Table 1.

Main studies analyzing the effects of EVOO on NAFLD/MAFLD/MASLD.

Study Model	Intervention and Used Compounds	Dose	Population	Effect of Treatment	Reference
Cellular model of NASH (palmitic and oleic acid-treated HepG2 cells co-cultured with THP1-derived M1 macrophages and LX2 cells).	Cellular model: co-culture with Tyr at increasing dosages diluted in complete medium.	Cellular model: 0.5, 1, 2, and 5 μM	HepG2 and LX2 cells	Cellular model: reduction of FA accumulation in HepG2 cells and modulation of LX2 activation and macrophage differentiation.	[158]
Mouse model of NASH (high fructose—high-fat diet combined with CCl4 treatment).	Mouse model: Tyr administered daily by oral gavage for 10 weeks.	Mouse model:10 mg/kg	Male C57Bl6 mice	Mouse model: reduction of steatotic and fibrotic areas and hypertrophic hepatocytes without modification of Tg contents. Reduction of proinflammatory cells and reduction of prooxidant enzyme NOX1 and the mRNA expression of TGF-β1 and IL6.
Iron-induced NAFLD mouse model.	Iron-rich diet (200 mg iron/kg diet) vs. a control diet (50 mg iron/kg diet) with alternate EVOO supplementation for 21 days.	100 mg/day for 21 days	Male Wistar rats	Iron-rich diet-induced liver steatosis, oxidative stress, mitochondrial dysfunction, loss of PUFAs, increasing expression of lipogenic enzymes, and reducing those involved in FA oxidation. EVOO supplementation prevented iron-rich diet effects.	[159]
HFD-induced NAFLD mouse model.	Lard-based HFD vs. EVOO-based HFD vs. phenolic compound-rich EVOO HFD.	EVOO-based HFD: 41% kcals fat from EVOO, 2.92 mg of polyphenols/kg of mouse body weight/day	Female Ldlr−/−. Leiden mice	No differences were proven for liver steatosis. Both EVOO diets improved mice body weight and insulin sensitivity without effects on liver transaminases or increasing LDL liver collagen content. EVOO did not show effectiveness in preventing liver inflammation or fibrosis in gene expression analysis.	[160]
		Phenolic compound-rich EVOO HFD: 41% kcals fat from EVOO, 6.08 mg of polyphenols/kg of mouse body weight/day
Fish model: spotted seabass juveniles.	Fish model: normal-fat vs. HFD vs. normal fat + HT vs. HFD + HT.	Fish Model: HT 200 mg/kg for 8 weeks	Spotted seabass juveniles	Fish model: HT prevented HFD-increased fat deposition and oxidative stress in the liver.	[161]
Cellular model: zebrafish liver cell line.	Cellular model: Culture with addition of HT, cyclosporin A, and compound C.	HT: 50 µM for 24 h	Zebrafish liver cell line	Cellular model: HT promoted mitochondrial function and activated PINK1-mediated mitophagy. These processes were blocked by both cyclosporin A (mitophagy inhibitors) and compound C (AMPK inhibitor).
HFD-induced NAFLD mouse model.	Control diet vs. HFD vs. HFD plus n-3 LCPUFA vs. HFD plus EVOO vs. HFD plus n-3 LCPUFA and EVOO.	n-3 LCPUFA 100 mg/kg/die for 12 weeks; EVOO 100 mg/kg/die for 12 weeks	Male C57BL/6J mice	HFD caused liver steatosis (increased total fat, Tg, and free FA contents), glucose and lipid metabolism impairment (glucose, insulin, total cholesterol and Tg serum levels, and HOMA-IR), activation of inflammation (higher TNF-α and IL-6 serum levels), liver and plasma oxidative stress enhancement (decrease of GSH levels), depletion of n-3 LCPUFA hepatic content, increased lipogenic enzyme (ACC and FAS), and reduced lipolytic (CPT-1) enzyme activity. These modifications were either reduced or normalized to control diet values in mice subjected to HFD supplemented with n-3 LCPUFA and EVOO but not in mice subjected to HFD supplemented with n-3 LCPUFA or EVOO alone.	[162]
HFD-induced NAFLD mouse model.	Control diet vs. HFD vs. HFD plus oleacein supplementation.	20 mg/kg for 5 weeks	Male C57BL/6JolaHsd mice	Compared to HFD, mice who received HFD plus oleacein had glucose, cholesterol, and Tg serum levels, as well as liver histology similar to control diet mice. Oleacein positively increased insulin sensibility by modulating protein levels of FAS, SREBP-1, and phospho-ERK in the liver and by reducing body weight.	[163]
Iron=induced NAFLD mouse model.	Iron=rich diet (200 mg iron/kg diet) vs. a control diet (50 mg iron/kg diet) with alternate EVOO supplementation for 21 days.	100 mg/day for 21 days	Male Wistar rats	Compared to control diet, the iron-rich diet increased hepatic total fat, Tg and free FA contents, and serum transaminase levels; in addition, iron-rich diet reduced n-6 and n-3 LCPUFA hepatic and extrahepatic content, increasing n-6/n-3 ratios, and decreasing unsaturation index and Δ5-D and Δ6-D activity. All these changes were prevented by concomitant AR-EVOO supplementation.	[164]
