Zhen Yang
Lin Wang- Department of Hepatobiliary Surgery, Xi-Jing Hospital, The Fourth Military Medical University, Xi’an, China
Non-alcoholic fatty liver disease (NAFLD) has been identified as the most common chronic liver disease worldwide, with a growing incidence. NAFLD is considered the hepatic manifestation of a metabolic syndrome that emerges from multiple factors (e.g., oxidative stress, metabolic disorders, endoplasmic reticulum stress, cell death, and inflammation). Non-alcoholic steatohepatitis (NASH), an advanced form of NAFLD, has been reported to be a leading cause of cirrhosis and hepatic carcinoma, and it is progressing rapidly. Since there is no approved pharmacotherapy for NASH, a considerable number of therapeutic targets have emerged with the deepening of the research on NASH pathogenesis. In this study, the therapeutic potential and properties of regulating metabolism, the gut microbiome, antioxidant, microRNA, inhibiting apoptosis, targeting ferroptosis, and stem cell-based therapy in NASH are reviewed and evaluated. Since the single-drug treatment of NASH is affected by individual heterogeneous responses and side effects, it is imperative to precisely carry out targeted therapy with low toxicity. Lastly, targeted therapeutic agent delivery based on exosomes is proposed in this study, such that drugs with different mechanisms can be incorporated to generate high-efficiency and low-toxicity individualized medicine.
1 Introduction
In general, chronic liver disease (CLD) comprises drug-induced liver disease, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), viral hepatitis, liver cirrhosis, and liver cancer (Tomedi et al., 2018). NAFLD is the most common chronic liver disease, encompassing a spectrum of liver injuries ranging from fatty liver disease to liver cirrhosis (Setiawan et al., 2016; Younossi et al., 2018). The prevalence of NAFLD has been increasing (25.24% in 2015 to 29.38% in 2021) (Younossi et al., 2016; Liu et al., 2022), accounting for 45.8% of the total causes of death from chronic liver disease (Golabi et al., 2022). Non-alcoholic steatohepatitis (NASH) refers to an advanced form of NAFLD, and it is characterized by the presence of hepatic steatosis with hepatocellular injury (hepatocyte ballooning) and inflammation, which is one of the main causes of cirrhosis and hepatocellular carcinoma (Goldberg et al., 2017).
Overnutrition is the initiating event of NAFLD, resulting in the expansion of fat depots and accumulation of ectopic fat. The imbalance of lipid metabolism leads to the production of toxic metabolites in the setting of insulin resistance, thus resulting in oxidative stress and endoplasmic reticulum (ER) stress, lipid peroxidation and inflammasome activation, apoptotic cell death, and tissue fibrosis and regeneration (Sanyal, 2019). NAFLD is affected by a multitude of pathogenic pathways (e.g., metabolic disorders, hormones secreted from the adipose tissue, overnutrition, oxidative stress, and genetic and epigenetic factors) (Buzzetti et al., 2016). The pathogenesis and clinical manifestations of NAFLD are highly heterogeneous, which may be correlated with different pathogenic factors (Alonso et al., 2017). Furthermore, disease progression and response to treatment are not universally identical. Nutrition and energy balance takes on a critical significance in the development of NAFLD and NASH. Diet quality is highly important in the management and prevention of NAFLD (Ma et al., 2018). Adherence to a Mediterranean dietary pattern and energy restriction exhibit a favorable effect on the reduction of liver fat accumulation and steatohepatitis (Trovato et al., 2015; European Association for the Study of the Liver EASL et al., 2016). Physical activity is conducive to the prevention of NAFLD and ameliorates fatty liver (Sung et al., 2016; Kwak et al., 2017). Weight loss is capable of significantly ameliorating hepatic steatosis and even fibrosis (Vilar-Gomez et al., 2015). Lifestyle intervention contributes to the prevention of the progression of NAFLD to NASH, and it is fundamental to other therapies. Metformin refers to an antihyperglycemic agent with lipid-lowering effects. However, there is insufficient evidence to support metformin’s therapeutic effect on NAFLD (Iogna Prat and Tsochatzis, 2018). Even though no convincing evidence implicates that improvement in hepatic histology emerges under statin therapy, patients with NASH have a marked reduction in the risk of major cardiovascular events (Dongiovanni et al., 2015; Tziomalos et al., 2015). Bile acid metabolism is closely correlated with NASH; perhaps bile salt-based therapies have a significant treatment landscape (Donkers et al., 2019; Okushin et al., 2020).
