| Objective:To discover the pathology changes in hippocampal tissue of medial temporal lobe epilepsy (mTLE) rat model and after blocking the ubiquitin-proteasome system (UPS), to analyze the expression changes of ubiquitin, Nedd4-2and C1C-2during every stages, and to investigate the contribution of Nedd4-2/ClC-2ubiquitin signal pathway in the pathogenesis of medial temporal lobe epilepsy rat model.Methods:Taking75SD rats (clean) which are25days old, were randomly divided into3groups, control group, model group and MG-132(A kind of proteasome inhibitors) pretreatment group.The rats of model group and pretreatment group were built by injecting corresponding drugs (pilocarpine, lithium chloride, MG-132, etc.) into abdominal cavity respectively, then induced status epilepticus, and the control group was used the same amount of normal saline instead. After the success of establishing rat model, the three groups mentioned above were randomly divided into3subgroups again according to different time of obtainning specimens, these subgroups were named acute group, latent period group and chronic group respectively.(1) In control group and model group, using immune coprecipitation technology and fluorescence immunoassay double standard combined with laser confocal technique to reveal if there is a relationgship between ubiquitin and Nedd4-2, in the hippocampus of rats.(2) In each subgroup of control group, model group and MG-132pretreatment group, we observed the behavioral changes of each rats model. HE and Nissl’s staining was used to discover the loss of neurons in the hippocampus of rats’brain tissue. Timm staining was used to found the mossy fiber budding. Then we detected the expression levels of ubiquitin, Nedd4-2and C1C-2in the rats’hippocampus by using Western blot technique.Results:In control group, rats’ behavior and histopathology were normal. Then in model group and MG-132pretreatment group,90.0%of rats had been successfully induced mTLE by intraperitoneal injection of lithium chloride-pilocarpine. The total mortality was24.1%,73.7%of the survival rats were found to suffer from chronic spontaneous seizures after8weeks. In the hippocampal of model group, the loss of neurons was obvious, and moss fiber sprouting could also be found. The pathological changes of MG-132pretreatment group was more significant when comparing with model group, and the differences between two groups had statistical significance (P<0.05).By using Co-IP and immunofluorescence double standard combined with laser confocal technique, we proved that Ubiquitin was correlated with nedd4-2. Western bolt detected the expression level changes of ubiquitin, Nedd4-2and CIC-2both in model group and MG-132pretreatment group, ubiquitin expression:model group had no changes when compared with control group. On the other hand, when compared with control group and control group, MG-132decreased the expression of ubiquitin in acute and latent phase, and activated the expression in chronic phase (P<0.05). Nedd4-2expression:compared with control group, Nedd4-2expression of model group was downregulated in both acute and latent phase,whereas the expression was upregulated in the chronic phase (P<0.05). When MG-132pretreatment group compared with control group, Nedd4-2expression of three periods all decresed (P<0.05). When compared with model group, the expression was downregulated in the acut and chronic phase, but upregulated in the chronic phase (P<0.05). CIC-2expression:in comparison with control group, the expression of model group was coincident with Nedd4-2. MG-132pretreatment group compared with control group, MG-132decreased the expression of CIC-2in acute and chronic phase, and on the contrary in the chronic phase (P<0.05). When compared with model group the expression was consistent with ubiquitin.Conclusion:(1) Our results strongly suggest the exsistence of the ubiquitin mediated signaling pathway in the hippocampus of both normal and mTLE rats. Nedd4-2is the key role of this pathway which is acted as a kind of ubiquitin-protein ligases (E3s), and CIC-2is worked as the target protein of Nedd4-2.(2) The Nedd4-2/C1C-2ubiquitin signal pathway has a regulatory effect on the pathogenesis of mTLE. We can promote the occurrence and development of mTLE by blocking the pathway, and fuether illustrate that the Nedd4-2/C1C-2ubiquitin signal pathway plays a important role in the thepathogenesis of medial temporal lobe epilepsy. |
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