MC4R激动剂缓解继发性腹腔高压症中肠道及脑损伤的作用研究

发布时间：2018-06-21 22:16

本文选题：腹腔高压症 + 失血性休克　；参考：《第三军医大学》2016年博士论文

【摘要】：研究背景严重创伤性患者在失血性休克、烧伤、腹部创伤等,在救治过程中常发生腹腔高压症(intra-abdominal hypertension,IAH),甚至进展为腹腔间隙综合征(abdominal compartment syndrome,ACS),累及多种腹腔内脏器(肠道、胃、肝和肾)及腹腔外器官(脑、心及肺)等。IAH/ACS包括:原发性、继发性和复发性3类。原发性IAH/ACS是由腹腔/盆腔疾病或损伤所引起;继发性IAH/ACS并不由腹腔/盆腔疾病或损伤引起,如全身炎症反应综合征、缺血休克和大面积烧伤等;复发性IAH/ACS是指前期IAH/ACS经手术或药物治疗缓解后再次发生。IAH发生的危险因素包括:创伤后大量输液、复苏后毛细血管渗漏、腹腔内容物增加和腹壁顺应性下降等,而其中创伤后大量输液是主要原因。IAH的发病机制和病理进展过程目前尚不完全明确。目前治疗IAH的方法依然集中在剖腹减压等外科技术的应用上,但依然存在如减压后综合征造成的再灌注损伤等问题。由于肠道水肿等损伤是腹内压升高的重要因素,本研究以如何减缓肠道水肿等损伤的发生,以及如何缓解腹内高压影响的重要腹腔外器官—脑部的损伤为主要内容。目前越来越受到人们关注的黑色素皮质激素(melanocortin,MC),MC通过其不同受体在多器官中发挥调节色素沉着,能量稳态,炎症控制和免疫调节等功能,有研究发现MC4受体(melanocortin 4 receptor,MC4R)激动剂和其人工合成类似物可以在失血性休克、缺血性卒中和创伤性颅脑损伤中发挥保护作用,并通过胆碱能抗炎通路发挥快速抗炎的作用,但具体机制尚不清楚。本课题旨在探寻MC是否可以起到缓解继发性IAH中肠和脑功能障碍的作用,并进一步探寻其机制,以期为临床腹腔高压症患者的治疗提供新的策略。为了检验上述假设,本研究首先建立继发性IAH大鼠模型,该模型应便于操作、可重复性高并可在一定程度上较好的模拟IAH的发病过程。其次,观察给予RO27-3225(MC4R激动剂)后可否减轻IAH诱导的肠道损伤,该保护作用通过何种机制发挥效果。最后探讨RO27-3225是否对于IAH诱发的腹腔外器官损害--脑损伤的具有保护效果,并判断胆碱能信号通路在其中的作用。研究方法1.采用股动脉插管后放血法,诱导平均动脉压(mean arterial pressure,MAP)分别达到:40mm Hg(维持120 min);30mm Hg(维持90min)和25mm Hg(维持60min),而后均合并IAP=20mm Hg(维持4 h)以建立大鼠继发性IAH模型,利用干湿比法检测肠道和脑组织含水量,利用HE染色法在光镜下观察大鼠肠道组织形态学和分析不同组间生存率,从中选择一种既可造成明显损伤又可保证生存率以供后续试验可顺利进行的大鼠休克后IAH模型进行下述观察。2.在继发性IAH大鼠模型(30mm Hg维持90min合并IAP=20mm Hg维持4h)中,通过观察大鼠MAP的变化程度;采用real-time PCR法检测肠壁全层炎症因子(TNF-α和IL-1β)变化;使用干湿比法检测肠道含水量变化;利用HE染色法在光镜下观察大鼠肠道组织形态学组织变化;利用酶联免疫吸附方法检测血浆内肠脂肪酸结合蛋白变化;通过检测异硫氰酸荧光素以反映肠道渗透性变化和使用免疫印迹法检测影响肠道渗透性的关键因子ROCK1及肌球蛋白轻链磷酸化水平以及检测肠道内超氧化物歧化酶变化等指标探讨IAH对肠道的损伤效果。并在此基础上进一步观察RO27-3225是否可以发挥保护肠道的作用。3.在继发性IAH大鼠模型中,利用干湿比法检测脑含水量变化;通过检测伊文思蓝外渗法反映血脑屏障渗透性、使用real-time PCR法和酶联免疫吸附方法检测脑内炎症因子(TNF-α和IL-1β)变化、采用酶联免疫吸附方法及免疫印迹法检测水通道蛋白4及金属基质蛋白酶9表达变化,以及利用免疫组化法检测神经胶质细胞表达的变化等指标探讨IAH对脑的损伤效果,进一步观察RO27-3225是否可以发挥保护脑组织的作用。研究结果1.选取MAP为30mm Hg持续90 min合并IAP=20 mm Hg持续4 h这一条件可诱导大鼠肠道出现明显组织学损伤,导致肠道含水量明显上升,并可保证其生存率以确保后续试验可以进行;2.RO27-3225可在一定程度上稳定MAP变化,降低IAH所诱发的肠道内炎症因子(TNF-α和IL-1β)升高,缓解IAH导致的肠道内ROCK 1蛋白表达和肌球蛋白轻链磷酸化水平下降,降低血浆内肠脂肪酸结合蛋白水平,减轻肠水肿、肠道渗透性和肠道组织形态学等结果的异常变化,从而起到缓解IAH导致的肠道损伤的作用。而该保护作用可被其特异性阻滞剂(HS024)和烟碱型乙酰胆碱受体拮抗剂(氯异桑大明)所阻止。3.RO27-3225可减轻IAH导致的脑水肿,降低炎症因子、水通道蛋白4及金属基质蛋白酶9的表达,从而发挥缓解IAH中脑损伤的作用。而该保护作用可被其特异性阻滞剂(HS024)和烟碱型乙酰胆碱受体拮抗剂(氯异桑大明)所阻止。但对IAH诱导的神经胶质细胞表达增多并无影响。结论1.IAH可导致腹腔内脏器-肠道产生明显水肿及炎症反应,并出现明显组织学损伤,此外IAH也可诱导腹腔外器官-脑组织产生明显炎症、水肿并导致血脑屏障完整性损害;2.选择性MC4R激动剂(RO27-3225)可以稳定机体血流动力学,并通过调节ROCK1表达和肌球蛋白轻链磷酸化水平以缓解肠道渗透性增高,并可降低炎症反应程度,而有效缓解IAH诱导的肠道损伤。该保护作用可能是通过胆碱能通路发挥效果;3.选择性MC4R激动剂可以通过降低金属基质蛋白酶9表达以缓解血脑屏障破坏,通过作用水通道蛋白4以降低脑水肿程度,并可减轻脑组织炎症反应有效改善IAH导致的脑组织损伤,该保护作用可能是通过胆碱能通路发挥效果。但其在急性期内对神经胶质细胞数量无明显影响。
[Abstract]:The research background of severe traumatic patients in hemorrhagic shock, burns, abdominal trauma, and so on. In the course of treatment, intra-abdominal hypertension (IAH) often occurs, and even abdominal compartment syndrome (ACS), involving a variety of abdominal viscera (intestine, stomach, liver and kidney) and external organs of the abdomen (brain, .IAH/ACS includes 3 types: primary, secondary and recurrent. Primary IAH/ACS is caused by abdominal / pelvic diseases or injuries; secondary IAH/ACS is not caused by abdominal / pelvic diseases or injuries, such as systemic inflammatory response syndrome, ischemic shock and large area burn, and recurrent IAH/ACS refers to prophase IAH/ACS through surgery or medicine The risk factors for the recurrence of.IAH after the treatment of remission include: massive infusion after trauma, capillary leakage after resuscitation, increase of intraperitoneal content and decrease of abdominal wall compliance, and the pathogenesis and pathological progress of.IAH are not completely clear at present. At