SIRT1在创伤性脑损伤后的功能及机制研究

发布时间：2018-07-16 07:44

【摘要】：研究背景创伤性脑损伤（traumatic brain injury, TBI）是一种常见的中枢神经系统损伤性疾病，具有极高的致死率及致残率，已经引起国际卫生组织的广泛关注。研究发现，引起创伤性脑损伤的主要机制包括谷氨酸兴奋性毒性、DNA损伤、氧化应激的产生以及促凋亡因子的调节等等。目前临床上对于TBI治疗有效的神经保护性药物很少，主要原因是对TBI后分子代谢及调控的机制还不十分透彻，常常影响伤者的治疗及预后。因此，积极探索可以激活内源性的存活信号及修复机制的关键分子，成为TBI治疗的重要策略。研究发现，NAD（nicotinamide adenine dinucleotide）-依赖的去乙酰化酶Sir2（silence informationregulator two）与酵母的长寿有关。SIRT1是哺乳动物中和酵母Sir2最接近的亚型，其在脑组织中的表达明显高于其他器官。通过药理学或基因调控的方法调节SIRT1的活性及表达在许多神经代谢性疾病中发挥神经保护作用。然而，一些研究发现使用SIRT1的抑制剂也具有神经保护的作用，提示SIRT1可能在一些模型中发挥有害的作用。这一看似矛盾的结论使SIRT1在神经元损伤中的作用更加不明朗。因此，阐明SIRT1在TBI中的生物学功能及相关的分子调控机制对于利用SIRT1作为神经保护治疗的靶点非常重要。实验目的（1）明确SIRT1在TBI后表达的变化规律及生物学功能；（2）探索SIRT1与MAPK/ERK通路之间的相互调节机制及在TBI中的作用；（3）探索SIRT1和homer1a之间在TBI后的相互调控及发挥保护作用的可能机制。实验方法（1）细胞培养：胎鼠皮层神经元原代培养及鉴定；（2）模型建立：建立离体机械性损伤模型和小鼠重物下落闭合性脑损伤模型模拟离体及在体条件下的创伤性脑损伤；（3）分子表达及定位：分别采用Westernblot、免疫荧光及免疫组化等方法检测各种分子的表达及空间定位；（4）神经元损伤评估：采用LDH试剂盒和PI/Hoechst活细胞染色检测神经元损伤程度；（5）脑损伤评估：利用神经功能学评分（）检测TBI后小鼠脑损伤的程度；（6）细胞凋亡：cleaved Caspase-3检测或TUNEL染色检测细胞凋亡；（7）SIRT1调控：利用SIRT1抑制剂salermide及siRNA的方法下调SIRT1在神经元的表达，采用SIRT1激动剂白藜芦醇上调SIRT1的活性或表达；（8）MAPK/ERK通路调控：使用ERK特异性阻断剂PD98059和U0126抑制MAPK/ERK通路的激活；（9）Homer1a调控：利用慢病毒包装homer1a过表达质粒转染神经元上调Homer1a在离体培养的神经元中的表达。实验结果实验一：离体神经元机械性损伤模型可显著增加TBI后神经元中LDH的释放；TUNEL染色显示：TBI后24h神经元中的凋亡细胞数明显增加；Western blot检测活化的caspase-3的表达在损伤后早期即出现明显升高，并在损伤后期逐渐回落至正常水平。在小鼠重物下落闭合性脑损伤模型中：TBI后小鼠的血糖水平从损伤后3h开始显著升高，在损伤后5天恢复正常；TUNEL染色显示TBI后12h损伤侧皮层中TUNEL染色阳性的神经元数量明显增加；活化的caspase-3在损伤后3h表达增加，并在3d后恢复正常水平；免疫荧光染色显示TBI后损伤侧皮层中活化的caspase-3表达较对照组显著升高，进一步证实了TBI后损伤侧皮层中凋亡神经元的数量增加。此外，离体和在体的研究均显示：TBI后神经元中SIRT1的表达显著增加，之后逐渐恢复到正常水平。