不同链长脂肪酸对胎盘滋养细胞氧化应激和炎症反应的影响

Study on the oxidative stress and inflammation in trophoblast cells stimulated by different chain length fatty acids

目的探讨不同链长脂肪酸对胎盘滋养细胞氧化应激和炎症反应的影响。方法体外培养胎盘滋养细胞，将细胞分为5组，每组加入不同链长的脂肪酸孵育细胞，即分别为无游离脂肪酸（FFA—free，FFFA）组、短链脂肪酸（SC—FFA）组、中链脂肪酸（MC—FFA）组、长链脂肪酸（LC—FFA）组、极长链脂肪酸（VLC—FFA）组。每组再分别以DMEM培养基、还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶抑制剂（NADPH—I）和p38丝裂原活化蛋白激酶（p38MAPK）抑制剂（p38MAPK—I）孵育细胞。采用实时荧光定量PCR技术和蛋白印迹法检测各组细胞p38MAPK和环氧合酶2（COX-2）mRNA和蛋白的表达变化。结果（1）LC—FFA组＋DMEM、VLC—FFA组＋DMEM、LC—FFA组＋NADPH—I、LC—FFA组＋p38MAPK—I、VLC—FFA组＋NADPH—I、VLC—FFA组＋p38MAPK—I胎盘滋养细胞p38MAPKmRNA的表达量分别为4．56±0．28、22．65±2．40、0．87±0．06、1．02±0．15、19．87±1．93、10．22±0．75，蛋白的表达量分别为0．79±0．02、0．93±0．10、0．43±0．06、0．44±0．19、0．79±0．10、0．81±0．14。LC—FFA组＋DMEM、VLC—FFA组＋DMEM细胞p38MAPKmRNA和蛋白的表达量升高（P〈0．05）。与LC—FFA组＋DMEM比较，LC—FFA组＋NADPH-I、LC—FFA组＋p38MAPK—I细胞p38MAPKmRNA和蛋白的表达量明显降低（P〈0．05）。与VLC—FFA组＋DMEM比较，VLC—FFA组＋NADPH—I细胞p38MAPKmRNA和蛋向的表达量差异无统计学意义（P〉0．05）；VLC—FFA组＋p38MAPK—I细胞p38MAPKmRNA的表达量明显降低（P〈0．05），而蛋白的表达量无差异（P〉0．05）。（2）LC—FFA组＋DMEM、VLC—FFA组＋DMEM、LC—FFA组＋NADPH—I、LC—FFA组＋p38MAPK—I、VLC—FFA组＋NADPH—I、VLC—FFA组＋p38MAPK—I细胞COX-2mRNA的表达量分别为3．97±0．03、39．08±0．63、0．99±0．13、0．98±0．18、20．93±3．70、13．46±2．31，蛋白的表达量分别为1．32±0．20、1．33±0．25、0．594-0．13、0．58±0．30、0．88±0．18、0．91±0．24。与其他组比较，COX-2mRNA和蛋白的表达量在LC—FFA组＋DMEM、VLC—FFA组＋DMEM细胞明显升高（P〈0．05）。与LC—FFA组＋DMEM比较，LC—FFA组＋NADPH—I、LC—FFA组＋p38MAPK—I细胞COX-2mRNA和蛋白的表达量明显降低（P〈0．05）。与VLC—FFA组＋DMEM比较，VLC—FFA组＋NADPH—I、VLC—FFA组＋p38MAPK—I细胞COX-2mRNA和蛋白的表达量均降低（P〈0．05）。（3）相关性分析显示，在LC—FFA组中p38MAPK与COX-2在mRNA和蛋白水平上均呈正相关（r=0．657、0．916，P〈0．05）。在F．FFA、SC—FFA、MC—FFA、VLC—FFA组中，p38MAPK与COX-2在mRNA水平上无相关性（P〉0．05），而在蛋白水平上呈正相关（P〈0．05）。结论Lc—FFA和VLC—FFA刺激下的胎盘滋养细胞中存在氧化应激和炎症反应，该过程有p38MAPK信号通路参与。

