3.4 Antioxidant enzymes activity and oxidative stress status
No significant differences were observed in antioxidant enzymes activity
and OS status between groups at baseline (Table 5). Mean levels of MDA
(-0.70 nmol/mL, -18.75%), 8-iso-PGF2α (-6.65 pg/ml, -19.55%) reduced
significantly in the intervention group but not in the placebo group. A
significant elevation was observed in the mean levels of TAC (0.35
mmol/L, 31.82%), and SOD (165.50 U/g Hb, 10.22%) in the intervention
group compared with the placebo group. Changes in the levels of uric
acid (0.40 mg/dL, 6.66%), GSH-Px (2.15 U/g Hb, 6.42 %), and catalase
(6.81 U/g Hb, 10.20%), were not statistically significant in the
intervention group compared with the placebo group (P<0.05,
adjusted for baseline values, sex, weight changes and energy intake).
TAC, SOD, GSH-Px, 8-iso-PGF2α and MDA levels were significantly altered
in the intervention group compared with baseline, but catalase and uric
acid remained unchanged (P<0.05, paired T-test). No
significant within group differences were observed for the oxidative
stress/antioxidant parameters in the SFO group (P > 0.05,
paired Student t -test).
4. Discussion
To the best of our knowledge, our study was the first randomized
clinical trial to assess the combined effects of caloric restriction and
CSO on NAFLD penitents. Our findings showed that CSO, as a rich plant
source of OM3FA may exert beneficial effects on glycemic indices,
inflammation, ME and oxidative stress/antioxidant status in NAFLD
patients. Dietary intake of CSO significantly decreased energy,
carbohydrate, and fat intake, body weight, BMI, insulin, HOMA-IR,
hs-CRP, LPS, 8-iso-PGF2α, MDA, and significantly increased TAC and SOD
in our study. However, changes of FPG, QUICKI, catalase, GSH-Px, and
uric acid were not significant in the intervention group
Weight management has been suggested as one of the most applicable
strategies for NAFLD treatment. It has been reported that a 5%
reduction in BMI results in a 25% decrease in the lipid content of the
liver.29 Our results on body weight and BMI are in
agreement with Rezaei et al. 4 and with a systematic
review and meta-analysis of FSO supplementation on body weight in
overweight and obese adults.30 However, some trials
did not report the beneficial impacts of the CSO on anthropometric
indices. In a study which investigated the effect of CSO and canola oil
on anthropometric indices in postmenopausal women for six weeks, a
significant changes in WC observed in both groups and a significant
reduction was found in waist-to-hip ratio only in the CSO group. While,
weight and BMI changes were not significant.31 The
weight-decreasing effect of CSO may be attributed to a modulation of the
GM, ME, leptin, ghrelin, adiponectin, peroxisome proliferator-activated
receptors (PPARs) gene expression, gut hormones, including PYY and GLP-1
and short chain fatty acid (SCFA) production, fatty acid synthesis and
oxidation through up-regulating β-oxidation gene expression, like
carnitine palmitoyltransferase-1 (CPT-1) and peroxisome
proliferator-activated receptor α (PPARα) and repressing lipogenic genes
expression such as sterol regulatory element binding proteins (SREBPs),
carbohydrate-responsive element-binding protein (ChREBP) and PPA32 which regulate food intake and energy
expenditure.31
The results of our study showed a significant improvement in insulin
levels and HOMA-IR after 12 weeks of supplementation with CSO. While, no
significant reduction was observed in FPG and QUICKI levels at the end
of the intervention. The effects of dietary intervention with CSO on
glycemic indices has been reported in subjects with
IGM.19 A systematic review reported a 0.2 reduction in
IR after OM3FA supplementation.33 In agreement with
the present study, Hutchins et al reported that the FSO supplementation
as another OM3FA rich plant oil reverses IR.13However, Schwab et al reported that CSO oil did not affect fasting, post
load plasma glucose or serum insulin concentrations in
IGM.19 Furthermore, in a study by Hajiahmadi et al.
significant improvements in FPG and insulin concentrations were found in
pre-diabetic patients who were supplemented with 2000 mg FSO for 14
weeks.34 These conflicting findings may be attributed
to designed diet, omega-6 to omega-3 ratio in diet, placebo group, basal
levels of glycemic indices, trial duration, type and dosage of
supplementation, as well as medical condition of patients.
IR as a main cause of NAFLD, augments lipid aggregation, and stimulates
inflammation in hepatocytes 29. On the other hand, it
was reported that development of IR and consequently NAFLD are
associated with dysbiosis. In the presence of dysbiosis, there is an
increased production of LPS from Gram-negative bacteria or ME1 that exacerbates IR via enhancement of the
expression of pro-inflammatory cytokines such as tumor necrosis factor
(TNF-α), interleukin (IL)-1, and IL-6 35, reducing
insulin action through inhibiting phosphorylation of the insulin
receptor substrate (IRS)-1 and IRS-2 and reducing or even suppressing
the IRS-1 and IRS-2 expression.36 It was reported that
OM3FA improve ME 37 via SCFAs
production.38 SCFAs as byproduct of OM3FA may
alleviate IR via inducing GLP-1 secretion from L cells in the colon39, activating intestinal gluconeogenesis, and
exerting beneficial effects on host glucose and energy homeostasis.
Thus, CSO supplementation may promote hypoglycaemic effects via reducing
energy intake, weight, LPS levels, and SCFAs production.
