The Effects And Mechanism Of High Mobility Group Box 1 In Hippocampal Injury Of Epileptic Mice

BackgroundEpilepsy is a chronic neurological disease characterized by unpredictable,recurring seizures that vary between different extents.It has influenced over 65 million people worldwide.Epilepsy can lead to physical injuries due to loss of autonomy for activities.And patients with epilepsy suffer from psychological pressure and social discrimination.In rural area of China,it has been identified to be associated with mortality risk in cohort with epilepsy.About 30%of epilepsy patients are recognized as refractory and have displayed ineffectiveness to pharmacological therapies.Risk factors of epilepsy include daily stress,fatigue and attention-deficit/hyperactivity disorder(ADHD),all of which are presumed to be triggering factors for seizures.Moreover,a study on twins suggests that genetic factor may contribute to the etiology of epilepsy.Hypothesis suggests that inflammatory reaction in brain may mediate the activation of seizure in epilepsy.Yet the pathogenesis of epilepsy is not well-understood at present.Autophagy is a cell clearance process in eukaryotic cells which function as an intracellular homeostasis maintaining mechanism through dynamic degradation of damaged organelles and aberrant proteins.Three different forms of autophagy have been identified so far:macroautophagy,microautophagy,and chaperone-mediated autophagy,among which macroautophagy is the most extensively studied(referred to as "autophagy" in this study).The mechanism of macroautophagy is usually described as formation of autophagosome through an isolated fragment of membrane engulfing protein or organelles,which subsequently fuses with lysosome for degradation.Normally,autophagy is a carefully regulated pathway and occurs at a relatively low basal level.Autophagy level is reported to be upregulated in response to different metabolic stress like hypoxia,infection and nutrient deprivation.Although autophagy has already been reported to be implicated in many neurodegenerative diseases such as Huntington disease,Alzheimer disease and Parkinson's disease,only until recently that studies come up with the postulation that altered autophagy could be involved in the epileptogenesis.Nevertheless,the specific role of autophagy in epilepsy is still unclear.High mobility group box-1 protein(HMGB1)is a non-histone,conserved chromosome binding protein.Commonly acting as a transcription regulator,it has been reported to possess "cytokine-like" properties when released into the extracellular milieu.Significant elevation of HMGB1 in serum has been reported in both mouse models and patients with sepsis.Additionally,research has discovered that HMGB1 is a critical inducer of autophagy when it translocated into cytosolic space.Li Fu et al.report that HMGB1 antibody can effectively reduce epileptic seizure and inhibit hippocampus cell apoptosis in pilocarpine-treated epilepsy mouse model.These effects have been attributed to the repression of the inflammatory effect of HMGB1.In the present study,we aimed to look into the role of autophagy and HMGB1 in epileptogenesis.Therefore,this study was designed to investigate whether HMGB1 has a direct influence on the neuronal autophagy and whether HMGB1 antibody can alleviate autophagy and apoptosis in the hippocampus of epileptic mouse model.Part 1:Study on the effects of HMGB1 in hippocampal injury of epileptic miceResearch PurposesTo investigate the changes of HMGB1 in hippocampus of mice with epilepsy induced by PILO injection at different time intervals and the effect of anti-HMGB1 neutralizing antibody on neuron damage caused by seizures.Research Method1.Epilepsy mice modeling and treatment:epilepsy models were established by PILO intraperitoneal injection.Select 8-week-old male mice were randomly divided into 4 groups:control group(normal mice),epilepsy group(epileptic mice injected PBS),IgG group(epileptic mice were injected IgG)and HMGB1 Ab group(epileptic micemice were injected with anti-HMGB1 neutralizing antibody).3.Real-time quantitative PCR(RT-PCR)and Western Blot were used to detect the dynamic changes of HMGB1 in hippocampus of epileptic mice.2.Observation of behavior and EEGs of mice.4.HE and Nissl staining neuronal morphology hippocampus of mice in each group.Research Result1.The behaviors and EEGs of mice in normal group were normal.According to Racine's seizure criteria,the epileptic seizure levels of mice in the epileptic group,IgG group and HMGB1 Ab group could reach about IV-V after the intraperitoneal injection of PILO,and the successful rate of epilepsy model-making and incubation period(the time from injection of PILO to SE)of the seizures of mice had no statistically significant difference in both groups(P>0.05).24 hours after the