Study On The Molecular Mechanism Of Neuronal Autophagy Mediated By NADPH Oxidase In Hippocampal CA1Subregion Following Diffuse TBI In Rats

Traumatic brain injury （TBI） is defined as critical damage to the brainthat resulted from external mechanical force. TBI is characterised with highincidence, high mortality and high morbidity. Most patients are youngs andadults, and the survivors suffered from TBI still display varying degrees ofneurological and psychological dysfunction, which bring huge economic andemotional burden to the society and their families. At present, there is noideally effective treatment for TBI, so to explore the pathophysiologicalmechanisms of TBI and to look for the new effective t treatment arget on TBIremains to be a hot and difficult challenge.The main pathological mechanism of further brain damage after TBI isthe secondary brain injury （SBI）. SBI occurred in few minutes to several daysor even longer period after TBI, which is the pathological changes graduallybased on the primary injury and evolves over time. It is the important causethat leads to neuronal cell injury and death. So the key strategy to improve theprognosis of TBI is to prevent and treat effectively on the SBI. Recent studieshave revealed that complex cascade reaction caused by oxyradical, especiallysuperoxide anion, is the core process of neuron damage. NADPH oxidase is anenzyme complext composing of multiple subunits. NOX₂, a catalytic subunitof NADPH oxidase, plays an important role in the process of maintaining theactivity of NADPH oxidae and the generation of oxygen free radical. Themechanisms that NADPH oxidase involves in TBI have not been fullyelucidated. Recent studies in cerebral ischemia-reperfusion found that thismechanism includes two aspects: one is the direct damage to biologicalmacromolecules by active oxygen, produced from the enchanced activity ofNADPH oxidase affected by kinds of factors, such as TBI. The other one is the involvement of neuronal death caused by reactive oxygen species incerebral ischemia through intracellular or intercellular signalling transductionpathways. Autophagy as one new form of neural cell death, recently gainesmore and more attentions, but the study on the role of autophagy and itsmechanism mediated by signal transduction pathway remains insufficient afterTBI. Whether its signal transduction pathway is mediadte or not by NADPHoxidase are not yet very clear after TBI.In this paper, rat diffuse TBI models were reproduced by using theimproved Marmarou’s falling method to explore the expression of NOX₂, acatalytic subunit of NADPH oxidase, and to investigate NADPH oxidase’srelationship to autophagy expression. NADPH oxidase specfic inhibitorApocynin and Akt specfic inhibitor LY294002were used as drugs forpretreatment further to investigate the expression NOX₂protein and thechange of NADPH oxidase activity, and to assess the roles of NADPH oxidasein the secondary brain injury after TBI. To identify whether or not the NADPHoxidase is involved in the regulation of the occurrence and development ofautophagy in neurons of hippocampal CA1region and to demonstrate whetheror not the Akt/mTOR signaling pathway is mediated by NADPH oxidase andis involved in the process of neuronal autophagy in hippocampus. Aim to lookfor the key target of neuronal autophagy at the upstream of signalling pathwayafter TBI, and to provide new theoretical basis and treatment strategies forclinical prevention against secondary brain injury. This paper is divided intothree parts to elaborate the idea.Part I Changes of NADPH oxidase activity and catalytic subunit NOX₂expression and its roles in rats following diffuse TBIObjective: To explore the changes of the activity of NADPH oxidase andits catalytic subunit NOX₂protein expression in hippocampal CA1subregion,and to identify its role in secondary brain injury after diffuse TBI in rats.Methods: A total of240male SD rats were used and divided into fourgroups by randomized block method: Sham group, TBI group, DMSO group,and Apocynin group. TBI models were reproduced according to the modified Marmarou’s falling method. Apocynin was injected intraperitoneally30minprior to TBI at dose of50mg/kg on body weight as specific inhibitor ofNADPH oxidase. Each group was further divided into6h,12h,24h,48hand72h subgroups after TBI. To observe the following indexes: Hï¹ E stainsto observe morphological changes. The cellular localization and expression ofNOX₂protein were observed by immunohistochemistry. NOX₂protein wasevaluated by Western blotting. NADPH oxidase activity was determined bycolorimetric method. Brain water content was measured by wet/dry method.Another40rats undergo morris water maze test to detect