There has not been any approved treatment for NASH. An increasing number of novel targets and strategies have emerged with the development of the pathogenesis of NASH. Notably, NASH has a rapid progression, and there is little evidence that NASH is always secondary to NAFLD, such that existing drug research and development is focused on NASH. In this review, the focus is placed on the summary of therapeutic potential and utility of metabolic regulation, the gut microbiome, antioxidant, microRNA, inhibiting apoptosis, targeting ferroptosis, and stem cell-based therapy in the progression of NASH. Furthermore, the potential and value of exosome-mediated targeted therapeutic agent delivery in the treatment of NASH are evaluated.
2 Therapy with regulating metabolism in NASH
Generally, NASH is considered a hepatic manifestation of the metabolic syndrome, and metabolic disorders are involved throughout the course of NASH (Neuschwander-Tetri and Caldwell, 2003; Yki-Järvinen, 2014; Masoodi et al., 2021; Tilg et al., 2021). Maintaining and improving metabolic homeostasis has been proven as a therapeutic strategy for NASH. Several therapies for improving metabolism are shown in the following (Figure 1).
FIGURE 1. Targets for correcting metabolic disorders in NASH.
2.1 Peroxisome proliferator-activated receptor agonists
Peroxisome proliferator-activated receptors (PPARs), a group of nuclear receptors and heterodimers with retinoid X receptors, are capable of regulating multiple metabolic processes through transcription. PPARs comprise three subtypes (i.e., PPARα, PPARβ/δ, and PPARγ), with different targeting ligands, and they are present in several tissues at different levels. PPARs exhibit vital regulatory effects as lipid sensors in response to fatty acid transport, gluconeogenesis, and β-oxidation (Lalloyer and Staels, 2010; Fougerat et al., 2020).
PPARα is mostly expressed in the liver and brown adipose tissue, which improves lipid metabolism by regulating fatty acid transport and β-oxidation. The activation of PPARα can increase NASH-related intestinal permeability and improve liver inflammatory injury (Velkov, 2013; Régnier et al., 2020). Moreover, the activation of PPARα attenuates plasma levels of proinflammatory cytokines, such as IL-1, IL-6, and TNF, and improves triglyceride (TG) metabolism via upregulating the expression of lipoprotein lipase (Mansouri et al., 2008) and suppressing hepatic apolipoprotein C-Ⅲ (apoC-Ⅲ) secretion (Qu et al., 2007). The histological improvement of NASH is correlated with an increased expression of PPARα and its target gene, but not PPARβ/δ and PPARγ (Francque et al., 2015). PPARγ is mainly distributed in the immune system and adipose tissue, where its functions are immunomodulation and ameliorating insulin sensitivity (Croasdell et al., 2015). The peroxisome proliferator-activated receptor-γ coactivators (PGC1s) have the potential for treating NAFLD as regulators of mitochondrial metabolism and immune responses (Piccinin et al., 2019). Binding of β-arrestin-1 to PPARγ suppresses adipogenesis and inflammation, and ameliorates systemic insulin sensitivity (Zhuang et al., 2011). PPARβ/δ also has the function of regulating glucose and lipid metabolisms. PPAR agonists that have made progress in clinical studies are summarized and discussed as follows.
Pemafibrate (K-877) is a novel selective PPARα modulator that improves dyslipidemia, plasma transaminase level, and the pathological condition of NASH in animal trials (Honda et al., 2017). Pemafibrate regulates glucose oxidation and facilitates fatty acid oxidation by inducing the expression of key target genes, including 3-hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) and pyruvate dehydrogenase kinase 4 (Sasaki et al., 2019). Clinical research studies show that pemafibrate can effectively and safely decrease plasma TG, VLDL cholesterol, remnant cholesterol, and apoC-III levels (Das Pradhan et al., 2022; Ginsberg et al., 2022). A previous study indicated that pemafibrate significantly decreased magnetic resonance elastography-based liver stiffness without reducing liver fat content (Nakajima et al., 2021). The adverse effects of pemafibrate include adverse renal events and venous thromboembolism (Das Pradhan et al., 2022). Further studies are required to determine whether pemafibrate can alleviate NASH progression and repair histological damage.
