大鼠弥漫性脑创伤中JNK介导的P53通路对神经元自噬的分子机制研究
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摘要
脑创伤的发病率逐年升高,如何降低致死率和致残率就显得尤为重要。TBI引起的一系列生化生理和病理方面反应可以导致神经元持续数天到数周时间的细胞死亡和神经功能障碍。以前在TBI引发的神经功能障碍与细胞坏死和凋亡方面研究较为深入。自噬作为神经细胞死亡的一种形式,近期越来越受到关注,但是在TBI中的作用机制研究不足。目前国内外研究焦点集中于创伤后的神经元自噬过程中信号转导通路和标志性基因方面。
细胞内外的很多刺激可以诱导自噬的激活,其是一系列细胞分子事件的级联反应的结果。近来研究发现MAPK家族的JNK基因在神经元自噬发生后表达升高,同时其下游因子p53同时表达也升高,这是否与外伤导致神经元自噬的激活有关,目前不得而知。文献报道在研究肺癌肿瘤细胞的自噬之中发现,自噬基因DRAM是p53的靶基因,对于癌细胞的自噬存在调节作用,同时p53对于自噬标志基因beclin-1与bcl-2/bcl-xl复合体的解聚存在调节作用。说明肿瘤抑制因子p53在肺癌中可以调控自噬。我们前期的实验提示在TBI后存在自噬的激活,自噬标志因子LC3、 beclin-1表达升高。那么JNK通路是否能通过对p53的调节来激活神经元自噬,在TBI中是这条通路是否成立这也是本研究的目的,本论文分三部分来阐述。
第一部分大鼠弥漫性脑创伤后自噬相关蛋白的表达
目的：本部分研究应用雄性SD大鼠复制Marmarou闭合性弥漫性颅脑创伤模型,检测大鼠弥漫性脑创伤后是否存在神经元自噬。
方法：采用本实验室改良Marmarou方法。大鼠随机分成：①假手术组(n=48),②脑创伤组(n=48),取伤后1h、6h,12h、24h、48h、72h6个时间点。检测①HE染色；②免疫组学法检测beclin-1的蛋白表达；③Western-blot法检测LC3II/LC3I的比值和beclin-1的蛋白表。应用Image Proplus6.0和Gel-Doc分析系统定量分析测定。实验中所得数据均用x±s表示,用SPSS11.5统计软件进行重复方差分析,以单侧P<0.05,表示差异有统计学意义。
结果：
1光镜下观察大鼠脑创伤模型切片
1.1伤后大鼠脑组织大体观察伤后可见蛛网膜下腔出血,伴随少量脑室出血；点状出血于脑实质多见,多低位脑干的背侧,脑表面的血管充血明显,未见局部脑挫伤。
1.2HE染色观察组织形态学的改变Sham组脑组织结构正常,血管管腔正常,管壁光滑,神经细胞数量多,形态完整；TBI组神经元胞体皱缩呈三角形,胞浆嗜色性染色减弱,核仁皱缩浓染,细胞周围出现间隙,出现神经元细胞变性坏死改变。
2海马和皮层部位beclin-1免疫组化检测结果beclin-1定位于神经元细胞的胞质,Sham组偶见阳性细胞,着色浅淡,免疫反应性不随时间发生变化。TBI组与sham组比较,伤后6h beclin-1阳性细胞显著增多,着色加深,免疫反应性增强(P<0.05),伤后48h达高峰(P<0.05),72h开始下降(P<0.05),TBI组24h和72h的结果与48h结果比较差异有统计学意义(P<0.05)。
3海马和皮层部位LC3的Western blot检测结果Sham组LC3Ⅱ/LC3Ⅰ的比值和LC3Ⅰ与LC3Ⅱ的蛋白表达量在各时间点无变化；TBI组LC3Ⅱ和LC3II/LC3I的比值随着时间点依次升高,TBI组与Sham组比较,伤后6h开始LC3II/LC3I的比值显著升高(P<0.05),伤后表达持续升高,高表达状态持续至伤后72h(P<0.05)。
4海马和皮层部位beclin-1的Western blot检测结果Sham组beclin-1蛋白表达量在各时间点无变化；TBI组与Sham组比较,伤后6h beclin-1蛋白量显著增高(P<0.05),伤后48h达高峰,高表达状态持续至伤后72h(P<0.05),伤后72h开始下降(P<0.05)；TBI组24h和72h的结果与48h结果比较差异有统计学意义(P<0.05)。
