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.
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.
(1) mRNA expression of LCHAD: the Δct 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 ± 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 ± 0.41. 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 < 0.05). 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 ± 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 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).
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.