Pioglitazone (a synthetic ligand of PPARγ) which is used clinically for the treatment of type 2 diabetes mellitus (T2DM) mitigates steatosis, inflammation, and fibrosis in subjects with NASH (Belfort et al., 2006; Aithal et al., 2008); however, it increases the patient’s weight and risk of bladder cancer (Sanyal et al., 2010; Tang et al., 2018). Rosiglitazone, a selective PPARγ activator, did not ameliorate NASH, but inhibited lipogenesis in the rosiglitazone therapy (FLIRT 2) extension trial (Ratziu et al., 2010).
Elafibranor (GFT505) is a PPARα/δ dual agonist that avoids problems, such as weight gain, edema, and induction of heart failure, associated with the activation of PPARγ. A phase Ⅱ trial showed that elafibranor improved NASH without deteriorating hepatic fibrosis (Ratziu et al., 2016). Nevertheless, another phase Ⅲ trial reported that elafibranor failed to reach the predefined histological endpoint since there was no significant difference in the rate of histological resolution (Ratziu et al., 2016).
Saroglitazar (a PPARα/γ dual agonist) is an anti-metabolic syndrome drug developed locally in India by the company named Zydus Cadila in 2001, which can improve insulin sensitivity, lipid, and glycemic parameters (Gowda et al., 2022). Saroglitazar ameliorates steatohepatitis and lowers lipid levels in diet-induced mice (Kumar et al., 2020). It improves histological features and reduces the liver fat content in NASH models (Jain et al., 2018). A phase Ⅱ trial demonstrated that saroglitazar significantly reduced serum ALT levels, insulin resistance, dyslipidemia, liver fat content, markers of hepatocyte injury, and fibrosis in NASH patients (Gawrieh et al., 2021). Saroglitazar administered to NASH patients resulted in improvement in histological changes (Siddiqui et al., 2021). Further investigation is required to determine whether it can reach the intended histological endpoint of NASH resolution.
The activation of individual receptors of PPARs has a limited effect, and the widespread activation of PPARs may have a more effective and radical therapeutic potential for NASH through multiple pathological mechanisms. Lanifibranor (IVA337) is a pan-PPAR agonist with moderate and balanced effects on PPARα (on hepatocytes), PPARδ (on macrophages), and PPARγ (on stellate cells) that can ameliorate all aspects of NAFLD (Lefere et al., 2020). A phase Ⅱb trial involving patients with active NASH showed that lanifibranor remarkably improved NASH without worsening fibrosis (Francque et al., 2021). The adverse effects of lanifibranor include diarrhea, nausea, anemia, and weight gain (Francque et al., 2021). The aforementioned findings provided support for the phase Ⅲ clinical trial of lanifibranor.
2.2 Farnesoid X receptor agonists
The farnesoid X receptor (FXR) refers to a nuclear receptor stimulated by bile acids (BAs) distributed in the liver, kidneys, intestines, and adrenal glands. It has the capability of maintaining metabolic homeostasis by decreasing liver fat accumulation through the FXR-SREBP1 signaling pathway (Schmitt et al., 2015; Liu et al., 2020) and regulating glucose metabolism (Ma et al., 2006). The FXR is also expressed in liver resident macrophages, Kupffer cells, natural killer cells, and dendritic cells and plays an immunomodulatory role (Fiorucci et al., 2022). The latest study found that FXR inhibition can prevent SARS-CoV-2 infection by reducing angiotensin-converting enzyme 2 (ACE2) (Brevini et al., 2022). Nevertheless, activating the FXR selectively inhibits a hepatic inflammatory reaction mediated by NF-κB and impairs the aggregation of macrophages into body tissues by downregulating monocyte chemoattractant protein-1 (MCP-1) levels (Wang et al., 2008; Li et al., 2015). An FXR agonist is capable of inhibiting asymmetric dimethylarginine (ADMA) accumulation through activating dimethylarginine dimethylaminohydrolase-1 (DDAH-1) expression, thereby increasing the levels of endogenous nitric oxide that regulates NASH progression (Hu et al., 2006; Cunningham et al., 2021). As revealed by a recent study, withaferin A as a dual liver X receptor-α (LXR-α) and FXR agonist ameliorated NAFLD-related hepatitis and liver fibrosis through multiple pathways, including inhibiting NF-κB and TGF-β signaling (Shiragannavar et al., 2023). Accordingly, FXR agonists have become a focus of research for NASH treatment.