treatment,the electroencephalogram illustrated abnormal manifestations in the acute phase in the epilepsy group,the IgG group,and the HMGB1 Ab group:in the beginning,the background wave was abnormal slow activity;24 hours after SE,electroencephalogram appeared explosive,persistent,and cluster multi-spikes with high amplitude and high rhythm,and there was no significant difference among the groups.After 4 weeks,the electroencephalograms of these three groups of mice still showed abnormally typical spikes,but the amplitude of the electroencephalogram in the HMGB1 Ab group was significantly lower than that of the epilepsy group and the IgG group2.RT-PCR results indicated that HMGB1 mRNA level in hippocampus tissue of epileptic group mice was significantly higher than that of normal group(P<0.05)at12h after the status epilepticus(SE).At 24h,72h,4w after SE,the expression level of HMGB1 mRNA in epilepsy group was extremely significantly higher than that in normal group(P<0.01),and it increased with the the time of epilepsy.Western Blot results showed,24h after SE,HMGB1 protein level in epilepsy group was significantly up-regulated compared with normal mice,P value was less than 0.01 calculated by statistical analysis,and the HMGB1 protein expression level was enhanced with SE time increased.3.HE staining displayed that the pyramidal and granular cell layers of the hippocampus of normal mice were band-shaped,arranged neatly,without cell loss.The neurons were arranged tightly,the shape was complete and regular,the cytoplasm was transparent,the nucleus was round or oval shape,the chromatin was evenly distributeded,and the morphology of nucleolus was clear.Normal neurons of hippocampus were damaged severely in epilepsy group and IgG group mice,which were swollen and broken,the structure was incomplete,the space was enlarged,the arrangement was irregular,and the nuclei were rigid and small.However,most of the pyramidal cells of the hippocampal CA1 region have clear edges and normal morphology in HMGBI Ab mice.The number of normal neurons is significantly increased compared with the epilepsy group and IgG group.Nissl staining of hippocampal tissue section results showed that the number of Nissl body per field in epileptic mice was less than 50,while that in normal mice was130 per field.Whereas,the number of Nissl body in HMGBI Ab group mice was much more than epilepsy group and IgG group,the difference was statistically significant(P<0.01).ConclusionHMGB1 expression levels of hippocampal tissue increased in epileptic mice induced by PILO,along with the obvious hippocampal neuronal damage.HMGB1 Ab can improve the abnormal electroencephalogram of mice and reduce the damage of hippocampal neurons in CA1 area.It was confirmed that HMGB1 Ab has protective effect on hippocampus of epileptic mice.Part 2:Study on the mechanism of autophagy and apoptosis of anti-HMGBl neutralizing antibody in hippocampal neurons of epileptic miceResearch PurposesTo investigate the effects of anti-HMGB1 neutralizing antibody on hippocampal autophagy activity and apoptosis in PILO-induced epileptic mice.Research Method1.Epileptic mice modeling and treatment:epilepsy models were established by PIOL intraperitoneal injection.Select 8-week-old male mice were randomly divided into 4 groups:control group(normal mice),epilepsy group(epileptic mice injected PBS),IgG group(epileptic mice were injected IgG),HMGB1 Ab group(epileptic mice were injected with anti-HMGB1 neutralizing antibody)and 3-MA group(epileptic mice were injected with 3-Methyladenine).2.Observation of autophagy-related ultrastructure of hippocampal neurons by transmission electron microscope3.RT-PCR method was used to detect the changes of transcription levels of hippocampal autophagy related genes in each group of mice.4.Western Blot was used to detect the changes of HMGB1 protein,autophagy and apoptosis-related protein expression in hippocampus of each group of mice.5.Immunofluorescence double staining was used to detect the expression of LC3 and NeuN in hippocampus of mice in each group.6.TUNEL-NeuN double staining detected the apoptosis of mice hippocampal neuron in each group.Research Result1.Effects of HMGB1 Ab on autophagy of hippocampal neurons in epileptic mice1.1 Effect of HMGB1 Ab on autophagy-related ultrastructure of hippocampal neurons in epileptic miceThe structure of hippocampal CA1 region was clear and complete in normal mice group,organelle was abundant,ultrastructure is normal and autophagic vacuoles barely been observed.In the epilepsy group and the IgG group,the number of autophagolysosomes and autophagosomes increased,the rough endoplasmic reticulum was swollen,mitochondria was sparse and swollen,mitochondrial ridges were disappeared,organelles were decreased,and structures were damaged.Different size of vacuoles were