5LC3和PI的共聚焦检测结果
在TBI组中神经元细胞胞浆中可见大量绿色和红色荧光聚集,此时细胞核和胞浆清晰可见；TBI组伤后12h中LC3在神经元细胞胞浆中阳性细胞数量明显增多,阳性强度增强,此时细胞核仁清晰。
小结：改良的Marmarou弥漫性脑创伤模型满足了本实验对神经元自噬的研究；大鼠脑创伤后,beclin-1和LC3持续升高证明存在神经元自噬。
第二部分JNK介导p53通路在TBI中对大鼠海马神经元自噬的影响
目的：本部分实验通过应用JNK抑制剂SP600125,检测INK、p-p53、 DRAM和beclin-1的表达来证明JNK介导p53通路在TBI中调控大鼠海马神经元自噬是否成立。
方法：大鼠模型同上,将296只成年雄性SD大鼠随机分成4组：①假手术组(n=74),②脑创伤组(n=74)③DMSO组(n=74)④抑制剂组(n=74)。术后时间点同上。取80只大鼠行水迷宫测试,每组划分为伤后7d、8d、9d及10d四个时间点。检测①HE染色；②免疫组化法和共聚焦法检测p53、 beclin-1和DRAM的蛋白表达定位情况；③Western-blot法检测JNK. p53. beclin-1和DRAM的蛋白表达量。定量分析和统计同上
结果：
1HE染色组织形态学改变光镜下伤后24h可见Sham组和TBI组脑组织结构同前TBI+SP600125组病理形态改变较TBI组有不同程度减轻,可见神经元数量多,排列整齐,形态完整,核质均匀,核仁清晰；TBI+DMSO组与TBI组比较变化不大。
2JNK的Western blot检测结果Sham组JNK蛋白表达量在各时间点无变化；TBI组与Sham组比较,伤后1h JNK蛋白量显著增高(P<0.05),伤后12h达高峰,高表达状态持续至伤后24h(P<0.05),伤后24h开始下降(P<0.05); TBI组与TBI+DMSO组比较有差异无统计学意义(P>0.05);TBI+SP600125组JNK蛋白表达时程与TBI组相似,但表达量减少,与TBI组比较,以伤后6h、12h和24h差异有统计学意义(P<0.05)。
3p-P53和DRAM的Western blot检测结果Sham组p-p53和DRAM蛋白表达量在各时间点无变化；TBI组与Sham组比较,伤后1h p-P53蛋白量显著增高(P<0.05),伤后24h达高峰,高表达状态持续至伤后48h(P<0.05),伤后48h开始下降(P<0.05)；TBI组与TBI+DMSO组比较有差异无统计学意义(P>0.05); TBI+SP600125组p-p53和DRAM蛋白表达时程与TBI组相似,但表达量减少,与TBI组比较,以伤后12h、24h、48h和72h差异有统计学意义(P<0.05)。
4Beclin-1的Western blot检测结果Sham组beclin-1蛋白表达量在各时间点无变化；TBI组与Sham组比较,伤后1h beclin-1蛋白量显著增高(P<0.05),伤后48h达高峰,高表达状态持续至伤后48h(P<0.05),伤后72h开始下降(P<0.05); TBI组与TBI+DMSO组比较有差异无统计学意义(P>0.05); TBI+SP600125组beclin-1蛋白表达时程与TBI组相似,但表达量减少,与TBI组比较,以伤后6h、12h、24h、48h和72h差异有统计学意义(P<0.05)。
5DRAM和beclin-1免疫组化检测结果光镜下DRAM和beclin-1标记为棕黄色颗粒,定位于胞浆。Sham组偶见阳性细胞,着色浅淡,免疫反应的数量和强度不随时间点发生变化。TBI组与Sham组比较,伤后6h DRAM和beclin-1阳性细胞显著增多,着色加深,免疫反应性增强(P<0.05),伤后分别于24h和48h达高峰(P<0.05); TBI组与TBI+DMSO组比较有差异无统计学意义(P>0.05); TBI+SP600125组DRAM和beclin-1免疫阳性反应性时间点与TBI组相似,但是免疫反应性减弱,以12h、24h、48h和72h,差异有统计学意义(P<0.05)。
