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

Whittingham-Dowd, JK

Abstract

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.

Citation

Whittingham-Dowd, J. Investigating the use of omega three fish oils as protective agents against cytokine-induced skeletal muscle damage. (Thesis). University of Salford

• Thesis Type: Thesis
• Deposit Date: Jul 30, 2021
• Award Date: May 1, 2012