Sawai T, Drongowski R A, Lampman R W, Coran A G, Harmon C M
Section of Pediatric Surgery, University of Michigan, Mott Children's Hospital, Ann Arbor, MI 48109-0245, USA.
Pediatr Surg Int. 2001 May;17(4):269-74. doi: 10.1007/s003830100592.
The activity of phospholipase A2 (PLA2) is elevated in the intestinal epithelia of patients with inflammatory bowel disease (IBD). We recently reported that PLA2 mediates hydrolysis of phosphatidylcholine (PC) to lysophosphatidylcholine (L-PC) when both are applied to the apical surface of cultured EC monolayers, resulting in increased bacterial translocation (BT) and decreased transepithelial electrical resistance (TEER). Free fatty acids (FFA) are the other products of this reaction, however, their effect on Caco-2 cell permeability has not been reported. In addition to PC, other luminal phospholipids are present at the surface of the enterocyte. PLA2 may also mediate the hydrolysis of luminal phospholipids other than PC. The aim of this study was to examine the effects of phospholipids other than PC and common FFA on intestinal epithelial permeability and BT. Human Caco-2 enterocytes were grown to confluence on porous filters in the apical chamber of a two-chamber cell-culture system. Monolayer integrity and tight-junction permeability were measured as TEER. First, common FFA released by PC hydrolysis were determined using thin-layer chromatography (TLC). In separate experiments, monolayers were treated with phosphatidylethanolamine (PE), lysophosphatidylethanolamine (L-PE), or palmitoleic acid, oleic acids, linoleic acids, and arachidonic acid solubilized in solution with PC. The magnitude of BT was determined 2 h after treatment by adding Escherichia coli C25 to the apical chamber followed by quantitatively culturing basal-chamber samples. Statistical analysis was by the Kurosaki-Wallis test. TLC of PC samples incubated with PLA2 on the apical surface of Caco-2 monolayers demonstrated the production of palmitoleic acid, oleic acids, linoleic acids, and arachidonic acid. L-PE significantly decreased TEER compared to controls, but to a lesser degree than L-PC alone. L-PE had no effects on BT. Palmitoleic acid and oleic acid likewise significantly decreased TEER compared to controls, however, less than L-PC. All FFA tested had no effect on BT. Phospholipids applied to the apical surface of enterocytes, such as those found in vivo in mucus, can be hydrolyzed by the enzyme PLA2 resulting in lysophospholipid and FFA species that can alter enterocyte monolayer permeability. However, FFA and L-PL, other than L-PC, appear to have no effect to stimulate BT. This observation may have clinical implications in the pathogenesis and treatment strategies for IBD patients in whom enterocyte PLA2 activity has been shown to be elevated.
炎症性肠病（IBD）患者肠上皮中磷脂酶A2（PLA2）的活性升高。我们最近报道，当将磷脂酰胆碱（PC）和溶血磷脂酰胆碱（L-PC）都应用于培养的内皮细胞单层的顶端表面时，PLA2介导PC水解为L-PC，导致细菌易位（BT）增加和跨上皮电阻（TEER）降低。游离脂肪酸（FFA）是该反应的其他产物，然而，它们对Caco-2细胞通透性的影响尚未见报道。除了PC外，肠上皮细胞表面还存在其他腔内磷脂。PLA2也可能介导PC以外的腔内磷脂的水解。本研究的目的是研究PC以外的磷脂和常见FFA对肠上皮通透性和BT的影响。人Caco-2肠上皮细胞在两室细胞培养系统顶端室的多孔滤器上生长至汇合。单层完整性和紧密连接通透性通过TEER来测量。首先，使用薄层色谱法（TLC）测定PC水解释放的常见FFA。在单独的实验中，单层用磷脂酰乙醇胺（PE）、溶血磷脂酰乙醇胺（L-PE）或溶解在含PC溶液中的棕榈油酸、油酸、亚油酸和花生四烯酸处理。处理2小时后，通过向顶端室加入大肠杆菌C25，然后对基底室样品进行定量培养来测定BT的大小。采用Kurosaki-Wallis检验进行统计分析。在Caco-2单层顶端表面用PLA2孵育PC样品的TLC显示产生了棕榈油酸、油酸、亚油酸和花生四烯酸。与对照组相比，L-PE显著降低了TEER，但程度小于单独的L-PC。L-PE对BT没有影响。与对照组相比，棕榈油酸和油酸同样显著降低了TEER，然而，低于L-PC。所有测试的FFA对BT均无影响。应用于肠上皮细胞顶端表面的磷脂，如在体内黏液中发现的那些磷脂，可被PLA2酶水解，产生溶血磷脂和FFA，它们可改变肠上皮细胞单层通透性。然而，除L-PC外，FFA和L-PL似乎对刺激BT没有影响。这一观察结果可能对IBD患者的发病机制和治疗策略具有临床意义，在IBD患者中，肠上皮细胞PLA2活性已被证明升高。
