Diazoxide Protects Against Cell Death Of Hippocampal Neurons Of Rats With Seizures Induced By Lithium-Pilocarpine And The Related Mechanism

Epilepsy is a common nervous system, and it brings a heavy burden to social, family and patients. There are about35million patients in the world; including more than9million patients are suffering in china. About25%of patients suffer with intractable epilepsy. Temporal lobe epilepsy (TLE) presents the most prevalent refractory epilepsy and the pathogenesis of TLE has not yet been fully elucidated. The usual administration of antiepileptic drugs (AEDs) and surgical therapy is often difficult to obtain significant curative effects. The alteration of behavior and the hippocampal neuronal injury in pilocarpine-induced seizures in rats is similar to that in TLE patients, so pilocarpine-induced seizures have been one of the most frequently used models to research status epilepticus (SE) and TLE.SE is the serious state of seizures, it means continues seizures lasting at least30min or at least2times successive seizures with not fully recover the normal state of consciousness during the seizures. SE can make the selective neuronal death in hippocampus or other regions. The selective neuronal death could promote the formation and progression of epilepsy. In view of the complexity of epilepsy etiology and pathogenesis, exploring new neuroprotective drugs and their potential mechanism is current epilepsy research focus.The mitochondrial ATP-sensitive potassium (mitoKATP) channel was reported to be involved in cerebral and myocardial ischemic preconditioning. Moreover, the level of mitoKATP channels is at least six-fold higher in brain than heart cells, which implies that mitoKATP channels may play an essential role in the function of the central nervous system. ATP-sensitive potassium (KATP) channels are involved in several physiological functions. Their open and close in response to changes in intracellular ATP/ADP ratios. Low ATP levels open the channels, thus allowing K+efflux and cell hyperpolarization. Recently, the expression of functional KATP channels was found to inhibit seizure responses and possibly limit the release of excitatory neurotransmitters such as glutamate. For epilepsy, mitoKATP channel openers, particularly diazoxide (DZ), protects against status epilepticus-induced neuron damage during diabetic hyperglycemia. However, the molecular mechanisms of the neuroprotective effect of DZ have not been fully elucidated. Studies proved that the protective mechanisms of the mitoKATP channel may involve several aspects, including Bcl-2-dependent mechanism, inhibiting the mitochondrial permeability transition pore and so on. Whereas, the specific mechanism by which the activation of mitoKATP channel exerts protection of cell death has not yet been fully established.The anti-apoptotic and neuroprotective effects of DZ have been widely recognized, but there are few researches on whether it has protective effects on hippocampus neuronal damage in pilocarpine-induced seizures and the possible mechanism has not yet report. At present study, we established epilepsy model of rats induced by lithium-pilocarpine, to observe the pathology characteristics of hippocampal neurons damage in seizure; after pilocarpine administrated, DZ administrated, DZ with mitoKATP inhibitor5-hydroxydecanoic acid (5-HD) administrated, and DZ with phosphatidylinositol-3-kinase (PI3K) inhibitor wortmannin (WTN) administrated, we observe the changes of signal transduction pathway, mitochondrial apoptosis-related protein in hippocampus and the effects of free radicals in rats. As to further explore the potential molecular mechanisms in seizures, investigate the antiepileptic effects and the potential mechanisms. The study is divided into three parts: ObjectiveTo study the effects of mitoKATP opener DZ on behavior, hippocampal neuronal injury in lithium-pilocarpine-induced seizures in rats, and to explore the neuroprotective effects of DZ on neurons after seizures and the potential mechanisms.MethodsAdult male Wistar rats were randomly divided into control group (control),24h after seizures (pilo), DZ preconditioning group (pilo+DZ), DZ+5-HD group (pilo+DZ+5-HD), and DZ+WTN group (pilo+DZ+WTN). DZ,10mg/kg, injected intraperitoneally30min before pilocarpine injection;5-HD,5mg/kg, was injected intraperitoneally15min before DZ; WTN,15μg/kg, injected intraperitoneally15min before DZ, rats were monitored by video recordings to assure development of seizures. We recorded latency to stage Ⅳ seizures and percentage of animals with seizure episodes. Seizures were allowed to last for60min and then were terminated by administration of diazepam. The rats were perfused with paraformaldehyde and the brains were fixed with paraffin and cut at24h after SE. HE and Nissl staining was used to study the pathological changes in hippocampal neurons induced by pilocarpine.Results1. Behavior:Rats showing stage Ⅳ-Ⅴ convulsive seizures according Racine standard were included in the experimental group which demonstrated bilateral forelimb clonus, twitching and comprehensive tonic-clonic seizures, Tonic hind limbs with the body upright, the back curved trunk rigidity, falls. The percentage of animals reaching stage IV SE was38.9%, and the percentage of animals reaching stage V SE was61.1%in pilocarpine-induced seizures. The latency period with DZ injection was significantly longer (67.8±9.6min)(p<0.05) and the percentage of animals reaching stage V SE was considerably lower (19.9%)(p<0.05) than with pilocarpine alone (42.3±6.2min). In addition,5-HD or WTN treatment reversed the DZ-mediated protection of seizures as compared with DZ treatment alone. Control animals did not exhibit any behavioral seizure activity.2. HE staining and Nissl staining showed pyramidal cell structure was clear and complete tightly packed. Nuclear structure of normal, evenly distributed chromatin, cytoplasm