乌司他丁对脓毒血症肾损伤肾小球血管内皮糖萼的保护作用及分子机制研究

发布时间：2018-04-17 20:16

本文选题：脓毒血症 + 肾损伤　；参考：《浙江大学》2015年博士论文

【摘要】：背景：严重感染(severe sepsis)所继发的脓毒血症和多脏器功能障碍综合征(multiple organ dysfunction syndrome, MODS)是当前重症监护病房(intensive care unit, ICU)内的主要死亡原因,也是当前重症医学面临的主要焦点及难点。虽然对脓毒症的病理生理学机制和临床治疗进行了大量研究,然而迄今为止患者的发病率和病死率仍然居高不下。脓毒血症发生后肾脏往往是最容易遭受损伤的器官,根据医学界已基本达成共识的急性肾损伤(Acute kidney injury, AKI)标准(RIFLE标准),有近8%的住院病人及50%以上的ICU患者出现AKI。因此,AKI已成为现代医院及ICU的主要临床难题。此外,有确凿证据表明,严重感染及脓毒血症是导致危重症患者发生AKI的最主要原因,在ICU中,大约50%以上的AKI由此引发。虽然目前临床上脏器的支持治疗和液体复苏治疗水平正日益增强,但由脓毒血症所诱发的AKI的发生率及死亡率未有下降,这可能与其确切的发病机制尚未明确有关。新近的研究发现脓毒血症的主要病理生理机制——微循环障碍可能与内皮表面糖萼(glycocalyx,多糖-蛋白复合物)的状态密切相关。越来越多的研究已认识到血管内皮糖萼对心血管疾病具有重要的意义。糖萼是衬于血管内皮细胞管腔膜侧,覆盖着一种富含多糖的绒毛状细胞结构,作为心血管系统的重要屏障,将血液与组织分隔开来。结构完整的糖萼具有维持血管通透性,抑制细胞间粘附等血管保护作用,否则将最终导致组织水肿和脏器功能障碍,并激发中性粒细胞与内皮粘附后所产生的炎症爆发。既往研究已经发现脓毒血症可导致肾小球毛细血管内皮糖萼破坏并伴有蛋白尿,同时该现象的发生与脓毒血症后机体产生大量的(tumor necrosis factor-a,TNF-a)可能有关。然而这其中的确切机制尚不清楚,对于TNF-a的来源,自身的作用,与中性粒细胞贴壁之间的关系,其产生的调节机制以及其损伤糖萼的作用机制仍不清楚。乌司他丁(urinary trypsin inhibitor,UTI):其以两种前体分子形式存在于血浆中,inter-a-inhibitor (IαI)和pre-a-inhibitor(Pal),其结构包括两个Kunitz结构域,O-糖链、硫酸软骨素,N-糖链,C端K区：抑制多种水解酶,N端K区：抑制组织蛋白酶G和弹性蛋白酶,O-硫酸软骨素糖链：能与细胞、钙等结合,与稳定溶酶体、抑制炎性细胞释放有关,N-糖链：调节内皮细胞与单核细胞、粒细胞、淋巴细胞的结合力。乌司他丁主要是通过两个Kunitz结构域,分别能抑制多种酶的活性,尤其是减少丝氨酸蛋白酶(包括组织蛋白酶,粒细胞弹性蛋白酶,纤溶酶,激肽原酶,胰蛋白酶或糜蛋白酶,类胰蛋白酶等)活性,通过抑制中性粒细胞,淋巴细胞,巨噬细胞,肥大细胞等过度激活,抑制纤维蛋白降解,抑制缓激肽生成而导致血管扩张,阻止蛋白酶活化受体激活,从而减少炎性细胞活化,将炎性反应局限化,减轻对正常组织的损伤,其直接作用于细胞前体,减少细胞因子的分泌,稳定溶酶体膜,减少酶的释放,达到缓解炎症反应,改善微循环的作用。目前尚未发现乌司他丁对脓毒血症的肾血管内皮糖萼破坏的保护作用及其分子机制的相关研究。因此,本研究将选用脂多糖内毒素(lipopolysaccharide, LPS)诱导脓毒血症的肾损伤大鼠在体模型,计划对糖萼破坏与AKI发生相关的机制展开研究,并进一步探讨肾脏血管内皮糖萼在脓毒血症导致AKI的机制中的作用与地位——仅仅是炎症作用的“靶子”,抑或是兼具炎症爆发的“炸药桶”。并拟从肾脏血管内皮糖萼的病理生理变化及相关分子水平阐述乌司他丁对脓毒血症肾损伤的保护作用和具体机制,旨在从药物治疗脓毒血症后肾血管糖萼破坏提供基础实验依据。