Tocotrienol-rich Fraction Modulated Genes Responsible for Inflammation in Lipopolysaccharide-stimulated RAW 264.7 Macrophages
Sitti Rahma Abd Hafid *
Malaysian Palm Oil Board, No.6 Persiaran Institusi, Bandar Baru Bangi, Kajang Selangor D.E., Malaysia.
Maliya Azilah Mohammad Aini
Malaysian Palm Oil Board, No.6 Persiaran Institusi, Bandar Baru Bangi, Kajang Selangor D.E., Malaysia.
Nabiha Iran
Malaysian Palm Oil Board, No.6 Persiaran Institusi, Bandar Baru Bangi, Kajang Selangor D.E., Malaysia.
Irmaliayana Norisam
Malaysian Palm Oil Board, No.6 Persiaran Institusi, Bandar Baru Bangi, Kajang Selangor D.E., Malaysia.
Khairul Adzfa Radzun
Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Selangor, Malaysia.
Ammu Kutty Radhakrishnan
Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia.
*Author to whom correspondence should be addressed.
Abstract
Background: Inflammation plays a vital role in the pathogenesis of chronic non-communicable diseases (NCDs), the leading health issue worldwide. An earlier study reported that tocotrienol-rich fraction (TRF) showed better anti-inflammation effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.
Aim: This study aimed to investigate the anti-inflammatory effects of tocotrienol-rich fraction at the molecular level by looking at the genes that were differentially regulated and pathways affected in LPS-stimulated macrophages exposed to TRF using the microarray approach.
Methods: A microarray study was carried out in LPS-stimulated RAW 264.7 macrophages. Total ribonucleic acid (RNA) was extracted from the RAW 264.7 cells treated with TRF (10µg/mL), alpha-tocopherol (10 µg/mL) or LPS (10 ng/mL). Untreated cells served as control. Enrichment analyses, such as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), were conducted for genes listed in the differentially expressed genes (DEGs).
Results: The microarray analysis showed that the expression of five genes [Hamp, Interleukin-1a (IL-1a), IL-b, C-X-C motif chemokine ligand 2 (CXCL2) and colony-stimulating factor 3 (CSF3)] and one gene (SLC1A4), an amino acid transporter, was modulated (fold change 2, P< 0.05) in the TRF-treated cells. With a more stringent analysis (fold change 3, P < 0.05), only one gene (CSF3) was downregulated in the TRF-treated in RAW 264.7 cells. Analysis using the GO and KEGG pathways revealed interactions between pro-inflammatory agents such as tumor necrosis factor-alpha (TNFα) and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-B), as well as signaling pathways of interleukin (IL)-1 and IL-17.
Conclusion: TRF modulated the expression of genes responsible for acute and chronic inflammation that were part of the lipoxygenase (LOX) and cyclooxygenase (COX) inflammatory pathways. Further investigation on the effects of TRF in different cell lines and in vivo studies should be conducted in the future.
Keywords: Tocotrienol-rich fraction (TRF), inflammatory response, macrophages, gene regulation, ontology, KEGG pathway