Obeticholic acid (OCA), a derivative of chenodeoxycholic acid (the major component of human bile acid), serves as a potent agonist of the FXR with minimal activity to a G-protein-coupled bile acid receptor (Pellicciari et al., 2004). OCA suppresses the progression of NASH by protecting against disruption of the intestinal epithelial and gut vascular barriers (early events in the development of NASH) (Mouries et al., 2019). In clinical studies, OCA significantly improved histological features of NASH without worsening fibrosis in a higher proportion of patients than placebo (Neuschwander-Tetri et al., 2015; Younossi et al., 2019). However, OCA has not gained approval from the Food and Drug Administration (FDA) for treating NASH since its adverse effects outweigh its benefits (Mullard, 2020). The adverse effects comprise pruritus, elevated low-density lipoprotein cholesterol, and liver failure (Mullard, 2020). Tropifexor (LJN452) is a highly potent FXR agonist and induces a target gene following very low concentrations (Tully et al., 2017). Its clinical effect needs further investigation. There are several FXR agonists in the development of NASH treatment.
2.3 Thyroid hormone receptor β agonists
The thyroid hormone receptor (THR) is a member of the nuclear hormone receptor family that links and regulates transcription factors including two subtypes, THRα and THRβ. THRs are associated with multiple functions, such as development, differentiation, growth, and metabolism (Gionfra et al., 2019). THRβ is highly expressed in hepatocytes, and THRβ agonists decrease the levels of serum cholesterol, triglycerides, and intrahepatic lipid contents (Sinha et al., 2019). Interestingly, hepatic hypothyroidism occurs with the downregulated expression of deiodinase 1 and the upregulated expression of deiodinase 3 in patients with NAFLD (Bohinc et al., 2014). Resmetirom (MGL-3136), a liver-directed selective THRβ agonist, contributes to profound reduction in hepatic steatosis, plasma alanine aminotransferase activity, blood glucose, inflammatory, and fibrosis makers (Kannt et al., 2021). A phase Ⅱ clinical trial showed that resmetirom treatment distinctly reduced hepatic fat and improved hepatic inflammation in patients with NASH. There were no significant side effects among the groups, except for transient diarrhea and nausea (Harrison et al., 2019). VK2809 (MBD7811) undergoes the hepatic first pass effect to become VK2809A (a negatively charged THRβ agonist) and has high selectivity toward liver tissues (Erion et al., 2007). It has significant potential in the treatment of NASH. Based on the aforementioned findings, the THRβ agonist in patients with NASH and fibrosis is currently being investigated.
2.4 Glucagon-like peptide-1 receptor agonists
GLP-1 is an incretin hormone secreted by ileum cells after food intake. GLP-1 controls blood glucose by stimulating insulin secretion from islet β cells and inhibiting glucagon secretion from islet α cells. In addition to acting on the pancreas, existing research has suggested that GLP-1 improves peripheral insulin sensitivity, suppresses hepatic lipogenesis, delays gastric emptying, and reduces appetite (Knudsen and Lau, 2019). An increase in energy expenditure stimulated by GLP-1 receptor agonists through hypothalamic AMP-activated protein kinase (AMPK) pathways assists in weight reduction (Beiroa et al., 2014). Long-acting GLP-1 analogs have also been reported to alleviate hepatic steatosis (Armstrong et al., 2016). Tirzepatide is a novel dual GLP-1 and glucose-dependent insulinotropic peptide (GIP) receptor agonist, and its therapeutic efficacy in improving NASH has been reported in a clinical trial (Hartman et al., 2020). A previous study has confirmed that tirzepatide dose-dependently reduces NASH-related biomarkers and increases adiponectin (Hartman et al., 2020). The most common side effects of tirzepatide comprise emesis, nausea, decreased appetite, abdominal bloating, and diarrhea, which are considered mild to moderate in severity (Coskun et al., 2018). Semaglutide, another GLP-1 receptor agonist, exhibited powerful improvements against NASH without worsening fibrosis at a dose of 0.4 mg compared to placebo in a phase Ⅱ trial (Newsome et al., 2021). GLP-1 has served as a well-established target in diabetes treatment and is also a promising factor in treating NASH.
2.5 Fibroblast growth factor analogs
The family of fibroblast growth factor (FGF) peptides has 22 members and binds to five receptors (FGFR1–4 and FGFRL1). To be specific, FGF19 (a peptide directly targeting the liver) and FGF21 (a peptide mainly secreted by the liver) are promising targets for the treatment of NAFLD for their immunoregulatory effects, hepatoprotective properties, and regulatory capacity of metabolisms (Ocker, 2020). Alternatively, recent research has suggested that previously unappreciated FGF4 mitigates NAFLD and NASH through AMPK-caspase 6 signaling pathways (Song et al., 2022). The progress of FGF in NASH is described as follows.
