Olson Alicia, Diebel Lawrence N, Liberati David M
From the Department of Surgery, Wayne State University, Detroit, Michigan.
J Trauma Acute Care Surg. 2014 Oct;77(4):570-5; discussion 576. doi: 10.1097/TA.0000000000000378.
The incidence and severity of Clostridium difficile colitis have increased dramatically in the last decade. Disease severity is related to C. difficile virulence factors, including toxins A and B, as well as the patient's immune status. The intestinal mucus is an important component of innate barrier function in the intestine. Phosphatidylcholine (PC) is a key constituent of the intestinal mucus barrier, and exogenous PC administration has had therapeutic efficacy in patients with ulcerative colitis. We studied the protective function of exogenous PC on C. difficile toxin effects on the intestinal barrier in vitro.
Mucus-producing (HT29-MTX strain) and non-mucus-producing (HT29 strain) intestinal epithelial monolayers were cocultured with PC and C. difficile toxin A added to the apical media. Basal chamber culture supernatants were subsequently obtained, and tumor necrosis factor and interleukin 6 were quantitated by enzyme-linked immunosorbent assay. In other experiments, HT29 toxin A uptake, intestinal monolayer permeability, necrosis, and actin microfilament disruption were determined.
There was a threefold to fourfold decrease in tumor necrosis factor and interleukin 6 levels and similar decreases in toxin A uptake and permeability changes in intestinal epithelial cells with mucus or PC versus control. Intestinal epithelial cell necrosis was reduced by more than 50% with either mucus or PC versus control. The integrity of HT29 cell cytoskeleton was demonstrated by both the mucus layer of the HT29-MTX strain and by exogenous PC administration by phalloidin staining of actin microfilaments.
PC supplementation was effective in improving intestinal barrier defense against C. difficile toxin A challenge. PC administration may be a useful therapeutic adjunct in severe cases of C. difficile colitis or in patients who do not improve with conventional treatment.
在过去十年中，艰难梭菌结肠炎的发病率和严重程度急剧上升。疾病严重程度与艰难梭菌毒力因子有关，包括毒素A和B，以及患者的免疫状态。肠道黏液是肠道固有屏障功能的重要组成部分。磷脂酰胆碱（PC）是肠道黏液屏障的关键成分，外源性PC给药对溃疡性结肠炎患者具有治疗效果。我们在体外研究了外源性PC对艰难梭菌毒素对肠道屏障影响的保护作用。
将产生黏液的（HT29-MTX菌株）和不产生黏液的（HT29菌株）肠道上皮单层与添加到顶端培养基中的PC和艰难梭菌毒素A共培养。随后获得基底室培养上清液，并通过酶联免疫吸附测定法定量肿瘤坏死因子和白细胞介素6。在其他实验中，测定HT29毒素A摄取、肠道单层通透性、坏死和肌动蛋白微丝破坏情况。
与对照组相比，有黏液或PC的肠道上皮细胞中肿瘤坏死因子和白细胞介素6水平降低了三到四倍，毒素A摄取和通透性变化也有类似程度的降低。与对照组相比，黏液或PC均可使肠道上皮细胞坏死减少超过50%。HT29-MTX菌株的黏液层和通过肌动蛋白微丝的鬼笔环肽染色外源性给予PC均证明了HT29细胞细胞骨架的完整性。
补充PC可有效改善肠道屏障对艰难梭菌毒素A攻击的防御能力。在严重的艰难梭菌结肠炎病例或对传统治疗无改善的患者中，给予PC可能是一种有用的治疗辅助手段。
