Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide, and it is probably to become the leading cause of end-stage liver disease in the next decades ^[1]. The disease encompasses a broad spectrum of liver damage, from steatosis, that refers to more than 5% ectopic fat accumulation in the liver, to steatohepatitis, and leads to more severe complications including fibrosis, cirrhosis, and even hepatocellular carcinoma ^[2]. Over the years, NAFLD was indicated as the hepatic manifestation of metabolic syndrome. Recently, an international panel of experts have proposed the change of the terminology of this disease, right now NAFLD being defined like metabolic-associated fatty liver disease (MAFLD) ^[3][4]. This new terminology intents to comprise the strict connection between hepatic steatosis and metabolic disorders such as T2DM, obesity, dyslipidemia, and hypertension, which increase the risk of disease progression and development of NASH and fibrosis ^[5].

NAFLD became a public health issue due to the fact that almost 25% of the world’s population is affected ^[6]. The increased rates of obesity and overweight in the last decades has led to the increase in the worldwide prevalence of NAFLD, especially in obese patients in which the prevalence is up to 75% ^[7]. A worrying phenomenon is the increase of NAFLD prevalence among children, estimated to be around 7.6%, rising up to 50–70% among obese children ^[8].

Initially, the pathogenesis of NAFLD started from the “two-hit theory” ^[9]. Based on current knowledge, the widely accepted theory is the “multifactorial theory” ^[10]. “Multiple-hit” hypothesis states that the pathogenesis of NAFLD results from a combination of genetic predisposition and environmental factors, like high-fat diet, sedentary lifestyle, sleep deprivation, along with lipotoxicity, altered gut-microbiome, cytokines, and adipocytokines ^[11][12]. The first step in the disease is represented by the fat accumulation in the liver ^[10]. Simultaneously, there is a complex network of interrelations between various organs, the initial stage of NAFLD development not being limited to liver involvement. The second hits in NAFLD includes the effect of inflammatory cytokines, adipokines, mitochondrial destruction, as well as changes in the gut microbiota and promotes the effect of oxidative stress, all this leading to steatohepatitis and fibrosis ^[11][12]. Among the many parallel strokes, oxidative stress is the main factor contributing to the development of liver damage and disease progression. Both exogenous factors—viruses, alcohol, an unbalanced diet, drugs-, and endogenous—such as insulin resistance, obesity, and diabetes—may promote oxidative stress in liver disease ^[13]. These parallel hits have been studied as potential therapeutic targets of NAFLD ^[14][15][16].