FGF19 produced primarily in the ileum is significantly elevated after the meal via the BA-FXR pathway (Lundåsen et al., 2006; Somm and Jornayvaz, 2018). FGF19 is generally effective, requiring binding to FGFR1-4 and βKlotho (KLB) (Kurosu et al., 2007; Lin et al., 2007). It is capable of regulating hepatic protein synthesis and glucose metabolism mediated by the mitogen-activated protein kinase (MAPK) signaling pathway instead of the insulin-related pathway (Kir et al., 2011). FGF19 plays a cytoprotective role against ER stress by activating FGFR4-glycogen synthase kinase-3β (GSK3β)-nuclear factor E2-related factor 2 (Nrf2) signaling cascade (Teng et al., 2017). FGF19 and its analog modulate bile acid levels and ameliorate steatosis by inhibiting the hepatic expression of CYP7A1 and CYP27A1 which encodes the key enzymes for bile acid synthesis (Zhou et al., 2016). Aldafermin (NGM282) is a manufactured non-tumorigenic analog of FGF19 that binds to FGFR4/KLB. In a phase Ⅱ trial in NASH patients, aldafermin decreased liver fat content and the levels of serum markers, such as 7α-hydroxy-4-cholesten-3-one, alanine, and aspartate aminotransferases (Harrison et al., 2021a). More recently, aldafermin alleviated hepatic fibrosis without worsening NASH and had no apparent dose-dependent response (Harrison et al., 2022).
FGF21 activates multiple targets with binding FGFR1-3 and KLB (Kurosu et al., 2007), thereby initiating downstream signaling. FGF19 and FGF21 exhibit similar physiological effects. The downregulation of FGF21 is correlated with loss of hepatic fat following high-protein diet, and FGF21 may serve as the biomarker of metabolic improvement (Markova et al., 2017). FGF21 can improve PPARα-related lipid metabolism (Badman et al., 2007) while regulating adiponectin-mediated energy metabolism and insulin sensitivity in the liver (Lin et al., 2013). ER stress can upregulate FGF21 expression, such that ER stress can be ameliorated via the AMPK-sirtuin1 (SIRT1) pathway (Chau et al., 2010; Kim et al., 2015). FGF21 reduces hepatic steatosis and peroxidation damage in NASH by regulating the activation and oxidation of fatty acids in the liver (Fisher et al., 2014). Hence, FGF21 analog is expected to become a therapeutic agent for NASH. Pegbelfermin (BMS-986036), a PEGylated analog of FGF21, has been shown to safely reduce hepatic fat fraction in patients with NASH (Sanyal et al., 2019), as has efruxifermin (AKR001), another long-acting Fc-FGF21 fusion protein (Harrison et al., 2021b).
2.6 Sodium–glucose cotransporter-2 inhibitors
Sodium–glucose cotransporter-2 (SGLT-2) has been reported as a critical mediator of epithelial glucose transport in charge of the majority of glucose reuptake in the kidney (Rieg and Vallon, 2018). SGLT-2 inhibitors as new hypoglycemic agents improve blood glucose and lower weight by preventing SGLT-2 from renal reabsorption of sodium and glucose (Kaneto et al., 2016). An SGLT inhibitor can activate AMPK phosphorylation and inhibit the mammalian target of rapamycin (mTOR) phosphorylation, thereby improving hepatic steatosis (Luo et al., 2021a). The SGLT-2 inhibitor also exhibits cytoprotective properties, reduces hepatic lipid contents, and attenuates oxidative stress (Honda et al., 2016; Kabil and Mahmoud, 2018). Furthermore, the promotion of autophagy and reduction of ER stress and hepatocyte apoptosis are mediated by SGLT-2, alleviating the progression of NAFLD (Nasiri-Ansari et al., 2021). In a clinical study, empagliflozin (SGLT-2 inhibitor) significantly reduced hepatic fat content in patients with type 2 diabetes and NAFLD (Kuchay et al., 2018). Thus, SGLT-2 inhibitors have been proposed as treatment options for NASH.
