Abstract
Pervious study showed that diets with high levels of docosahexaenoic acid (DHA) cause growth retardation in Megalobrama amblycephala. In order to explore the mechanisms, a feeding trial and a primary hepatocyte culture experiment were designed. In vivo, fish (average weight 26.40±0.11 g) were randomly divided into groups and fed three levels of DHA (0%, 0.2% and 1.6% diets, respectively.) for 8 weeks. The results showed that the final body weight and weight gain of fish fed 1.6% DHA were significantly lower than that of fish fed 0.2% DHA (P < 0.05). P53 gene and protein expression levels were significantly increased (P < 0.05), while expression of downstream Cyclin D1 and Cyclin E1 was significantly inhibited in the 1.6% DHA group when compared with the 0% DHA and 0.2% DHA groups (P < 0.05). In vitro, primary hepatocytes isolated from Megalobrama amblycephala incubated with 500 μM DHA showed significantly increased cell cycle arrest and apoptosis (P < 0.05), significantly increased P53 gene and protein levels (P < 0.05), and significantly decreased Cyclin D1 and Cyclin E1 levels (P < 0.05), when compared with other groups. When primary hepatocytes were incubated with DHA and a P53 inhibitor (pifithrin-α), P53 expression and P53-mediated signaling were inhibited, cell cycle progression recovered and apoptosis was reduced. In summary, high levels of DHA activated the P53/Cyclin pathway to induce cell cycle arrest. Inhibition of P53 activity may be a potential way of reducing the side effects of DHA on the growth of Megalobrama amblycephala.
Highlights
Docosahexaenoic acid (C22:6n-3; docosahexaenoic acid (DHA)) is an essential n-3 polyunsaturated fatty acid (n-3 PUFA) that is commonly used as a feeding supplement for aquatic animals (Hossain et al, 2012; Hong et al, 2017; Wang et al, 2019), pigs (Gabler et al, 2007; Li et al, 2009), chickens (Allen and Danforth, 1998; Howe et al, 2002), and other species
Protein Expression in Liver Compared with the control group, fish fed 1.6% DHA showed significantly higher hepatic ataxia telangiectasia mutated (ATM) and P53 protein levels (Figures 3A,C), whereas Cyclin D1, Cyclin E1, and Cyclin A2 protein levels were significantly lower (P < 0.05; Figures 3D,E,G)
DHA significantly influenced the ATM and P53 gene expression levels, which peaked at 500 μM DHA and decreased when the DHA concentration was increased to 1,000 μM
Summary
Docosahexaenoic acid (C22:6n-3; DHA) is an essential n-3 polyunsaturated fatty acid (n-3 PUFA) that is commonly used as a feeding supplement for aquatic animals (Hossain et al, 2012; Hong et al, 2017; Wang et al, 2019), pigs (Gabler et al, 2007; Li et al, 2009), chickens (Allen and Danforth, 1998; Howe et al, 2002), and other species. DHA supplementation at appropriate levels is beneficial to animals and humans, but an excess may produce adverse effects. DHA, a highly unsaturated fatty acid, is susceptible to attack by active free radicals, resulting in lipid peroxidation, cell membrane damage, and various physiological abnormalities and disease (Tappel, 1973). Some studies have focused on the damage caused by the peroxidation of DHA. A previous study reported that high DHA levels led to increased plasma urea concentration and fish redness (Glencross and Rutherford, 2011). There have been very few studies focusing on the damage caused by unoxidized DHA. Investigating whether there is possible damage from high levels of unoxidized DHA is important for human and animal nutrition
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