) To observe whether mitochondria signaling pathway participated in anti-apoptotic activity of PTE, cell mitochondrial membrane potential was examined by FCM after Dioc6 staining, in parallel with Caspase-3 molecular expression was detected by Western blot anaylsis.(3) To explore if BMPs signaling pathway mediated the anti-apoptotic activity of PTE, Western blot analysis was applied to detect the BMPs signaling pathway moleculars expression. Bio-Rad Quantity One gel analysis system was used for semi-quantitative analysis of bands. Further, anti-apoptotic activity of PTE against serum starvation-induced PC12 apoptosis was examined after BMP4 siganling pathway blocked or overexpressed.(二) Screen which compounds in PTE can inhibit serum starvation-induced apoptosis in PC12 cells and to detect its mechanism.(1) Apoptosis of PC12 cells was continued to establish by serum starvation-induced for three days. Cells were randomly divided into normal control group, model group and drug group (30 and 100μg/ml of cholesterol myristate, Palmitic acid methyl esters, Stearic acid ethyl ester, stearic acid, (+)-cholesten-3-one, cholesterol). Which compounds exerts the ability to inhibit serum starvation-induced apoptosis in PC12 cells was examined. Cell optical density was determined by MTT, cell morphology was observed by optical microscope, ratio of cell apoptosis was examined by Annexin-V/PI double stain FCM.(2) A dual luciferase reporter system was applied to detect the effects of different concentrations of Cholesterol myristate in Idl promoter reporter activity in PC12 cells at the indicated times following transfection Idl promoter reporter plasmid. Further, Idl promoter reporter activity was examined after BMP4 signaling pathway blocked by BMP4 Neutralizing antibody. Western blot and RT-PCR were used to detect BMPs signaling pathway molecular, Id1, Bcl-x/L expression; ratio of apoptosis was examined using Annexin-V/PI double staining flow cytometry after blocking BMP4 signaling pathway.Results(1) Results of MTT and the optical microscope observation showed that, PTE can increase the viability of serum starvation-induced PC12 cells in a dose-dependent manner, while Palmitic acid methyl esters, Stearic acid ethyl ester, stearic acid, (+)-cholesten-3-one, cholesterol had no effects in viability of serum starvation-induced PC12 cells. Annexin-V/PI double staining flow cytometry found that, PTE inhibited apoptsis of serum starvation induced PC12 cells dose dependently. PTE (3μg/ml) and PTE (30μg/ml) compared with model group, the difference was statistically significant.(2) Dioc6 tag flow cytometry results showed that mitochondrial membrane potential decreased significantly in model group compared with the control group, while PTE group can significantly increased mitochondrial membrane potential compared with Model group. Western blot results showed that PTE can reduce the expression of Caspase-3 in a dose dependent manner.(3) Western blot results showed that, PTE can increase BMP4, BMPRIA, p-Smadl/5/8 expression, There is difference between PTE (3μg/ml), PTE (30μg/ml) group and model group. PTE had no effects in BMP2, BMP7, and BMPR II expression in PC12 cells, BMPR-IB expression was not detected. When BMP4 signal pathway was blocked by neutralizing BMP4 antibody, FCM results showed that apoptotic ratio increased and antiapoptotic effect of PTE was partially inhibited; when BMP4 signal pathway was overexpressed by transfection BMP4 plasmid, FCM results demonstrated that apoptotic ratio dereased and antiapoptotic activity of PTE was enlarged by BMP4 plasmid.(4) MTT results showed that Cholesterol myristate in PTE was the only compound to inhibit viability decreasing of serum starvation-induced apoptosis of PC12 cells. Palmitic acid methyl esters, Stearic acid ethyl ester, stearic acid, (+)-cholesten-3-one, cholesterol had no effect on its activity. FCM results showed that Cholesterol myristate was capable of inhibiting serum starvation-induced apoptosis in PC12 cells in a dose dependent manner.(5) Dual-Luciferase Reporter Assay System results showed that the Cholesterol myristate can increase the Idl promoter activity in PC12 cells in a dose and time dependent manner. And similar compounds of Cholesterol myrisatate including Stearic acid ethyl ester, (+)-cholesten-3-one and cholesterol did not increase the promoter activity of Idl in PC12 cells. Results of Western blot and RT-PCR results showed that the Cholesterol myristate can increase BMP4, BMPRIA expression; Western blot results also showed that Cholesterol myristate can up-reulate p-Smadl/5/8 expression. The effects of Cholesterol myrisatate in esters of BMP2, BMPRⅡexpression were not changed, BMPR-IB expression was not detected. When BMP4 signal pathway was blocked by neutralizing BMP4 antibody, FCM results showed that apoptotic ratio increased and antiapoptotic effect of PTE was partially inhibited, in addition, the ability of Cholesterol myristate increasing Idl promoter activity in PC12 cells was partially increased.ConclusionPTE can inhibite serum starvation induced apoptosis in PC12 cells, and this effect was dose dependent. In addition, its mechanism may be related to (partial) activation of the expression of BMP4 signaling pathway, thereby inhibiting the downstream mitochondrial pathway. Cholesterol myristate was the only compound in PTE could inhibit serum starvation-induced PC12 cell injury; myristate maybe the active structure that play anti-apoptotic ability. The anti-apoptotic activity of Cholesterol myristate maybe (partially) associated with activating BMP4-Idl signaling pathway. Myristate maybe the active structure that Cholesterol myristate increases Idl promoter activity.
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