The Function And Mechanism Of Autophagy In 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 oxidative 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.Autophagy is the regulated process by which cytoplasmic organelles and long-lived proteins are delivered for lysosomal degradation.There are 3 major forms of "autophagy".These forms of autophagy share in common the delivery of intracellular cargo for degradation within lysosomes.Macroautophagy involves the sequestration of cytoplasmic proteins and organelles into a double-membrane vesicle,followed by stepwise maturation involving dissolution of the inner membrane,acidification, delivery to and fusion with lysosomes.Chaperone mediated autophagy(CMA) involves recognition of protein motifs by trans-membrane lysosomal proteins,and their direct import across the lysosomal membrane.Microautophagy involves rearrangement of the lysosomal membrane to engulf portions of adjacent cytoplasm or nucleus.Under normal condition,autophagy is at basal level in most cells.Autophagy is induced ten times under starvation,differentiation,and hormone stimulation to maintain cellular homeostasis and survival.The reported studies showed that oxidative stress is an important inducer of autophagy.As a signal molecule,active oxygen can activate autophagy.Simultaneously,it has also been evident that autophagy can trigger a form of cell death distinct from apoptosis in neurons,known as typeⅡprogrammed cell death.To our knowledge,the role of autophagy has not been studied in oxidative stress induced by seizures in animal model of epilepsy produced by pilocarpine(PILO).The effects of antioxidant vitamin E and autophagy inhibitor wortmanin(WM) in on activated autophagy in seizures have not been determined.Thus,in this study we try to investigate whether seizures promote autophagy including macroautophagy and CMA.In addition,we also examined whether vitamin E and wortmannin might attenuate hippocampal neuron damage after seizures induced by pilocarpine. PartⅠThe role of macroautophagy in rat hippocampus injury induced by seizures and the effect of vitamin E on macrautophagyObjectiveTo explore the pathology features of rat hippocampus injury caused by PILO-induced seizures.To investigate the changing patterns of autophgy and neuroprotective effects of antioxidant vitamin E on rat hippocampus injury induced by seizures.Method1.Seizures were induced by PILO through intraperitoneal injection.To detect the time courses of autophagy activity,rats were randomly divided into control group and groups at 2,8,16,24 and 72 h after PILO treatment.Next,to study the neuroprotective effects of vitamin E or pathology features of hippocampus injury,rats were randomly divided into control,PILO 24h,vitamin E+saline and vitamin E+PILO groups.2.The animal behaviors were observed including the incidence rate of animals with status epilepticus(SE),the latent period from PILO injection to initiation of SE,and mortality rate of animals with SE at 24h after induction of SE.The neuroprotective effects of vitamin E on animal behavior were also studied.3.To evaluate pathology features of hippocampal neurons of every experiment groups,Haematoxylin & Eosin staining and Nissl staining by Toluidine Blue were performed.4.Electron microscope was used to detect the ultrastructure of damaged neurons and the changes of autophagic vacuole.5.Western blot analysis was used to detect the ratio of light chain(LC3)Ⅱto LC3Ⅰand beclin 1 which were respectively related to the macroautophagy activity at different experiment group.Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect beclin 1 mRNA at different experiment group.6.Furthermore,the effect of vitamin E on macroautophagy was investigated by Western blot examination. Result1.The incidence rate of SE induced by pilocarpine was 93.33%.The vitamin E pretreatment only slightly decreased the incidence rate of SE(the incidence rate of SE in vitamin E+PILO group was 80%(p＞0.05).The latent period from PILO injection to initiation of SE was(35.64±13.77 min,n=14),but the latent period in vitamin E+PILO group was(54.27±22.16 min,n=12),which was significantly longer than PILO group(t=0.027,p＜0.05).The mortality rate of animals with SE at 24h after induction of SE was 50%(7 from 14),vitamin E pretreatment significantly increased the mortality rate of animals with SE at 24h,which was 8.3%(1 out of 12)(x~2=5.26, p＜0.05).2.Neurons in CA1 of control rats were clear and complete with normal nucleolus, well-distributed karyotin and rich nissl bodies in kytoplasm.PILO-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.Most pyramid cells were normal and only a few neurons showed fuzzy outline and agglomerated karyotin in vitamin E+PILO group.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 that was associated with cytoplasmic hyperchromasia,rough endoplasmic reticulum dispersion, mitochondrial swelling and cristaeolysis.Many autophagic vacuoles were seen obviously.On the contrary,autophagic vacuoles were rarely seen in neurons from hippocampus CA1 of rats in control group under electron microscope.Vitamin E also reduced autophagic vacuoles obviously.3.The surviving neuron number at 24h was sharply decreased in the PILO-treated group(75.5±5.92) compared with control(110.67±18.56)(P＜0.05).Moreover, vitamin E significantly decreased the neuron loss induced by seizures(94.67±13.03) compared with the PILO group(P＜0.05). 