| Objective:Apoptosis mediates the precise and programmed natural death of neurons and is a physiologically important process in neurogenesis during maturation of the central nervous system. However, premature apoptosis and/or an aberration in apoptosis regulation is implicated in the pathogenesis of neurodegeneration, a multifaceted process that leads to various chronic disease states, such as Alzheimer's disease(AD). Abnormal metabolism and deposition ofβ-amyloid protein(Aβ) in AD brain is a key factor in the occurrence and development of the disease. In vitro and in vivo experiments,it is demonstrated that neuronal apoptosis is closely related with Aβ, and Aβcan induce neuronal apoptosis. Neuronal apoptosis is considered as one of the main factor resulting in a great deal of neuronal loss in AD. Therefore, if what can reduce Aβ-induced neuronal apoptosis,the prognosis of AD may be improved. Recent studies suggest that triptolide (TP) can inhibit the immunoinflammatory response induced by Aβin brains of AD model rats and improve the learning and memory ability of AD model rats through its anti-immunoinflammatory effect.In this experiment,the effect of TP on the neuronal apoptosis and the expression of apoptosis-related proteins in hippocampus of AD model rats induced by Aβis studied to further clarity the protective effect of TP on neurons of AD model rats and its mechanism.Method:1. Healthy male SD rats were randomly divided into control group, AD model group and TP-treated group. The AD model group was made with bilateral microinjection of Aβ1-40 into hippocampus in rats and Morris water maze test was used to judge that the model succeeds to or not. The control group was made with bilateral microinjection of normal saline into hippocampus in rats.The TP-treated group rats were administered TP (0.4mg/kg.d) intraperitoneally for 15d after The AD model was made successfully.2. By Toluidine Blue staining, we observed the structural changes of hippo- campus near the injected site in each group.3. By flow cytometry, we observed the changes in the rate of neuronal apoptosis in hippocampus of each group.4. By TUNEL staining, we observed the changes in the number of positive cells in hippocampus of each group.5.By transmission electron microscopy, we observed the changes in apoptosis- related ultrastructure of neurons in hippocampus of each group.6.By immunohistochemical staining, we observed the changes of cytochrome c and Bcl-2 protein expression in hippocampus of each group.Results:1. Toluidine Blue staining results showed that there is no obviously abnormal change in hippocampus of the control group, and there is a great deal of neuronal loss and glial infiltration near the injected site in hippocampus of the AD model group, and the hippocampal neuronal damage was reduced in hippocampus of the TP-treated group.2. Flow cytometry demonstrated that the apoptosis rate of hippocampal neurons in the AD model group was significantly higher than the control group (P <0.01), and the rate in the TP-treated group was decreased significantly, compared with the AD model group (P <0.01).3. TUNEL staining results showed that the cell number of positive staining in hippocampus of the AD model group were significantly higher than the control group (P <0.01), and compared with the AD model group, the number in the TP-treated group was significantly decreased(P <0.01).4. Transmission electron microscope results showed that the neuronal ultrastructure were generally normal in hippocampus of the control group, and the nuclear chromatin of hippocampal neurons are unevenly distributed with margination to the nuclear membrane and forming irregular clumps of high electron density in hippocampus of the AD model group, which showed signs of early apoptosis, and the margination of nuclear chromatin was not obvious in hippocampus of the TP-treated group.5. Immunohistochemically , the number and average optical density of cytochr- ome c positive products in hippocampus of the AD model group were significantly higher than the control group (P <0.01), and the number and average optical density of cytochrome c positive products in hippocampus of the TP-treated group were obviously lower than the AD model group (P <0.01).6. Immunohistochemically , the cell number and average optical density of Bcl-2 positive staining in hippocampus of the AD model group were significantly lower than the control group (P <0.01), and the cell number and average optical density of Bcl-2 positive staining in hippocampus of the TP- treated group were obviously higher than the AD model group (P <0.01).Conclusion:TP can inhibit the neuronal apoptosis in hippocampus of the AD model group.Its mechanism may concern increasing the Bcl-2 expression and reducing the release of cytochrome c in hippocampus of the AD model group. |
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