A Novel Nutraceuticals Mixture Improves Liver Steatosis by Preventing Oxidative Stress and Mitochondrial Dysfunction in a NAFLD Model

Abstract

Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disease globally, and represents a health care burden as treatment options are very scarce. The reason behind the NAFLD progression to non-alcoholic steatohepatitis (NASH) is not completely understood. Recently, the deficiency of micronutrients (e.g., vitamins, minerals, and other elements) has been suggested as crucial in NAFLD progression, such that recent studies reported the potential hepatic antioxidant properties of micronutrients supplementation. However, very little is known. Here we have explored the potential beneficial effects of dietary supplementation with FLINAX, a novel mixture of nutraceuticals (i.e., vitamin E, vitamin D3, olive dry-extract, cinnamon dry-extract and fish oil) in a NAFLD model characterized by oxidative stress and mitochondrial function impairment. Steatosis was firstly induced in Wistar rats by feeding with a high-fat/high-cholesterol diet for 4 weeks, and following this the rats were divided into two groups. One group (n = 8) was treated for 2 weeks with a normal chow-diet, while a second group (n = 8) was fed with a chow-diet supplemented with 2% FLINAX. Along with the entire experiment (6 weeks), a third group of rats was fed with a chow-diet only as control. Statistical analysis was performed with Student’s T test or one-way ANOVA followed by post-hoc Bonferroni test when appropriate. Steatosis, oxidative stress and mitochondrial respiratory chain (RC) complexes activity were analyzed in liver tissues. The dietary supplementation with FLINAX significantly improved hepatic steatosis and lipid accumulation compared to untreated rats. The mRNA and protein levels analysis showed that CPT1A and CPT2 were up-regulated by FLINAX, suggesting the enhancement of fatty acids oxidation (FAO). Important lipoperoxidation markers (i.e., HNE- and MDA-protein adducts) and the quantity of total mitochondrial oxidized proteins were significantly lower in FLINAX-treated rats. Intriguingly, FLINAX restored the mitochondrial function, stimulating the activity of mitochondrial RC complexes (i.e., I, II, III and ATP-synthase) and counteracting the peroxide production from pyruvate/malate (complex I) and succinate (complex II). Therefore, the supplementation with FLINAX reprogrammed the cellular energy homeostasis by restoring the efficiency of mitochondrial function, with a consequent improvement in steatosis.

The Mitochondrial Trigger in an Animal Model of Nonalcoholic Fatty Liver Disease

“…In the present study, all tested diets led to a decrease in SOD2 and PrxIII amounts, which was significant in the HF-F group and was not prevented in the HF-F-D rats, thus demonstrating the mitochondrial oxidative stress induced by all diets. These findings agree with other reports showing marked oxidative stress inside mitochondria because of the obesogenic diets administered [ 23 , 31 ]. In particular, in liver from genetically modified mice, placed on an obesogenic diet, the diminished expression and activity of SOD2 have been described [ 32 ].The reduction of the mitochondrial scavenger protein PrxIII has been reported in various conditions featuring oxidative stress inside the organelles [ 33 ].…”

“…Other extra FFAs derive from hepatic “de novo” lipogenesis, through which the surplus dietary energy, mostly introduced as carbohydrates and fat, is partially used, leading to the massive deposition of triglycerides and resulting steatosis [ 30 ]. The imbalance of ETC activity in NAFLD models and patients has been largely described in the literature and thoroughly examined [ 31 ]. In the latter study, the activities of ETC complexes were analyzed and found to decrease for all complexes but Complex II in the NAFLD model.…”

Involvement of Ceramides in Non-Alcoholic Fatty Liver Disease (NAFLD) Atherosclerosis (ATS) Development: Mechanisms and Therapeutic Targets

“…The lock micronutrients have been reported as crucial in NAFLD progression [147]. Sangineto et al [148] explored the probable beneficial effects of dietary supplementation with FLINAX, an innovative composition of nutraceuticals (vitamin E, vitamin D3, olive dry-extract, cinnamon dry-extract and fish oil) in a NAFLD model characterized by oxidative stress and mitochondrial function damaged. The Flinax group significantly presented lower hepatic fat accumulation compared to non-supplemented.…”

“…Reduced: serum ceramide content reduced, (p < 0.05), hepatic triglyceride, ALT, AST, hepatic inflammation, amount of inflammatory cell; Bcl-2 expression restored, (p < 0.05); 2019 [87] Fenretinide Mice Lowered: plaque area 50.8% (p < 0.05), plasma lipid levels by 20.1%, (p < 0.05), and plasma ceramides; 2020 [156] Diet Mice Flinax reduced lipoperoxidation markers, hepatic fat accumulation restores complex I, III, V (ATP-synthase), lower peroxides levels, (p < 0.05), no difference in complex IV, and higher production of CPT1A and CPT2; 2021 [148] Alphamangostin Mice Inhibits: ceramide content, and, Inhibites aSMase activity, 2021 [146] Farnesoid X receptor Mice Lower: ceramide content, hepatic cholesterol levels, mRNA levels of Smpd3 elevates hepatic Cyp7a1 mRNA levels Repressed lesion areas in aortas and smaller atherosclerotic lesions; 2021 [120] Liraglutide Mice Gene expression Sptlc2, cerS4, and cerS6 decreased; C16 and C24 accumulation was limited (p < 0.05); Unchanged: saturated fatty acid, phospholipids with long chains-reduced, phospholipids with very long chains 2021 [132]…”

Preliminary Results of CitraVes™ Effects on Low Density Lipoprotein Cholesterol and Waist Circumference in Healthy Subjects after 12 Weeks: A Pilot Open-Label Study

“…Recently, various nutraceuticals with lipid-lowering effects have attracted much attention [ 18 , 19 ], including citrus-derivatives such as citrus peel extracts, citrus flavonoids, and citrus polymethoxyflavones [ 20 ]. Furthermore, epidemiological studies have demonstrated that the intake of citrus flavonoid-containing foods is associated with a decreased incidence of CVD [ 21 , 22 ].…”