急性重度失血性休克大鼠GRP58表达变化及其影响因素研究

【作者】 李晓峰；

【导师】 周虹；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 失血性休克（hemorrhagic shock,HS）是最为常见的休克类型之一,是因急性血液或血浆的大量丢失导致循环血量骤减引起,常继发于创伤或其他疾病,属低血容量性休克（hypovolemic shock）的一种。失血性休克是战伤死亡的首要因素之一,也是和平时期健康人群致死的主要因素。失血性休克是一个危急的进行性的病理过程,救治时机至关重要。失血性休克的有效救治时间为伤后1小时,称为“黄金时间”,很多伤者因为超过了救治的“黄金时间”而死亡。这主要是因为在急性失血性休克条件下会造成机体应激反应过度,从而引发一系列的“炎性连锁反应”,机体会产生大量的炎性细胞因子,进而产生自由基等物质,全身血液灌流障碍,血流减缓又会造成微循环障碍。由于细胞损伤的不断加重,出现多器官功能障碍,造成休克的恶性循环,此时全身血管反应性降低,使休克向难治性阶段不可逆发展,亦即转变成了使用常规治疗失血性休克的方法和手段都无效的难治性休克。可见,目前在急性失血性休克中因单纯低血容量问题导致的死亡已不常见,炎性损伤、自由基损伤及其导致的多器官功能障碍成为重度创伤性休克的常见并发症和主要死亡原因。因此在急性失血的早期寻找到分子靶标,给予有效的抗炎、抗自由基损伤的药物,阻断炎性瀑布的启动,打断休克的恶性循环,防止休克后器官功能障碍成为创伤休克研究的当务之急。本课题组主要从事失血性休克相关机理和防治药物的研究,通过系统生物学和生物信息学的方法,从蛋白质组学、基因组学、代谢组学等多个方面对失血性休克损伤机制展开了研究。在前期的蛋白质组学研究中发现:急性重度失血性休克（acute severehemorrhagic shock,ASHS）大鼠去除高丰度蛋白的血浆和肝脏组织总蛋白质中有7个相同的表达差异的蛋白质斑点,其中葡萄糖调节蛋白58（Glucose-regulated protein 58,GRP58）是肽段匹配分值（224）和氨基酸序列覆盖率最高（41%）的蛋白,并且通过Western-blot发现:在正常条件下,肝脏组织总蛋白中存在三个分子量相同、等电点不同的GRP58蛋白斑点;而在ASHS组中,这三个蛋白斑点中较低等电点的GRP58蛋白表达显著下调。葡萄糖调节蛋白58（glucose regulated protein58,GRP58）,又名ERp61、ER-60、ERp57、PDI-Q2和HIP70,与蛋白二硫化物异构酶（Protein disulfide isomerase,PDI）家族具有高度同源性,被认为是PDI家族成员之一。它具有巯基依赖性氧化还原酶活性,可以作为一种分子伴侣在应激蛋白的折叠和转运、细胞内的信号转导、肿瘤的治疗等方面具有重要的作用;它也是一种非常重要的应激蛋白,在多种应激损伤下大量表达以维持内质网的稳定,保护细胞。研究表明,GRP58在某些肿瘤细胞中呈高表达,对于肿瘤细胞的抗化疗药物的性质及抗原表达有重要意义。我们前期结果显示GRP58作为一种重要应激蛋白,与文献中报道的在多种病理状态下表达上调相反,在大鼠ASHS状态下显著下调,因此本研究的目的是对前期结果进行进一步鉴定,分析ASHS状态下GRP58在细胞内分布是否发生改变,并利用细胞模型研究GRP58表达水平与细胞抗损伤能力的关系,初步探讨GRP58对ASHS的影响。ASHS大鼠肝脏GRP58的质谱分析及细胞内分布研究通过Western-blot、MALDI-TOF-MS、MS/MS实验对前期的结果作了进一步确证,并发现GRP58的150位丝氨酸残基发生了磷酸化修饰,且ASHS状态大鼠肝脏GRP58磷酸化程度增加,曾有研究报道GRP58磷酸化可激活JAKs-STAT3信号通路发挥抗损伤与凋亡作用,因而我们推测ASHS状态下,作为应激反应大鼠肝脏GRP58发生磷酸化,激活抗炎通路发挥抗休克损伤功能。此外,通过免疫荧光组化实验,我们发现在正常条件下肝脏组织中的GRP58广泛存在于细胞质、细胞膜、细胞核中,而在ASHS条件下,GRP58仅在细胞质中表达,因而,我们推测GRP58向胞浆迁移有利于其发挥抗失血性休克损伤的功能。但是,Western-blot、RT-PCR与免疫荧光组化结果表明ASHS大鼠肝脏GRP58转录及翻译水平均表现为下调,原因有待于进一步研究分析。有研究报道,低糖、低氧、低Ca²⁺等可诱导GRP58表达,而高糖则抑制其表达,我们课题组前期研究表明,ASHS大鼠急性期氧分压没有明显减低,而曾有人报道失血性休克会造成机体短时间内血糖升高,后期才表现为低糖低氧,我们认为本研究中GRP58下调可能是由于取材时ASHS大鼠处于正常氧分压和高糖状态,高糖抑制了GRP58的表达,而且这种抑制作用强于GRP58上调以抵抗休克损伤的应激反应。因此第二部分我们进行了失血性休克病程与血糖的相关性研究,以证实我们的推测。失血性休克大鼠血糖分析及GRP58表达对细胞抗损伤能力的影响通过检测正常对照组（sham hemorrhage shock,SHS）（n=10）和急性轻度失血性休克组（acutelight hemorrhagic shock,ALHS）、急性中度失血性休克（acute mild hemorrhagic shock,AMHS）、急性重度失血性休克（acute severe hemorrhagic shock,ASHS）（n=10）大鼠血糖变化与失血时间以及失血程度的关系,我们发现机体的失血程度与血糖关系密切,随失血性休克程度加重,血糖明显升高,30min达到峰值,随后开始下降,尤其ASHS大鼠血糖异常升高,30min约为正常血糖的4倍,至60min没有明显下降,即ASHS大鼠较长时间内处于高糖状态,这可能是GRP58表达下调的主要原因。另一方面,通过RT-PCR、Western-blot实验,我们发现BRL和QSG7701细胞GRP58合成能力有显著差异,BRL细胞可合成GRP58,而QSG7701细胞合成能力极低或缺失。对这两组细胞在高糖、低氧等条件下进行细胞活力、凋亡率及生化指标等检测发现,BRL细胞抗高糖、低氧损伤能力明显高于QSG7701细胞,Western-blot、免疫荧光组化及RT-PCR实验,发现BRL细胞中GRP58受高糖及低氧影响,且应激状态下会向胞浆移位,推测GRP58合成能力的差异有可能是导致BRL和QSG7701细胞抗高糖低氧损伤能力不同的原因之一,初步证明GRP58在ASHS状态下具有抗损伤功能。根据以上研究结果,我们推测大鼠发生急性重度失血性休克后,肝脏GRP58磷酸化水平增高且向胞浆迁移,发挥抗损伤功能,但休克造成的应激性高糖抑制了GRP58的表达,使其蛋白水平下降,结果导致机体的抗休克损伤能力下降,炎性反应加重,加速脏器衰竭,形成难治性休克,直至机体死亡。在初步证实GRP58在ASHS状态下具有抗损伤功能的同时也从另一侧面提示我们,ASHS造成的持续高糖是机体损伤的重要因素,如何在实施恢复血液灌流等复苏救治的同时尽快降低血糖,防止高糖损伤及高糖对GRP58等应激蛋白的抑制可能为改进失血性休克（尤其是ASHS）救治方案提供新的思路。更多还原