2. Animal Models’ Studies

PSs were investigated for diminishing inflammation through modulation of the immune cell’s inflammatory activity ^[17]. One of the first studies about the role of PSs in liver inflammation associated in NASH was performed by Plat et al. in 2014 ^[18]. Their study started from in vitro cells, continuing with mice and human analysis, and the studying hypothesis was that plant sterol/stanol esters reduces hepatic inflammation. The in vitro study showed that isolated bone-marrow derived macrophages incubated with LPS and sitostanol produced less Tnf-α, concluding that plant stanols could directly affect macrophages inflammation, independent of lipid metabolism ^[18]. In the animal model, supplementation of a high-fat diet (HFD) with plant sterol and stanol esters showed reduced inflammation in mouse liver, with less infiltrating macrophages (Mac-1) and neutrophils (NIMP) on immunohistochemical staining. The study also analyzed pro-inflammatory markers genes expression of the Cd68, Mcp-1, IL-1β, Tnf-α, ICAM in the hepatic tissue. These markers were significantly lowered in HFD enriched with plant sterol or stanol ester compared to the HFD alone. Moreover, synergic administration of plant sterols and HFD in mice showed a reduction of the serum and hepatic cholesterol precursors lathosterol/desmosterol levels compared with HFD fed mice ^[18]. Han H et al., 2014, studied the effects of flaxseed oil containing flaxseed oil ester of plant sterols on hepatic steatosis and the underlying molecular mechanisms. Male apolipoprotein-E knockout mice were fed with a high fat diet, and two groups received also flaxseed oil-treated (FO) or flaxseed oil combined with its ester of plant sterols-treated (FO-PS). The results showed a decrease of serum and liver triglycerides levels, serum levels of ALT and AST, reactive oxygen species (ROS) production, in mice fortified with FO-PS compared with the significantly higher values exhibited in HFD-fed mice only. Furthermore, the plasma levels of IL-6, TNF-α, MCP-1, and sICAM-1 were significantly lower in combined treatment than in high-fat diet. Moreover, an up-regulation of the genes expression of hepatic proliferator activated receptor alpha (PPARα) and genes involved in cholesterol efflux (ABCA1, LXR, SR-BI) was observed ^[19]. In 2017, Song et al. suggested that PSs would play an important role in reducing the evolution of NAFLD, already knowing the effects in lowering cholesterol, blood sugar, and antioxidants serum levels ^[20]. In his study, four-week-old male Sprague Dawley rats received a hyperlipidemic diet daily; the administered milk was enriched with different doses of phytosterol esters (PSE). During the study, hepato-renal function, lipid profile, histopathological liver changes, and the impact on proinflammatory cytokines, oxidative markers, and the gene related to lipid metabolism and inflammation were evaluated. The results of the study showed that PSE has changed favorably liver transaminases and lipid profile, correlated with improved histopathological status in rats with NAFLD. The human-rat equivalent of low and medium doses of PSE has been shown to have a protective effect on the onset and progression of NAFLD, by decreasing oxidative stress, serum LDL-C, and by modulating the cytokines involved. Thus, the need for additional studies in humans to confirm the therapeutic role of PSE in this pathology was emphasized ^[20]. Another aspect of NAFLD’s development is the association with intestinal dysbiosis. Song et al. investigated the impact of phytosterol esters (PSEs) on the intestinal microbiota by comparing the administration of high and low doses in adult Sprague Dawley rats. Those rats that received high doses of phytosterols in daily administration had a richer intestinal flora in Bacteroidetes and Anaerostipes. The researchers argue that the effect on the intestinal microbiota and fecal metabolites is beneficial. The findings opens up new opportunities for prevention and therapy in NAFLD ^[21]. Knowing that different forms of PSs have different implications in improving the lipid profile, Simin Feng et al. compared the effects of a pure stigmasterol and β-sitosterol dietary combination in experimental animals that developed NAFLD after receiving a high-fat Western-style diet (HFWD) ^[22]. The doses of phytosterols used were in accordance with those recommended by the FDA for human use. The researchers hypothesized that PSs influence lipid absorption and metabolism by altering the reabsorption of cholesterol and acid bile. Total cholesterol, HDL-C, non-HDL-C, TG, and ALT levels were periodically dosed from serum; liver histological lesions were also assessed using a histological scoring system, as well as the degree of inflammation associated. Further evaluations were performed to assess the effects of genes involved in lipid and bile acid metabolism. The determination of lipidomics is an efficient and sensitive approach to the biological changes caused by lipid variations in the evaluation of NAFLD. The results showed that stigmasterol is more effective than β-sitosterol in reducing body weight gain, hepatic lipid accumulation, and alleviation of HFWD-induced NAFLD, secondary to decreasing the reabsorption or absorption of cholesterol, bile acids, and dietary lipids; these effects can be explained by altering the expression of some lipogenic genes and lowering circulating CM levels ^[22]. Another in vivo study published in the same year confirm the positive influence of stigmasterol and β-sitosterol on NAFLD induced by a high-fat Western diet in mice. The long-term (33 weeks) supplementation of 0.4% stigmasterol or β-sitosterol in the diet of mice included in the study had antilipidemic activity: reducing triacylglycerols, hepatic cholesterol, and disruption in liver-free fatty acids ^[23]. Most of these effects are the results of a decreased absorption of the lipid. In 2020, Gumede et al. published two studies on the benefits of β-sitosterol in lipid metabolic disorders, as well as anti-obesogenic and antidiabetic effects. One study evaluated the protective potential of β-sitosterol in the onset and progression of NAFLD in growing rats fed with high levels of fructose; the researchers correlated the developmental pattern in baby mice exposed to a diet unsuitable for physiological growth, with children exposed to obesogenic diets ^[24]. Markers of serum liver function were evaluated, which showed an increase in total liver lipids. At the end of the study, a macro and microscopic examination of the liver showed an increase in liver mass, the presence of areas of inflammation and lesions of micro- and macrovesicular hepatic steatosis. The groups of rats that received additional β-sitosterol/fenofibrate diet showed a favorable liver evolution compared to the groups with a high fructose diet, which reinforces the idea that β-sitosterol has a favorable effect in preventing/attenuating diet-induced NAFLD ^[24]. Knowing the favorable effects in ameliorating lipid metabolism disorders of PSs, Gumede et al. publishes another study on growing female rats, who received a diet rich in fructose; that type of diet is known as a risk factor for disorders of lipid metabolism and the development of metabolic syndrome, NAFLD, cardiovascular pathology. Basically, they mimicked the pediatric population that consumes a diet predisposing to obesity. Serum levels were assessed for the following parameters: glucose, insulin, cholesterol, triglycerides, adiponectin; the visceral fat was also weighed. The results obtained after the evaluation of the group that received a diet rich in fructose with supplementation of β-sitosterol, suggest that β-sitosterol could be used in the prevention of metabolic disorders induced by a diet rich in fructose. This favorable result is due to its proven effect in preventing hypertriglyceridemia, hypoadiponectemia, and visceral fat ^[25]. Endoplasmatic Reticulum (ER) stress, according to literature studies, represents a key factor in the onset and progression of NAFLD. The direct effect of ER stress is the activation of ER oxidoreductin 1, the consequence being the overproduction of Reactive Oxygen Species (ROS), followed by disorder in the β-oxidation of fatty acids, with intracellular storage for cholesterol and saturated fatty acids. In a study conducted by Hao Han et al. on laboratory animals receiving a high fat and high cholesterol diet, administration of plant sterol ester of α-linolenic acid (PS-ALA) decreased liver size and body mass index, significantly improved liver tissue degradation, reduced oxidative stress, inhibited Sterol Regulatory Element-Binding Protein (Srebp) pathway activation, ameliorated lipid metabolism and thus intracellular fat accumulation, as well as inflammatory reaction (IL-1β, IL-18) and hepatocytolysis. The researchers concluded that supplementation with PS-ALA leads to improvement of β-oxidation of fatty acids, by inhibiting ROS overproduction and enhanced mitochondrial biogenesis, which means it plays an important role in improvement of NAFLD ^[26].

