5.1. Lifestyle Changes and MedicationA strict diet control seems to be an attractive and safe method for treating NASH. Both the Mediterranean and ketogenic diets advocate a reduced intake of carbohydrates. In addition, more physical exercise routinely can prevent and relieve NAFLD by improving lipid homeostasis. Further, statins (lipid-lowering drugs) present some benefits for the liver.
The Mediterranean diet is characterized by decreased intake of sugars and refined carbohydrates and enhanced intake of monounsaturated and n-3 FAs [96]. Different types of lipids (e.g., n-3 and n-6) may have opposite net influence on inflammation, and therefore, the final biological net effect is determined by their relative proportion [57]. Unlike n-6, n-3 PUFAs have important anti-inflammatory effects; they reduce adipogenesis and increase fatty acid oxidation (FAO), leading to a decrease in hepatic steatosis [97–99]. Fish and n-3 PUFA intake are reported to be lower in NAFLD patients than in nonfatty liver patients. Similarly, a downward trend of n-3 FA, eicosapentaenoic acid (20 : 5n-3) and docosahexaenoic (22 : 6n-3), intake is observed in these patients considering multiple lipids [98, 99]. These two n-3 FAs have significant antiproliferative, anti-inflammatory, and modulatory effects on the metabolic and immune systems [57]. The observed decreasing trends in the intake of these key n-3 FAs may promote steatosis, inflammation, dyslipidemia, cell damage, and carcinogenic risk in NASH patients [100, 101]. This reveals the theoretical basis for NASH treatment with n-3 FAs [73]. To date, most studies have used the methods of complementing patients with n-3 PUFAs. In 2012, a meta-analysis of nine studies revealed that n-3 PUFA supplementation has a beneficial influence on the liver fat and liver enzyme level [102]. The Mediterranean diet results in reduced calories, which is acceptable by patients and should be encouraged.
Ketogenic diets (KDs) are very low in carbohydrates and high in fats and/or proteins compared with various diets and, therefore, have gained popularity [103]. Low-calorie, especially low-carbohydrate, KD quickly reduces liver fat content and related metabolic abnormalities [104]. KD has been reported to promote weight loss, reduce intrahepatic triglyceride content, and alleviate metabolic parameters in obese patients. In addition, KD was reported to provoke weight loss in rodents. However, maintaining a long-term KD stimulated the progression of NAFLD and systemic glucose intolerance in mice. Thus, the relationship between KD and systemic insulin resistance in humans and rodents remains to be elucidated [105].
Physical activity (PA) is an integral part of any therapeutic strategy for weight loss, and it may play an important role in preventing NAFLD [106–108]. Multiple cohort studies revealed that change in body weight was correlated with both the development and remission of NAFLD [109, 110]. In men, initiation of an exercise regimen was remarkably associated with NAFLD remission [111]. The role of PA in delaying NAFLD progression has been demonstrated to be beneficial in the presence and absence of one or more metabolic syndromes [112]. In addition, an increase in PA prevents and/or retards NAFLD-related disease progression independent of weight loss [113, 114]. Dietary counseling and appropriate exercise should be combined and adjusted according to individual circumstances, targeting a gradual weight loss of 7%–10% [115, 116].
Atorvastatin and rosuvastatin [117] are widely used for treating dyslipidemia; however, they have not been well established as specific treatments for NAFLD. The guidelines of the European Association for the Study of the Liver (EASL)/European Association for the Study of Diabetes (EASD)/European Association for the Study of Obesity (EASO) [106] consider that statins have not been thoroughly tested. However, a large number of animal and human studies have demonstrated that the use of statins was safe in NAFLD, without an increase in the risk of hepatotoxicity, and may even significantly reduce aminotransferases. Animal data suggested that statins had certain beneficial effects on liver histology in NASH models [118]. Three post hoc analyses of randomized controlled trials in humans revealed that the use of atorvastatin had a beneficial effect on NAFLD in terms of liver enzyme reduction and ultrasonography improvement [118]. Statins may be the valuable option to be considered in patients with NAFLD/NASH, as it significantly reduces the risk of cardiovascular disease and liver cancer simultaneously [119–148].
5.2. Targeted TherapyNAFLD is characterized by ectopic toxic lipid accumulation, which is due to an extensive derangement in hepatic lipid metabolism [149–151]. Underlying these abnormalities is a wide range of disorder of nuclear transcription factors that adjust lipid metabolism, inflammation, and fibrogenesis, which consist of CD36; peroxisome proliferator-activated receptor- (PPAR-) α, PPAR-δ, and PPAR-γ; farnesoid X receptor (FXR); and sterol regulatory element binding protein 1 (SREBP-1), which are ideal targets for NAFLD treatment [152, 153].
CD36 is a FA receptor that plays a significant role in regulating lipid and glucose use, and the upregulation of CD36 expression is associated with NASH [154]. The RNA expression of nuclear factor kappa-B (NF-κB), a key regulator involved in the inflammation process, can be affected by the manipulation of CD36 expression [155]. Several studies have indicated that abnormal expression of CD36 in the liver was markedly associated with insulin resistance, hyperinsulinemia, and steatosis in NAFLD patients [154]. CD36 expression was reported to be elevated in mouse models with genetic obesity and high-fat diet- (HFD-) induced fatty livers [37]. Hence, treatment strategies designed to reverse this process by restoring normal levels of CD36 may provide a new method for treating NAFLD. A study reported that the absence of CD36 in the liver remarkably retarded the development of hepatic steatosis, although FA level increased. In addition, CD36 deletion affected the blood FA composition and improved the serum markers of hepatic inflammation [156]. Further, hepatocyte-specific loss of CD36 was found to significantly improve whole body insulin sensitivity in HFD-fed mice [156]. Briefly, CD36 not only is a disease marker but also plays an active role in FA uptake and has a significant influence on insulin sensitivity and hepatic lipid content and composition [157].
