The Function And Mechanism Of Poly(ADP-Ribose) Polymerase On Rat Hippocampus Injury Induced By Seizures

BackgroundEpilepsy is one of the most common chronic neurological disorders affecting people of all ages.The striking characteristic of epilepsy is a tendency to recurrent, unprovoked seizures.At present,there are about 9 million of epilepsy patients in China.They suffer a lot from this disease and become big burdens to the family and society.As many as 20%～30%of medicated epilepsy patients are still far from seizures-free control.In the end,they will become patients with intractable epilepsy. Temporal epilepsy usually turns out to be intractable epilepsy syndrome.Although great progress has been made on the etiology,pathology and treatment of temporal epilepsy,the mechanism of the disease is still far from clear.The main pathology feature of temporal epilepsy is hippocampus sclerosis including the loss of neurons and hyperplasia of astrocytes.As known,seizures are associated with a variety of pathophysiological alterations and can result in neuronal degeneration in hippocampus.It has been indicated that seizures-induced neuronal death is associated with glutamate release and N-methyl-D-asparate(NMDA)receptor activation.When NMDA receptor is stimulated,calcium influxes into cells and activates nitric-oxide synthase,which elevates oxygen-free radical levels and causes DNA damage.Until now,the mechanism underlying the neuron death is still unclear.Moreover,there are still limited strategies to protect neuronal death and the subsequent epileptogenesis.Poly(ADP-ribose)polymerase(PARP)is a ubiquitous DNA repair-associated enzyme that is presented in the nuclei of numerous cell types.It is proved to be involved in a variety of cellular processes including DNA duplication,DNA transcription,DNA repair,gene expression and cell death.PARP can convert nicotinamide adenine dinucleotide(NAD)into long branched polymers of poly(ADP-ribose)(PAR)and attached them to numerous of nuclear DNA binding proteins.Massive DNA damage induces the overactivation of PARP which consumes intracellular NAD,depletes ATP stores,triggers activation of cell signaling molecules, ultimately,leads to cell death.Recently,emerging data indicate PARP is involved in the regulation of tumor,inflammation,and autoimmune diseases.Knockout of PARP-1 in mice or inhibition PARP through pharmacological approaches leads to a significant reduction in tissue damage during ischemia,brain trauma,excitotoxitic injury or inflammation.Recently,cumulative evidences show that PARP may regulate apoptosis inducing factor(AIF)dependent cell death signaling,Akt mediated surviving signaling and nuclear factorκB(NF-κB)dependent transcription of inflammatory factors.To our knowledge,the role of PARP inhibitor(3-aminobenzamide,3-AB)has not been studied in any animal model of epilepsy yet and the mechanism whereby PARP is deleterious in seizures has to be determined.Thus,in this study we try to investigate whether 3-AB might attenuate hippocampal neuron damage after seizures induced by kainic acid(KA).In addition,we also examined the potential protective mechanisms triggered by 3-AB including AIF dependent cell death pathway,Akt cell survival signaling and NF-κB dependent inflammatory responses.PartⅠThe effect of poly(ADP-ribose)polymerase on rat hippocampus injury induced by seizuresObjectiveTo explore the pathology features of rat hippocampus injury caused by KA-induced seizures.To investigate the changing patterns and neuroprotective effects of PARP on rat hippocampus injury induced by seizures.Method1.Seizures were induced by KA through intracerebroventricular injection by a stereotaxic apparatus.To detect the time courses of PARP activity,rats were randomly divided into control group and groups at 2,6,12,24 and 72 h after KA treatment. Next,to study the neuroprotective effects of 3-AB or pathology features of hippocampus injury,rats were randomly divided into control,KA,KA+saline and KA+3-AB groups.2.To evaluate pathology features of hippocampal neurons of every experiment groups,Haematoxylin & Eosin staining and Nissl staining by Toluidine Blue were performed.3.Electron microscope was used to detect the ultrastructure of damaged neurons.4.Western blot analysis and immunohistochemistry staining were used to detect PARs which were the products of PARP activity at different experiment groups. Furthermore,the inhibition effect of 3-AB on PARP activity was investigated by Western blot examination.Result1.KA-induced seizures led to obvious neuron damage in rat hippocampus.The surviving neurons showed round and palely stained nuclei,meanwhile,the dead neurons in hippocampus showed pyknotic nuclei and shrunken plasma body.There were small and irregular chromatin clumps in the dead cells.Under electron microscope,the dead neurons displayed rupture of nuclear and cytoplasmic membrane,organelle swelling and a high number of vacuoles in plasma.There were numerous and irregular clumps distributed throughout the nucleus which was associated with cytoplasmic hyperchromasia,rough endoplasmic reticulum dispersion, mitochondrial swelling and cristaeolysis.2.The surviving neuron number was sharply decreased in the KA-treated group (47.22±4.95;53.26±8.08)compared with control(85.23±6.71;131.91±14.24) (P＜0.05).Moreover,3-AB significantly decreased the neuron loss induced by seizures(62.37±6.57;106.28±9.52)compared with the KA+saline group(50.07±5.91;54.31±8.02)(P＜0.05).3.Little immunoreactivity of PAR was detected at control group.PAR immunoactivity began to increase at 2 