6p-P53与DRAM和beclin-1的激光共聚焦检测结果
p-P53阳性细胞胞浆为FITC标记的绿色荧光,DRAM和beclin-1阳性细胞胞浆为TRITC标记的红色荧光。TBI组可见p-p53与DRAM和beclin-1在神经元细胞胞浆中可见大量绿色和红色荧光聚集,此时细胞核清晰可见；TBI+SP600125组中p-p53与DRAM和beclin-1在神经元细胞胞浆中阳性细胞数量明显减少,阳性强度减弱,此时细胞核仁清晰度降低。
7神经功能评分结果
Sham组评分为24分；TBI组评分7-9分；TBI+SP600125组评分15-18分。与sham组比较,在TBI组和TBI+SP600125组中,大鼠在后肢抓持反射、触须诱发前肢定位及侧向垫步试验中均表现异常,评分下降,差异有统计学意义(P＜0.05); TBI+SP600125组与TBI组比较,评分明显提高(P<0.05)。
8Morris水迷宫结果
TBI组和TBI+DMSO组伤后第9天及第10天搜索安全岛潜伏期较sham组明显延长,存在差异有统计学意义(P<0.05)。TBI+SP600125组伤后第9天及第10天搜索安全岛潜伏期较TBI组明显缩短存在差异有统计学意义(P<0.05)。
小结：大鼠弥漫性脑创伤后JNK通路激活引起p53磷酸化,自噬基因DRAM和beclin-1的表达升高；p-p53对DRAM有显著的调控作用；脑创伤前给予JNK抑制剂SP600125可以降低继发性神经损伤,具有神经保护的作用,改善了脑创伤后大鼠学习和记忆功能。
第三部分大鼠弥漫性脑创伤后p53的表达对beclin-1和bcl-2的调控机制研究
目的：本实验应用p53抑制剂PFT-α通过应用激光共聚焦、免疫印迹和免疫共沉淀的检测方法来检测p-p53、 p-bcl-2、 bcl-2、bcl-xl和beclin-1的表达来验证是否脑创伤后p53变化调控复合物解离。
方法：将240只成年雄性SD大鼠随机分成4组：①假手术组(n=60),②脑创伤组(n=60)③DMSO组(n=60)④抑制剂组(n=60)。每组又分别划分为伤后6h、12h、24h、48h等4个时间点。水迷宫测试同上部分。检测①激光共聚焦方法检测beclin-1与bcl-2和p53的蛋白表达定位情况③IP和Western-blot法检测beclin-1、 bcl-2、 bcl-xl、 p-bcl-2和p-p53的蛋白表达量。分析和统计同前。
结果：
1p-bcl-2和p-p53的Western blot检测结果TBI组和TBI+DMSO组各个时间点与Sham组比较,伤后6h p-bcl-2和p-p53蛋白量显著增高(P<0.05),伤后24h达高峰,伤后48h表达量下降(P<0.05); TBI+PFT-α组与Sham组比较p-bcl-2和p-p53蛋白表达量逐渐减弱,以伤后6h、12h和24h差异有统计学意义(P<0.05)。
2IP及Western blot法检测TBI组和TBI+DMSO组的beclin-1、 bcl-xl和bcl-2的结果Beclin-1的表达在TBI组和TBI+DMSO组各个时间点与Sham组比较,伤后由12h开始beclin-1蛋白量显著降低(P<0.05); Bcl-xl和bcl-2的表达在TBI组和TBI+DMSO组各个时间点与Sham组比较,伤
后由6h开始bcl-xl和bcl-2蛋白量显著降低(P<0.05)；同时实验发现伤
后12h、24h和48h bcl-2蛋白量变化较bcl-xl蛋白量变化幅度更大,有统
计学差异(P<0.05)。3IP及Western blot法检测TBI+PFT-a组的beclin-1.bcl-xl和bcl-2
的结果Beclin-1的表达在TBI+PFT-α组各个时间点与Sham组比较,伤
后由48h开始beclin-1蛋白量降低有统计学意义(P<0.05)；Bcl-xl和bcl-2
的表达在TBI+PFT-a组各个时间点与Sham组比较,伤后由48h开始bcl-xl
和bcl-2蛋白量降低统计学意义(P<0.05)；同时实验发现伤后48h bcl-2
