| Endophytic fungi refers to those fungus that live in the plant tissue without causing significant symptoms for a period of its life. Vatica genus plants are rich of resveratrol, which has been reported that can prevent oxidative stress-induced apoptosis in PC12cells. Based on the symbiosis theory, endophytic fungi of Vatica genus plants can likely have the same or similar active ingredients as resveratrol. We screened several compounds in solid fermented product of Phomopsi sp. and found that Cytochalasin H(CytH)might have potential protective effect on PC12cell injury. This activity of CytH has not been reported yet. So the studies of their function and mechanism are very necessary and meaningful.Parkinson’s disease(PD) is a degenerative disease of the central nervous system.The dopamine(DA) neurons progressive deletions may be its causes. Whereas apoptosis is the main form of its neuronal loss. One of the main pathogenesis of PD is oxidative stress. CytH and resveratrol are both from Phomopsi sp., whether it has similar anti-oxidative stress function and have the beneficial effects on PD or not?So, we used1-methyl-4-phenylpyridinium(MPP+) to prepare PD cell model to observe the effects of CytH on its damage and apoptosis.And we also carried out a preliminary study of its mechanism. The main results were as follows:1. Effects of Cytochalasin H on MPP+-induced neurotoxicity in PC12cellsMethods:PC12cell survival rate was detected by MTT assay. LDH release rate was determined by colorimetric determination. Grouping and dosing:(1) the control group:0.01%DMSO solvent;(2) the model group:500μmol/L MPP+;(3) the positive drug group:100umol/L selegiline;(4)the test drug groups:CytH of different concentrations. Tested drugs and positive drug were dosed by three methods:①Pretreatment:CytH was added4h before adding MPP+;②After intervention:CytH was added4h after adding MPP+;③Simultaneous intervention: CytH and MPP+were added simultaneously. MPP+role of each group all lasted for48h.Results:(1) Cytotoxicity of CytH:Compared with the control group, cell survival rates were not significantly reduced in0.00125～2μmol/L CytH group. When CytH concentrations were0.05,0.1,0.2μmol/L, survival rates of PC12cells were increased to118.22%±4.29%, 119.00%±7.33%,118.48%±6.85%, resectively. Compared with the control group, they were statistically significant difference (P<0.01). Results showed that CytH not only had no cytotoxicity, but also could promote nerve cell proliferation.(2) Effects of CytH pretreatment on MPP+-induced PC12cell survival rate and LDH release rate:The cell survival rate was increased within the range of0.001～0.01μmol/L after CytH pretreatment, and it was concentration-depended (r=0.939, P<0.01). When CytH concentration was0.01μmol/L, survival rate was103.7%±0.3%(P<0.001). The LDH release rate was decreased after CytH (0.0025～0.01μmol/L) pretreatment, and it was concentration-depended (r=-0.946, P<0.01).Compared with the model group, the difference was significant (P<0.01,0.001).(3) Effects of CytH post-intervention on MPP+-induced PC12cell survival rate and LDH release rate:The cell survival rate was increased within the range of0.01～0.15μmol/L after CytH post-intervention, and it was concentration-depended (r=0.890, P<0.01).When CytH concentration was0.15μmol/L, survival rate was reached to104.3%±2.4%(P<0.001). The LDH release rate was decreased after CytH (0.01～0.2μmol/L) post-intervention, and it was concentration-depended (r=-0.858, P<0.01). Compared with the model group, the difference was significant (P<0.01,0.001).(4) Effects of CytH simultaneous intervention on MPP+-induced PC12cell survival rate and LDH release rate:The cell survival rate was increased within the range of0.05～0.2μmol/L after CytH simultaneous intervention, and it was concentration-depended(r=0.885, P<0.01). When CytH concentration was0.2μmol/L, survival rate was reached to96.6%±4.6%(P<0.001). The LDH release rate was decreased after CytH (0.05～0.2μmol/L) simultaneous intervention, and it was concentration-depended (r=-0.913, P<0.01).Compared with the model group, the difference was significant (P<0.01,0.001).Conclusion:CytH had certain prevention, treatment and repair function on MPP+-induced cell damage in PC12cells.2. Protective effects of Cytochalasin H on MPP+-induced apoptosis in PC12cellsMethods:It was grouped as above and dosed as simultaneous intervention. TEM was used to observe cell ultrastructure. AO/EB staining was used to observe cell morphology. Flow cytometry was used to determine apoptosis ratios.Results:(1) Ultrastructure of cells under TEM:TEM results showed that the ultrastructural changes of cell apoptosis was visible after MPP+intervention:the microvilli on the cell surface was reduced or disappeared. Nuclear membrane was shrinked. Nuclear volume was deflated. Chromatin condensed and lumped together on the nucleus periphery. Mitochon-dria were seen significant swelling in cytoplasm, even seen vacuoles. After0.05,0.1,0.2μmol/L CytH intervention, compared with the model group, the ultrastructure of cells became normal, nuclear membrane was seen clearly, nuclear chromatin was relatively normal, normal and intact cell organelles were also visible especially in high concentrations group. The results showed that CytH could improved ultrastructural changes in MPP+-induced PC12cells, reduce the lose of cell organelles. All the results showed that CytH could improve MPP+-induced ultrastructural changes in PC12cells.