2.7 Acetyl-CoA carboxylase inhibitors
Conversion of acetyl-CoA to malonyl-CoA mediated by acetyl-CoA carboxylase (ACC) is one of the vital steps of adipogenesis. The ACC inhibitor can improve fatty degeneration of the liver and insulin resistance, such that it is promising for the treatment of NASH (Harriman et al., 2016). PF-05221304, an ACC inhibitor that targets the liver, alleviates inflammation and fibrosis in NASH (Ross et al., 2020). However, PF-05221304 may elevate serum triglyceride levels by increasing SREBP1c expression and hepatic VLDL exportation (Kim et al., 2017). PF-05221304 coadministered with a diacylglycerol acyltransferase 2 (DGAT2) inhibitor is one pathway used to resolve this issue (Calle et al., 2021). DGAT2 is highly expressed in liver and adipose tissues, which catalyzes the terminal step of de novo lipid synthesis, particularly the esterification of fatty acids with diacylglycerol to form triglycerides. The combination of PF-05221304 and DGAT2 inhibitors dramatically lowers hepatic fat content with good safety and tolerability (Calle et al., 2021). Arachidonate 12-lipoxygenase (ALOX12) protects ACC1 from lysosomal degradation to promote the occurrence of NASH (Zhang et al., 2021b). IMA-1, identified as a small molecule, has been confirmed to be capable of interrupting the ALOX12–ACC1 axis via directly binding to a pocket in ALOX12, thereby preventing the progression of NASH with milder side effects (Zhang et al., 2021a). Additionally, ACC inhibitors can suppress the activation of HSCs to inhibit NASH-related fibrosis (Bates et al., 2020).
3 The gut microbiome as a therapeutic target for NASH
The mutualism between the gut microbiome and humans is a well-confirmed symbiosis, and the gut microbiome is beneficial to the health of the host (Sekirov et al., 2010; Lin and Zhang, 2017). There are different degrees of gut microflora dysbiosis in different stages of NAFLD. Some studies indicate that the overgrowth of Bacteroides and Prevotella genera may play a key role in the development of NASH (Mouzaki et al., 2013; Wong et al., 2013; Sobhonslidsuk et al., 2018). The gut microbiome affects liver disease and cancer by altering intestinal permeability and activating the innate immune system. The effect of the gut microbiome on host lipid metabolism is mediated by the metabolites generated from the gut microbiome (e.g., short-chain fatty acids, secondary bile acids, and trimethylamine), or by the components of bacteria themselves (e.g., lipopolysaccharides, peptidoglycans, and DNA), which interact with host hepatocytes through the portal vein (Ji et al., 2019). The disruption of gut–liver axis homeostasis leads to a variety of diseases, including NASH.
Probiotics are living microorganisms that provide health benefits to the host when consumed in sufficient amounts (Hill et al., 2014). Probiotics have the capability of increasing the diversity of intestinal flora and the relative abundance of beneficial bacteria, inhibiting lipid synthesis, reducing liver inflammation, and increasing antioxidant enzyme activity (Park et al., 2020; Luo et al., 2021b). Prebiotics are organic substances that selectively promote the metabolism and proliferation of beneficial bacteria without being digested and absorbed by the host, thereby improving the health of the host. Prebiotics possess high potential to ameliorate NASH, as do probiotics (Carpi et al., 2022). Fecal microbial transplantation (FMT) refers to the transplantation of functional microflora from the donor’s feces into the patient’s gut in an attempt to improve the original intestinal flora imbalance and rebuild the normal intestinal microecology, achieving the treatment of disease (Khoruts, 2018). Following FMT therapy, gut microbiota dysbiosis was corrected, the abundance of beneficial bacteria was increased, and steatohepatitis was attenuated in mice with high-fat diet (HFD) (Zhou et al., 2017). A clinical study has suggested that FMT improves intestinal permeability in patients with NAFLD, while there is no increase in insulin sensitivity or decrease in intrahepatic lipid content (Craven et al., 2020). The efficacy of FMT is closely related to the characteristics of the donors, especially the richness and diversity of fecal microbiota. Therefore, whether FMT can be used in the treatment of NASH still needs a large number of trials to provide evidence. Furthermore, several antibiotics are used to ameliorate gut microbiome dysbiosis associated with NASH. Rifaximin is a non-systemic antibiotic that is barely absorbed in the gastrointestinal tract and does not significantly affect the diversity of the gut microbiome (Kaji et al., 2017). However, the effect of rifaximin in the treatment of human NASH remains debatable (Cobbold et al., 2018; Jian et al., 2022). Triclosan and solithromycin are antibiotics that improve intestinal homeostasis and hepatic steatosis (Sumida and Yoneda, 2018; Sun et al., 2022). On one hand, antibiotics regulate the disorder of intestinal flora to achieve the purpose of treatment, and on the other hand, it leads to the occurrence of bacterial resistance and produce new flora disorders when used in the long-term. Antibiotics must be used with caution for treating NASH. In the following period, the gut microbiome will be a promising therapeutic target for NASH therapy.