HFD=induced NAFLD mouse model.	Control diet vs. HFD supplemented with DHA or EVOO or DHA + EVOO.	DHA (50mg/kg/day) for 12 weeks; EVOO (50mg/kg/day) for 12 weeks; DHA (50mg/kg/day) + EVOO (50mg/kg/day) for 12 weeks	Male C57BL/6J mice	DHA + EVOO supplementation in HFD mice significantly reduced hepatic steatosis (histologically proven, total fat, Tg, and cholesterol hepatic level reduction), oxidative stress (increase of total serum antioxidant capacity, reduction of liver GSH and 8-isoprostanes levels, and TBARS serum and liver levels), systemic inflammation (TNF-α and IL-6 reduction), and insulin resistance (HOMA-IR reduction) compared to DHA or EVOO supplementation alone. In addition, DHA + EVOO supplementation reduced the activation of lipogenic enzyme (ACC and FAS) and increased it for lipolytic (CPT-1) enzyme.	[165]
HFD=induced NAFLD mouse model.	Control diet vs. HFD supplemented with placebo or water with NO2- or EVOO or water with NO2- and EVOO	NO2- 150 μM; 10% (w/w) EVOO for 12 weeks; 150 μM NO2- and 10% (w/w) EVOO for 12 weeks	Female C57Bl/6J mice	EVOO consumption reduced body and liver weight; hepatic fat accumulation increased nitro-FA levels in plasma (higher in water with NO2- and EVOO group compared with the EVOO alone group) and improved hepatic mitochondrial function (enhancement of both complex II and complex V activity).	[166]
HFD=induced NAFLD mouse model.	Control diet vs. HFD vs. HFD + HOPO vs. HFD + EVOO.	HOPO 10% total fat intake for 12 weeks; EVOO 10% total fat intake for 12 weeks	Male SD rats	Both EVOO and HOPO HFDs reduced body weight gain, HOMA-IR, and liver steatosis (histologically proven, reduction of Tg liver levels) compared to HFD alone. HOPO + HFD reduced total cholesterol, LDL, and Tg serum levels. Both HOPO and EVOO significantly increased microbiota diversity and abundance of Bifidobacterium.	[167]
Osteoarthritis mice model obtained by anterior cruciate ligament transection.	Control diet vs. sicilian EVOO supplemented diet vs. Tunisian EVOO supplemented diet vs. Tunisian EVOO and leaf extract-supplemented diet.	All supplements were provided as 2.5 mL/100 g of chow (i.e., 2.25 g/100 g of chow of EVOO) for 7 days	Male Wistar rats	No differences in liver steatosis were found between groups.	[168]
HFD-induced NAFLD mouse model.	Regular diet vs. regular diet + vitamin D supplementation vs. regular diet + vitamin D restriction vs. HFD-butter + vitamin D supplementation vs. HFD-butter vs. + vitamin D restriction vs. HFD-EVOO + vitamin D supplementation vs. HFD-EVOO vs. + vitamin D restriction	Vitamin D supplementation: 4000 U.I./Kg; Vitamin D restriction: 0 U.I./Kg; HFD-butter and HFD-EVOO 41% energy intake from fats (no more specific data in terms of EVOO supplementation)	Sprague/Dawley male rats	All groups showed a NAFLD activity score between 0 and 2 (not diagnostic of steatohepatitis). Collagen I levels were greater in both HFD-butter + vitamin D supplementation and HFD-butter vs. + vitamin D restriction groups compared to other groups. IL-1 was mostly expressed in all vitamin D-restricted groups. IGF-1 and DKK-1 were reduced in all HFD-butter and HFD-EVOO diets. EVOO supplementation seemed to reduce collagen-1 liver production.	[169]
HFD-induced NAFLD mouse model.	Control diet vs. HFD-lard-based vs. HFD-EVOO-based vs. HFD-based on phenolics-rich EVOO.	Control diet: 13% energy from fat for 24 weeks; HFD-lard-based: 49% energy from fat for 24 weeks; HFD-EVOO-based: 49% energy from fat, 41.7% from EVOO for 24 weeks; HFD-based on phenolics-rich EVOO: 49% energy from fat, 41.7% from EVOO for 24 weeks	C57BL/6J mice	Compared with the HFD-lard-based mice, HFD based on phenolic-rich EVOO reduced total cholesterol and LDL (p < 0.001), increasing HDL (p < 0.01), whereas EVOO-based HFD reduced Tg (p < 0.001). Both EVOO-based diets reduced IFN-γ levels in serum and epididymal adipose tissue, whereas only HFD based on phenolic-rich EVOO reduced IL-6 levels compared to lard-based HFD. Both EVOO-based diets reduced NAFLD activity score, reducing hepatic lipid accumulation (p < 0.05) and modulating lipid metabolism (increased PNPLA3 expression (p < 0.05)) and composition (increased MUFAs (p < 0.05)).	[170]