Another outcome of CSO supplementation in patients with NAFLD was
inflammation reduction via modulation of ME and inflammatory biomarkers.
ME proposed as a triggering factor for the systemic
inflammation.40 Limited animal studies reported
beneficial effects of OM3FA on endotoxin levels. It has been shown that
post-prandial ME caused by coconut oil decreased in pigs following fish
oil supplementation.41 In another animal study,
feeding high levels of omega-6 in mice led to increased levels of ME,
which were dramatically reduced in transgenic mice with the ability to
convert omega-6 to OM3FA.14 In a recent animal study,
dietary FSO in diabetic rats improved ME via modulating GM as well as
SCFAs. Additionally, a negative relationship was reported between ME
with Bacteroidetes and Alistipes and a positive relationship with
Blautia and Firmicutes.38 It is believed that exposure
to LPS are related to activating the NF-κB pathway leading to
inflammation in NAFLD patients.1,5 Probably, LPS
binding to TLR4 resulted in production of inflammatory cytokines such as
NFkB, IL-6 and interferon gamma (IFNγ) and ME.42Possibly CSO, as a source of OM3FA decreases systemic inflammation and
subsequently ME 43 via inhibiting the growth of
Bilophila wadsworthia and increasing the growth of A. muciniphila and
bifidobacteria, decreasing postprandial lipaemia involved in
chylomicron-LPS complex transport, influencing lipids transportation
through the intestinal barrier cells phospholipid membranes, increasing
endogenous activity of intestinal ALP involved in LPS production and
intestinal permeability improvement, and inhibiting the TLR4-induced
signaling pathway through modulation of the G protein-coupled receptor
(GPR)-120.37 Furthermore, anti-inflammatory effects of
OM3FA may be related to the suppression of the formation of
omega-6-derived inflammatory lipid derivatives, production of
omega-3-derived endocannabinoids, ethanolamides and oxylipins, as well
as endocannabinoid system modulation.5,44
In the current study, another effect of CSO in patients with NAFLD was
the modulation of OS biomarkers. Our study is in agreement with a recent
preclinical study investigating the CSO effect on OS parameters in mice
with irritable bowel syndrome (IBS). The authors found that CSO
intervention caused a significant reduction in MDA levels, as well as
increases in SOD and GHP-x levels.45 The effect of CSO
on OS has been examined only in one clinical trial and reported no
remarkable changes in urinary prostanoids in patients with
IGM.22 Han et al. found that FSO consumption led to a
significant reduction in MDA levels and a significant increase of GSH
and SOD in mice.17 The underlying mechanisms of the
effect of CSO on OS is not fully explored. Several studies indicated
that ROS contributes to development of NAFLD via increasing IR, lipid
peroxidaition, inflammation and ME.46 OM3FA may reduce
OS by suppressing the IκB kinase (IKK) responsible for dissociation of
nuclear factor-kappa B (NF-κB) from IKB-α as a modulator of
pro-inflammatory cytokine production.47 Furthermore,
ALA is a precursor of long-chain docosahexaenoic acid. It has been shown
that docosahexaenoic acid increases the activity of glutathione
reductase, GSH-Px, and SOD and decreases MDA concentration in mice with
nonalcoholic steatohepatitis.48 Additionally,
favorable effects of intervention on OS biomarkers can be attributed to
the presence of high levels of natural antioxidants such as tocopherols,
carotenoids and phytosterols in CSO 11 that may exert
inhibitory effects on lipid peroxidation and ROS production. Other
possible underlying mechanism associated with changes in the GM.
Costantini et al.
reported that OM3FA intake stimulated the growth of A. muciniphila,
Bifidobacteria, Lactobacilli, Faecalibacterium, Roseburia, and inhibited
some pathogenic bacteria, such as the Enterobacteriaceae, Clostridium,
and Streptococcus.49 It was reported that A.
muciniphila alleviated OS in diabetic rats 50 due to
having thiol specific antioxidant proteins such as typical 2‐Cys
Peroxiredoxins as a family of thioredoxin (Trx)-scaffold enzymes and
ubiquitous.51 Bifidobacterium longum and Lactobacillus
acidophilus inhibited lipids peroxidation by scavenging ROS52, reducing MDA levels and increasing SOD and TAC
levels.5 Also, Clostridium perfringens, probably,
triggers OS by α-toxin production, and activation of mitogen-activated
protein kinase/ERK kinase/extracellular-signal-regulated kinase
(MEK/ERK), protein kinase C, and NF-κB pathways.53Short-chain fatty acids such as butyrate reduces ROS production via
suppression of IKK 54, increases in plasma antioxidant
enzymes production, modulation of FOXO3A and MT2 transcription through
histone deacetylases inhibition 55, reduction of
colonic myeloperoxidase activity 56 and the MAPK/ERK,
p38/MAPK and c-Jun N-terminal kinase activation.57Finally, CSO may protect against IR, OS damage, ME, and inflammation by
improving gut microbial dysbiosis (Fig 2).
Limitations in this study included lack of assessment of gut and fecal
microbial composition, serum fatty acids, glucose clamp, serum SCFA, and
other inflammatory/oxidative stress biomarkers. However, this study is
the triple-blind, placebo-controlled, randomized clinical design and
stratification by BMI, gender and age factors, which eliminates
inter-individual variation. Also, it was the first study to evaluate the
impact of CSO on glycemic indices, inflammatory and OS parameters, and
ME in NAFLD patients.