present, the methods for the treatment of IAH However, it is focused on the application of surgical techniques such as laparotomy, but there are still problems such as reperfusion injury caused by post decompression syndrome. Because of intestinal edema, such as intestinal edema is an important factor in intraperitoneal pressure increase, this study can reduce the occurrence of intestinal edema and other injuries, as well as the important external organs such as how to relieve the effect of intraabdominal hypertension. The damage of the brain is the main content. Melanocortin (MC) is becoming more and more concerned. MC plays the functions of regulating pigmentation, energy homeostasis, inflammatory control and immunoregulation through its different receptors in multiple organs. The research has found that the MC4 receptor (melanocortin 4 receptor, MC4R) agonists and their artificial agents have been found. Synthetic analogues can play a protective role in hemorrhagic shock, ischemic stroke and traumatic brain injury, and play a rapid and anti inflammatory role through the cholinergic anti-inflammatory pathway, but the specific mechanism is not clear. The purpose of this study is to explore whether MC can play a role in alleviating intestinal and brain dysfunction in secondary IAH and further explore. The mechanism is expected to provide a new strategy for the treatment of patients with clinical abdominal hypertension. In order to test the above hypothesis, this study first established the secondary IAH rat model. This model should be easy to operate, can be highly reproducible and can simulate the pathogenesis of IAH to a certain extent. The second, the observation is given RO27-3225 (MC4R agonist) can be reduced. The protective effect of the protective effect of light IAH on the intestinal damage and the effect of the protective effect. Finally, the effect of RO27-3225 on IAH induced intraperitoneal organ damage, brain injury, and the role of cholinergic signal pathway in it were judged. Method 1. the mean arterial pressure (mean AR) was induced by the method of blood release after femoral artery intubation. Terial pressure, MAP), 40mm Hg (maintain 120 min), 30mm Hg (maintain 90min) and 25mm Hg (maintain 60min), and then all merged (maintain 4) to establish a secondary rat model, use the dry wet ratio method to detect the water content of the intestine and brain tissue, and observe the intestinal morphology and analysis of the rat's intestines under the light microscope. The survival rate of the same group was selected from the IAH model of rats after shock, which could both cause obvious damage and guarantee survival rate for subsequent experiments. The following observation was made by.2. in the secondary IAH rat model (30mm Hg maintenance 90min combined with IAP=20mm Hg to maintain 4h), by observing the degree of change of MAP in rats; real-time PCR method was used. The changes in the whole intestinal wall inflammatory factors (TNF- - and IL-1 beta) were detected, the changes in the intestinal water content were detected by the dry wet ratio method, and the changes of the intestinal tissue in the rats were observed by the HE staining method, and the changes in the intestinal fatty acid binding protein were detected by the enzyme linked immunosorbent assay, and the isothiocyanate fluorescent protein was detected to reflect the intestinal tract. The key factors affecting the permeability of the intestinal tract, ROCK1, the level of myosin light chain phosphorylation, and the changes of the superoxide dismutase in the intestinal tract were detected by Western