rich in Nissl bodies in hippocampal CA1and CA3regions of normal rats. Part of the neuron loss, disordered cells, cell vague outline, visible part of the cytoplasmic concentration, nuclear condensation, cytoplasmic Nissl bodies reduced were observed in hippocampal CA1region and CA3region at24h after pilocarpine-induced seizures. The surviving neurons showed round and palely stained nuclei, meanwhile, the dead neurons in hippocampus showed pyknotic nuclei and shrunken plasma body. Moreover, DZ pretreatment significantly attenuated the neuronal loss induced by seizures at24h, and the protective effect was attenuated by preconditioning with5-HD.ConclusionsThe changes on behavior, hippocampal neuronal injury in lithium-pilocarpine-induced seizures in rats proved that lithium-pilocarpine could induce acute seizures in rats (SE), and then induce hippocampal neuronal injury. DZ pretreatment could improve the changes on behavior, hippocampal neuronal injury in lithium-pilocarpine-induced seizures in rats, which showed that DZ have neuroprotective effect on hippocampal neurons in pilocarpine-induce seizures. ObjectiveTo observe the effect of the diazoxide (DZ), a mitochondrial ATP-sensitive potassium channel (mitoKATP channel) opener, on PI3K/AKT signal transduction pathway and mitochondrial apoptosis-related proteins such as apoptosis-inducing factor (AIF), cytochrome c (CytC) and caspase-3in hippocampus in epileptic rats induced by lithium-pilocarpine, and explore the mechanism of the neuroprotective effect of DZ on epilepsy.MethodsAdult male Wistar rats were randomly divided into control group, SE2h,8h,16h,24h and72h group. Next, to study the neuroprotective effects of DZ on hippocampus neuron, rats were randomly divided into control group (control),24h after seizures (pilo), DZ preconditioning group (pilo+DZ), DZ+5-HD group (pilo+DZ+5-HD), and DZ+WTN group (pilo+DZ+WTN). Rats were killed by decapitation at the corresponding time after seizures, and then hippocampus was separated and proteins were extracted. The express of phospho-Akt (p-akt), apoptosis-inducing factor level in mitochondria fraction (mito-AIF), apoptosis-inducing factor level in nucleus fraction (nu-AIF), cytochrome c level in mitochondria fraction (mito-CytC), cytochrome c level in cytosolic fraction (cyto-CytC) and activated caspase-3proteins in rat hippocampus were detected respectively by Western blotting.Results1. Compared with control, the level of p-akt was increased at2h (p<0.05) and then decreased thereafter in rat hippocampus after seizures. AIF translocated from mitochondria to the nucleus in the hippocampus after seizures beginning at2h and peaking at24h after seizures. As well, CytC level decreased in the mitochondrial fraction after seizures (p<0.05). Moreover, the active cleavage product of caspase-3appeared at24h after seizures and increased until72h.2. Compared with pilocarpine treatment, DZ pretreatment sharply upregulated the phospho-Akt level at24h after seizures. Pretreatment with DZ significantly suppressed the translocation of AIF, CytC release and caspase-3activation at24h after seizures (p<0.05).3. Compared with DZ pretreatment, the increased p-akt expression markedly reduced (p<0.05), the decreased AIF in the nuclear fraction and CytC activation in the cytosolic fraction and caspase-3activity significantly increased (p<0.05) in pilo+DZ+5-HD group and pilo+DZ+WTN group.ConclusionsDZ can open mitoKATP channel, activate PI3K/AKT pathway, suppress the translocation of AIF, CytC release and caspase-3activation, and then protect hippocampal neurons from apoptosis in epileptic rats induced by lithium-pilocarpine. DZ has Neuroprotective effects against hippocampal neuronal damage induced by seizures, and these beneficial effects may suggest that this could be a possible new target for neuroprotective treatment of epilepsy. This study illustrated the molecular mechanisms of hippocampal neurons injury after seizures, and provided the neuroprotective treatment of epilepsy a theoretical basis. ObjectiveTo observe the effect of DZ, a mitoKATP channel opener, on oxidative stress in hippocampal neuronal of epileptic rats induced by lithium-pilocarpine, and to further explore the neuroprotective effect of DZ and the mechanism of it on epilepsy.MethodsAdult male Wistar rats were randomly divided into①control group (control);②24h after seizures (pilo);③DZ preconditioning group (pilo+DZ);④DZ+5-HD group (pilo+DZ+5-HD);⑤DZ+WTN group (pilo+DZ+WTN);⑥5-HD alone (5-HD);⑦WTN alone (WTN). For groups4and5, pilocarpine was given30min after DZ injection and for groups6and7; pilocarpine was given45min after5-HD or WTN injection. Rats were induced by lithium-pilocarpine and the rats were sacrificed and the hippocampus was removed. The contents of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured.Results1. MDA level started to increase at2h after seizures and peaked at7days. SOD activity was significantly decreased at24h after SE (p<0.05), with no difference in levels from controls at2h,72h and7d.2. DZ pretreatment markedly decreased MDA level (p<0.05) and increased SOD activation (p<0.05) as compared with no pretreatment.5-HD, when administered with DZ, completely prevented the DZ-mediated decrease in MDA activity and increase in SOD level (p<0.05). Inhibiting PI3K activation by WTN increased MDA activity (p<0.05), and decreased SOD level (p<0.05), as compared with DZ treatment alone. Similarly,5-HD treatment alone and WTN treatment alone could aggravate seizure-induced oxidative damage (p<0.05).ConclusionsDZ may protect hippocampal neurons against cell death through PI3K/Akt signal transduction pathway to downregulate MDA and upregulate SOD, thus preventing oxidative stress in rat model of epileptic seizures induced by pilocarpine. DZ could be a potential treatment for hippocampal neuron demise caused by seizures. Diazoxide; PI3K/Akt; Seizure; Malonaldehyde (MDA); Superoxide dismutase (SOD)...