蛋白量变化较bcl-xl蛋白量变化幅度更大,有统计学差异(P<0.05)。4Beclin-1与bcl-2和p-p53的激光共聚焦检测结果Bcl-2和p-p53阳
性细胞胞浆为FITC标记的绿色荧光,beclin-1阳性细胞胞浆为TRITC
标记的红色荧光。TBI组可见beclin-1与bcl-2和p-p53在神经元胞浆中可
见大量绿色和红色荧光聚集,此时细胞核清晰可见；TBI+PFT-α组中
beclin-1与bcl-2和p-p53在神经元胞浆中阳性细胞数量明显减少,阳性强
度减弱,此时细胞核仁清晰度降低。5神经功能评分结果Sham组评分为24分；TBI组评分在7-10分之间；TBI+PFT-α组评
分16-20分之间。与TBI组比较在TBI+PFT-a组中,大鼠在后肢抓持反
射、触须诱发前肢定位及侧向垫步试验中表现均明显改善,评分明显提高,
差异有统计学意义(P<0.05)6Morris水迷宫结果TBI组和TBI+DMSO组伤后第9天及第10天搜索安全岛潜伏期较
sham组明显延长,存在差异有统计学意义(P<0.05)。TBI+PFT-α组伤后第
9天及第10天搜索安全岛潜伏期较TBI组明显缩短存在差异有统计学意
义(P<0.05).小结：大鼠弥漫性脑创伤后beclin-1-bcl-2/bcl-xl复合物解离,导致神
经元自噬水平升高；p53调控beclin-1-bcl-2/bcl-xl复合物解离,间接影响
脑创伤后神经元自噬；脑创伤前给予p53抑制剂PFT-a可以降低继发性神
经损伤,具有神经保护的作用,改善了脑创伤后大鼠学习和记忆功能。结论：
在大鼠弥漫性脑创伤中,JNK通路可以通过介导p53的磷酸化调控自噬因子DRAM、 beclin-1的表达,达到调控神经元自噬的目的,同时阻断JNK的传导通路可以有效的抑制脑创伤后神经元的自噬,具有神经保护作用,改善了脑创伤后大鼠学习和记忆功能。
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 TBI
Objective: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 TBI
Objective: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.
细胞内外的很多刺激可以诱导自噬的激活,其是一系列细胞分子事件的级联反应的结果。近来研究发现MAPK家族的JNK基因在神经元自噬发生后表达升高,同时其下游因子p53同时表达也升高,这是否与外伤导致神经元自噬的激活有关,目前不得而知。文献报道在研究肺癌肿瘤细胞的自噬之中发现,自噬基因DRAM是p53的靶基因,对于癌细胞的自噬存在调节作用,同时p53对于自噬标志基因beclin-1与bcl-2/bcl-xl复合体的解聚存在调节作用。说明肿瘤抑制因子p53在肺癌中可以调控自噬。我们前期的实验提示在TBI后存在自噬的激活,自噬标志因子LC3、 beclin-1表达升高。那么JNK通路是否能通过对p53的调节来激活神经元自噬,在TBI中是这条通路是否成立这也是本研究的目的,本论文分三部分来阐述。
第一部分大鼠弥漫性脑创伤后自噬相关蛋白的表达
目的：本部分研究应用雄性SD大鼠复制Marmarou闭合性弥漫性颅脑创伤模型,检测大鼠弥漫性脑创伤后是否存在神经元自噬。
方法：采用本实验室改良Marmarou方法。大鼠随机分成：①假手术组(n=48),②脑创伤组(n=48),取伤后1h、6h,12h、24h、48h、72h6个时间点。检测①HE染色；②免疫组学法检测beclin-1的蛋白表达；③Western-blot法检测LC3II/LC3I的比值和beclin-1的蛋白表。应用Image Proplus6.0和Gel-Doc分析系统定量分析测定。实验中所得数据均用x±s表示,用SPSS11.5统计软件进行重复方差分析,以单侧P<0.05,表示差异有统计学意义。
结果：
1光镜下观察大鼠脑创伤模型切片
1.1伤后大鼠脑组织大体观察伤后可见蛛网膜下腔出血,伴随少量脑室出血；点状出血于脑实质多见,多低位脑干的背侧,脑表面的血管充血明显,未见局部脑挫伤。