their changes of theability of learning and memory in space at8d and10d after TBI.Immunohistochemical stained sections were analyzd with IOD/Area valuesdetected by Image proplus6.0digital medical image analysis systems. Thefilms of western blot were scanned and quantified using the Gel-Doc imageanalysis software. Results were analyzed by SPSS15.0software. Statisticalsignificance between multiple groups was determined using a one-wayANOVA. Q-test was used to determine the statistical significance between twogroups. A p value of less than0.05was considered statistically significant.Results:1Pathological changes in hippocampal CA1region: Light microscopylevel of Hï¹ E indicated that the histomorphology of sham-operated rat brainwas normal and integrity with abundance of integrity neuron cells. While inTBI and DMSO group there were severe neurons swelling, cytoplasmacidophilic stain weaken, nucleolus shrinkage and hyperchromaticsignificantly, a larger gap existed around cells, and there were neuronaldegeneration and necrosis in hippocampal CA1region. When treated withApocynin, these changes were weakened at the corresponding time pointsafter TBI （P<0.05）.2NOX₂results in immunochemistry in hippocampal CA1subregion:NOX₂located in the membrane of neural cells, and was stained yellow andbrown in immunochemistry. In sham group, we can seldom see positive cells,and the positive cell was light stained. NOX₂positive cells began to increase significantly in hippocampal CA1subregion from6h after TBI, with colordeepened and immune reactivity enhanced and peaked at24h after TBI. InDMSO group the change of NOX₂expression was similar with TBI group,and there was no significant difference between the two groups （P0.05）.While the change of NOX₂immune reactivity in Apocynin treatment groupwas similar to that in TBI group and DMSO group, but the immune reactivitydeclined significantly （P<0.05）.3NOX₂result in western blot: Western blot results indicated that Shamgroup has only a little expression of NOX₂and there were no changes in eachpoint. The NOX₂protein in TBI group began to increase obviously at6h withpeaking at24h （P<0.05）, then decreased, but still higher than the basic leveltill to72h （P<0.05）. The comparison of datas between TBI group and DMSOgroup had no statistically significance （P＞0.05）. When treated withApocynin, expression of NOX₂was weakened compared with TBI groups atcorresponding time points （P<0.05）.4The activity of NADPH oxidase: The activity of NADPH oxidase waslower in Sham group, and did not change over time. The activity of NADPHoxidase increased significantly from6h in TBI group and DMSO group afterTBI, with peaked at24h, and then began decreased, and did not dropped tonormal level till72h. The comparison of datas in TBI group and DMSOgroup had no statistically significant（P＞0.05）. When treated with Apocynin,these changes of NADPH oxidase activity were down regulated significantly（P<0.05）.5Brain water content measurements: The brain water content increasedin from6h to72h after TBI. The brain water content of percentage atdifferent time point in TBI group respectively were:77.23士0.82,78.51士0.92,80.62±0.79,80.75±1.06and80.92±0.87. DMSO treatment did notreduce brain water content compared with TBI group. But in the two groupsthe brain contents at each time point were higher than74.62±0.76in the Shamgroup. Apocynin treatment significantly attenuated brain water contentcompared with the TBI and DMSO groups. 6Changes of learning and memorizing ability: Morris water maze testwas use to detect the changes of learning and memorizing ability. Obviousspace learning ability deficit and memorizing obstruction existed in TBI andDMSO groups compared with Sham group and the delitescence prolongedobviously at8d and10d after TBI （P<0.01）. Search moving tracks showedthat search strategy altered more ideally with time in Apocynin group thanthat in TBI group and DMSO group and there was significant differencebetween these groups （P<0.05）.Summary: The level of NOX₂expression and the activity of NADPHoxidase are significantly increased, which aggravates the secondary braininjury following TBI. Inhibition of NADPH oxidase activation can attenuatebrain edema, improve learning and memory ability and afford neuroprotectiveeffects.Part II The effect of NADPH oxidase on neuronal autophagy inhippocampal CA1subregion after diffuse TBI in ratsObjective: To investigate the expression of autophagy in neurons ofhippocampus CA1subregion, and to explore whether or not the neuronalautophagy is mediated by NADPH oxidase after TBI.Methods: The modified Marmarou’s falling method was used toreproduce TBI model. Rats were randomly divided into four groups: Shamgroup （n=45）; TBI group （n=45）; DMSO group （n=45）; Apocynin group（n=45）. Each group was further divided into6h,12h,24h,48h and72h atotal of5subgroups. The cellular localization and expression of Beclin-1wasobserved by immunohistochemistry. LC3â…¡/LC3â… and Beclin-1, markersproteins of autophagy, were used to evaluated autophagy expression byWestern blot. LC3and NeuN （Neuronal marker） double labelling wereobserved by immunofluorescence microscope. Quantitative analysis andstatistical method are the same as mentioned in the first part.Results:1Beclin-1result in immunochemistry in hippocampal CA1subregion:The positive cells of Beclin-1were stained yellow and brown, and mainly located in the cytoplasm of neural cells. In Sham group, we can seldom see apositive cell, and the positive cell was light stained. The immunoreactivity didnot change over time. The expression of Beclin-1increased significantly at6hafter TBI, and the immunoreactivity enhanced, with peaked at24h, and beganto decline from24h after TBI. That in DMSO group was similar to TBI group,and there were no significantly difference between the two groups （P0.05）.The tendency of Beclin-1expression in Apocynin treated group was similar tothat in TBI group and DMSO group, but the positive expression and immunereactivity decreased significantly （P<0.05）.2Beclin-1results in western blot: Western blot indicated that the shamgroup had only a little expression of Beclin-1and there were no changes atcorresponding time. The Beclin-1expression in TBI group began to increaseobviously at6h and peaked at24h, then decreased, but still higher than thebasic level till to72h （P<0.05）. The comparison of datas in TBI group andDMSO group had no statistically significant （P＞0.05）. When treated withApocynin, the expression of Beclin-1decreased at each time points （P<0.05）.3LC3result in western blot in hippocampus: Western blot indicated thatthe sham group has only a little expression of LC3â…¡ and there were nochanges of LC3â…¡/LC3â… in each point. The LC3â…¡/LC3â…  ratio in TBIgroup began to increase obviously at6h and peaked at24h （P<0.05）, thendecreased, but still higher than that in Sham group（P<0.05）. The comparisonof datas in TBI group and DMSO group had no statistically significance（P＞0.05）. When treated with Apocynin, these changes were weakened atcorresponding time points （P<0.05）.4LC3and NeuN double labeling result in immunofluorescence inhippocampal CA1subregion: Obeseved with confocal microscopy, we couldclear see the red fluorescent of LC3labeled by TRITC accumulated in thecytoplasm and the green fluorescent of NeuN labeled by FITC located inneuronal nucleus. In hippocampal CA1subregion of TBI group, we can seethere were large number of overlapping of green and red fluorescent,presented as yellow color at24h after TBI, indicateing that autophagy is located in neurons; while when treated with Apocynin, the overlapping ofgreen and red fluorescent decreased signifcantly, illustrating that the level ofneuronal autophagy in hippocampal CA1region decreased.Summary: Neuronal autophagy could be detected in hippocampal CA1subregion at6h after TBI, and autophagy expression gradually increased, withpeaked at24h after TBI. Pretreatment with Apocynin can significantlyattenuate the level of neuronal autophagy expression. The results indicatedthat NADPH oxidase mediated autophagy expression in neurons ofhippocampal CA1subregion after TBI.Part III Study on the molecular mechanisms on neuronal autophagymediated by NADPH oxidase in hippocampal CA1subregion after diffuseTBI in ratsObjective: Apocynin, NADPH oxidase specific inhibitor, and LY294002,Akt specific inhibitor, were used as pretreatment drugs. To identify whether ornot the Akt/mTOR signaling pathway is involved in neuronal autophagymediated by NADPH oxidase in hippocampal CA1subregion after TBI.Methods: The modified Marmarou’s falling method was used toreproduce TBI models. A total of270male SD rats were randomly dividedinto six groups: Sham group; Sham+LY294002group; TBI group; DMSOgroup; Apocynin group; Apocynin+LY294002group. Each group was furtherdivided into6h,12h,24h,48h and72h subregion. Hï¹ E stains to observemorphological changes. Beclin-1, LC3, p-AktSer-473and p-P70S6KThr-389protein expression were evaluated by Western blotting analysis. The cellularlocalization and expression of LC3and p-AktSer-473marked respectively withNeuN were observed by immunofluorescence microscope. Quantitativeanalysis and statistics are the same as mentioned in the first part.Results:1Histomorphology changes of Hï¹ E stains: Histomorphology changesunder light microscopy in Sham and TBI group are the same as mentionedpreviously. While there were severe changes in Apocynin+LY294002group.There were