Objective: To investigate the effects of short chain fatty acid (SCFA) on barrier disruption of human intestinal epithelial cell induced by endotoxin/lipopolysaccharide (LPS) and the related mechanism. Methods: The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. Cells were divided into control group, LPS group, and SCFA+ LPS group according to the random number table. Cells in control group were only routinely cultured with DMEM medium. Cells in LPS group were cultured with DMEM medium and LPS with final mass concentration of 10 μg/mL. Cells in SCFA+ LPS group were cultured with DMEM medium, LPS and SCFA (consisting of 0.5 mmol/L acetate, 0.01 mmol/L propionate, and 0.01 mmol/L butyrate) with final mass concentration of 10 μg/mL. At post culture hour (PCH) 0, 1, 2, 6, 12, and 24, transepithelial electrical resistance (TER) of cells was determined with an ohmmeter, with sample number of 72. Another portion of cells were divided and treated as above, and then Western blotting was employed to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, and claudin-1 at PCH 24, with sample number of 6. Another portion of cells were divided and treated as above and then immunofluorescence was used to observe cellular morphology and distribution of ZO-1. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) Compared with that in control group, TER of cells in LPS group was significantly reduced from PCH 1 to 24 (P<0.01), while TER of cells in SCFA+ LPS group showed no obvious change (P>0.05). TER of cells in SCFA+ LPS group was significantly higher than that in LPS group from PCH 1 to 24 (P<0.01). (2) Compared with the protein expressions of ZO-1, occludin, and claudin-1 of cells in control group (1.25±0.10, 1.17±0.04, and 1.24±0.20), those of cells in LPS group (0.74±0.23, 0.76±0.11, and 0.77±0.11) at PCH 24 were significantly decreased (P<0.05), while those of cells in SCFA+ LPS group (1.23±0.46, 1.05±0.09, and 1.01±0.13) showed no significant differences (P>0.05). Protein expressions of occludin and claudin-1 of cells in SCFA+ LPS group were significantly higher than those in LPS group at PCH 24 (P<0.05). Protein expression of ZO-1 of cells in SCFA+ LPS group was higher than that in LPS group at PCH 24 with no significant difference (P>0.05). (3) At PCH 24, cells in control group were compact in arrangement with pebble-like appearance, and ZO-1 was distributed smoothly and continuously along the cell membrane. In LPS group, cells were sparse in arrangement with change in appearance, and ZO-1 was distributed uncontinuously along the cell membrane with curls and breaks. In SCFA+ LPS group, the appearance of cells and distribution of ZO-1 were remarkably ameliorated compared with those in LPS group. Conclusions: SCFA can alleviate the barrier disruption of human intestinal epithelial cell induced by LPS through interdicting the abnormal distribution of ZO-1 and decrease of TER and tight junction proteins' expressions.
目的： 探讨短链脂肪酸(SCFA)对内毒素/脂多糖(LPS)引起的人肠上皮细胞屏障功能损害的作用及相关机制。 方法： 建立人肠上皮细胞株Caco－2单层细胞培养模型。取细胞按随机数字表法分为对照组、LPS组及SCFA＋LPS组。对照组细胞仅采用DMEM培养液常规培养，LPS组细胞在DMEM培养液中另加入终质量浓度为10 μg/mL的LPS，SCFA＋LPS组细胞在DMEM培养液中另加入终质量浓度为10 μg/mL的LPS和SCFA(由0.5 mmol/L乙酸、0.01 mmol/L丙酸、0.01 mmol/L丁酸组成)的混合液培养。于培养0、1、2、6、12、24 h，取细胞采用电阻测定仪测定细胞的跨上皮电阻(TER)，样本数为72。另取细胞同前分组及处理后，采用蛋白质印迹法于培养24 h后检测带状闭合蛋白1(ZO－1)、咬合蛋白及闭合蛋白1表达，样本数为6。另取细胞同前分组及处理后，采用免疫荧光法检测细胞形态及ZO－1的分布。对数据行析因设计方差分析、单因素方差分析、LSD检验及Bonferroni校正。 结果： (1)与对照组相比，LPS组细胞培养1～24 h的TER均明显降低(P<0.01)，SCFA＋LPS组细胞培养1～24 h的TER无明显变化(P>0.05)。SCFA＋LPS组细胞培养1～24 h的TER均较LPS组明显升高(P<0.01)。(2)与对照组的1.25±0.10、1.17±0.04、1.24±0.20相比，LPS组细胞培养24 h ZO－1、咬合蛋白及闭合蛋白1的表达量(0.74±0.23、0.76±0.11、0.77±0.11)均明显降低(P<0.05)，而SCFA＋LPS组细胞培养24 h ZO－1、咬合蛋白及闭合蛋白1的表达量(1.23±0.46、1.05±0.09、1.01±0.13)均与之相近(P>0.05)。与LPS组相比，SCFA＋LPS组细胞培养24 h的咬合蛋白和闭合蛋白1表达量均明显增加(P<0.05)，ZO－1表达量增加但差异无统计学意义(P>0.05)。(3)培养24 h对照组细胞排列紧密，近似鹅卵石样外观；ZO－1沿细胞膜呈连续、规则排列。LPS组细胞排列稀疏且形态改变；ZO－1沿细胞膜不规则排列，且偶有断裂和卷曲。SCFA＋LPS组细胞形态和ZO－1排列均较LPS组明显改善。 结论： SCFA可通过阻断LPS引起的TER下降、紧密连接蛋白表达下降和ZO－1分布异常，从而减轻LPS对人肠上皮细胞屏障的损害。.
Keywords: Barrier function; Claudins; Fatty acids; Human intestinal epithelial cell; Lipopolysaccharides.