4 Targeting oxidative stress in the treatment of NASH
Oxidative stress caused by ROS is an important contributing factor in the progression of NASH. As a powerful biological antioxidant, vitamin E has a protective effect on cell membranes and lipoproteins, preventing lipid peroxidation. Vitamin E reduces liver injury by ameliorating mitochondrial oxidative damage and inhibiting apoptosis (Soden et al., 2007). Studies have revealed that vitamin E reduced steatosis, lobular inflammation, and hepatocyte ballooning, but not fibrosis, in NASH (Sanyal et al., 2010). However, the safety of vitamin E has raised a concern as a result of multitudinous adverse effects, such as increasing all-cause mortality, incidence of prostate cancer, and risk of hemorrhagic stroke (Miller et al., 2005; Lippman et al., 2009; Schürks et al., 2010). Vitamin E as a hydrophobic compound is unable to translocate across the membrane into the cytoplasm and mitochondria, thereby limiting the antioxidant effect. Superoxide dismutase (SOD) plays an antioxidant role by catalyzing superoxide anions to hydrogen peroxide and molecular oxygen in vivo. rMnSOD is a recombinant humanized molecule that can cross the cell membrane to exhibit antioxidant effects (Borrelli et al., 2016). Meanwhile, rMnSOD has an excellent biodistribution in the liver and reduces portal pressure (Guillaume et al., 2013). It can safely and effectively halt the progression of NASH by ameliorating oxidative stress (Borrelli et al., 2018). It is certain that rMnSOD is full of potential in NASH treatment.
5 Treatment with microRNA
miRNAs are classes of endogenous non-coding RNAs consisting of about 22 nucleotides, activating or silencing target gene expression via regulating transcription and post-transcriptional processes, including the regulation of the non-coding RNA transcriptome, alternative mRNA splicing, translation, and mRNA degradation (Catalanotto et al., 2016). Thus, miRNAs are involved in multiple biological processes like cell proliferation, differentiation, apoptosis, tumorigenesis, and energy homeostasis (Jovanovic and Hengartner, 2006; Hwang and Mendell, 2007; Carrer et al., 2012).
The levels of hepatic miR-122 undergo a dynamic change in the downward trend during the progression of NAFLD to cirrhosis, and the ameliorative function of miR-122 on NASH is controversial (Hochreuter et al., 2022). Increased expression of PPARα and SIRT1 mediated by silencing miR-34a led to the improvement of hepatic steatosis and lipid metabolism (Ding et al., 2015). Lipophagy, mitochondrial biogenesis, and energy metabolism were inhibited following the activation of miR-34a and miR-let-7a mediated by LXRα (Kim et al., 2021b). There were no detectable NASH-related phenotypic alterations in normal/miR-21 knockout mice under normal dietary conditions, but miR-21 deficiency improved insulin resistance, steatosis, and lipid oxidation in mice with HFD, reducing inflammation and fibrosis in NASH (Calo et al., 2016; Loyer et al., 2016). Hepatocytes absorbed neutrophil-derived extracellular vesicles (EVs) filled with miR-223, mediated by a low-density lipoprotein receptor (LDLR) on hepatocytes and apolipoprotein E (APOE) on EVs, ameliorating hepatic inflammation and NASH in mice (He et al., 2021). The secretion of miR-223-enriched exosomes from macrophages facilitated by IL-6 altered gene expression in hepatocytes via an exosomal transfer (Hou et al., 2021). Kupffer cell-derived miR-690 also were shuttled to other cells like hepatocytes, recruited hepatic macrophages, and HSCs to improve insulin sensitivity, inflammation, and fibrosis in NASH mice through exosome-mediated transfer (Ying et al., 2021; Gao et al., 2022a). In addition to the aforementioned research, various miRNAs are involved in NASH progression.
