ABSTRACT
AIM:To compare the synthesis efficiency of n3 and n6 very long chain polyunsaturated fatty acid ( VLC-PUFA ) by overexpressing ELOVL4 protein, providing guidance for treating Stargardt-like macular dystrophy (STGD3). METHODS:To establish recombinant adenovirus with the ELOVL4 protein and green fluorescent protein, transferred into cultured PC12 cells. The cells were divided into 3 groups: PC12, PC12 + Ad- GFP and PC12 + Ad-ELOVL4, former two groups serve as controls. ELOVL4 gene expression was quantified by qRT-PCRs. ELOVL4 protein was analyzed by Western - Blot ( WB ) . The transduced cells were treated with both EPA and AA (1:1). After 48h of incubation, cells were collected, total lipids extracted and fatty acid methyl esters prepared and analyzed by gas chromatography-mass spectrometry ( GC-MS) . RESULTS:When supplemented together, 20:5n3 (EPA) and 20:4n6 ( AA) were efficiently taken up at almost the same amounts in the PC12 cells regardless of ELOVL4 expression. The ELOVL4-expressing cells elongated both EPA and AA to a series of n3 and n6 VLC-PUFAs. From 20:5n3/EPA, 34:5n3 and 36:5n3 account for 0. 71% and 1.6%, respectively. From 20:4n6/DHA, 34:4n6 and 36:4n6 were only 0. 46% and 0. 61%, respectively. The total relative mol% of n3 VLC-PUFAs synthesized from EPA was almost two times that of n6 VLC-PUFAs synthesized from AA. CONCLUSION: ELOVL4 protein preferentially elongates n3 PUFA to VLC - PUFAs over n6 PUFA. Dietary supplementation of appropriate n3/n6 PUFAs may provide STGD3 patients with some therapeutic benefits.
ABSTRACT
Background Researches determined that ELOVL4 gene is a disease-causing gene of Stargard tmacular dystrophy and is a elongation enzyme of very long chain fatty acids.Stargardt type Ⅲ(STGD3) may be associated with the metabolism of extensive enzyme of very long chain fatty acids.To explore the effect of ELOVL4 in STGD3 and its relationship with the metabolism of extensive enzyme of very long chain fatty acids is of important clinical significance.Objective This study was to determine the role of ELOVL4 gene for the pathogenesis of STGD3.Methods Recombinant adenovirus type 5 carrying mouse ELOVL4 gene and green fluorescent protein (GFP) was transfected into pheochromocytoma cells (PC12 cells),and then the cells were divided into PC12 group,PC12+Ad5-GFP group and PC12+AdS-ELOVL4 group.Ad-GFP or Ad-ELOVL4 was added into the culture medium for 24 hours with the virus plasmid 1 × 104-2× 104 pfu/ml.Expression of ELOVL4 mRNA in the PC12 was quantified by quantitative real time PCR(qRT-PCR) and was described as relative value to the expression of RPL19.ELOVL4 protein was assayed by Western blot.The transfected cells were treated with arachidonic acid (AA,20:4n6),eicosapentaenoic acid (EPA,20:5n3) and docosahexaenoic acid (DHA,22:6n3) individually for 48 hours.The cells were collected,and total lipids were extracted,and fatty acid methyl esters were prepared and analyzed by gas chromatography-mass spectrometry (GC-MS).Results The relative expression levels of ELOVL4 mRNA in PC12 cells in the PC12+Ad5-ELOVL4 group,PC12+Ad5-GFP group and PC12 group were 0.833± O.138,0.027t±0.002 and 0.024 ±0.002,with a significant difference among the 3 groups (F =102.700,P =0.000),and relative expression levels of PC12+Ad-ELOVL4 were 30-40 folds more than those in the PC12 group and the PC12+Ad-GFP group.Western blot assay showed a stronger response band for ELOVL4 protein in the PC12+Ad-ELOVL4 group.GC-MS found that abundant polyunsaturated fatty acids (C28-C38) were synthesized by PC12 cells in the PC12+Ad-ELOVL4 group,with the more levels in C34 and C36.Conclusions ELOVL4 can promote the synthesis of C28-C38 polyunsatured fatty acid in PC12 cells,which offers a novel clue for the treatment of STGD3.