1.2HE染色观察组织形态学的改变Sham组脑组织结构正常,血管管腔正常,管壁光滑,神经细胞数量多,形态完整；TBI组神经元胞体皱缩呈三角形,胞浆嗜色性染色减弱,核仁皱缩浓染,细胞周围出现间隙,出现神经元细胞变性坏死改变。
2海马和皮层部位beclin-1免疫组化检测结果beclin-1定位于神经元细胞的胞质,Sham组偶见阳性细胞,着色浅淡,免疫反应性不随时间发生变化。TBI组与sham组比较,伤后6h beclin-1阳性细胞显著增多,着色加深,免疫反应性增强(P<0.05),伤后48h达高峰(P<0.05),72h开始下降(P<0.05),TBI组24h和72h的结果与48h结果比较差异有统计学意义(P<0.05)。
3海马和皮层部位LC3的Western blot检测结果Sham组LC3Ⅱ/LC3Ⅰ的比值和LC3Ⅰ与LC3Ⅱ的蛋白表达量在各时间点无变化；TBI组LC3Ⅱ和LC3II/LC3I的比值随着时间点依次升高,TBI组与Sham组比较,伤后6h开始LC3II/LC3I的比值显著升高(P<0.05),伤后表达持续升高,高表达状态持续至伤后72h(P<0.05)。
4海马和皮层部位beclin-1的Western blot检测结果Sham组beclin-1蛋白表达量在各时间点无变化；TBI组与Sham组比较,伤后6h beclin-1蛋白量显著增高(P<0.05),伤后48h达高峰,高表达状态持续至伤后72h(P<0.05),伤后72h开始下降(P<0.05)；TBI组24h和72h的结果与48h结果比较差异有统计学意义(P<0.05)。
5LC3和PI的共聚焦检测结果
在TBI组中神经元细胞胞浆中可见大量绿色和红色荧光聚集,此时细胞核和胞浆清晰可见；TBI组伤后12h中LC3在神经元细胞胞浆中阳性细胞数量明显增多,阳性强度增强,此时细胞核仁清晰。
小结：改良的Marmarou弥漫性脑创伤模型满足了本实验对神经元自噬的研究；大鼠脑创伤后,beclin-1和LC3持续升高证明存在神经元自噬。
第二部分JNK介导p53通路在TBI中对大鼠海马神经元自噬的影响
目的：本部分实验通过应用JNK抑制剂SP600125,检测INK、p-p53、 DRAM和beclin-1的表达来证明JNK介导p53通路在TBI中调控大鼠海马神经元自噬是否成立。
方法：大鼠模型同上,将296只成年雄性SD大鼠随机分成4组：①假手术组(n=74),②脑创伤组(n=74)③DMSO组(n=74)④抑制剂组(n=74)。术后时间点同上。取80只大鼠行水迷宫测试,每组划分为伤后7d、8d、9d及10d四个时间点。检测①HE染色；②免疫组化法和共聚焦法检测p53、 beclin-1和DRAM的蛋白表达定位情况；③Western-blot法检测JNK. p53. beclin-1和DRAM的蛋白表达量。定量分析和统计同上
结果：
1HE染色组织形态学改变光镜下伤后24h可见Sham组和TBI组脑组织结构同前TBI+SP600125组病理形态改变较TBI组有不同程度减轻,可见神经元数量多,排列整齐,形态完整,核质均匀,核仁清晰；TBI+DMSO组与TBI组比较变化不大。
2JNK的Western blot检测结果Sham组JNK蛋白表达量在各时间点无变化；TBI组与Sham组比较,伤后1h JNK蛋白量显著增高(P<0.05),伤后12h达高峰,高表达状态持续至伤后24h(P<0.05),伤后24h开始下降(P<0.05); TBI组与TBI+DMSO组比较有差异无统计学意义(P>0.05);TBI+SP600125组JNK蛋白表达时程与TBI组相似,但表达量减少,与TBI组比较,以伤后6h、12h和24h差异有统计学意义(P<0.05)。
3p-P53和DRAM的Western blot检测结果Sham组p-p53和DRAM蛋白表达量在各时间点无变化；TBI组与Sham组比较,伤后1h p-P53蛋白量显著增高(P<0.05),伤后24h达高峰,高表达状态持续至伤后48h(P<0.05),伤后48h开始下降(P<0.05)；TBI组与TBI+DMSO组比较有差异无统计学意义(P>0.05); TBI+SP600125组p-p53和DRAM蛋白表达时程与TBI组相似,但表达量减少,与TBI组比较,以伤后12h、24h、48h和72h差异有统计学意义(P<0.05)。