PPAR-α, a transcription factor, is mainly expressed in metabolically active tissues and regulates FAO. Lipid accumulation owing to FAO inhibition indirectly accelerates fibrogenesis by promoting inflammation. Agonists of PPAR-α presented beneficial effects of reversing deficiencies in FAO and improving NAFLD progression in animal and cell models [157, 158]. A randomized controlled trial demonstrated that pharmacological modulation of the PPAR-α nuclear receptor leads to substantial histological improvements in NASH patients, including the improvement of steatohepatitis and alleviation of cardiometabolic risk profile, with a sound security profile [157]. Several studies have emphasized on developing dual agonists against PPAR-α and PPAR-δ. The effects of PPAR-α promoted FAO [158], and those of PPAR-δ reduced de novo lipogenesis and inhibited inflammation [159, 160]. One such agent, elafibranor (GFT505), improved diet-induced NASH in rodents [160]. Elafibranor has been reported to improve hepatic and peripheral insulin sensitivity in humans [161]. In addition, improvements in NASH, insulin resistance, and dyslipidemia to some extent have been reported in long-term studies [157]. In summary, PPAR-α is important in reversing deficiencies in FAO and alleviating NAFLD progression.
The most widely studied drugs with potential benefits for NASH are thiazolidinediones (TZDs). TZDs such as pioglitazone and rosiglitazone activate the nuclear receptor, PPAR-γ, allowing preadipocyte differentiation into insulin-sensitive, fat-storing adipocytes [106]. It is noteworthy that the PPAR-γ ligands attenuate liver fibrosis by inhibiting transdifferentiation of liver stellate cells into activated myofibroblasts, suggesting a direct hepatoprotective influence. In addition, they present anti-inflammatory effects and increase circulating adiponectin, which is an adipokine that resists adipogenesis, and insulin sensitization [106]. Similarly, a clinical research study reported that they ameliorate glycemic control and NASH-associated parameters [162–164]. However, rosiglitazone has been withdrawn from the market in most of the countries due to the deficiency and the possible long-term treatment. We hold the opinion that it is necessary to overcome this obstacle [108, 117]. According to the current guidelines, pioglitazone is useful and is advocated for elderly patients with advanced fibrosis, confirmed by biopsy, who are unable to adopt or maintain lifestyle interventions and have persistent metabolic risk factors; however, pioglitazone should be administered to patients with T2DM and/or heart failure with caution [162, 165, 166]. Meta-analyses have revealed that rosiglitazone and pioglitazone were remarkably better than placebo in relieving balloon formation, lobular inflammation, and steatosis [167, 168]. Pharmacological inhibition of PPAR-γ resulted in the amelioration of NAFLD development [169]. For instance, in HFD-fed mice, hepatocyte/macrophage-specific PPAR-γ knockout protected against hepatic steatosis and PPAR-γ knockdown induced by RNA interfering-adenovirus vector injection improved a fatty liver [10, 170, 171]. In summary, TZDs improved hepatic steatosis and alleviated NASH and liver fibrosis by increasing insulin sensitivity in skeletal muscle and adipose tissue, thus overcoming the direct steatogenic influence on liver cells [171–173].
Bile acids (BAs) and their receptors (e.g., BA nuclear receptor and FXR) play indispensable roles in regulating systemic metabolism and hepatic lipid homeostasis. FXR is a nutrient-sensing nuclear receptor in the gut and liver that regulates glucose and fat metabolism [174]. These functions of FXR were assessed through a quantitative proteomic analysis of mouse liver tissue [175]. Studies had found that FXR regulates amino acid catabolism and detoxification of ammonium in the livers of mice through ureagenesis and glutamine synthesis. Further, the synthesis of ceramide pools throughout the body was reported to be regulated by the BA/FXR axis in the ileum and cecum [176]. Intestinal specific genetic or pharmacological inhibition of FXR led to a decrease in circulating ceramide levels, increase in browning of adipose tissue, and amelioration of liver insulin resistance and liver injury in HFD-induced obese mice [177]. Activation of hepatic FXR has been shown to reduce liver glucose, adipogenesis, and steatosis in animal models [178]. FXR is important for liver inflammation and has been proven to be a potential therapeutic target for NASH [179, 180]. FXR activation reprograms arachidonate metabolism in mice [181] and stimulates 1-deoxysphengolipid catabolism and thereby attenuates the cytotoxic effects [182]. A phase 2 randomized double-blind placebo control trial in Japan showed that compared with placebo, high doses of obeticholic acid (OCA) intake remarkably resolved NASH [178]. In the FXR Ligand OCA in NASH Treatment (FLINT) trial, OCA induced NASH remission in 22% of patients, whereas placebo induced NASH remission in 13% of patients [157]. In summary, FXR regulates circulating ceramide levels and improves hepatic insulin resistance and liver damage in NAFLD/NASH patients.
Hepatic de novo lipogenesis is stimulated by activation of the nutrient-sensing mTorc1 pathway, a substrate of insulin-Akt signaling under physiological conditions [179]. For example, treatment of hepatocytes with rapamycin, an allosteric inhibitor of mTorc1, inhibited insulin activation of the lipogenic transcription factor SREBP-1c [183, 184]. In addition, specific knockout of the mTorc1-defining component, raptor, in the liver alleviated HFD-induced hepatic steatosis, which may be due to reduced lipogenesis [185]. It has been demonstrated that Notch antagonism uncouples Akt from mTor activation, implying that NAFLD can be treated by Notch antagonists [186].