目的：本研究探讨乌司他丁对脂多糖内毒素诱导的脓毒血症大鼠肾脏血管内皮的保护作用和分子机制,旨在从新的保护血管内皮细胞糖萼破坏的的药物途径上提供基础实验依据。方法： 1)脓毒血症打击模型建立及血清学参数检测通过腹部皮下注射水合氯醛对大鼠实施麻醉,对其右侧颈静脉进行置管。通过尾静脉注射脂多糖,监测平均动脉压(mean artery pressure, MAP)及在0,180,360分钟进行血清学(creatinine, Cr),(blood usea nitrogen, BUN)等相关检测。 2)肾脏微血流灌注评估-激光多普勒技术应用激光多普勒仪(Moor Instruments, Axminister, England)检测法在活体状态下观察肾脏皮质微循环血流状态及灌注并进行实时计算。 3)肾脏血管内皮细胞膜侧糖萼的结构分析通过电镜直接观察脓毒血症大鼠肾小球毛细血管内皮糖萼结构及厚度,通过检测外周血,肾静脉血(heparan sulfate, HS)和syndecan-1水平来间接评估肾小球毛细血管内皮糖萼的破坏。 4)肾脏内皮细胞TNF-α合成相关的分子信号通路研究免疫组化检测肾小球毛细血管内皮细胞浆内(p38mitogen-activated protein kinase, p38MAPK)、(nuclear factor of kB,NF-kB)分子表达：通过对外周血和肾静脉血中TNF-α炎性介质(流式细胞仪检测)的浓度及肾小球毛细血管内皮细胞内(Mitogenactivatedproteinkinase,MAPK)家族分子及NF-κB分子表达的检测。结果： 1)大鼠尾静脉注射LPS(10μg/g)后,内毒素组大鼠平均动脉压进行性下降,生理盐水干预组与乌司他丁组经补液复苏及血管活性药物支持下,平均动脉压呈现逐步上升,而肾脏微循环血流监测显示仍呈下降趋势。 2)正常标准对照组大鼠肾脏毛细血管糖萼层完整连续,厚约200nnm；脓毒血症后肾脏毛细血管糖萼不连续,结构松散,变薄。生理盐水干预组和乌司他丁药物干预组大鼠肾脏毛细血管糖萼结构同样存在不连续,较正常组松散,肿胀。脓毒血症发生后1小时大鼠肾脏静脉及外周血中HS、syndecan-1浓度即出现明显的升高,且其较高浓度维持的时间较长(3小时达到高峰值) 3)脓毒血症发生后0.5小时大鼠肾脏静脉血中TNF-α浓度即出现明显的升高,且其较高浓度维持的时间较长(3小时后迅速下降),而外周血TNF-a浓度的峰值明显低于肾静脉血。相对于标脓毒血症组及生理盐水干预组大鼠,乌司他丁能够降低肾静脉血和外周血中TNF-a的高峰浓度及其持续时间(P0.05)。 4)脂多糖内毒素诱导的脓毒血症肾损伤模型组大鼠肾小球毛细血管内皮细胞浆内NF-kB分子表达比正常对照组显著升高(P0.01),而乌司他丁药物治疗组较脓毒血症组也表现为明显降低(P0.05),其余组间比较无显著性差异。 5)脂多糖内毒素诱导的脓毒血症肾损伤模型组大鼠肾小球毛细血管内皮细胞浆内P38MAPK分子表达比正常对照组显著升高(P0.01),而乌司他丁药物治疗组较脓毒血症组表现为明显降低(P0.05),其余组间比较无显著性差异。结论：乌司他丁能减轻脂多糖内毒素诱导的脓毒血症的肾损伤微循环屏障破坏,尤其是减轻了肾小球血管内皮细胞糖萼的破坏,从而提高肾血流,抑制肾小球血管内皮细胞膜侧的炎性反应。糖萼的破坏可能导致了脓毒血症后肾脏微循环障碍损伤,肾脏血管内皮细胞NF-κB/TNF-α分子机制可能参与了糖萼的破坏。以糖萼作为治疗靶点,乌司他丁可能成为修复糖萼损伤而改善微循环障碍的药物选择并具有潜在的治疗效果。
[Abstract]:Background :

Severe sepsis and multiple organ dysfunction syndrome ( MODS ) are the main causes of death in intensive care unit ( ICU ) .