4Beclin-1的Western blot检测结果Sham组beclin-1蛋白表达量在各时间点无变化；TBI组与Sham组比较,伤后1h beclin-1蛋白量显著增高(P<0.05),伤后48h达高峰,高表达状态持续至伤后48h(P<0.05),伤后72h开始下降(P<0.05); TBI组与TBI+DMSO组比较有差异无统计学意义(P>0.05); TBI+SP600125组beclin-1蛋白表达时程与TBI组相似,但表达量减少,与TBI组比较,以伤后6h、12h、24h、48h和72h差异有统计学意义(P<0.05)。
5DRAM和beclin-1免疫组化检测结果光镜下DRAM和beclin-1标记为棕黄色颗粒,定位于胞浆。Sham组偶见阳性细胞,着色浅淡,免疫反应的数量和强度不随时间点发生变化。TBI组与Sham组比较,伤后6h DRAM和beclin-1阳性细胞显著增多,着色加深,免疫反应性增强(P<0.05),伤后分别于24h和48h达高峰(P<0.05); TBI组与TBI+DMSO组比较有差异无统计学意义(P>0.05); TBI+SP600125组DRAM和beclin-1免疫阳性反应性时间点与TBI组相似,但是免疫反应性减弱,以12h、24h、48h和72h,差异有统计学意义(P<0.05)。
6p-P53与DRAM和beclin-1的激光共聚焦检测结果
p-P53阳性细胞胞浆为FITC标记的绿色荧光,DRAM和beclin-1阳性细胞胞浆为TRITC标记的红色荧光。TBI组可见p-p53与DRAM和beclin-1在神经元细胞胞浆中可见大量绿色和红色荧光聚集,此时细胞核清晰可见；TBI+SP600125组中p-p53与DRAM和beclin-1在神经元细胞胞浆中阳性细胞数量明显减少,阳性强度减弱,此时细胞核仁清晰度降低。
7神经功能评分结果
Sham组评分为24分；TBI组评分7-9分；TBI+SP600125组评分15-18分。与sham组比较,在TBI组和TBI+SP600125组中,大鼠在后肢抓持反射、触须诱发前肢定位及侧向垫步试验中均表现异常,评分下降,差异有统计学意义(P＜0.05); TBI+SP600125组与TBI组比较,评分明显提高(P<0.05)。
8Morris水迷宫结果
TBI组和TBI+DMSO组伤后第9天及第10天搜索安全岛潜伏期较sham组明显延长,存在差异有统计学意义(P<0.05)。TBI+SP600125组伤后第9天及第10天搜索安全岛潜伏期较TBI组明显缩短存在差异有统计学意义(P<0.05)。
小结：大鼠弥漫性脑创伤后JNK通路激活引起p53磷酸化,自噬基因DRAM和beclin-1的表达升高；p-p53对DRAM有显著的调控作用；脑创伤前给予JNK抑制剂SP600125可以降低继发性神经损伤,具有神经保护的作用,改善了脑创伤后大鼠学习和记忆功能。
第三部分大鼠弥漫性脑创伤后p53的表达对beclin-1和bcl-2的调控机制研究
目的：本实验应用p53抑制剂PFT-α通过应用激光共聚焦、免疫印迹和免疫共沉淀的检测方法来检测p-p53、 p-bcl-2、 bcl-2、bcl-xl和beclin-1的表达来验证是否脑创伤后p53变化调控复合物解离。
方法：将240只成年雄性SD大鼠随机分成4组：①假手术组(n=60),②脑创伤组(n=60)③DMSO组(n=60)④抑制剂组(n=60)。每组又分别划分为伤后6h、12h、24h、48h等4个时间点。水迷宫测试同上部分。检测①激光共聚焦方法检测beclin-1与bcl-2和p53的蛋白表达定位情况③IP和Western-blot法检测beclin-1、 bcl-2、 bcl-xl、 p-bcl-2和p-p53的蛋白表达量。分析和统计同前。
结果：
