Acute Severe Hemorrhagic Shock In Rats Grp58 Expression Changes And Its Influencing Factors

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.