Effect Of Tacrolimus On The Expression Of PKCδ In The Hippocampus Of Rats With Status Epilepticus

Backgroud and Objective:Epilepsy (EP) is a nervous system dysfunction syndrome,which is due to cerebral neurons excessive synchronous abnormal discharge. Status epilepticus (SE) refers to frequent clinical seizures without complete recovery of consciousness or the occurrence of a single unremitting seizure lasting more than30minutes to stop on its own,which can lead to acute or persistent changes of cognition and behavior and is a medical emergency of department of neurology.Protein kinase C (PKC) participates in physiological and pathological processes,such as the growth、differentiation and apoptosis of neurons. PKCδ,a member of a new kind of PKC isoforms,can activate caspase3under the effects of various stimulus,resulting in apoptosis. Calcineurin (CaN) is a member of serine/threonine protein phosphatase families. Our previous research shows that CaN can cause refractory epilepsy,and tacrolimus (FK506,a specific inhibition of CaN) is able to control epilepsy development、reverse neuronal injury. It has been found that in the model of cerebral ischemia,CaN can activate PKC,causing intracellular signal transduction. In the pathophysiological process of epilepsy,the interaction between PKC and CaN and neuronal damage is still short of related research.Our this study,we use Wistar rats to build lithium chloride-pilocarpine-induced status epilepticus model to observe the morphologic changes of the hippocampus, the expression of protein kinase Cδ and the influence of FK506on it,and explore the relationship between CaN and PKCδ-dependent apoptotic pathway.Methods:1. Animal models:One hundred and eight healthy adult male Wistar rats were randomly divided into the control group,the epilepsy group and the FK506group (36rats in each group). SE group and FK506group were divided into six groups, espectively3,6,12,24,72hours and7days after SE were sacrificed. At the same time, control group rats without being established epilepsy model, were sacrificed at corresponding time points.2. Behavioral observation:Observe the epileptic seizure of rats on the basis of Racine classification, and record seizures incubation period and the onset level.3. Observation of brain tissue pathology:Observe the hippocampal neuronal damage7days after SE by HE staining.4. RT-PCR:Detect the expression of PKCδ mRNA in the hippocampus at different time points (3,6,12,24,72h) after SE.5. Immunohistochemistry(SP):Detect the expression of PKCδprotein in the hippocampus at different time points (3,6,12,24,72h) after SE.Results:1. FK506inhibited seizures:After injection of pilocarpine,the SE group and the FK506rats had seizures,the onset at a lesser extent and gradually increased until the status epilepticus. Incubation period and the onset level of the two groups were different. Compared with the SE group,incubation period of the FK506group was significantly longer (P<0.01), and the onset level lower (P<0.05). The control group did not have seizures.2. FK506reduced SE hippocampal neurons injuries:Under the light microscope, the control group neurons arranged in neat rows, had normal morphology and had no neuronal loss in the hippocampal CA3region of rats7days after SE. In the SE group, the neurons arranged in disorder and lost normal structure; compared with the control group,the neurons lost more (P<0.05). Compared with the control group,the neurons number of the FK506group decreased,but compared with the SE group,survival neurons increased significantly (P<0.05).3. FK506suppressed the expression of PKC8:There were a few expressions of PKCδ mRNA and protein in the control group. Compared with the control group, the expressions of PKC8in the epilepsy group were increased observably (P<0.05),the expression of PKCδ mRNA arrived at a peak6h after SE,the expression of PKCδ protein arrived at a peak12h after SE(P<0.01), and both of PKCδ mRNA and protein were still more than the level of the control group72h after SE (P<0.05). Compared with the epilepsy group,pre-treatment with FK506could remarkably decrease the expression of PKCδ and also obviously alleviate hippocampal neuronal damage (P<0.05)Conclusions:FK506may regulate the PKCδ-dependent apoptotic pathway, reduce neuronal damage after SE and exert anti-epileptic and cerebral protection effects by inhibiting the CaN specifically,which provide a new drug target for the clinical application of immunosuppressive to treat epilepsy.