Effect Of Tacrolimus On Apoptosis Cells, MMP And AIF Of Rats With Epilepticus

Background and Objective:Epilepsy is a common form in adults.It is harm on people's healthy and life and has high fatality rate. In recent years, although the studies of epilepsy have made great progress, its pathogenesis is still unclear. Status epilepticus (SE) is one of the most common neurological emergency, its pathogenesis and drug therapy has become the focus of research in recent years.SE generally refers to the occurrence of a single unremitting seizure with a duration longer than 30 minutes or frequent clinical seizures without an interictal return to the baseline clinical state. The studies from epileptic patient and animal model showed that SE may induce local or diffuse neurodegeneration. The change, if irreversible, may lead to permanent neurological impairments.SE is a medical emergency and results in subcellular changes that lead to neuronal damage and death in the hippocampus. Now current research is focus on the specific mechanisms and the drug treatment of epilepsy.Large studies indicated that status epilepticus can lead to the generation of neuronal apoptosis. Now our research focus on the role of mitochondrial in apoptosis. Studies reported that the transmembrane potential is necessary to maintain the normal function of mitochondria, the decline in mitochondrial membrane potential may occuerd before morphological changes when cells apoptosis, The change of mitochondrial permeability transition pore opening is irreversible,it may led to changes in mitochondrial volume of mitochondria.The apoptosis-related factors may released into the cytoplasm and caused cell apoptosis. An immunosuppressive agent, FK506, has been used in clinical settings for the prevention of allograft rejection. Recent studies in vitro and vivo have demonstrated that FK506 not only induces some neurological diseases such as focal cerebral ischemia, but also plays a role in neuroprotection and neurotrophy. Despite the protective effect of FK506 on neuron was observed in experimental cerebral ischemia and seizure model, the mechanism by which FK506 targets neuron remains unclear.Our current study was designed to establish lithium chloride-pilocarpin-induced SE (LPCSE) rat models to investigate the effects of tacrolimus on apoptosis cells, MMP and AIF of rats with epilepticus and also to reseach the anti-epilepsy chanisms of FK506.Methods:1. Pilocarpine was intraperitoneally injected to make modles of SE. The rats were randomly divided into 3 groups (36 rats in each group, n=108):control group, pilocarpine group (pilo group) and FK506 group. In FK506 group, the animals were further treated with FK506 (Fujisawa, Japan) (2mg/kg,i.p) 24h and 1h before pilocarpine administration. In addition, rats in control group were treated as FK.506 group except that FK506 was replaced by the same dose of 0.9% saline. The rats were killed 24h later, isolated fresh hippocampus,-80 degrees saved, for TUNEL apoptosis, mitochondrial membrane potential and immunohistochemistrial techniques.2. The seizure activity of rats induced by pilocarpine was scored using a modified Racine scale.3. To detecte the apoptotic cells by One Step TUNEL Apoptosis Assay Kit, and the pathological changes of neuronal apoptosis were observed with the confocal microscope (OLYMPUS FV1000), each slice to take two 400-fold vision which the apoptotic cells show green.4. The flow cytometry was used to detect the mitochondrial membrane and the size of mitochondria.Extracting the mitochondria from fresh hippocampus with mitochondrial extraction kit, and using the flow cytometry and CELL Quest software to analysis.5. To check the apoptosis inducing factor in hippocampus by Immunohistochemistry, the tissue slices was fixed and treated with paraffin. using citrate buffer to microwave repair. The first antibody concentration is 1:300. The AIF was detected by the SABC method, dyed by TAB, used PBS to replace the first antibody and second antibody in control group. The sections were analyzed with HPIAS-1000 high-resolution color pathological image analysis system. The data showed that positive cells of AIF were seen with brown colour.6. The data are shown as the means±SEM. Statistical analysis was performed using t test and variance analysis with SPSS 10.0 software. Statistical significance was accepted at the conventional p<0.05 level.Results:1. Behavioral episodes induced by pilocarpine injections showed typical increases in their intensity and duration, gradually progressing towards status epilepticus. The rats treated with FK506 showed highly significant differences in their latency period reaching to stage IV-V (P<0.05) and the profile of proportions of rats suffering from seizures of different intensities. The latency period of the group with FK506 was significant longer and the percentage of animals reaching stage V was considerably lower compared with pilo group (P<0.05). Control animals did not exhibit any behavioral seizure activity.2. FK506 reduced the neuronal apoptosis after seizures:With the confocal microscope, we found that the apoptotic cells in pilo group were significantly increased compared with control group (P<0.05). We also observed that FK506 remarkably decreased the SE-induced apoptotic cells (P<0.05). This suggested that FK506 could inhibit apoptosis.3. FK506 decreased the mitochondrial swelling:Compared with control group, mitochondrial swelling was a significant increase in pilo group (P<0.05). With treatment of FK506, mitochondrial swelling showed a significant decrease(P<0.05).4. FK506 decreased the expression of the positive cells of AIF. The data showed that positive cells of AIF were seen in the cytoplasm and nucleus with brown colour, especially in nucleus. Compared with control group, the positive cells of AIF were significantly increased in rats induced with pilocarpine(P<0.05). While with traet-ment of FK506, the positive cells of AIF were significantly decreased compared with pilo group (P<0.05). The results indicated that FK506 had effect of anti-apoptosis.Conclusions:1. Epilepsy could damage the nerve cells induced by epilepsy, while FK506 reduced the neuronal apoptosis after seizures,it demonstrated that FK506 plays a role in neuroprotection and neurotrophy.2. The mitochondrial membrane potential was decreased and the mitochondrial showed swelling after epilepsy. This suggested that FK506 plays a role in protecting mitochondrial induced by pilocarpine.3. FK506 decreased the expression of the positive cells of AIF for cell apoptosis induced by epilepsy. The findings indicated FK506 had potential protection against apoptosis.