Department of Surgery, Division of Trauma, Emergency Surgery and Surgical Critical Care, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
J Surg Res. 2010 Mar;159(1):474-81. doi: 10.1016/j.jss.2009.04.011. Epub 2009 May 13.
The initial management of a poly-trauma patient requires evaluation for potential hemorrhage and ongoing monitoring to assess the efficacy of treatment and avoid complications related to massive blood loss. Certain serum protein levels may be altered in response to hemorrhagic shock, and may serve as useful biomarkers to guide diagnosis, prognosis, and therapeutics in traumatic hemorrhagic shock (HS). Treatment with valproic acid (VPA) has been shown to up-regulate various survival pathways and improve outcome. Here we determine whether these changes would result in altered serum biomarkers.
Wistar-Kyoto rats underwent hemorrhagic shock (60% blood loss) followed by treatment with or without VPA (300 mg/kg). Using surface enhanced laser desorption-time of flight mass spectrometry (SELDI or SELDI-TOF MS) technology, we screened serum samples obtained from five rats at different time points (baseline, post-hemorrhagic shock, and post-VPA treatment) in a lethal model of hemorrhagic shock (HS). Additionally, we used isobaric tag labeling for relative quantitation (iTRAQ) to identify potential biomarkers in the serum. Western blots were performed to validate iTRAQ-identified biomarker from independent serum samples, and to analyze protein biomarker levels in the intestine during hemorrhagic shock and treatment.
HS and treatment with VPA affected serum levels of many proteins. One such protein with a mass spectrum around 22.7 kDa was detected in all five rats. The same serum samples subjected to iTRAQ resulted in our identification of claudin-3, a 23 kDa tight junction protein. HS elevated serum claudin-3 protein levels, which was reversed by VPA treatment in a pattern similar to the SELDI-TOF MS studies. Further validation with independent serum and intestine samples from individual rats by Western blots confirmed that HS increased the protein level of claudin-3 in serum and decreased its level in the intestine. Treatment with VPA reversed the hemorrhagic shock-induced alteration in claudin-3 to sham levels.
HS causes an acute rise in serum claudin-3 protein levels and a concurrent decrease in intestinal claudin-3 protein expression. VPA treatment attenuates these alterations and stabilizes intestinal claudin-3 levels. Our results demonstrate for the first time that claudin-3 is a potential biomarker in HS and treatment.
多发伤患者的初始治疗需要评估潜在的出血，并进行持续监测，以评估治疗效果并避免与大量失血相关的并发症。某些血清蛋白水平可能会因出血性休克而改变，并且可能作为有用的生物标志物来指导创伤性出血性休克（HS）的诊断、预后和治疗。已证明使用丙戊酸（VPA）治疗可上调多种存活途径并改善预后。在这里，我们确定这些变化是否会导致血清生物标志物改变。
Wistar-Kyoto 大鼠接受出血性休克（失血 60%），然后用或不用丙戊酸（300mg/kg）治疗。使用表面增强激光解吸时间飞行质谱（SELDI 或 SELDI-TOF MS）技术，我们筛选了在致命性出血性休克模型中不同时间点（基础、出血性休克后和 VPA 治疗后）从五只大鼠获得的血清样本（基线、出血性休克后和 VPA 治疗后）。此外，我们使用等压标签标记相对定量（iTRAQ）来鉴定血清中的潜在生物标志物。进行 Western blot 以验证 iTRAQ 鉴定的生物标志物的独立血清样本，并分析出血性休克和治疗期间肠道中的蛋白生物标志物水平。
HS 和 VPA 治疗会影响许多蛋白质的血清水平。在所有五只大鼠中都检测到一种分子量约为 22.7kDa 的蛋白质。同样的血清样本进行 iTRAQ 分析导致我们鉴定出紧密连接蛋白 3（claudin-3），一种 23kDa 的紧密连接蛋白。HS 增加了血清 claudin-3 蛋白水平，VPA 治疗以类似于 SELDI-TOF MS 研究的方式逆转了这种增加。用来自单个大鼠的独立血清和肠样本进行进一步验证Western blot 确认 HS 增加了血清中 claudin-3 的蛋白水平，并降低了其在肠道中的水平。VPA 治疗逆转了出血性休克诱导的 claudin-3 改变，使其恢复到假手术水平。
HS 导致血清 claudin-3 蛋白水平急性升高，并同时降低肠道 claudin-3 蛋白表达。VPA 治疗减轻了这些改变并稳定了肠道 claudin-3 水平。我们的结果首次证明 claudin-3 是 HS 和治疗的潜在生物标志物。
