| The incidence of traumatic brain injury increased year by year, how to reduce mortality and morbidity is particularly important. A series of chemical, biological, physiological and pathological reactions caused by TBI can lead to cell death and neurological dysfunction of neurons lasted for several days to several weeks. The study in TBI-induced neurological dysfunction, cell necrosis and apoptosis have more in-depth. Autophagy as a form of neuronal cell death, recently more and more attention, but the studies in TBI were poor. The present research focused on the signal transduction pathways and signs gene.Intracellular and extracellular stimuli can induce the activation of autophagy, which is the result of a series of cellular and molecular events. Recent studies found that the expression of JNK gene in MAPK family raised after activation of neuron autophagy, while expression of its downstream effectors-p53increased,but the activation of neuron autophagy is currently unknown to be concerned with TBI. The reports in lung cancer cells were found that DRAM, a p53target genes, regulated autophagy and p53regulated depolymerization of beclin-l-bcl-2/bcl-xl complex. Description of the tumor suppressor p53in lung cancer can be modulated by autophagy. Our preliminary experiments suggest that the expression of LC3and beclin-1elevated after TBI, but JNK-medated p53would activate the neuron autophagy after TBI is unknown, which is also the aim of this study. This paper is divided into three parts to elaborate the idea. Objective:According to Marmarou's falling model in1994, this part of study detects whether there is autophagy in neuron.Method:A total of96male Sprague-Dawley (SD) rats (300-350g) were used in this study. By randomized block method rats were randomly divided into four groups:sham-operated (n=48), TBI (n=48). Each group was further divided into1h,6h,12h,24h,48h and72h. LC3II/LC3I and Beclin-1, were evaluated by Western blotting analysis. H&E staining observes morphological changes. The cellular localization and expression of Beclin-1was observed by immunohistochemistry.The comparisons of two groups were conducted using analysis of variance (ANOVA). P values of less than0.05were considered statistically significant. Immunohistochemical stained sections were analyzd with IOD/Area values detected by Image proplus6.0digital medical image analysis system. The films of western blot were scanned and quantified using the Gel-Doc image analysis software.Result:1Pathological changes:After injury, the majority of rats had a transient decerebration and respiration suppression. Light microscopy level of H&E indicated that brain tissue was normal in sham group while there were severe changes in TBI group,such as hemorrhage, yellow-stained, brain tissue edema, vascular congestion. In addition, there were signicant swollen, necrotic and denaturaious neurons in brain tissue after trauma, Which were correspondenced with the pathological changes in Marmarou model.2Beclin-1result in immunochemistry The production of beclin-1, located in the cytoplasm of neural cells, was stained yellow and brown in immunochemistry. In sham group, we can seldom see a positive cell, and the positive cell we see was light stained, there was significant difference between TBI group and sham groups(P<0.05):in the TBI group there is the peak of48h, which compares with the expression of24h and72h (P<0.05).3LC3and beclin-1result in western blot In sham group, we can seldom see the positive change of beclin-1and LC3II/LC3I in all points of time. The value of LC3II/LC3I is gradually raising and peak of beclin-1is 48h(P<0.05), which compares with data of sham group. In the TBI group the peak of beclin-1at48h compares with the expression of24h and72h about which there is statistically significant(P<0.05).4LC3and PI result in immunofluorescence. The green fluorescent of LC3positive cells labeled by FICT accumulated in the cytoplasm and the red fluorescent of nucleus labeled by PI in TBI group, and the form of the nucleus was regular,and the nucleolus was clearly visible. At24h after TBI, compared with the sham group, LC3positive expression in numbers and immune strength were increased.Conclusion:Marmarou's model modified fits this study; this part of study detects that there is autophagy in neuron.The second part JNK-mediated p53phosphorylation enhancing neuron autophagy after TBIObjective:This part of study apply with SP600125of JNK inhibitor and detect the expression of JNK, p-p53, DRAM and beclin-1, which would show whether JNK-mediated p53phosphorylation might be an important mechanism for enhancing neuron autophagy in response to TBI.Method:A total of296male SD rats were used in this study. By randomized block method rats were randomly divided into four groups:sham group (n=74), TBI group(n=74), TBI+DMSO(n=74), and TBI+SP600125(n=74). Each group was further divided into1h,6h,12h,24h,48h and72h. JNK was treated with SP600125, a specific JNK inhibitor. JNK, p-p53, beclin-1and DRAM were evaluated by Western blotting analysis. The cellular localization and expression of p53, Beclin-1and DRAM were observed by immunofluorescence and immunohistochemistry. Another80rats undergo morris water maze test. Quantitative analysis and statistics are the same mentioned previously.Result:1Pathological changes:Light microscopy level of H&E indicated that brain tissue was normal in sham group while there were severe changes in TBI group. There were signicant swollen, necrotic and denaturaious neurons in brain tissue at24h after trauma. When treated with sp600125, these changes were weakened in TBI groups at difference time points(P<0.05).2JNK and beclin-1result in western blot In sham group, we can seldom see the positive change of beclin-1and JNK in all points of time. The peak of beclin-1and JNK were repectively the48h and12h, which compares with data of sham group(P<0.05). The comparison of datas in TBI group and TBI+SP600125group are not statistically significant(P>0.05). Expression of beclin-1in TBI+SP600125group