免疫组化染色显示TBI后SIRT1表达较对照组显著增加，主要定位在损伤侧皮层和海马部位神经元的胞浆和轴突中。实验二：创伤性脑损伤可引起离体神经元和动物脑组织中MAPK/ERK通路的激活。在离体机械性损伤模型中：加入不同浓度的SIRT1抑制剂salermide后可显著降低神经元的存活率，同时神经元中LDH的释放显著增加且呈浓度依赖性；Western blot检测发现加入salermide抑制了神经元中SIRT1的表达后，ERK通路的激活受到显著抑制，同时活化的caspase-3的表达显著升高；采用siRNA的方法干涉离体神经元中SIRT1的表达后，ERK通路的激活受到抑制，同时活化的caspase-3显著增加,这一结果和神经元加入抑制剂后的结果一致。采用立体定向的方法向小鼠侧脑室注射不同浓度的salermide后建立在体TBI模型，发现TBI后小鼠损伤侧皮层神经元中SIRT1的表达抑制后，ERK通路的激活也受到明显抑制，而小鼠的NSS评分显著增加。为了观察TBI后MAPK/ERK通路的激活是否调节SIRT1的表达，我们采用ERK通路的抑制剂PD98059预处理离体培养的神经元中后行机械性损伤，当神经元中MAPK/ERK通路的激活受到显著抑制后，SIRT1的表达和对照组相比也被明显抑制，同时活化的caspase-3的表达也受到抑制。TUNEL染色显示在加入PD98059的神经元中，TBI后TUNEL染色阳性的细胞数较加入DMSO组显著降低。在体的实验同样证实：抑制TBI后ERK通路的激活可以显著降低损伤侧皮层SIRT1的表达，同时小鼠的NSS评分也显著降低。此外，关于TBI后高糖血症的研究显示：TBI后，小鼠尾静脉血中血糖水平明显升高，5d后恢复至正常水平。采用侧脑室注射的方式抑制小鼠脑组织中ERK通路的激活可以显著降低TBI后的血糖水平的升高，，而侧脑室注射SIRT1抑制剂salermide后小鼠尾静脉血中的血糖水平较对照组明显升高。第三部分：离体和在体的研究均证实：TBI后神经元中即早基因homer1a的表达明显增高，在达到高峰后逐渐恢复至正常水平，而homer1b/c的表达比较稳定。离体和在体的免疫荧光染色显示TBI后homer1a和p-ERK主要表达在神经元中，且共定位于神经元的胞浆和轴突。采用siRNA抑制SIRT1表达后，homer1a的表达明显受到抑制，同时β-catenin的表达也受到抑制；而使用SIRT1的激动剂白藜芦醇预处理神经元后，SIRT1表达增加的同时，homer1a及β-catenin的表达也显著增加。此外，离体和在体的研究证实，抑制MAPK/ERK通路的激活可以显著降低TBI后Homer1a表达的增加。同时我们使用慢病毒包裹的homer1a过表达质粒转染神经元发现，机械性损伤后离体培养的神经元中TUNEL染色阳性的细胞数明显减少，提示homer1a过表达具有神经保护的作用。Western blot检测发现，转染过表达质粒组的神经元中homer1a和β-catenin的表达增加，而SIRT1和p-ERK的表达受到明显抑制。提示外源性的homer1a可能对TBI后神经元中SIRT1的表达存在负反馈的作用。实验结论本课题的研究结果证实，TBI后SIRT1和homer1a作为内源性的神经保护因子，其表达升高发挥神经保护的作用；SIRT1可能通过与MAPK/ERK通路的相互作用来调节homer1a的表达，SIRT1与Homer1a相互作用调节β-catenin进一步保护TBI引起的神经元凋亡。该研究结果不但阐明了神经元存在的一种自我保护机制，同时为治疗创伤性脑损伤提供了潜在的药物干预靶点和新的治疗策略。
[Abstract]:Background of the study