【Abstract】 Hemorrhagic shock(HS),as the most common type of shock,results from the sharp decrease of circulation volume caused by the acute and great missing of blood or plasma. HS belongs to one type of the hypovolemic shock,often secondary to trauma or other diseases.HS is one of the primary factors of the wounds and deaths in wartime,as well as the unexpected deaths of healthy crowd in peacetime.The pathologic process of HS is a critical and progressive,so the remedy time is very important.The effective remedy time of HS is 1 hour after the damage,called "the Golden Hour",and if the patients were given the treat beyond "the Golden Hour",they may die. The overresponses to the ASHS lead to "the Inflammation cascade reaction";then body will give rise to many Inflammation cytokine,and further free redical and so on.These will cause the disorder of total body hemoperfusion and slow-down of blood flow,which will again result in microcirculation disturbance.As the cellar damage is growing serious, multi-organs dysfunction happens,resulting in vicious cycle orris.The total body’s vascular reactivity descends,which drives the HS to refractory and inreversible phase—Refractory Shock(in which the common remedy do not work).It is clear that the fatal factors of HS is not only due to the merely hypovolemia,but also because of the inflammation and free redical damages as well as multi-organs dysfunctions.Therefore,the urgent priority of ASHS study is to prevent the happening of multi-organs dysfunctions, which calls for finding out the target at the early phase of ASHS,treating with effective anti-inflammation and anti-free redical drugs,preventing the start of inflammation cascade reaction and interrupting the vicious cycle of HS.In a previous study of analyzing differential expression proteins in the rat liver under acute severe hemorrhagic shock,seven protein spots with significant difference were found between sham hemorrhage shock group and acute severe hemorrhagic shock group,and up-regulated or down-regulated or appeared after the stimulation of refractory hemorrhage, The current study focussed on one of these newly recognized proteins,namely ERp57,in order to determine its function during acute severe hemorrhagic shock and its relevance in hemorrhage shock.GRP58/ERp57,also known as ERp61,ER-60 and HIP70,a member of the PDI family,is also an important component involved in the calnexin/calreticulin system. It contains two thioredoxin motifs and acts as a thiol oxidoreductase to catalyze the disulfide bond formations of the loaded glycoproteins;It acts also as molecular chaperones, and is,therefore,part of a quality-control system for the correct folding of the proteins in the same subcellular compartment,acts also as stress protein in the endoplasmic reticulum (ER).MS analysis and cellar distribution of Rats liver GRP58 under ASHS In the acute severe hemorrhagic shock,hepatic cells occupying in high glucose condition will lead its intracellular signal transduction,mass conveying...to change significantly,while the lost 40%of blood volume should not have evident impact in body weight of rat. Therefore,in this experiment we speculate that the reason that the protein expression of GRP58 in ASHS is less than its expression in SHS,should be directly related that high glucose inhibits the synthesis of GRP58,when organism occupies in the state of high glucose after the acute severe hemorrhagic shock.As an important molecular chaperone in endoplasmic reticulum,GRP58 is a critical regulatory substance,which can promote cell to be sophisticated and maintain cell function in the normal growth conditions.When the organism occupies in this stress state under the acute severe hemorrhagic conditions,a large number of needed stress-polypeptide will be synthetized in liver,which is extremely important in maintaining normal metabolism,GRP58 contains two thioredoxin motifs