Neureprotective Effects Of Histonedeacetylases Inhibitor 11R Inlithium-Pilocarpine Induced Seizures On Rats

With 65 million people affected worldwide, epilepsy is the most common, chronic, serious neurological disease, which entails a major burden to family and social as well as the patients. There are about 9 million people suffer from this disease,30% of which were diagnosed with refractory epilepsy. Temporal lobe epilepsy (TLE) is the most common type in refractory epilepsy.The behavioral and pathological changes of hippocampal neurons in lithium - pilocarpine-induced seizures in rats is very similar with temporal lobe epilepsy in human. Therefore, this model is widely used in status epilepticus and TLE study. In the past decade, important advances have been made in the understanding of the pathophysiological mechanisms of the disease and factors aff ecting its prognosis. But its precise mechanism has not studied thoroughly. As a result, although epilepsy can be successfully treated in most cases, about a third of patients remain resistant to medical treatment. So further study on exact pathogenesis of epilepsy and exploration on new neuroprotective drugs have very important significance.Acetylation and deacetylation of histone proteins associated with chromatin plays a pivotal role in the epigenetic regulation of transcription and other functions in cells Histone has a variety of covalent modification methods, of which acetylation is an important mechanism for the regulation of transcription. Chromatin structure remodeling caused by post-translational modifications of histone plays an important role in the regulation of gene expression in eukaryotes. The acetylation status of chromatin is determined by the activities of two classes of enzymes, histone acetyltransferases (HATs) and HDACs. HATs is used to promot protein acetylation and enhance transcriptional activity; In contrast, HDACs function to deacetylate and suppress transcription. Any shift in the balance of acetylation on chromatin may result in changes in the regulation of patterns of gene expression, which affects the growth, differentiation and death of cells. Considerable research activity has focused on histone deacetylase (HDAC) inhibitors as novel therapeutics in models of ischemic stroke, intracerebral hemorrhage and Huntington’s disease.11r, a novel N-hydroxycinnamamide-based derivative,was reported on Medicinal chemistry by Xiaoyang Li et al for the first time, which has been shown to be potent dual HDAC 1/3 selective inhibitor and be excellent growth inhibition in multiple tumor cell lines. However, no data have been reported regarding its effects on epilepsy. Therefore, this study established lithium-pilocarpine induced induced seizures in rats, observe behavior and pathological changes in lithium-pilocarpine induced seizures in rats, detect levels of histone acetylation in CA1 and CA3 regions of rats hippocampal and evaluate the spatial memory and cognitive function in rats by Morris water maze, in order to explore whether histone deacetylases inhibitor 11r has neuroprotective effects in lithium-pilocarpine induced seizures in rats. This study is divided into two parts:PART I A study of behavior and pathological changes in lithium-pilocarpine induced seizures on ratsObjectiveTo observe the behavioral changes in lithium-pilocarpine induced seizures in rats, and to investigate pathological changes in hippocampus caused by seizures.MethodsAdult Sprague Dawley rats (male, healthy) were given lithium-pilocarpine to induce SE intraperitoneally, to observe the behavioral changes in rats. Seizures were terminated by an injection of diazepam after lasting for 60 min. The rats were sacrificed at 24 hours after epilepsy induced and made paraffin, to observe pathological changes induced by seizures.Results1. Depending on Racine, the rats showing stage IV or V convulsive seizures were defined as developping SE successfully. The incidence of Lithium-pilocarpine-induced SE was 90.0%.Latency to the time to SE was 36.3±10.8min.The mortality rate was 13.0% within 72 h.2. HE and Nissl staining were used to observe neuronal loss and degeneration induced by pilocarpine in hippocampus CA1 and CA3.The hippocampus CA1 and CA3 of rats in control group had a large number of pyramidal cells and granule cells,which tightly packed,and the nucleus of which were round or oval with clear nucleolar and even chromatin distribution.Nissl bodies were abundant in the cytoplasm.However,in pilocarpine group, the neurons in hippocampus CA1 and CA3 of rats arranged disorderly and loose, which was swelling, cracking and vague outline.The neurons which degenered and necrotized were triangular in shape with concentrated and deeply stained cytoplasm and pyknotic nucleus.Nissl bodies were decreased in the cytoplasm.ConclusionsLithium-pilocarpine could induce SE according to the alteration of behavior. Lithium-pilocarpine could lead to degeneration and damages of hippocampal neurons in rats.PART II Neuroprotective effects of histone deacetylases inhibitor 11r in lithium-pilocarpine induced seizures on ratsObjectiveTo investigate the neuroprotective effects of histone deacetylases inhibitor 11r in lithium-pilocarpine induced seizures in rats, and observe its effect on histone acetylation levels on hippocampal CA1 and CA3 regions in rats, in order to explore the potential neuroprotective mechanism of 11r.Methods40 adult Sprague Dawley rats were randomly divided into five groups (n= 8):The control group, pilocarpine group, treatment group Ⅰ, group treatment Ⅱ,11r pretreatment group. To observe the behavior performances of rats in each group. HE staining was performed to detect neuronal degeneration in hippocampus 24 hours after pilocarpine-induced seizure. At 72 hour after seizures, Nissl staining and immunohistochemical were used to evaluate the neuronal loss and histone acetylation levels of hippocampal CA1 and CA3 region in each group. Evaluate the spatial memory and cognitive function in rats by Morris water maze at 11 days after SE.Results1.Compared with pilocarpine group,11r pretreatment could delay pilocarpine-induced seizures (P<0.05) and reduced mortality (P＜0.05). The control rats did not occur seizures.2. The hippocampus CA1 and CA3 of rats in control group had a large number of pyramidal cells and granule cells,which tightly packed, and the nucleus of which were orbicular or oval with clear nucleolar and even chromatin distribution. Nissl bodies were abundant in the cytoplasm. However, in pilocarpine group, the neurons in hippocampus CA1 and CA3 of rats arranged disorderly and loose Nissl bodies decreased in the cytoplasm. The number of surviving neurons with pilocarpine treatment decreased significantly compared with controls and llr treatment (P<0.05). Moreover, 11r pretreatment significantly attenuated the neuronal loss induced by seizures (P<0.05), but the difference was not statistically significant between treatment group I and II (P> 0.05).3. Immunolabeled products were visible as blue or gray staining localized in the nucleus under the microscope. The positive cells in pilocarpine group was significantly less than 11r pretreatment group and two treatment groups, which means that the histone acetylation levels in pilocarpine group was significantly lower than the other three groups (P<0.05). Difference between two treatment groups had on statistics significance (P> 0.05). No immunostaining was detected in the minus primary antibody control condition. 4. Escape latency of each group of rats is getting shorter with the increase in the training days. Escape latency in pilocarpine group was significantly longer than the control group and llr pretreatment group (P<0.05). Escape latency in llr treatment group was significantly shorter than pilocarpine group but longger than control group. There was no significant difference between 11r pretreatment group and control group(P> 0.05).ConclusionsHDACs inhibitor 11r had a significant improvement on neuronal degeneration and cognitive abilities decline induced by seizures, in addition, we found it may be also have anticonvulsant effect, which showed that 11r could exert neuroprotective effects in the pilocarpine-induced epileptic model in rats. It seems that the anti-inflammatory effect of HDAC inhibitor plays an important role in this protective mechanism.