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.