Investigating the use of omega three fish oils as protective agents against cytokine-induced skeletal muscle damage

Whittingham-Dowd, JK 2012, Investigating the use of omega three fish oils as protective agents against cytokine-induced skeletal muscle damage , PhD thesis, University of Salford.

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Introduction Muscle injury and disease impacts on the lives of adults and children, contributing to severe morbidity and increased mortality e.g. Muscular dystrophy affects over 70,000 people UK wide (Muscular Dystrophy UK). Treatment of muscle disorders contribute to unemployment rates and cost the NHS billions of pounds per year. Growth factors and cytokines regulate skeletal muscle homeostasis and growth and cytokine dysregulation may cause irreversible damage. Currently, poorly effective therapies for muscle injury include non-steroidal/ steroidal anti-inflammatory drugs and physiotherapy. Previous research indicates omega three fish oils including Eicosapentaenoic acid (ERA), may offer protective anti-fibrotic properties and favoring useful repair processes. ERA decreases muscle protein degradation in cancer patients (Smith et al 2005) and increases the rate of protein synthesis in elderly patients (Smith et al 2011). In vitro studies show ERA down-regulates chronic inflammation via inhibition of NFKB activation (Huang et al 2011) and fibrosis via suppression of Transforming Growth Factor-Pi (TGF-pi,Das 2004). Our group showed ERA protection from cytokine (TNFa) induced muscular atrophy in a murine cell model (Magee et al 2008). Further, ERA may have intracellular signaling component interactions and be dependent on PPARy (Magee et al 2012). ERA and DMA interact with lipid rafts (Li et al 2005) therefore may be able to displace receptors from signaling domains. Development of omega 3 fatty acid for therapeutic uses in fibrosis and tissue repair requires a deeper understanding of additional intracellular signaling components, which is facilitated by a wider range of in vitro models. Objectives To establish whether ERA, and its relative Docosahexaenoic acid (DMA), could prevent cytokine-induced damage in models of atrophy and pathological fibrosis and identify some of the mechanisms involved. Methods Using the cell line, C2C12, a well established model of myogenesis, ERA and DMA were investigated to determine if they could attenuate TGFpi induced muscular fibrosis and TNFa/ TNF-related weak inducer of apoptosis (TWEAK) induced skeletal muscle inflammation/wasting. Myogenesis was assessed by morphology and myotube measurements or by qPCR of myogenic genes. Fibrosis was assessed by expression of fibrotic proteins by immunocytochemistry or fibrotic genes by qPCR. Inflammation (IL-6 release) was assessed by ELISA. Signaling mechanisms were investigated using antagonists to: Peroxisome Proliferator-Activated Receptor-y (PPARv) and the sphingosine kinase pathway. ERA and DMA effects on cytokine receptors were assessed by flow cytometry. Results ERA and DMA prevented decreases in myogenesis induced by all three cytokines that reduced the myotube diameter and myogenic index of differentiating and differentiated C2C12 cells. When used as a co-treatment or 2 hour pre-treatment, ERA and DMA returned myotube diameters to control levels and significantly increased myogenic index compared to cytokine alone (p<0.05). ERA alleviated TGFinduced myogenic gene expression changes. ERA and DMA also prevented TGFinduced increases in proliferation during low serum conditions. ERA and DMA attenuated TNFa and TWEAK induced IL-6 release and reduced TGFpi induced expression of fibrotic markers at both the level of gene and protein expression. PPARy blockade rendered ERA and DMA no longer effective in preventing TNFa and TWEAK-induced decreases in myotube diameter and myogenic index and increased IL-6 production. Blocking PPARy had no effect on ERA and DHA's ability to attenuate TGF(3-induced decreases in myogenesis. Antagonising PPARa had no effect on ERA and DMAs ability to attenuate TWEAK associated decreases in myogenesis or increased IL-6 production. ERA and DMA prevented TGFp-induced increases in the ratio of Sphingosine-1-phosphate receptor 3 (S1Pr3) to S1Pr1 gene expression and increased Sphingosine Kinase 1 gene expression There were no significant interactions of ERA and/or DMA with TGF(3, TNFa and TWEAK receptors (TGFpRII, TNFRI and Fn14 respectively). Conclusion Our findings suggest ERA and DMA are potentially useful 'neutriceuticals' that reduce fibrosis and improve muscle repair in vivo. Their mode of action is PPARy dependent in inflammatory skeletal damage (TNFa and TWEAK) but PPARy independent to profibrotic TGF(3. EPA and DMA attenuate inflammation PPARa independently. Taking this work further into other in vitro models or in vivo models will establish whether these omega-3 fatty acids have the ability to be used as therapeutic agents alone or in conjunction with other medicines to alleviate suffering caused by skeletal muscle damage.

Item Type: Thesis (PhD)
Contributors: Allen, J (Supervisor)
Schools: Schools > School of Environment and Life Sciences
Depositing User: Institutional Repository
Date Deposited: 30 Jul 2021 09:33
Last Modified: 04 Aug 2022 11:23

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