Objective To investigate the oxidative stress and i,fflammation in trophoblast cells stimulated by different chain length fatty acids. Methods Serum-free trophoblast cells cultured in vitro were divided into five groups, which were incubated with DMEM medium without free fatty acid （F-FFA）, short chain fatty acids （SC-FFA）, medium chain fatty acids （MC-FFA） , long chain fatty acids （LC-FFA）, very long chain fatty acids （VLC-FFA）. Then cells in each group were stimulated by DMEM medium, reduced form of nicotinamide adenine dinucleotide phosphate （NADPH） oxidase inhibitor （apocynin） and p38 mitogen-activated protein kinases （p38MAPK） inhibitor （SB203580） and were subdivided as each FFA plus-DMEM group, plus-NADPH-I and plus-p38MAPK-I groups. Expressions of mRNA and protein of p38MAPK and cyclooxygenase 2 （COX-2） in trophoblast cells were detected by real-time PCR and western blot. Results （1） The mRNA expression of p38MAPK in LC-FFA ± DMEM, VLC-FFA ± DMEM, LC-FFA ± NADPH-I, LC-FFA ± p38MAPK-I, VLC-FFA ± NADPH-I, VLC-FFA ± p38MAPK-I group were 4.56 ±_0. 28, 22.65 ±_2.40, 0.87 ±0.06, 1.02 ±0. 15, 19.87 ± l. 93, 10. 22 ±0.75 separately, and the protein expressions were O. 79 ± O. 02, 0. 93 ± O. 10, 0. 43 ± 0. 06, O. 44 ± 0. 19, 0. 79 ± 0. 10, 0. 81 ± O. 14. Compared with other groups, the mRNA and protein expressions of p38MAPK in LC-FFA ± DMEM, VLC-FFA ± DMEM group were increased （P 〈 O. 05）. Compared with LC-FFA ± DMEM group, mRNA and protein expressions of p38MAPK in LC-FFA ± NADPH-I and LC-FFA ± p38MAPK-I group were significantly decreased （ P 〈 0. 05 ） . Compared with VLC-FFA ± DMEM group, mRNA and protein expressions of p38MAPK had no difference in VLC-FFA ± NADPH-I group （P 〉 O. 05 ）, mRNA expression of p38 MAPK in VLC-FFA ± p38MAPK-1 group was significantly decreased （P 〈 0. 05 ）, but there was no difference in protein expression （P 〉 0. 05 ）. （2） The mRNA expression of COX-2 in LC-FFA ± DMEM, VLC-FFA ± DMEM, LC-FFA ± NADPH-I, LC-FFA ± p38MAPK-I, VLC-FFA ± NADPH-I, VLC-FFA ± p38MAPK-I group were 3.97 ±0.03, 39. 08 ±0.63, 0.99 ,±0.13, 0.98 ±0.18, 20. 93 ±3.70, 13.46 ±2.31 separately, and the protein expressions were 1.32 ± O. 20, 1.33 ± O. 25, 0. 59 ± O. 13, 0. 58 ± O. 30, O. 88 ± O. 18, 0.91 ± O. 24. Compared with other groups, mRNA and protein expressions of COX-2 in LC- FFA ± DMEM and VLC-FFA ± DMEM group were significantly increased （ P 〈 O. 05 ）. Compared with LC- FFA ± DMEM group, mRNA and protein expressions of COX-2 in LC-FFA ± NADPH-I and LC-FFA ± p38MAPK-I group were decreased （P 〈 O. 05） . Compared with VLC-FFA ± DMEM group, mRNA and protein expressions of COX-2 in VLC-FFA ± NADPH-I and VLC-FFA ± p38MAPK-I group were all decreased （ P 〈 0. 05 ）. （3） The eorrelation analysis showed that there were significantly positive correlations between the mRNA and protein expressions of p38MAPK and COX-2 in LC-FFA group （P 〈 O. 05 ）. There were significantly positive correlations in protein expression （ P 〈 O. 05 ） , but no correlation in the mRNA expression between p38MAPK and COX-2 in the F-FFA, SC-FFA, MC-FFA, VLC-FFA groups （P 〉0. 05）. Conclusions The oxidative stress and inflammation may exist in trophoblast cells which were stimulated by LC-FFA and VLC-FFA. p38MAPK signal transduction pathway may contributed in this process.