severe neurons swelling and necrosis, cytoplasm acidophilic stain ehchanced, nucleolus shrinkage, hyperchromatic in hippocampal CA1regionafter TBI.2P-AktSer-473results in western blot: Western blot indicated thatp-AktSer-473had only a little expression in sham group, and there were nochanges at each point. Compared with sham group, the protein of p-AktSer-473in TBI group increased significately at6h after TBI, and decreased from6h to24h, and then gradually increased from24h after TBI, presented as doublehump. In Apocynin group, these changes of p-AktSer-473protein were similar tothat in TBI group, but the expression of p-AktSer-473increased remarkably atthe corresponding time （P<0.05）.3P-P70S6KThr-389results in western blot: Western blot results indicatedthat p-P70S6KThr-389protein had only little expression in sham group, andthere were no changes in each point. Compared with the sham group, theprotein of p-P70S6KThr-389in TBI group increased significately at6h after TBI,decreased from6h to24h, and then gradually increased from24h. InApocynin treated group, these changes of p-P70S6KThr-389protein were similarto that in TBI group, but the expression of p-P70S6KThr-389enhancedremarkably at the corresponding time （P<0.05）.4Beclin-1results in western blot: The comparison of Beclin-1protein inSham group and Sham plus LY294002group had no statistically significanceat corresponding time （P＞0.05）. Compared with Sham group, the expressionsof Beclin-1in TBI increased gradually from6h after TBI, with peaked at24hand then decreased. The protein of Beclin-1in Apocynin group was similar tothat in TBI group, but its expression decreased remarkably （P<0.05）. Therewas significant statistic difference at every time between the two groups（P<0.05）. While treated with Apocynin combined LY294002, the expressionof Beclin-1increased significantly （P＞0.05）.5LC3results in western blot: Western blot result indicated that LC3â…¡/LC3â… ratio in Sham group and Sham+LY294002group had no statisticallysignificance （P＞0.05）. Compared with Sham group, LC3â…¡/LC3â… ratio inTBI increased markedly, with peaked at24h and then decreased. While in Apocynin group the LC3â…¡/LC3â… ratio decreased remarkably （P<0.05）.There was significantly statistic difference at every time between the twogroups （P<0.05）. When treated with Apocynin combine LY294002, the LC3â…¡/LC3â… ratio increased significantly compared with Apocynin and TBIgroup （P<0.05）.6LC3and NeuN double labeling results in immunofluorescence inhippocampal CA1subregion: Obeseved with confocal microscopy, we couldclear see the red fluorescent of LC3labeled by TRITC accumulated in thecytoplasm and the green fluorescent of NeuN labeled by FITC located inneuronal nucleus. In hippocampal CA1subregion of TBI group, we could seethere were a large number of overlappings of green and red fluorescent,presented as yellow color at24h after TBI, indicateing that autophagy islocated in neurons; while when treated with Apocynin, the overlapping ofgreen and red fluorescent decreased signifcantly, illustrating that the level ofneuronal autophagy in hippocampal CA1region was depressed; while ingroup treated with Apocynin combined LY294002, the red and greenfluorescence overlaps was obviously increased, indicating that the neuronalautophagy enhanced significantly.7P-AktSer-473and NeuN double labeling results in immunofluorescence inhippocampal CA1subregion: Via using confocal microscopy, we could clearsee the red fluorescent of p-AktSer-473positive cells accumulated in thecytoplasm and the green fluorescent of NeuN located in neuronal nucleus. Inhippocampal CA1subregion, we could see there were a large number ofoverlapping of green and red fluorescent at24h after TBI; while when treatedwith Apocynin, the overlapping of green and red fluorescent increasedsignifcantly, indicating that Apocynin promoted neuronal Akt phosphorylationby inhibition of NADPH oxidase in hippocampal CA1subregion.Summary: The enhanced phosphorylation of Akt/mTOR signallingpathway promoted by oxidative stress were depressed by the up regulatedactivity of NADPH oxidase after diffuse TBI. Inhibition of the activation ofNADPH oxidase with Apocynin could potentialize the activity of Akt/mTOR signaling pathway and negatively regulate neuronal autophagy following TBI.Conclusions:NADPH oxidase is involved in the secondary brain injury after diffuseTBI and can mediate the expression of neuronal autophagy in hippocampalCA1subregion via regulating the activity of Akt/mTOR signaling transductionpathway. Inhibition of the activity of NADPH oxidase with Apocynin coulddepress neuronal autophagy, attenuate brain edema, improve the learning andmemory ability and could afford neuroprotective effects.