3. Clinical Trials

The promising results in animal models on the beneficial effects of PSs in NAFLD encouraged straying the role of PSs supplementation in human patients. Cross-sectional studies have shown a correlation between decreased serum levels of plant sterols and NAFLD, suggesting that intrahepatic transport mechanisms may be affected. Tauriainen et al. studied the link between export mechanisms to the most common plant sterols and plant stanols present in humans and NAFLD ^[27]. The group included 138 obese individuals participating in the Kuopio Obesity Surgery Study (KOBS). Approximately one third of the participants did not have hepatic histological changes, the rest having characteristic features of NAFLD. Dietary phytosterol intake (DPI) was not different between the study groups. The following were evaluated: serum, hepatic, and bile plant sterols and stanols and liver mRNA expression of several genes regulating inflammation and lipid metabolism, as well as the evaluation of lipid metabolism in serum. The main idea was that liver sitosterol and sitostanol ratios to cholesterol vary in obese patients depending on the presence or absence of NAFLD, without correlation between serum levels of these biological compounds and histological lesions. The main observation was that in patients with NAFLD, hepatic and bile sitostanol levels were increased, showing a direct relationship with steatosis and lobular inflammation. For the future, it remains to be clarified by what mechanisms the disturbance of sitostanol metabolism intervenes in NAFLD ^[27]. In response to the aforementioned study, Plat et al. considers that there is a possibility that the results published in the study by Tauriainen et al. may be secondary to the applied analytical methodology. Thus, Plat et al. emphasized the need for placebo-controlled intervention studies to certify the effects of plant sitosterol and sitostanol on the liver, positive or negative ^[28]. They exemplify the study published by Javanmardi et al. ^[29] in which it was shown that the daily diet supplemented with phytosterols improved the biological profile of patients. In 2018, Javanmardi et al. performed a double-blind clinical trial with 38 patients with NAFLD, organized into two randomized groups. One of the groups received 1.6 g of phytosterol daily. The results obtained showed that daily supplementation with phytosterols had a positive impact on LDL-C, TNF-α, ALT, and AST, strengthening the idea that phytosterols could be an effective therapy in NAFLD ^[29]. Another study of Chen et al., 2015, mentioned that administration of 1.8 g of PSs daily may improve insulin resistance, reduces LDLc concentrations, and downregulates systemic inflammation ^[30]. These two studies highlight the idea that more than 1.6 g/day of PSs may have a beneficial role in preventing or ameliorating the damage in NAFLD in adult patients. A study, based on the favorable results of phytosterol supplementation on lipid metabolism and the evolution of NAFLD, evaluated in a randomized, double-blind, placebo-controlled trial the effects of the administrations of PS (3.3 g/d, equal to 2.5 g phytosterol in the free form/d) plus n-3 PUFA (450 mg EPA + 1500 mg DHA) ^[31]. Anthropometric evaluations (body mass index, Waist circumference, Hip circumference), blood samples (Serum TC, TAG, HDL-cholesterol, LDL-cholesterol, glucose, liver and kidney function markers, TNF-α, TGF-β), and CT-scanning (for quantify the liver:spleen attenuation ratio) were performed at the beginning and end of the study. The study included NAFLD patients aged 30–67 years, with an average liver:spleen attenuation ratio of ≤1·2 or liver attenuation ≤52 Hounsfield units (HU), overweight or dyslipidemic. The findings were encouraging, showing the synergism of the mechanisms of action of PS + DHA + EPA in daily administration, more effective compared to separate or placebo administration, in reducing serum TAG, TC, and LDL-Cholesterol, decreasing the level of pro-inflammatory cytokines and improving liver damage. However, the researchers emphasize the need for a large population trial with NAFLD subjects and simultaneous administration of phytosterol and n-3 Long-chain PUFA (especially EPA and DHA) ^[31]. Furthermore, the group of Song L et al. continued the study on the role of co-supplementation of n-3 PUFAs and PSE in metabolic disorders associated with NAFLD. After 12 weeks of co-supplementation of n-3 PUFAs and PSE in patients with NAFLD, they observed that serum levels of PUFA-containing phosphatidylcholine (PC), lysophosphatidylcholine (LysoPC), perillyl alcohol, and retinyl ester significantly increased. Therefore, metabolomic regulation may explain the potential role of co-supplementation of n-3 PUFAs and PSE in improving hepatic steatosis ^[32].