丙戊酸钠对严重烫伤后肠屏障功能的保护作用及机制研究

发布时间：2018-03-05 01:09

本文选题：烧伤　切入点：肠屏障　出处：《中国人民解放军医学院》2014年博士论文　论文类型：学位论文

【摘要】：目的肠粘膜上皮屏障功能障碍是严重烧伤后的常见并发症，也是诱发脓毒症和多器官功能障碍的重要危险因素。严重烧伤引起肠粘膜上皮屏障功能障碍的具体机制尚未完全清楚，但越来越多的研究提示，缺氧诱导因子1（hypoxia-inducible factor-1，HIF-1）及其下游靶基因血管内皮生长因子（vascular endothelial growth factor，VEGF）和肌球蛋白轻链激酶（myosinlight chain kinase，MLCK）的表达及功能在缺氧引起的细胞间屏障损害中起着关键作用。近年来研究表明，组蛋白去乙酰化酶抑制剂（histone deacetylaseinhibitor，HDACI）丙戊酸钠能提高细胞对缺氧、炎症等打击的耐受能力，保护机体各重要脏器功能，延长失血、烧伤及脓毒性休克动物的生存时间。此外，最近的研究还发现，丙戊酸钠能够抑制多种细胞间紧密连接（tight junction）蛋白的降解，，保护细胞屏障，减轻大鼠脊髓损伤或脑缺血-再灌注损伤后血脑和血脊髓屏障的通透性和组织水肿。本论文旨在研究和分析丙戊酸钠对严重烧伤后肠屏障的保护作用及其机制。方法 1.建立大鼠55%TBSAⅢ度烫伤模型并随机分为假烫+盐水组（Sham+NS）、假烫+丙戊酸钠组（Sham+VPA）、烫伤+盐水组（Scald+NS）、烫伤+丙戊酸钠组（Scald+VPA）。大鼠烫伤后即刻给予皮下注射丙戊酸钠（300mg/Kg）或等体积的生理盐水，于伤后2小时、6小时采用异硫氰酸荧光素标记葡聚糖检测肠上皮屏障的通透性变化；根据Chiu提出的肠屏障损害评分系统评价肠粘膜组织形态学变化；利用免疫荧光和（或）免疫印迹技术检测肠组织中组蛋白H3第9位赖氨酸的乙酰化（Ac-H3K9）水平以及HIF-1α、带状闭合蛋白1（zonaoccludens-1，ZO-1）、MLCK蛋白表达的变化；用酶联免疫吸附法检测肠组织VEGF蛋白表达的变化。 2.建立体外培养的单层Caco2肠上皮细胞模型，应用缺氧模拟剂氯化钴（1mM）刺激肠上皮细胞24小时，观察丙戊酸钠（2mM）对HIF-1α、VEGF、MLCK及ZO-1蛋白表达的影响；通过转染针对HIF-1α的siRNA特异性地抑制HIF-1α的表达，研究HIF-1α对VEGF、MLCK及ZO-1的影响，并分析丙戊酸钠保护肠上皮屏障功能的具体机制。结果 1.烫伤+盐水组大鼠伤后2小时肠屏障通透性、Chiu’s肠屏障损害评分、HIF-1α、VEGF及MCLK蛋白表达均明显增加，分别为假烫+盐水组的2.61倍、7.76倍、3.88倍、1.94倍和1.71倍（P均小于0.05）；而Ac-H3K9及ZO-1蛋白表达则明显减少，仅为烫伤+盐水组的0.73倍和0.65倍（P均小于0.05）；上述变化在伤后6小时更为明显，烫伤+盐水组上述指标分别为假烫+盐水组的4.80倍、17.33倍、4.42倍、2.43倍、2.80倍、0.48倍和0.43倍（P均小于0.05）。 2.给予丙戊酸钠治疗后，大鼠烫伤后2小时肠粘膜HIF-1α的蛋白水平即开始降低，而Ac-H3K9则有所升高，这些变化在伤后6小时更为明显（P均小于0.05）。丙戊酸钠治疗还能下调大鼠伤后6小时肠粘膜VEGF、MLCK的蛋白水平，同时抑制ZO-1降解，降低肠屏障通透性并减轻肠粘膜损害（P均小于0.05）。 3.氯化钴刺激24h后，肠上皮细胞HIF-1α、VEGF及MLCK的蛋白水平明显升高，而ZO-1蛋白水平则显著降低，分别为正常对照组的5.84倍、9.97倍，3.49倍和0.42倍（P均小于0.05）；丙戊酸钠治疗能显著降低肠上皮细胞HIF-1α、VEGF及MLCK的蛋白表达，同时增加ZO-1的蛋白水平（P均小于0.05）。 4.采用siRNA干扰技术特异性抑制HIF-1α能明显减少氯化钴刺激后肠上皮细胞VEGF及MLCK的蛋白表达及ZO-1蛋白的降解（P均小于0.05）。结论严重烧伤后肠粘膜组织组蛋白乙酰化及ZO-1水平降低，并引起肠屏障功能损害；丙戊酸钠能提高组蛋白乙酰化及ZO-1水平，保护肠上皮屏障功能，其作用机制与丙戊酸钠对HIF-1α及其下游靶基因VEGF和MLCK的抑制作用有关。
[Abstract]:objective
Intestinal mucosal barrier dysfunction is a common complication after severe burns, is an important risk induced by sepsis and multiple organ dysfunction caused by severe burn factors. Specific mechanism of intestinal mucosal barrier dysfunction is not entirely clear, but more and more studies suggest that hypoxia inducible factor 1 (hypoxia-inducible, factor-1, HIF-1) and its downstream target genes vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) and myosin light chain kinase (myosinlight chain, kinase, MLCK) plays a key role in the expression and function in hypoxia induced cell damage between the barrier. Recent studies show that histone deacetylase inhibitors (histone, deacetylaseinhibitor, HDACI) can enhance the cell of sodium valproate hypoxia, inflammation and other attack tolerance, protect the important viscera function, prolong blood loss, burn and sepsis Toxic shock animal survival time. In addition, recent studies also found that sodium valproate can inhibit a variety of intercellular tight junctions (tight junction) protein degradation, cell protection barrier, reduce rat spinal cord injury or cerebral ischemia reperfusion injury after cerebral blood and blood spinal cord barrier permeability and tissue edema. The aim of this paper is to protect the role of the research and analysis of sodium valproate on intestinal barrier and its mechanism.
Method