compared with that of TBI group are statistically significant (P<0.05) at6h,12h,24h,48h and72h. The same comparison about expression of JNK is at6h,12h and24h.3DRAM and p-p53result in western blot In sham group, we can seldom see the positive change of DRAM and p-p53in all points of time. The peak of DRAM and p-p53were the24h, which compares with data of sham group(P<0.05). The comparison of datas in TBI group and TBI+DMSO group are not statistically significant(P>0.05). Expression of beclin-1in TBI+SP600125group compared with that of TBI group are statistically significant(P<0.05) at12h,24h,48h and72h.4DRAM and beclin-1result in immunochemistry The production of DRAM and beclin-1, located in the cytoplasm and nucleus of neural cells, were stained yellow and brown. In TBI group, the peak of DRAM and beclin-1were repectively the24h and48h(P<0.05). The comparison of datas in TBI group and TBI+DMSO group are not statistically significant(P>0.05). When treated with SP600125, these changes were weakened in TBI+SP600125group at12h,24h,48h and72h (P＜0.05).5p-P53, DRAM and beclin-1result in immunofluorescence. The green fluorescent of p-p53positive cells labeled by FICT accumulated in the cytoplasm and the red fluorescent of DRAM and beclin-1labeled by TRITC accumulated in the cytoplasm in TBI group, and the form of the nucleus was regular,and the nucleolus was clearly visible. But their expression in the TBI+SP600125group, positive expression in numbers and immune strength were decreased.6Recommending score of neural function:sham group, TBI group and SP600125group each had a score of24,7-9,15-18respectively. As a result, there was significant difference between TBI group, SP600125group and sham group (P<0.05); there was also significant difference between TBI group and SP600125group (P<0.05).7Morris water maze result Morris water maze tests showed that obvious space leaming and memorizing obstruction existed in rats after injury, delitescence prolonged obviously9to10days later. Search moving tracks shows that search strategy altered more ideally with time in the TBI+SP600125group than in TBI group(P<0.05).Conclusion:This part of study detect that SP600125could raise learning and memorizing dysfunction in rats after injury; JNK-mediated p53phosphorylation might be an important mechanism for enhancing expression of DRAM and beclin-1in response to TBI.The third part The study of p53phosphorylation regulating beclin-1and bcl-2after TBIObjective:This part of study apply with PFT-aof p53inhibitor and detect the expression of JNK, p-p53, p-bcl-2, bcl-2, bcl-xl and beclin-1, which would show whether p53phosphorylation might be an important mechanism for dissociating the compound of beclin-1-bcl-2/bcl-xl after TBI.Method:A total of240male SD rats were used in this study. By randomized block method rats were randomly divided into four groups:sham group (n=60), TBI group(n=60), TBI+DMSO(n=60), and TBI+PFT-a (n=60). Each group was further divided into6h,12h,24h and48h. P53was treated with PFT-a, a specific PFT-ainhibitor. Beclin-1, p-p53, p-bcl-2, bcl-2and bcl-xl were evaluated by Western blotting analysis. Expression of p53, Beclin-1and DRAM were observed by immunoprecipitation and Western blot. The cellular localization and expression of beclin-1, p53and bcl-2were detected by immunofluorescence. Another80rats undergo morris water maze test. Quantitative analysis and statistics are the same mentioned previously.Result:1p-bcl-2and p-p53result in western blot The peaks of p-bcl-2and p-p53were the24h, which compare with data of sham group(P<0.05). Their expression in TBI+PFT-agroup are gradually decreasing compared with sham group and there are statistically significant P<0.05) at6h,12h and24h.2Beclin-1, bcl-2and bcl-xl result in TBI group and TBI+DMSO group by immunoprecipitation and Western blot Expression of beclin-1in TBI group and TBI+DMSO group is gradually decreasing compared with sham group and there are statistically significant P<0.05) at12h,24h and48h. Expression of bcl-2and bcl-xl are gradually decreasing from6h. At the same time we find that the changed quantity of bcl-2is the more great than that of bcl-xl (P<0.05).3Beclin-1, bcl-2and bcl-xl result in TBI+PFT-agroup by immunoprecipitation and Western blot Expression of beclin-1in TBI+PFT-agroup is decreasing compared with sham group and there are statistically significant P<0.05) at48h. Expression of bcl-2and bcl-xl are the same. At the same time we also find that the changed quantity of bcl-2is the more great than that of bcl-xl (P<0.05).4Beclin-1, bcl-2and p-p53result in immunofluorescence. The green fluorescent of bcl-2and p-p53positive cells labeled by FICT accumulated in the cytoplasm and the red fluorescent of beclin-1labeled by TRITC accumulated in the cytoplasm in TBI group, and the form of the nucleus was regular,and the nucleolus was clearly visible. But their expression in the TBI+PFT-agroup, positive expression in numbers and immune strength were decreased.5Recommending score of neural function:sham group, TBI group and TBI+PFT-a group each had a score of24,7-10,16-20respectively. As a result, there was significant difference between TBI group, TBI+PFT-agroup and sham group (P<0.05); there was also significant difference between TBI group and TBI+PFT-a group (P<0.05).6Morris water maze result Morris water maze tests showed that obvious space learning and memorizing obstruction existed in rats after injury, delitescence prolonged obviously9to10days later. Search moving tracks shows that search strategy altered more ideally with time in the TBI+PFT-a group than in TBI group(P<0.05).Conclusion:This part of study detect that PFT-a could raise learning and memorizing dysfunction in rats after injury; p53phosphorylation might be an important mechanism for dissociating the compound of beclin-1-bcl-2/bcl-xl after TBI.Conclusion:JNK-mediated p53phosphorylation might be an important mechanism for enhancing neuron autophagy in response to TBI. |
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