游离脂肪酸对滋养细胞线粒体脂肪酸氧化功能的影响及其与p38MAPK信号通路的相关性

Interaction mechanism and influence between fatty acid oxidation and p38MAPK signal transduction pathway in trophoblast cells incubated with fatty acids

目的探讨不同链长游离脂肪酸对胎盘滋养细胞中线粒体长链脂肪酸氧化功能影响及其与p38MAPK信号通路的相关性。方法分别以无游离脂肪酸和短、中、长、极长链脂肪酸孵育胎盘滋养细胞（F．FFA、SC-FFA、MC-FFA、LC-FFA、VLC-FFA组）。再分别以DMEM／F12培养基、烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NADPH）抑制剂和p38MAPK抑制剂孵育细胞。采用荧光实时定量PCR（ReahimePCR）和蛋白印迹法（Westernblot）检测各组LCHAD基因和蛋白表达变化。结果（1）LCHAD的mRNA表达变化：F．FFA＋DMED、SC．FFA＋DMEM、MC-FFA＋DMEM、LC-FFA＋DMEM、LC-FFA＋NADPH-I、LC-FFA＋p38MAPK-I、VLC-FFA＋DMEM、VLC-FFA＋NADPH-I、VLC-FFA＋p38MAPK-I组LCHADmRNA的△ct值为4．57±0．12、4．36±0．09、4．55±0．10、6．84±0．42、4．45±0．24、5．08±0．36、2．23±0．15、3．90±0．32、3．81±0．40。与F-FFA各组相比较，LC-FFA＋DMEM／p38MAPK-I组LCHADmRNA相对表达量明显降低，差异有统计学意义（P〈0．05），而VLC各组均有明显升高，差异有统计学意义（P〈0．05）；与LC-FFA＋DMEM组相比较，LC-FFA＋NADPH-I／p38MAPK-I组LCHADmRNA相对表达量明显升高，差异有统计学意义（P〈0．05）；与VLC-FFA＋DMEM组相比较，VLC-FFA＋NADPH．I／p38MAPK．I组中LCHADmRNA相对表达量明显降低，差异有统计学意义（P〈0．05）。（2）LCHAD蛋白表达变化：F-FFA＋DMED、SC-FFA＋D1VIE／VI、Mc-FFA＋DMEM、LC-FFA＋DMEM、LC-FFA＋NADPH-I、LC-FFA＋p38MAPK-I、VLC-FFA＋DMEM、VLC-FFA＋NADPH-I、VLC-FFA’p38MAPK-I组LCHAD／B-肌动蛋白比率分别为23．6±13．0、21．2±10．2、19．7±1．9、10．6±2．6、14．0±1．8、14．0±2．8、29．3±1.9、35．8±3．2、35．2±4．5。与F-FFA各组比较，LC-FFA组LCHAD蛋白相对表达量降低，差异有统计学意义（P〈0．05），VLC-FFA＋NADPH-I／p38MAPK-I组LCHAD蛋白相对表达量升高，差异有统计学意义（P〈0．05）。结论游离脂肪酸影响人滋养细胞线粒体脂肪酸13氧化循环LCHAD的基因蛋白表达变化；长链脂肪酸刺激下的胎盘滋养细胞中存在脂肪酸B-氧化代谢障碍，NADPH、p38MAPK抑制剂能够缓解脂肪酸氧化代谢障碍，提示p38MAPK信号通路可能参与了该过程；极长链脂肪酸与脂肪酸B-氧化代谢存在有一定的相关性，其相互作用机制还有待进一步研究。

Objective To explore the interaction mechanism and influence between fatty acids oxidation and p38MAPK signal transduction pathway in trophoblast cells stimulated by fatty acids of different chain lengths. Methods Serum-free trophoblast cells cultured in vitro were divided into 5 groups, i.e. incubation with DMEM/F12 medium without FFA （F-FFA）, short-chain fatty acids （SC-FFA）, medium-chain fatty acids （MC-FFA）, long-chain fatty acids （LC-FFA） and very long-chain fatty acids （VLC- FFA）. Then cells in each group were stimulated by DMEM/F12 medium, NADPH oxidase inhibitor （Apocynin） and p38MAPK inhibitor （SB203580） and were subdivided into FFA plus-DMEM group, plus- NADPH-I and plus-p38MAPK-I groups. Expressions of mRNA and protein of LCHAD in trophoblast cells were detected by real-time polymerase chain reaction （PCR） and Western blot. Results （1） mRNA expression of LCHAD： the Act of mRNA of LCHAD in F-FFA ± DMED, SC-FFA ± DMEM, MC-FFA ± DMEM, LC-FFA ± DMEM, LC-FFA ± NADPH-I, LC-FFA ± p38MAPK-I and VLC-FFA ± DMEM, VLC- FFA ± NADPH-I, VLC-FFA ± p38MAPK-I groups were 4. 57 ± 0. 12, 4. 36 ± O. 09, 4. 55 ± 0. 10, 6. 84 ± 0.42, 4.45±0.24, 5.08 ±0.36, 2. 23 ±0.15, 3.90±0.32, 3.81 ±0.41. Compared with the F-FFA groups, the relative mRNA expressions of LCHAD significantly decreased in LC-FFA ± DMEM/p38MAPK-I groups（P 〈0. 05） while increased in VLC-FFA groups（P 〈 0. 05 ）. Compared with the LC-FFA ± DMEM groups, the relative mRNA expressions of LCHAD increased in LC-FFA ± NADPH-I/p38MAPK-I groups （P 〈 O. 05 ）. The relative mRNA expressions of LCHAD in VLC-FFA ± NADPH-I/p38MAPK-I groups significantly decreased versus VLC-FFA ± DMEM group （ P 〈 0. 05 ）. （ 2 ） Protein expression of LCHAD ： The relative protein expressions of LCHAD in F-FFA ± DMED, SC-FFA ± DMEM, MC-FFA ± DMEM, LC- FFA ± DMEM, LC-FFA ± NADPH-I, LC-FFA ± p38MAPK-I and VLC-FFA ± DMEM, VLC-FFA ± NADPH- I, VLC-FFA ± p38MAPK-I groups were 23.6 -± β.0, 21.2 ± 10. 2, 19.7 ± 1.9, 10. 6 ±2. 6, 14. 0 -± 1.8, 14. 0 ± 2. 8, 29. 3 ± 1.9, 35.8 ± 3.2 and 35.2 ± 4. 5 respectively. Compared with the F-FFA groups, the protein expressions of LCHAD significantly decreased in LC-FFA groups（P 〈0. 05 ）while increased in VLC- FFA ± NADPH-I/p38MAPK-I groups（P 〈 0. 05 ）. Conclusion Free fatty acids affect the gene and protein expressions of mitochondrial β-oxidation enzyme of LCHAD in trophoblastic cells. Fatty acid β-oxidation is impaired in trophoblast cells incubated with long-chain fatty acid. NADPH oxidase and p38MAPK inhibitors may alleviate such an effect. Thus p38MAPK signal transduction pathway may participate in this process. The correlation between very long chain fatty acids and fatty acid β-oxidation is confirmed. But their interactions require further explorations.