65HFD-induced NAFLD mouse model.	Control diet vs. control diet + HT vs. HFD vs. HFD + HT	Control diet: 10% fat for 12 weeks; Control diet + HT: 10% fat + 5 mg/kg/day body weight for 12 weeks; HFD: 60% fat for 12 weeks; HFD + HT 60% fat + 5 mg/kg/day body weight for 12 weeks	C57BL/6J mice	HFD determined liver steatosis, inflammation (TNF-α, IL-6, and IL-1β), oxidative stress (GST), depletion of n-3 long-chain PUFAs (26% reduction), downregulation of PPARα and Nrf2, and upregulation of NF-κB. HT supplementation attenuated the metabolic alterations produced by HFD, normalizing the activity of Nrf2, reducing the drop in activity of PPARα, and attenuating the increment in NF-κB activation.	[171]
Prospective randomized human case-control study enrolling patients with high cardiovascular risk but no cardiovascular disease.	MD plus EVOO vs. MD plus mixed nuts vs. low-fat control diet.	EVOO: 1 L/week for 3 years; mixed nuts: 30 g/day (15 g walnuts, 7.5 g hazelnuts, and 7.5 g almonds) for 3 years	100 subjects (63 men aged 55–80 y and 37 women aged 60–80 y); MD enriched with EVOO: 34; MD supplemented with mixed nuts: 36; low-fat control diet: 30	Hepatic steatosis (assessed by magnetic resonance) was present in 8.8%, 33.3%, and 33.3% of the participants in the MD plus EVOO, MD plus nuts, and control diet groups, respectively (p = 0.027). Respective mean values of liver fat content were 1.2%, 2.7%, and 4.1% (p = 0.07). Median values of urinary 12(S)-hydroxyeicosatetraenoic acid/creatinine concentrations were significantly lower (p = 0.001) in the MD plus EVOO group (2.3 ng/mg) than in the MD plus nuts (5.0 ng/mg) and control (3.9 ng/mg) groups. No differences in adiposity or glycemic indexes were proven.	[172]
Prospective observational human study enrolling patients with NAFLD and metabolic syndrome.	MD plus EVOO with high concentration of oleocanthal.	EVOO: 32 g/day for 8 weeks	23 subjects (15 men and 8 women, age: 60 ± 11 years)	Intervention diet, compared to baseline, was associated with a reduction in body weight, waist circumference, BMI, alanine transaminase, and hepatic steatosis (evaluated by fatty liver index), as well as IL-6, IL-17A, TNF-α, and IL-1B, while IL-10 increased. Maximum subcutaneous fat thickness increased, with a concomitant decrease in the ratio of visceral fat layer thickness/subcutaneous fat thickness.	[173]
Prospective cohort human study (1-in-5 random sample study drawn from the electoral list of Castellana Grotte, Italy.	MD plus EVOO at different dosages according to standardized and validated food questionnaire.	EVOO consumption was categorized into four levels: low: <20 g/die; medium–low: 21–30 g/die; medium–high: 31–40 g/die; high: >40 g/die	2754 subjects divided according to EVOO consumption levels: low: 645 (male 340; age 46.38 ± 13.00); medium–low: 635 (male 353, age 52.15 ± 13.99); medium–high: 595 (male 346, age 56.9 ± 14.77); high: 879 (male 522, age 61.77 ± 13.54)	There was a significant negative effect on mortality for the whole sample when EVOO consumption was used, both as a continuous variable and when categorized. The protective effect was stronger in the sub-cohort with NAFLD (778 subjects), especially for the highest levels of EVOO consumption (hazard ratio = 0.58, p < 0.05).	[174]

ACC: acetyl-coa carboxylas; AMPK: adenosine monophosphate-activated protein kinase; BMI: body mass index; CPT-1: carnitine palmitoyltransferase 1; DHA: docosahexaenoic acid; DKK: DicKKopf-related protein; ERK: extracellular signal-regulated kinase; EVOO: extra virgin olive oil; FA: fatty acids; FAS: fatty acid synthase; GSH: glutathione; GST: glutathione-S-transferase; HFD: high-fat diet; HOMA-IR: homeostasis model assessment for insulin resistance; HOPO: high oleic acid peanut oil; HT: hydroxytyrosol; IFN: interferon; IGF: insulin-like growth factor; IL: interleukine; LCPUFA: long-chain polyunsaturated fatty acids; LDL: low-density lipoprotein; MAFLD: metabolic dysfunction-associated fatty liver disease; MASLD: metabolic dysfunction-associated steatotic liver disease; MD: Mediterranean diet; MUFAs: monounsaturated fatty acids; NAFLD: non-alcoholic fatty liver disease; Nrf2: nuclear factor erythroid 2–related factor 2; NASH: non-alcoholic steatohepatitis; PNPLA3: patatin-like phospholipase domain-containing protein 3; PPAR: peroxisome proliferator-activated receptor; PUFAs: polyunsaturated fatty acids; SREBP-1: sterol regulatory element-binding protein 1; TBARS: thiobarbituric acid reactive substances; Tg: triglycerides; TNF: tumor necrosis factor; Tyr: tyrosol.