Recent studies have found that the main pathological and physiological mechanisms of sepsis are closely related to the state of glycoccodextrin ( polysaccharide - protein complexes ) . More and more studies have recognized that vascular endothelial glycocal is of great importance to cardiovascular diseases . The glycocalycis is a vital barrier to the cardiovascular system . It can maintain vascular permeability , inhibit cell adhesion , etc . Otherwise , it will eventually lead to tissue edema and organ dysfunction , and trigger inflammation of neutrophils and endothelial cells .

Previous studies have found that sepsis can lead to the destruction of glomerular capillary endothelium and associated proteinuria , while the occurrence of this phenomenon may be associated with the occurrence of a large number of tumor necrosis factor - a , TNF - a after sepsis . However , the exact mechanism is unknown . However , the mechanism of regulation and the mechanism of its damage is still unclear for the origin of TNF - a .

Ulinastatin ( UTI ) , which is in the form of two precursor molecules in plasma , inter - a - inhibitor ( I.alpha . I ) and pre - a - inhibitor , the structure of which comprises two Kunitz domains , O - sugar chain , chondroitin sulfate , N - sugar chain , C - terminal K region , and N - sugar chain . In this study , we have not found the protective effect and molecular mechanism of Ulinastatin on the damage of renal vascular endothelium in septic rats , and further study the protective effect and specific mechanism of ullastatin on the renal injury of septic rats .

Purpose :

The purpose of this study was to investigate the protective effect and molecular mechanism of ullastatin on renal vascular endothelium in rats with sepsis induced by lipopolysaccharide ( LPS ) .

Method :

1 ) establishment of sepsis - resistant model and detection of serological parameters

The rats were anesthetized with subcutaneous injection of chloral hydrate , and the right jugular vein was placed . The mean artery pressure ( MAP ) was monitored by intravenous injection of lipopolysaccharide , and serum levels of creatinine ( Cr ) and BUN were monitored at 0,180 , 360 minutes .

2 ) Renal microperfusion assessment - laser Doppler technique

The renal cortex microcirculation blood flow state and perfusion were observed under the condition of living body by using laser Doppler instrument ( Moor Instruments , Axillary , England ) and real - time calculation was performed .

3 ) Structure analysis of vascular endothelial cell membrane side glycocalyon in kidney

The structure and thickness of glomerular capillary endothelium were directly observed by electron microscope . The damage of glomerular capillary endothelium was assessed indirectly by detecting peripheral blood , renal venous blood ( HS ) and syndecan - 1 level .

4 ) The expression of p38MAPK - activated protein kinase ( p38MAPK ) , ( p38MAPK ) , ( nuclear factor of kB , NF - kB ) in glomerular capillary endothelial cells was studied by immunohistochemistry .

Results :

1 ) After intravenous injection of LPS ( 10渭g / g ) in tail vein , the average arterial pressure of endotoxin group decreased gradually , and the average arterial pressure appeared gradually , while renal microcirculation blood flow monitoring showed a downward trend .

2 ) The normal control group rats were intact and continuous with the thickness of 200 nnm .
Compared with normal group , the concentration of HS and syndecan - 1 in the renal vein and peripheral blood of rats with septic shock increased significantly , and the concentration of HS and syndecan - 1 in the renal vein and peripheral blood increased significantly ( reaching the peak value in 3 hours ) .

3 ) The concentration of TNF - 伪 in the renal venous blood of rats after 0.5 hour after sepsis increased significantly , and the peak value of TNF - a in peripheral blood was significantly lower than that in renal venous blood ( P0.05 ) .

4 ) The expression of NF - kB in rat glomerular capillary endothelial cells was significantly higher than that in normal control group ( P0.01 ) .

5 ) The expression of P38MAPK in rat glomerular capillary endothelial cell was significantly higher than that in normal control group ( P0.01 ) .

Conclusion :

Ulinastatin can reduce the renal damage microcirculation barrier damage induced by lipopolysaccharides endotoxin , in particular to reduce the damage of the glycocalycis of the glomerular vascular endothelial cell , thereby improving the renal blood flow and inhibiting the inflammatory response on the membrane side of the glomerular vascular endothelial cell .

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R459.7

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