(2) Cell morphology by AO/EB staining:Most normal cells were in fusiform shape. Nucleoid were uniformly dyed into green, some cells fluorescence was dimmed. Cells in model group were mostly oval. Nucleoid were uniformly dyed into orange or bright yellow. CytH (0.05,0.1,0.2μmol/L) and positive drug could improve this phenomenon in varying degrees. Of which, high-dose group had the most significant effect close to selegiline group. The results showed that CytH had a protective effect on MPP+-induced apoptosis in PC12cells.(3) Apoptosis ratio analyzed by Flow cytometric:In the model group, early apoptotic cells was significantly higher than the control group (P<0.05), late apoptotic or necrotic cells (P<0.01) and total apoptosis rate (P<0.001) also showed a significant increase. Compared with the model group, the number of living cells was significantly increased (P<0.01,0.001) in each dose group of CytH (0.05,0.1,0.2μmol/L), total apoptosis rate decreased significantly (P<0.001), and they were dose-depended(r=-0.754, P<0.05). The results showed that CytH could reduce the apoptosis rate of MPP+-induced apoptosis in PC12cells.Conclusion:CytH could inhibit MPP+-induced apoptosis in PC12cells to certain extent.3. Effects of Cvtochalasin H on MPP+-induced oxidative stress in PC12cellsMethods:It was grouped as above and dosed as simultaneous intervention. Flow cytometry was applied to determine intracellular ROS contents.Thiobarbituric acid (TBA) method, xanthine oxidase method and dithio-dinitrobenzoic acid method were used to survey and evaluate MDA contents, SOD, GSH-PX enzyme activity in cells. Reduction method was used to detect total antioxidant capacity (T-AOC) in cells.Results:(1) Effects of CytH on intracellular ROS contents:Compared with the control group (ROS content ratio was1), ROS content ratio was increased to1.91±0.15(P<0.001) in the model group. ROS content ratios were decreased to1.78±0.23(P>0.05),1.07±0.30(P<0.05) and0.91±0.37(P<0.05) after CytH0.05,0.1,0.2μmol/L, respectively. ROS content ratio was decreased to1.18±0.13(P<0.01) in the positive group. Results showed that CytH could inhibit the increase of ROS caused by MPP+, and it was dose-depended (r=-0.780, P<0.05).(2) Effects of CytH on intracellular MDA contents:MDA content was24.48±3.51nmol/mgprot in the model group. It was significantly increased (P<0.01) compared with the control group. Compared with model group, MDA contents were significantly decreased (P<0.05) after CytH0.0125,0.025,0.05,0.1,0.2μmol/L and selegiline. In which CytH0.05μmol/L was the best, MDA content was only3.23±1.42nmol/mgprot. Results showed that CytH could inhibit the increase of MDA caused by MPP+in PC12cells.(3) Effects of CytH on T-AOC, SOD and GSH-PX activity in cells:T-AOC, SOD and GSH-PX activities were0.9±1.56U/mgprot (P,0.01),19.49±4.36U/mgprot (P<0.001) and507.6±67.3U/mgprot (P<0.001) in the model group, respectively..Compared with control group they were significantly decreased. Compared with the model group, CytH each dose group(0.05,0.1,0.2μmol/L) could significantly elevate T-AOC activity (P<0.01, P<0.001), increase SOD activity espacially in0.1,0.2μmol/L dose groups(P<0.05, P<0.001), elevate GSH-PX activity only significant differenceonly in0.2μmol/L dose group (P<0.001). In2μmol/L dose group of CytH, activities were as follws:T-AOC10.47±0.75U/mgprot (P<0.001), SOD81.25±3.79U/mgprot (P<0.001), GSH-PX1063.18±53.7U/mgprot (P<0.001). Results showed that CytH could significantly elevate T-AOC, SOD and GSH-PX activities in PC12cells.Conclusion:CytH could improve T-AOC, SOD and GSH-PX activity, reduce MDA and ROS contents in PC12cells. It was possibly had anti-oxidative stress function to inhibit MPP+-induced apoptosis in PC12cells.4. Research of molecular mechanism for the protective effect of Cytochalasin H on MPP+-induced apoptosis of PC12cellsMethods:It was grouped as above and dosed as simultaneous intervention. After that, the cells were processed to extract proteins. BCA method was utilized to quantitate protein amounts. Bcl-2and caspase3protein expression were measured by Western blot.Results:(1) Effect of CytH on Bcl-2expression in MPP+-induced PC12cells:Compared with the control group, Bcl-2expression was in a great decrease in the model group. After the intervention of CytH (0.05,0.1,0.2μmol/L), high dose group could inhibit the decrease of Bcl-2expression compared with the model group. Selegiline group also had the similar effect.(2) Effect of CytH on caspase-3expression in MPP+-induced PC12cells:Compared with the control group, the expression of caspase-3was significantly decreased in the model group. After the intervention of CytH (0.05,0.1,0.2μmol/L), caspase3expression was significantly increased compared with the model group. This indicated that MPP+could induce caspase-3activation, reduce caspase-3expression significantly, and trigger apoptosis effect. While CytH could reduce PC12apoptosis by reducing the activation of caspase-3.Conclusion:CytH could inhibit MPP+-induced apoptosis in PC12cells by increasing Bcl-2expression and inhibiting caspase-3activation. The molecular mechanisms for the protective effect of CytH on MPP+-induced of apoptosis in PC12cells might involve effects on Bcl-2family protein expression and caspase activation. |
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