| Hemorrhagic shock is one of the severe hypovolemia shocks, induced by extensive blood loss, or sudden and unexpected depletion of the circulating blood. Hemorrhagic shock is a major independent risk factor of the pathogenesis of trauma-related multiple organ failure and death. It is one of the most common causes of death all over the world today. There are various treatments on shock now. For example, the doctor can boost pressure, regulat microcirculation and re-perfuse the several important organs including heart, liver, spleen, lung, kidney and brain. They are not effective, however, hemorrhagic shock develops refractory hemorrhagic shock. The reason is that hemorrhagic shock is characterized by a loss in circulatory volume, which results in decreased venous return, decreased filling of the cardiac chambers, and hence a decreased cardiac output which leads to increase in the systemic vascular resistance. Hemorrhagic shock is one of four major categories of the syndrome of shock based on cardiovascular characteristics. It's pathomechanism is not clear, and need to be replenished in order to make perfect. Nowadays, the therapise of many kinds of diseases are being adjusted to research their molecule mechanisms. Proteomic approach is considered to be a powerful technology in the global analysis of protein expression and has been widely used in disease proteomics fields.Some of the latest studies propose that, vagus nerve and Ach interact with immune system to resist inflammatory responses. It is called the cholinergic anti-inflammatory pathway. Adrenocorticotrophin (ACTH) is one of the important activation agents in the pathway. Nowadays, there are not any reports, which investigate plasma and liver proteomic analysis in hemorrhagic shock, and which research proteomic analysis with ACTH in hemorrhagic shock.This study was to screen the differential expression proteins by proteome analysis technology in the rat plasma and liver under acute severe hemorrhagic shock (ASHS) and post-treatment with using ACTH. The differential expression proteins were cut from the gels, analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and identified through searching Swiss-prot database with Mascot software. These results provided scientific datum for screening the molecular biomarker used to diagnose and treat hemorrhagic shock, as well as seek out new target to elevate the patient's prognosis and provide new clue for the research on pathogenic mechanism of hemorrhagic shock. Establish rat model in ASHS and treatment method with using ACTH ASHS (?)t model: After lying quietly for 10 minutes, ASHS rats were induced by being withdrawn blood through the femoral arterial catheter in 15 minutes. The mean arterial blood pressure (MABP) decreased to 35-40 mmHg and stabilized MABP for 15 minutes. The MABP was maintained at this level for 1 hour. The total amount of blood withdrawn then was about 40-45% of rat blood volume. The animals were sacrificed at 80 minutes. The activity of SOD, MDA, AST, ALT, GST, LDH and TNF-αin plasma and HSP70 in liver were measured respectively by using the their test kits. And pathological changes of liver were observed by microscopy. These results suggested ASHS rat model had satisfactory stability. Then this model could be operated in ASHS research. At 50 minutes of ACTH model, we administered ACTH (3 ug·kg-1) intravenous injection, while we administered normal saline (equality volum of ACTH) in normal saline (NS) model. The results of survival time of SHS, ASHS, NS and ACTH suggested ACTH could remedy ASHS in some extent. Furthermore, the changes of the activity of SOD and the level of MDA in ASHS plasma might be positive correlation.Plasma proteomic analysis in ASHS Albumin, IgG,α1-Antitrypsin and a2-Macroglobulin count above 80 % in all the proteins of plasma, so we depleted them before 2-DE. About 570±0.88, 578 + 1.45 and 578 + 0.88 protein spots were respectively detected by Image Master analysis software in SHS, ASHS and ACTH. It was 85.4 % (493 pairs) that the ratio of protein spots matched between SHS and ASHS. There was 85.2 % (493 pairs) between ASHS and ACTH. Eight protein spots with significant difference were found, and upregulated or downregulated after ASHS. Seven of eight differential proteins were identified, and they were albumin, chain D of rat tansthyretin and semaphoring-3D precursor. With ACTH treating, there were five significant difference protein spots. Four of five differential proteins were identified, and they were albumin and semaphoring-3D precursor.Liver proteomic analysis in ASHS The 2-DE results of livers showed about 698±11, 700±13 and 696±18 protein spots were respectively detected in SHS, ASHS and ACTH. It was 91.7 % (641 pairs) that the ratio of protein spots matched between SHS and ASHS. There was 91.3 % (637 pairs) between ASHS and ACTH. Ten protein spots with significant difference were found, and upregulated or downregulated, or appeared after ASHS. All of ten differential proteins were successfully identified. These proteins included tumor rejection antigen gp96, glucose regulated protein, 58 kDa (GRP58), ATP synthase beta subunit, aldob protein, gapd protein, glutathione transferase, peroxiredoxin I , cytochrome B5 etc. With ACTH treating, there were two significant difference protein spots. The both of differential proteins were identified, and they were tumor rejection antigen gp96 and GRP58.Original research of GRPS8 in ASHS There were seven significant difference protein spots in plasma and liver proteomic analysis in ASHS with ACTH treating. GRP58 is the biggest (224) in paired protein score, and is the most (41 %) in sequence coverage. So GRP58 was chosen to investigate profoundly with western-blot, MALDI-TOF-MS, immunohistochemistry and RT-PCR. The results indicated that Ser150 of GRP58 was dephosphorylation after ASHS, while Ser150 was phosphorylation after treating with ACTH.In summary, we showed the changes of the activity of SOD and the level of MDA in ASHS plasma might be positive correlation. There were many of significant difference protein spots in plasma and liver proteomic analysis in ASHS. And Ser150 of GRP58 was dephosphorylation after ASHS. Therefore, all of these indicated albumin and GRP58 could be used as two new targets in diagnosing and treating hemorrhagic shock.
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