and acts as a thiol oxidoreductase to catalyze the disulfide bond combinations of the nascent polypeptide;GRP58 is involved in the proper folding and in the formation and reshuffling of the disulfide bridges of the proteins synthesized in the rough ER,imported in the lumen of this structure and destined to be secreted or incorporated in the cell membrane..In summary,as a result of high glucose inhibits the synthesis of GRP58,a larger number of nascent polypeptide can not import to the designated location via endoplasmic reticulum, so that the organism injuring is further increased;further aggravated injury in the body would lead the ability of synthesizing GRP58 to decreasing,so the protein expression of GRP58 is diminished.In acute severe hemorrhagic shock conditions,we have not only discovered the changes in the level of GRP58 protein,but also found that its gene exists at the transcriptional level changes.In addition,we found that GRP58 distributes in the cytoplasm,cell membrane and cell nucleus in SHS by Using immunofluorescence;but we found surprisely that GRP58 distributes in the cytoplasm,which expression is significantly reduced:Using MALDI-TOF-MS identification and Swiss-prot protein database analysis, we found that the finger printing amino acid sequence from 144 to 156 of GRP58 exists an obvious difference.The analysis of the relation between blood glucose and different degrees of SH & The investigate of GRP58 in cell level According to the results of rat blood glucose changing in SHS,ALHS,AMHS and ASHS(n=10) under different degrees and lasting time of SH,we can conclude that the blood glucose related SH closely,and the concentration of blood glucose will increase as SH is more serious,and in the compensation phase,the body is in high concentration of blood glucose.Two cell lines were chosen to service the next study:BRL cell(which can synthesize GRP58) and QSG7701 cell(which cannot synthesize GRP58).We carried out such experiments as MTT,cell-flow,the detection of main biochemical indicator,Western-blot,immunofluorescence and RT-PCR,and gained the same results as in the animal level,further to identify that GRP58 worked in the cytoplasm.To sum up,this study found that the concentration of blood glucose will increase as SH is more serious,and the body is in high concentration of blood glucose in the compensation phase.Some of the liver proteins such as GRP58 express differently under ASHS,and Ser150 of GRP58 is phosphorylated,which associated with the JAKs -STAT3 signaling pathway(having something with the anti-inflammatory injury and cell apoptosis). Consequently,it indicated that GRP58 may be a new target of the diagnosis and treatment of SH.In this experiment,we speculate in acute severe hemorrhagic shock conditions,40 percent of body’s blood is lost in a short time,the rats occupies rapidly in acute decompensated,which will excite major organ in the body to occupy in high glucose conditions.The protein expression of GRP58 is inhibited by high glucose.A larger number of nascent polypeptide can not import to the designated location,so that the organism injuring is further increased;Further aggravated injury in the body would lead the ability of synthesizing GRP58 to decreasing,Down-regulated expression of GRP58 enables the body to death that is a result of infernal circle.In addition,we also consider that a large number of nascent stress polypeptide can be only combined with GRP58 dephosphorylated in the stress state.更多还原