h,reached summit at 6 h and kept a high level until 24 h after seizures(P＜0.05).In addition,3-AB could effectively inhibit the overactivation of PARP activity induced by seizures(P＜0.05).Conclusion1.KA-induced seizures lead to severe hippocampal neuron damage.The damaged neuron mainly shows a feature of necrosis.2.KA-induced seizures can trigger the overexpression of PARP in hippocampus which is effectively inhibited by 3-AB.3.3-AB has the neuroprotective effect on hippocampus injury induced by seizures.PartⅡThe effect of PARP on apoptosis-inducing factor signaling and Akt pathway in epileptic ratObjectiveTo investigate the effects of PARP on AIF signaling and Akt/glycogen synthase kinase-3β(GSK-3β)pathway in rat hippocampus injury induced by seizures.Method1.Seizures were induced by KA through intracerebroventricular injection.To detect the time course changes of AIF,Akt and GSK-3βexpression in hippocampus,rats were randomly divided into control group and groups at 2,6,12,24 and 72 h after KA treatment.Next,to study the effects of PARP inhibitor on expression of AIF in mitochondria and nucleus,rats were randomly divided into control,KA,KA+saline and KA+3-AB groups.Last,to investigate the effects of PARP inhibitor on expression of phospho-Akt and phospho-GSK-3β,rats were randomly divided into control,KA, KA+saline,KA+3-AB,KA+saline+DMSO,KA+3-AB+LY294002 groups. 2.Western blot analysis was used to detect AIF expression in mitochondria or nucleus at various experiment groups.Next,the expressions of phospho-Akt and phospho-GSK-3βat each experiment groups were examined by Western blot analysis too.Lastly,the effects of 3-AB on expressions of AIF,phospho-Akt and phospho-GSK-3βwere also detected by Western blot examination.Result1.In the nuclear fraction,little AIF immunoreactivity was detected at 2 h after seizures.AIF began to accumulate in the nuclear fraction at 6 h and continued to increase up to 72 h after KA treatment(P＜0.05).Consistent with these,AIF immunoreactivity was found decreased in the mitochondrial fraction(P＜0.05).2.Akt phosphorylation or activation temporally increased at 2 h(P＜0.05),then returned to the control level at 6 h.On the contrary,GSK-3βphosphorylation or inactivation level was significantly decreased at 6 h after seizures(P＜0.05).In addition,3-AB sharply increased the phospho-Akt and phospho-GSK-3βlevels in KA treatment group(P＜0.05).Administration of LY294002,a PI3-K inhibitor,could prominently reverse 3-AB-mediated Akt and GSK-3βphosphorylation after the seizures(P＜0.05).Total Akt and GSK-3βprotein levels were unchanged in every group under our experimental conditions.Conclusion1.AIF is subjected to translocation from mitochondria to nucleus in hippocampus after seizures.2.Akt and GSK-3βphosphorylation in hippocampus are induced by seizures.3.PARP inhibition by 3-AB can promote Akt-mediated survival signaling and suppress AIF dependent cell death pathway in epileptic rat hippocampus. PartⅢThe effect of PARP on nuclear factor-kappa B and inflammatory factors in epileptic ratObjectiveTo investigate the effects of PARP on the activation of NF-κB and expression of inflammatory factors including interleukin-1β(IL-1β),cyclooxygenase-2(COX-2), matrix metalloproteinase-9(MMP-9)in epileptic rat hippocampus.Methods1.To detect the time course changes of NF-κB p65 expression in nucleus,rats were randomly divided into control group and groups at 2,6,12,24 and 72 h after KA treatment.Next,to study the effects of PARP inhibitor on expression of NF-κB p65 and inflammatory factors,rats were randomly divided into control,KA,KA+saline and KA+3-AB groups.2.Western blot analysis was used to detect NF-κB p65 expression at various experiment groups.Next,reverse transcriptase-polymerase chain reaction(RT-PCR) and Western blot analysis were performed to examine the effects of 3-AB on expressions of NF-κB p65 and inflammatory factors.Result1.Little immunoreactivity of NF-κB p65 was detected in nucleus at control group. NF-κB p65 began to accumulate in the nucleus at 2 h,reached summit at 6 h and kept rising at 12 h after seizures(P＜0.05).Then it returned to normal level at 24 h after seizures.When 3-AB was administrated,NF-κB p65 expression in nucleus was sharply decreased(P＜0.05).2.Weak mRNA expression of IL-1β,COX-2 and MMP-9 were apparent in the hippocampus tissues at control group.The mRNA levels increased significantly after seizures(P＜0.05).3-AB could significantly suppress on mRNA expression of IL-1β, COX-2 and MMP-9 induced by seizures(P＜0.05).Moreover,3-AB pretreatment also sharply blocked the increasing protein expression of these inflammatory molecules (P＜0.05)Conclusion 1.Inhibition of PARP activity by 3-AB may suppress NF-κB activation in hippocampus induced by seizures.2.3-AB can inhibit the expression of inflammatory molecules such as IL-1β, COX-2 and MMP-9 induced by seizures.SignificanceUsing a rodent epilepsy model induced by KA,we investigated and confirmed that PARP played an important role in seizures-induced hippocampal neuronal death. Furthermore,we showed that PARP inhibitor,3-AB could regulate AIF dependent cell death signaling,Akt dependent cell survival signaling and NF-κB dependent inflammatory responses.We did not find tha 3-AB could decrease seizures frequency. However,studies on PARP will undoubtedly help us to know more about the mechamism of seizures-induced neuronal injury or seizures-induced memory & learning ability damage.Thus,this study may provide new insights into therapeutic advances of 3-AB that may be neuroprotective or have memory improving ability.