Hossain Zakir, Kurihara Hideyuki, Hosokawa Masashi, Takahashi Koretaro
Division of Marine Biosciences, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, 041-8611, Japan.
Mol Cell Biochem. 2006 Apr;285(1-2):155-63. doi: 10.1007/s11010-005-9074-6. Epub 2006 Feb 14.
The influence of docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-enriched phosphatidylcholine (PC) on the permeability, transport and uptake of phospholipids was evaluated in Caco-2 cells. The cells were grown on permeable polycarbonate transwell filters, thus allowing separate access to the apical and basolateral chambers. The monolayers of the cells were used to measure lucifer yellow permeability and transepithelial electrical resistance (TEER). Transcellular transportation of diphenylhexatriene (DPH) labeled-PC small unilamellar vesicles (SUV) from the apical to basolateral chamber, and uptake of the same SUV was monitored in the cell monolayers. Cell-membrane perturbation was evaluated to measure the release of lactate dehydrogenase and to determine the cell viability with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl) -5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay. The lucifer yellow flux was 1.0 and 1.5 nmol/h/cm(2) with 50 microM PC, and 17.0 and 23.0 nmol/h/cm(2) with 100 microM PC when monolayers of Caco-2 cells were treated with DHA- and EPA-enriched PC, respectively. TEER decreased to 24 and 27% with 50 and 100 microM DHA-enriched PC, and to 25 and 30% with 50 and 100 microM EPA-enriched PC, respectively. Our results show that DHA- and EPA-enriched PC increases tight junction permeability across the Caco-2 cell monolayer whereas soy PC has no effect on tight junction permeability. Transportation and uptake of DHA- and EPA-enriched PC SUV differed significantly (P < 0.01) from those of soy PC SUV at all doses. We found that PC SUV transported across Caco-2 monolayer and was taken up by Caco-2 cells with very slight injury of the cell membrane up to 100 microM PC. Lactate dehydrogenase release and cell viability did not differ significantly between the treatment and control, emphasizing that injury was minimal. Our results suggest that DHA- and EPA-enriched PC enhance the permeability, transport and uptake of PC SUV across monolayers of Caco-2 cells.
在Caco-2细胞中评估了富含二十二碳六烯酸(DHA)和二十碳五烯酸(EPA)的磷脂酰胆碱(PC)对磷脂通透性、转运和摄取的影响。细胞生长在可渗透的聚碳酸酯Transwell滤膜上,从而可以分别接触顶侧和基底外侧腔室。细胞单层用于测量荧光素黄通透性和跨上皮电阻(TEER)。监测二苯基己三烯(DPH)标记的PC小单层囊泡(SUV)从顶侧到基底外侧腔室的跨细胞转运以及相同SUV在细胞单层中的摄取。通过测量乳酸脱氢酶的释放来评估细胞膜扰动,并使用2-(4-碘苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯基)-2H-四唑钠染料还原试验来确定细胞活力。当用富含DHA和EPA的PC处理Caco-2细胞单层时,荧光素黄通量在50μM PC时分别为1.0和1.5 nmol/h/cm²,在100μM PC时分别为17.0和23.0 nmol/h/cm²。富含50和100μM DHA的PC使TEER分别降至24%和27%,富含50和100μM EPA的PC使TEER分别降至25%和30%。我们的结果表明,富含DHA和EPA的PC增加了跨Caco-2细胞单层的紧密连接通透性,而大豆PC对紧密连接通透性没有影响。在所有剂量下,富含DHA和EPA的PC SUV的转运和摄取与大豆PC SUV有显著差异(P < 0.01)。我们发现PC SUV可穿过Caco-2单层并被Caco-2细胞摄取,直至100μM PC时细胞膜仅有非常轻微的损伤。处理组和对照组之间乳酸脱氢酶释放和细胞活力没有显著差异,强调损伤最小。我们的结果表明,富含DHA和EPA的PC增强了PC SUV跨Caco-2细胞单层的通透性、转运和摄取。