4.By Western blot,the ratio of LC3Ⅱto LC3Ⅰbegan to increase at 2 h,peaked at 24 h and kept at a high level until 72 h after seizures(P＜0.05).The immunoreactivity of beclin 1 was weak in control group and began to increase at 2h,peaked at 24 h and kept at a high level until 72 h after seizures(P＜0.05).In addition,the time-course change of beclin 1 mRNA was the same as beclin 1 protein.Vitamin E could effectively inhibit the overactivation of macroautophagy induced by seizures by inhibiting the transformation of LC3Ⅰto LC3Ⅱand the synthesis of beclin 1 in vivo (P＜0.05).Conclusion1.PILO-induced seizures lead to severe hippocampal neuron damage.The damaged neuron mainly shows a feature of necrosis.Macroautophagy was activated in damaged neuron.2.PILO-induced seizures can trigger the transformation of LC3Ⅰto LC3Ⅱand the up-regulation of beclin 1 in hippocampus that is partly inhibited by vitamin E.3.Vitamin E prolongs the latent period of SE and improves the survival rate of rats with SE,and has the neuroprotective effect on hippocampus injury induced by seizures.PartⅡThe role of CMA on rat hippocampus injury induced by seizures and the effect of vitamin E on CMAObjectiveTo investigate changes of CMA in rat hippocampus injury induced by seizures and effects of vitamin E on CMA.Method1.Seizures were induced by PILO through intraperitoneal injection.To detect the time courses of CMA activity,rats were randomly divided into control group and groups at 2,8,16,and 24 h after PILO treatment.Next,to study the effects of vitamin E on CMA injury,rats were randomly divided into control,PILO 24h,vitamin E+saline and vitamin E+PILO groups.2.The isolation of lysosomes and the preparation of lysosomal membranes and matrices.3.Western blot analysis was used to detect LAMP2a expression on lysosomal membranes and in lysosomal matrices at various experiment groups.Next,the mRNA of LAMP2a was evaluated by RT-PCR.Lastly,the effects of vitamin E on synthesis and expressions of LAMP2a were also detected by RT-PCR and Western blot examination.Result1.The levels of LAMP2a both on lysosomal membranes and in lysosomal matrices increased significantly at 8h after seizues,and continued to increase up to 24 h after PILO treatment(P＜0.05).The distribution of LAMP2a between on lysosomal membranes and in lysosomal matrices was not affected by seizures(p＞0.05).2.Vitamin E inhibited the increase of LAMP2a both on lysosomal membranes and in lysosomal matrices(P＜0.05),but did not affect the distribution of LAMP2a (p＞0.05)。3.RT-PCR showed that mRNA LAMP2a began to increase significantly at 8h after seizures,and continued to increase up to 24 h after PILO treatment(P＜0.05).Vitamin E also inhibited the synthesis of LAMP2a in hippocampus in vivo at 24h after seizures(P＜0.05).Conclusion1.PILO-induced seizures lead to activation of CMA that began at 8h,6h later than macroautophagy.Vitamin E can partly inhibit activated CMA by seizures.2.The activation of LAMP2a by seizures was achieved by increase of LAMP2a syhthesis in vivo,not by the redistribution of LAMP2a between on lysosomal membranes and in lysosomal matrices.Vitamin E did not affect the distribution of LAMP2a between on lysosomal membranes and in lysosomal matrices. PartⅢThe neuroprotective effect of wortmannin on epileptic rat by inhibiting macroautophagyObjectiveTo investigate the effects of wortmannin(WM) on the activation of macroautophagy in epileptic rat hippocampus,and the roles of phosphoinositide 3-kinase(PI3K) in macroautophagy and neuroprotective effects of WM on epileptic rats.Method1.Seizures were induced by PILO through intraperitoneal injection.To detect the time courses of macroautophagy activity,rats were randomly divided into control group and groups at 2,8,16,24 and 72 h after PILO treatment.Next,to study the neuroprotective effects of WM on hippocampus injury and macroautophagy,rats were randomly divided into control,PILO 24h,WM+PILO groups.2.The animal behavior were observed including the incidence rate of animals with status epilepticus(SE) induced by pilocarpine,the latent period from PILO injection to initiation of SE,and mortality rate of animals with SE at 24h after induction of SE. The neuroprotective effects of wortmannin on animal behavior were also studied.3.Western blot analysis was used to detect the ratio of LC3Ⅱto LC3Ⅰand phospho-PI3K at various experiment groups.Result1.WM pretreatment significantly decreased the incidence rate of SE(the incidence rate of SE in WM+PILO group was 60%)(p＜0.05).The latent period from PILO injection to initiation of SE was(34.75±12.76 min,n=14),but the latent period in WM+PILO group was(64.33±16.67min,n=9)(t=0.007,p＜0.01),which was significantly longer than PILO group.WM pretreatment significantly increased the mortality rate of animals with SE at 24h,which was 0(0 out of 9)(p＜0.01).2.Most pyramid cells were normal and only a few neurons showed fuzzy outline and agglomerated karyotin in WM+PILO group.3.The surviving neuron number at 24h was sharply decreased in the PILO-treated group(70.16±5.09) compared with control(110.67±18.56)(P＜0.05).Moreover, wortmannin significantly decreased the neuron loss induced by seizures (100.88±18.73) compared with PILO group(P＜0.05).4.The ratio of LC3Ⅱto LC3Ⅰand beclin 1 were significantly lower in WM+PILO group than in PILO group(P＜0.05).5.PI3K phosphorylation or activation temporally increased at 2 h and 8h(P＜0.05), began to decrease at 16h(P＜0.05),and returned to normal level at 72 h after seizures (P＜0.05).Conclusion1.WM can inhibit macroautophagy activated by seizures by reducing the transformation of LC3Ⅱto LC3Ⅰ.2.WM have neuroprotective effect on epileptic rats by reducing incidence rate of SE,prolonging the latent period of SE,decreasing the mortality rate of animals with SE and increasing the survival neurons in CA1 of rats with SE.3.PI3K may play a important role in activated macroautophagy induced by seizures.SignificanceUsing a rodent epilepsy model induced by PILO,we investigated and confirmed that autophagy played an important role in seizures-induced hippocampal neuronal death.Furthermore,we showed that antioxidant vitamin E and macroautophagy inhibitor,WM could regulate autophagy and play neuroprotective roles on epileptic rats.Although the mechanism underlying the effects of autophagy on neuron injury needs further study,studies on autophagy will undoubtedly help us to know more about the mechanism of seizures-induced neuronal injury.Thus,this study may provide new insights into therapeutic advances of vitamin E and WM that may be neuroprotective.