| Objective: To explore the protective effect of mesenchymal stem cells-conditioned medium(MSC-CM) on HD-induced injury on PC12 cells and its mechanism whether via inhibiting mitochondria-dependent caspase-3 pathway.Materials and Methods: Bone mesenchymal stem cells(BMSCs) were incubated and purified, then harvested to obtain the 5th passage conditioned medium. This experiment set up 4 groups:(1)blank control group: no drug or MSC-CM was added in the medium.(2)MSC-CMcontrol group: 40% MSC-CM was added in the medium.(3)HD expose group: PC12 cells were treated with 20 mmol/L HD for 24 hours.(4)MSC-CM protect group: 20 mmol/L HD exposed cells were added 10%, 20%, 40% MSC-CM. The viability was observed in PC12 cells with MTT; Apoptosis were estimated by Hoechst33342 staining and flow cytometry; Mitochondrial membrane potential(MMP) was examined byrhodamine 123;moreover, we investigated the expression of Bax, Bcl-2and NGF by real-time PCR, Bax, Bcl-2, cytochrome c and NGF estimated by western blotand NGF in BMSCs and MSC-CM estimated by ELISA; caspase-3 activity detection by caspase-3 activity assay kit.Results:1.MTT There was no significant difference in viability of PC12 cells between the control group and MSC-CM control group(p>0.05). However, viability of PC12 cells in HD exposure group was significantly lower than that in control group(p<0.05).On the other hand, viability of PC12 cells in the protective groups was significantly higher than that in HD exposure group(p<0.05) and scaled up with the increase of the treated dose of MSC-CM. 2. Cell apoptotic Hoechst 33342 showed that the nuclei of control cells were of a rounded shape with homogeneous intensity. However, the HD-exposed PC12 cells showed crescent-shaped nuclei and fragmentation with heterogeneous intensity in the nuclei, suggesting that these cells under went gross morphological change indicative of apoptosis. Compared with group received HD alone,the number of cells with condensation and fragmentation was decreased in the protective groups, indicating that MSC-CM reduced apoptotic morphological changes induced by HD. The rate of apoptosis in PC12 cells showed that apoptosis of PC12 cells exposed to HD significantly increased compared to control group. However, after treatment with MSC-CM the apoptosis rate of HD-exposed PC12 cells was reduced. 3.Bax and Bcl-2 m RNA expression in PC12 cells The m RNA expression of Bax in PC12 cells was significantly higher in HD exposure group than that in control group(p<0.05). However, after treating with MSC-CM, Bax m RNA expression in PC12 cellswas significantly lower in protective groups than that in HD exposure group(p<0.05) and decreased in a dose-dependent manner. On the other hand, the m RNA expression of Bcl-2 in PC12 cells exposed to HD was significantly lower than that in control group(p<0.05), while the m RNA expression of Bcl-2 in PC12 cellswas significantly higherin protective groups than that in HD exposure group(p<0.05). The expression ratio of Bax/Bcl-2 genes in PC12 cells was also analyzed. The ratio of Bax/Bcl-2 gene expression in PC12 cells significantly increased in HD exposure group compared with control group(p<0.05). However, the ratio of Bax/Bcl-2 gene expression in PC12 cells significantly decreased in protective groups compared with HD exposure group(p<0.05). 4. Bax and Bcl-2 protein expression in PC12 cells The protein expression of Bax in PC12 cells was significantly higher in HD exposure group than that in control group(p<0.05). However, after treating with MSC-CM, bax protein expression in PC12 cells was significantly lower in protective groups than that in HD exposure group(p<0.05) and decreased in a dose-dependent manner. On the other hand,the protein expression of Bcl-2 in PC12 cells exposed to HD was significantly lower than that in control group(p<0.05), while the protein expression of Bcl-2 in PC12 cells was significantly higher in protective groups than that in HD exposure group(p <0.05).The ratio of Bax/Bcl-2 protein expression in PC12 cells significantly increased in HD exposure group compared with control group(p<0.05). 5. The MMP was analyzed by rhodamine 123 PC12 cells exposure to HD resulted in a significant decrease in MMP of PC12 cells compared to control group(p<0.05). However, in the presence of MSC-CM, the MMP of PC12 cells was significantly increased in protective groups compared to HD exposure group(p<0.05). Moreover, the decreased MMP was attenuated from 24.28 % to 38.15%, 51.45% and 66.47%. 6. Cytochrome c protein expression in PC12 cells Protective effect of MSC-CM on expression of cytochrome c in PC12 cells exposed to HD showed that protein levels of cytochrome c significantly decreased in mitochondrial fraction and significantly increased in cytosolic fraction of HD-exposed cells compared to control group(p<0.05), that protein levels of cytochrome c significantly decreased in mitochondrial fraction and significantly increased in cytosolic fraction of HD-exposed cells compared to control group(p<0.05).7. caspase-3 viability The activation of caspase-3 protein showed that the activity of caspase-3in PC12 cells was significantly higher in HD-exposed cells when compared with control group(p<0.05). However, the activity of caspase-3in PC12 cells was significantly lower in protective groups compared to HD exposure group(p<0.05) and decreased with the increase in dose of the treated MSC-CM. 8. NGF level We performed real time RT-PCR, western blot and ELISA assays. By real time RT-PCR, we found that NGF m RNA expressed in BMSCs at passage 3 to passage 6, and the level was relatively high at passage 3. BMSCs expressed NGF protein all the time, and the expression pattern was similar to those of NGF m RNA. Furthermore, the amount of NGF protein in the cell culture medium of BMSCs at passage 3 was detected using a protein standard curve and the correlation coefficient was calculated by a linear correlation assay. The result showed the MSC-CM from passage 3 to 6 was 52.41, 49.17,24.11 and 23.38 pg/ml, respectively.Conclusion: HD exposure can induce PC12 cells apoptosis; MSC-CM have protective effect against HD induced apoptosis in PC12 cells; this protective effect may via mediating HD-disturbed mitochondria-dependent caspase-3 pathway; in the meantime,besides NGF, there are a large number of protective factors presented in MSC-CM, may also contribute to inhibit apoptosis-inducing pathways... |
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