Traumatic brain injury is a kind of common central nervous system injury disease . It has very high fatality rate and disability rate . It has attracted extensive attention from the international health organization . It has been found that the main mechanisms of traumatic brain injury include glutamate excitotoxicity , DNA damage , oxidative stress generation and regulation of pro - apoptotic factors .

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experimental purpose

( 1 ) To clarify the regularity of expression and biological function of SIRT1.In this study .
( 2 ) To explore the mechanism of mutual regulation between SIRT1 and MAPK / ERK pathway and its role in the treatment of the disease .
( 3 ) To explore the possible mechanism of mutual regulation and protective effect between SIRT1 and homeria in the aftermath .

experimental method

( 1 ) Cell culture : primary culture and identification of fetal rat cortical neurons ;
( 2 ) establishing the model of the isolated mechanical injury model and the mouse weight falling closed brain injury model to simulate the traumatic brain injury under the body condition ;
( 3 ) Molecular expression and localization : Western blot , immunofluorescence and immunohistochemistry were used to detect the expression and spatial location of various molecules .
( 4 ) Assessment of neuronal damage : The damage degree of neurons was detected by using LDH kit and PI / hoechst living cell staining ;
( 5 ) Assessment of brain injury : the degree of brain injury in mice after treatment with neurological score ( ) ;
( 6 ) Apoptosis was detected by Caspase - 3 or TUNEL staining .
( 7 ) SIRT1 : The SIRT1 inhibitor salermide and siRNA were used to downregulate the expression of SIRT1 in neurons .
( 8 ) MAPK / ERK pathway regulation : ERK specific blocker PD98059 and U0126 inhibit MAPK / ERK pathway activation ;
( 9 ) Homer1a Regulation : The expression of Homer1a in ex vivo cultured neurons was regulated by using slow virus packaged homer1a overexpression plasmid .

experimental results

The results showed that the mechanical injury model of ex vivo neurons could significantly increase the release of LDH in the neurons .
TUNEL staining showed that the number of apoptotic cells increased significantly in 24 h post - treatment neurons .
Western blot showed that the expression of caspase - 3 increased significantly in the early stage after injury , and gradually declined to normal level in the late injury period . In the model of mouse weight drop closed brain injury , the blood glucose level of mice after the injury increased significantly from 3 hours after injury , and returned to normal after 5 days after injury .
TUNEL staining showed that the number of TUNEL - positive neurons in the injured side cortex increased significantly at 12 h post - treatment .
The expression of caspase - 3 increased 3 hours after injury , and returned to normal after 3d .
Immunofluorescence staining showed that the expression of activated caspase - 3 in the injured side cortex increased significantly in the injured side cortex , and further confirmed that the number of apoptotic neurons in the injured side cortex increased significantly in the injured side cortex . In addition , the ex vivo and in vivo studies showed that the expression of SIRT1 increased significantly in the posterior neurons , and then gradually recovered to the normal level . Immunohistochemical staining showed that the expression of SIRT1 increased significantly in the injured side cortex and the axons of the neurons in the hippocampus site .

Experiment 2 : Traumatic brain injury can cause activation of MAPK / ERK pathway in isolated neurons and animal brain tissues .
Western blot showed that salermide inhibited the expression of SIRT1 in neurons , ERK pathway activation was significantly inhibited , and the expression of caspase - 3 was significantly increased .
In order to observe whether the activation of MAPK / ERK pathway in mice was inhibited significantly , the activation of ERK pathway was significantly inhibited after the activation of MAPK / ERK pathway in mice .

In the third part , the expression of homer1a and p - ERK in the neurons were significantly increased after reaching the peak , but homer 1b / c was stable . The expression of homer1a and p - ERK was inhibited in the neurons and the expression of 尾 - catenin was inhibited .
At the same time , the expression of homer1a and 尾 - catenin was significantly decreased after the pretreatment of the neuron with the agonist resveratrol of SIRT1.In addition , the expression of homer1a and 尾 - catenin was significantly decreased after the activation of the MAPK / ERK pathway . The expression of homer1a and 尾 - catenin was significantly inhibited in the neurons transfected with the slow virus .

experimental conclusion

The results of this study confirm that SIRT1 and homer1a , as endogenous neuroprotection factors , play a role in neuroprotection .
SIRT1 may regulate the expression of homer1a by interacting with MAPK / ERK pathway . The interaction between SIRT1 and Homer1a regulates 尾 - catenin to further protect neuron apoptosis . The results not only illustrate a self - protection mechanism in neurons , but also provide potential drug intervention targets and new treatment strategies for the treatment of traumatic brain injury .
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R651.15

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