blot, and the effects of IAH on the intestinal damage were investigated. On the basis of this, the effect of RO27-3225 on the protection of intestinal tract was further observed,.3. In the secondary IAH rat model, the changes in the water content of the brain were detected by the dry wet ratio method, and the blood brain barrier permeability was detected by Evans blue exosmosis. The changes of the brain inflammatory factors (TNF- and IL-1 beta) were detected by real-time PCR and ELISA, and the water channel eggs were detected by enzyme linked immunosorbent assay and Western blot. The changes in the expression of white 4 and metal matrix proteinase 9 and the changes of the expression of glial cells by immunohistochemical method were used to investigate the effect of IAH on brain damage, and further to observe the effect of RO27-3225 on the protection of brain tissue. Results 1. the results were selected as the condition of MAP for 30mm Hg holding 90 min with IAP=20 mm Hg continuous 4 h. It can induce obvious histological damage in the intestinal tract of rats, which can lead to an obvious increase in the water content of the intestine, and ensure the survival rate to ensure the follow-up test. 2.RO27-3225 can stabilize the MAP changes to a certain extent, reduce the increase of the intestinal inflammatory factors (TNF- A and IL-1 beta) induced by IAH, and alleviate the ROCK 1 protein table in the intestinal tract caused by IAH. The level of light chain phosphorylation of myosin and myosin decreases, reduces the level of intestinal fatty acid binding protein, reduces the abnormal changes in intestinal edema, intestinal permeability and intestinal histology, and thus plays a role in alleviating the intestinal damage caused by IAH, and the protective effect can be treated by its specific blocker (HS024) and nicotinic acetylcholate. The alkali receptor antagonist (cisisan Daming) prevents.3.RO27-3225 from the brain edema caused by IAH, and reduces the expression of inflammatory factors, aquaporin 4 and metal matrix protease 9, which can play a role in alleviating brain damage in IAH, and this protective effect can be treated by its specific blocker (HS024) and nicotinic acetylcholine receptor antagonist (chloroisoyl). Sang Daming) prevented. But it did not affect the increase of the expression of glial cells induced by IAH. Conclusion 1.IAH can cause obvious edema and inflammatory reaction in the visceral organs of the abdominal cavity, and there is obvious histological damage. In addition, IAH can induce obvious inflammatory disease in the external organ - brain tissue, edema and damage of blood brain barrier integrity; 2. Selective MC4R agonist (RO27-3225) can stabilize the hemodynamics of the body, and by regulating the expression of ROCK1 and the level of myosin light chain phosphorylation to alleviate the increase in intestinal permeability and reduce the degree of inflammation, and effectively alleviate the intestinal damage induced by IAH. The protective effect may be through the cholinergic pathway; 3. Sexual MC4R agonists can reduce the destruction of blood brain barrier by reducing the expression of matrix metalloproteinase 9, reducing the degree of brain edema through the action of aquaporin 4, and alleviating the inflammatory response of brain tissue to effectively improve the brain tissue damage caused by IAH. This protective effect may be achieved through the bile alkali energy pathway. There was no obvious effect on the number of glial cells.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R641

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