1p-bcl-2和p-p53的Western blot检测结果TBI组和TBI+DMSO组各个时间点与Sham组比较,伤后6h p-bcl-2和p-p53蛋白量显著增高(P<0.05),伤后24h达高峰,伤后48h表达量下降(P<0.05); TBI+PFT-α组与Sham组比较p-bcl-2和p-p53蛋白表达量逐渐减弱,以伤后6h、12h和24h差异有统计学意义(P<0.05)。
2IP及Western blot法检测TBI组和TBI+DMSO组的beclin-1、 bcl-xl和bcl-2的结果Beclin-1的表达在TBI组和TBI+DMSO组各个时间点与Sham组比较,伤后由12h开始beclin-1蛋白量显著降低(P<0.05); Bcl-xl和bcl-2的表达在TBI组和TBI+DMSO组各个时间点与Sham组比较,伤
后由6h开始bcl-xl和bcl-2蛋白量显著降低(P<0.05)；同时实验发现伤
后12h、24h和48h bcl-2蛋白量变化较bcl-xl蛋白量变化幅度更大,有统
计学差异(P<0.05)。3IP及Western blot法检测TBI+PFT-a组的beclin-1.bcl-xl和bcl-2
的结果Beclin-1的表达在TBI+PFT-α组各个时间点与Sham组比较,伤
后由48h开始beclin-1蛋白量降低有统计学意义(P<0.05)；Bcl-xl和bcl-2
的表达在TBI+PFT-a组各个时间点与Sham组比较,伤后由48h开始bcl-xl
和bcl-2蛋白量降低统计学意义(P<0.05)；同时实验发现伤后48h bcl-2
蛋白量变化较bcl-xl蛋白量变化幅度更大,有统计学差异(P<0.05)。4Beclin-1与bcl-2和p-p53的激光共聚焦检测结果Bcl-2和p-p53阳
性细胞胞浆为FITC标记的绿色荧光,beclin-1阳性细胞胞浆为TRITC
标记的红色荧光。TBI组可见beclin-1与bcl-2和p-p53在神经元胞浆中可
见大量绿色和红色荧光聚集,此时细胞核清晰可见；TBI+PFT-α组中
beclin-1与bcl-2和p-p53在神经元胞浆中阳性细胞数量明显减少,阳性强
度减弱,此时细胞核仁清晰度降低。5神经功能评分结果Sham组评分为24分；TBI组评分在7-10分之间；TBI+PFT-α组评
分16-20分之间。与TBI组比较在TBI+PFT-a组中,大鼠在后肢抓持反
射、触须诱发前肢定位及侧向垫步试验中表现均明显改善,评分明显提高,
差异有统计学意义(P<0.05)6Morris水迷宫结果TBI组和TBI+DMSO组伤后第9天及第10天搜索安全岛潜伏期较
sham组明显延长,存在差异有统计学意义(P<0.05)。TBI+PFT-α组伤后第
9天及第10天搜索安全岛潜伏期较TBI组明显缩短存在差异有统计学意
义(P<0.05).小结：大鼠弥漫性脑创伤后beclin-1-bcl-2/bcl-xl复合物解离,导致神
经元自噬水平升高；p53调控beclin-1-bcl-2/bcl-xl复合物解离,间接影响
脑创伤后神经元自噬；脑创伤前给予p53抑制剂PFT-a可以降低继发性神
经损伤,具有神经保护的作用,改善了脑创伤后大鼠学习和记忆功能。结论：
在大鼠弥漫性脑创伤中,JNK通路可以通过介导p53的磷酸化调控自噬因子DRAM、 beclin-1的表达,达到调控神经元自噬的目的,同时阻断JNK的传导通路可以有效的抑制脑创伤后神经元的自噬,具有神经保护作用,改善了脑创伤后大鼠学习和记忆功能。
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 TBI
Objective: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 TBI
Objective: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.
引文
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