formed in cytoplasm,cell membrane was collapsed,and cell contents leaked,but aboved pathological changes in the HMGB1 Ab group and 3-MA group were relieved,and autophagolysosomes and autophagosomes were decreased.1.2 Effect of HMGB1 Ab on transcription levels of autophagy-related genes in hippocampus of epileptic miceAt 4 weeks after SE,the relative expression change of Beclinl,ATG5 and ATG7 mRNA were same in each treatment group.The Beclinl,ATG5,and ATG7 mRNA expression of hippocampus in epileptic mice was significantly higher than the normal group(P<0.01).Moreover,these three genes of transcription level in 3-MA group was significantly lower than epileptic mice(P<0.01),and decreased mRNA level of these three gene in HMGB1 Ab mice was statistically significant compared to epilepsy and IgG group(P<0.05)1.3 Effect of HMGB1 Ab on the expression of autophagy-related proteins in hippocampus of epileptic miceWestern Blot results showed that compared with the normal group,the expression levels of HMGB1 protein,autophagy-related proteins Beclin 1,ATG5 and ATG7,and the ratio of LC3 II/I in the epilepsy group were significantly increased(P<0.01).On the contrary,p62 proteins was low expression levels(P<0.01).After HMGB1 Ab and 3-MA treatments,the HMGB1,autophagy related proteins and LC3?/? ratio were decreased significantly(P<0.01),while the expression level of p62 protein was increased(P<0.01).1.4 Detection of LC3 and NeuN in CA1 region of hippocampus by immunofluorescence double stainingIt can be detected LC3 fluorescence signal diffusely around the nucleus is very low in CA1 region of normal mice,and it was shown that LC3 fluorescent signal around the nucleus clearly enhanced in epilepsy group and IgG groups,which suggested that the number of autophagosomes was significantly increased.After the intervention with HMGB1 Ab and 3-MA respectively,the fluorescence signal of LC3 was significantly weakened than that of the epilepsy group,and the fluorescence signal of NeuN was recovered.Compared with the normal group,the proportion of LC3 neurons in total neurons in epileptic mice was significantly higher(P<0.01).The ratio of neurons expressed autophagosome in HMGB1 Ab group or 3-MA group was significantly reduced(P<0.01).2.Effect of HMGBI Ab on hippocampal apoptosis in epileptic mice2.1 TUNEL-NeuN double staining detection of apoptosis of hippocampal neuronsIn 4 weeks after SE,a large number of TUNEL fluorescent signal in CA1 region of epileptic mice,and the distribution is basically consistent with NeuN signal of hippocampus.The NeuN signal of mature neurons of epileptic mice treated with HMGBI Ab or 3-MA was enhanced,while the apoptotic signal was weakened,which means the percentage of apoptotic neurons decreased to 1/3 of epilepcy group.2.2 Effects of HMGB1 Ab on apoptosis-related proteins in hippocampal neurons of epileptic miceWestern Blot results of poptosis related protein in the hippocampus of mice showed Bcl-2 protein expression was decresed and Bax and cleaved-caspase 3 protein were increased in epileptic mice.We treated epileptic mice with HMGB1 Ab and found that HMGBI Ab could upregulate the Bcl-2 protein expression and inhibit Bax and cleaved-caspase 3 protein expression.ConclusionThe expression of HMGB1 in hippocampus was increased in epileptic mice induced by PILO,accompanied by severe cellular autophagy and neuronal damage.HMGB1 Ab can inhibit over-activated cell autophagy,reduce the number of apoptotic cells,and protect damaged neurons of mice.Therefore,we speculated that HMGB1 is involved in the autophagy process of epilepsy and can be an important target for epilepsy.Part 3:Study on protective effect and mechanism of anti-HMGBl neutralizing antibody in spatial memory impairment of epileptic miceResearch PurposesTo investigate the mechanism of anti-HMGB1 neutralizing antibody on spatial memory impairment in PILO-induced epileptic mice.Research Method1.Epileptic mice modeling and treatment:the model was induced according to the method in part 2.2.Morris water maze experiments investigated the behavioral effects of HMGB1 Ab in memory impairment of epilepsy mice.3.Timm staining was used to observe the mossy fiber sprouting(MFS)in the hippocampal CA3 region.Research Result1.Morris water maze experiment found that HMGB1 Ab can significantly improve the spatial memory learning ability of epileptic mice.2.Timm staining displayed moderate to severe MFS was appear in the hippocampus of epilepsy group and IgG group,while after HMGB]Ab treatment,the degree of MFS was relieved obviously in hippocampus CA3 region.ConclusionHMGB1 Ab can reduce the degree of MFS in the hippocampal CA3 region,and significantly improve the spatial memory learning ability of mice.Therefore,we speculated that HMGB 1 can be an important target for memory disorders caused by epilepsy.