Supplementary information
Nimbolide attenuate the lipid accumulation-induced oxidative stress and antioxidant in primary rat hepatocytes
Ghedeir M. Alshammari*, Aristatile Balakrishnan and Thirunavukkarasu Chinnasamy*
Department of Food Science and Nutrition, College of Food and Agricultural Science, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia.
*Corresponding authors
Email: 1. or (GA);
2. (AB)
3. (TC; orcid id 0000-0001-6799-6234.)
Abstract
Background: Nimbolide is a bioactive compound found in Azadirachta indica. This work was devised to investigate the potential effects of nimbolide on intracellular lipid deposition and its associated redox modulation in primary hepatocytes (Heps).
Methods and Results:Lipid accumulation was induced in Heps cells by supplementing 1mM oleic acid (OA) for 24h which was marked by significant accumulation of lipids. The results demonstrated that nimbolide can decrease intracellular cholesterol, free fatty acids and triglycerides. Nimbolide may also improve hepatocytes function through itsantioxidanteffects by inhibiting oxidative DNA damage and lipid peroxidation by curtailing the reactive oxygen species levels. Further it also restore the mitochondrial potential, improving the endogenous antioxidant levels such as GSH and antioxidant enzymes activities. Nimbolide increased (P0.05) liver X receptor-α (LXRα), peroxisome proliferator-activated receptor-γ (PPARγ) and sterol regulatory element-binding protein-1(SREBP1) gene expression in Heps. The biological significance of nimbolide may involvehypolipidemiceffect, lipid peroxidation inhibition, DNA damage inhibition, ROS inhibition, restoring mitochondrial function, increases in GSH and SOD & CAT activities, and direct regulation of LXRα, PPARγ and SREBP1 gene expression.
Conclusions: Nimbolide may be used as effective lipid lowering compound and lipid deposition-induced Heps changes, however further in vivo studies are warranted.
Key words: Hypolipid; Nimbolide; Hepatocytes; Oxidative stress; DNA damage.
Supplementary table 1. Primer sequences used for real-time polymerase chain reaction (PCR)
S. No. / Gene name / Sequence (5'-3') / Product Size (bp)1 / LXRα / F: TTGCTCTGCTCA TAGCCA TC
R:ATGGAGACATAGGCATGCAG / 118
2 / PPARα / F:TGAAAGATTCGGAAACTGC
R: TTCCTGCGAGTATGACCC / 111
3 / SREBP-1c / F: CGCTACCGTTCCTCTATCAATGAC
R:AGTTTCTGGTTGCTGTGCTGTAAG / 140
4 / GAPDH / F: GCAAGTTCAACGGCACAG
R: GCCAGTAGACTCCAC GACAT / 140
Supplementary figure 1. Schematic representation of the mechanism of hypothesis.