Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan 48109-5033, USA.
J Surg Res. 2011 Jan;165(1):128-35. doi: 10.1016/j.jss.2009.06.012. Epub 2009 Dec 6.
Burn injury is frequently complicated by bacterial infection. Following burn injury, exposure to endotoxin produces a measurable decrease in cardiomyocyte sarcomere contractile function. Lipopolysaccharide-binding protein (LBP) is an acute phase protein that potentiates the recognition of lipopolysaccharide (LPS) by binding to the lipid A moiety of LPS. In this study, we sought to determine the effect of recombinant rat LBP (rLBP) on cardiomyocyte sarcomere function after burn or sham injury in the presence or absence of bacterial endotoxin.
Rats underwent a full-thickness 30% total body surface area scald or sham burn. At 24 h post-injury, cardiomyocytes were isolated, plated at 50,000 cells/well, and incubated with 50 μg/mL LPS and rLBP or chloramphenicol acetyltransferase (BVCat, an irrelevant control protein produced using the same expression system as rLBP) at concentrations by volume of 1%, 5%, 10%, and 30%. Subsets of cardiomyocytes were incubated with 5% rat serum or 30% rLBP and blocking experiments were conducted using an LBP-like synthetic peptide (LBPK95A). In vitro sarcomere function was measured using a variable rate video camera system with length detection software.
Co-culture of burn and sham injury derived cardiomyocytes with high-dose rLBP in the presence of LPS resulted in a significant reduction to the functional impairment observed in peak sarcomere shortening following exposure to LPS alone. LBP-like peptide LBPK95A at a concentration of 20 μg/mL, in the presence of LPS, abolished the ability of 30% rLBP and 5% rat serum to restore peak sarcomere shortening of cardiomyocytes isolated following burn injury to levels of function exhibited in the absence of endotoxin exposure.
In the setting of LPS challenge following burn injury, rLBP at high concentrations restores cardiomyocyte sarcomere contractile function in vitro. Rather than potentiating the recognition of LPS by the cellular LPS receptor complex, rLBP at high concentrations likely results in an inhibitory binding effect that minimizes the impact of endotoxin exposure on cardiomyocyte function following thermal injury.
烧伤常伴有细菌感染。烧伤后，内毒素暴露会导致心肌细胞肌节收缩功能明显下降。脂多糖结合蛋白（LBP）是一种急性期蛋白，通过与脂多糖（LPS）的脂质 A 部分结合，增强 LPS 的识别。在这项研究中，我们试图确定重组大鼠 LBP（rLBP）在烧伤或假烧伤后存在或不存在细菌内毒素的情况下对心肌细胞肌节功能的影响。
大鼠进行全层 30%体表面积烫伤或假烫伤。伤后 24 小时，分离心肌细胞，铺板于 50,000 个细胞/孔，用 LPS 和 rLBP 或氯霉素乙酰转移酶（BVCat，一种使用与 rLBP 相同表达系统产生的无关对照蛋白）以体积浓度 1%、5%、10%和 30%孵育。部分心肌细胞用 5%大鼠血清或 30%rLBP 孵育，并使用 LBP 类似合成肽（LBPK95A）进行阻断实验。使用可变速率视频摄像机系统和长度检测软件测量体外肌节功能。
烧伤和假伤衍生的心肌细胞与高剂量 rLBP 在 LPS 存在下共培养，导致单独暴露于 LPS 后观察到的峰值肌节缩短功能障碍明显减少。在 LPS 存在下，浓度为 20 μg/mL 的 LBP 类似肽 LBPK95A 可消除 30%rLBP 和 5%大鼠血清恢复烧伤后分离的心肌细胞峰值肌节缩短的能力，使其功能恢复至无内毒素暴露的水平。
在烧伤后 LPS 挑战的情况下，高浓度 rLBP 可恢复体外心肌细胞肌节收缩功能。高浓度的 rLBP 不是增强细胞 LPS 受体复合物对内毒素的识别，而是可能导致抑制性结合作用，最大限度地减少热损伤后内毒素暴露对心肌细胞功能的影响。