1. establishment of rat 55%TBSA model and scald were randomly divided into sham + saline group (Sham+NS), sham group (Sham+VPA), sodium valproate + saline group (Scald+NS), scald + scald + sodium valproate group (Scald+VPA). The scalded rats given immediately after subcutaneous injection of sodium valproate (300mg /Kg) or saline etc. the volume, in 2 hours after injury, 6 hours by the permeability of fluorescein isothiocyanate dextran for detecting intestinal epithelial barrier; intestinal barrier damage according to the Chiu's scoring system to evaluate the morphological changes of intestinal mucosa by immunofluorescence; and (or) acetylation detected by immunoblotting technique in intestinal tissue of histone H3 lysine ninth acid (Ac-H3K9) and the level of HIF-1 alpha, zona occluden 1 (zonaoccludens-1, ZO-1), the changes of the expression of MLCK protein; adsorption changes in expression of VEGF protein was detected in intestinal tissue by ELISA.
Caco2 intestinal epithelial cell monolayer model 2. in vitro culture, application of hypoxia mimetic cobalt chloride (1mM) stimulation of intestinal epithelial cells 24 hours, observation of sodium valproate (2mM) on VEGF, HIF-1 alpha, the expression of MLCK and ZO-1 protein expression in HIF-1; alpha siRNA specifically inhibited by transfection of HIF-1 alpha. HIF-1 alpha on VEGF, MLCK and ZO-1, and analyze the specific mechanism of sodium valproate protect intestinal epithelial barrier function.
Result
1. scald + saline group rats 2 hours after injury of intestinal barrier permeability, intestinal barrier damage score Chiu 's, HIF-1 alpha, VEGF expression and MCLK protein were significantly increased, respectively 2.61 times, sham + saline group 7.76 times, 3.88 times, 1.94 times and 1.71 times (P < 0.05); the expression of Ac-H3K9 and ZO-1 protein were significantly reduced, only 0.73 times the scald + saline group and 0.65 times (P < 0.05); the change in 6 hours after injury is more obvious, 4.80 times, scald + saline group the indicators were sham + saline group 17.33 times, 4.42 times, 2.43 times, 2.80 times, 0.48 times and 0.43 times (P < 0.05).
2. VPA treated rats after burn 2 hours of intestinal mucosal protein level of HIF-1 alpha began to decrease, while Ac-H3K9 increased, the changes in 6 hours after injury was more significant (P < 0.05). Sodium valproate in the treatment of rats can cut 6 hours after injury of intestinal mucosal VEGF and protein levels of MLCK at the same time, the inhibition of ZO-1 degradation, reduce intestinal barrier permeability and reduce the damage of the intestinal mucosa (P < 0.05).
3. cobalt chloride after 24h stimulation of intestinal epithelial cells HIF-1 alpha protein levels of VEGF and MLCK increased significantly, while the level of ZO-1 protein decreased significantly, respectively 5.84 times, 9.97 times of the normal control group, 3.49 times and 0.42 times (P < 0.05); sodium valproate treatment can significantly reduce the intestinal epithelial cells HIF-1 a, the expression of VEGF and MLCK protein also increased the protein level of ZO-1 (P < 0.05).
4., using siRNA interference technology to specifically inhibit HIF-1 alpha can significantly reduce VEGF and MLCK protein expression and ZO-1 protein degradation in intestinal epithelial cells after CO chloride stimulation (P is less than 0.05).
conclusion
Intestinal mucosa histone acetylation and the level of ZO-1 decreased, and cause the impairment of intestinal barrier function; sodium valproate can improve the histone acetylation and the level of ZO-1, protect the intestinal epithelial barrier function, the inhibitory effect and its mechanism of sodium valproate on HIF-1 alpha and its downstream target genes VEGF and MLCK.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R644

【参考文献】