Antagomirs are chemically engineered oligonucleotides silence-specific miRNAs in vivo, and they have been demonstrated as powerful tools to inhibit miRNA in diseases (Krützfeldt et al., 2005). The efficacy of miRNA-based therapy is primarily determined by effectively delivering miRNA or its inhibitor to targets. There are various oligonucleotide delivery strategies delivering drugs to specific cells and tissues, which comprise bioconjugation, lipid conjugates, liposomes, exosomes, and spherical nucleic acids (Roberts et al., 2020). Thus, miRNAs are highly promising in the treatment of NASH.
6 Therapeutic strategy using apoptosis inhibitors
Apoptosis is a strictly ordered programmed cell death characterized by programmed degradation of DNA, chromatin pyknosis, cell shrinkage, fragmentation, and apoptotic body formation (Kerr et al., 1972; Nagata, 2018), initiated by caspase (McIlwain et al., 2015). Apoptosis can be activated by a mitochondrial-dependent pathway and the extrinsic pathway mediated by TNF signaling pathway (Locksley et al., 2001; McIlwain et al., 2015). Apoptosis is closely related to the progression of NASH, and the role of inhibiting apoptosis in the treatment of NASH has also been confirmed (Kanda et al., 2018).
The TNF signaling pathway can cause apoptosis mainly depending on whether the anti-apoptotic signals such as NF-κB are inhibited (Tang et al., 2001). TNF-α induces the activation of apoptosis signal-regulating kinase 1 (ASK1), which activates p38/JUN N-terminal kinase (JNK) signaling, leading to apoptosis (De Smaele et al., 2001; Brenner et al., 2013). Cellular repressor of E1A-stimulated genes (CREG) and glutathione S-transferase Mu 2 (GSTM2) inhibits the phosphorylation of ASK1 to block its subsequent downstream signaling, thereby improving insulin resistance and hepatic steatosis (Zhang et al., 2017; Lan et al., 2022). The inhibition of ASK1-mediated activation of p38/JNK cascades delays the progression of NASH (Xiang et al., 2016). Some previous research studies have suggested that selonsertib (an inhibitor of ASK1) reduces fibrosis in NASH patients with fibrosis, whereas the improvement appears to be limited (Loomba et al., 2018; Harrison et al., 2020).
Caspase-8 plays a central role in apoptosis, which initiates apoptosis by activating caspase-3, caspase-6, and caspase-7 (Galluzzi et al., 2012; Yuan et al., 2016). A study showed that the ablation of caspase-8 protected against hepatic steatosis, accumulation of ROS, apoptosis, liver inflammation, and fibrosis in NASH mice (Hatting et al., 2013). In contrast, some studies have demonstrated that emricasan (a pan-caspase inhibitor) has no efficacy for NASH-related cirrhosis (Garcia-Tsao et al., 2020; Frenette et al., 2021). The possible reason for this result is that caspase-8 has a protective effect on preventing excess activation of necroptosis, and the key molecules of necroptosis may be the more potential targets for the treatment of NASH (Gautheron et al., 2014; Schwabe and Luedde, 2018). Consequently, the inhibition of RIPK1 which is the initiator of necroptosis has shown significantly therapeutic effects on NASH (Majdi et al., 2020).
7 Targeted therapeutic strategy of ferroptosis in NASH
Ferroptosis is an iron-dependent non-apoptotic form of cell death characterized by accumulation of reactive oxygen species (ROS) (Stockwell et al., 2017). Aberrant iron distribution leads to hepatic metabolic diseases. Hepatocytes have been confirmed to be iron deficient, and hepatic stellate cells have been demonstrated to be overloaded with iron in NAFLD (Gao et al., 2022b). Programmed cell death triggered by ROS accumulation in hepatocytes is a possible cause of liver injury and inflammation in NASH. Correspondingly, ferroptosis is the earliest form of cell death in NASH (Tsurusaki et al., 2019). Excess ferrous iron produces hydroxyl radicals through the Fenton reaction, thus leading to cytotoxicity including lipid peroxidation (Dixon et al., 2012; Jhelum et al., 2020). System xc- (cystine/glutamate antiporter) promotes the uptake of cystine to increase the levels of glutathione (GSH), which is the main antioxidant in vivo (Mandal et al., 2010). The depletion of GSH indirectly leads to the inactivation of glutathione peroxidase 4 (GPX4), which causes disorders of lipid metabolism and ferroptosis (Yang et al., 2014). As discussed previously, iron disorders, lipid peroxidation, and disruption of the system xc--GSH-GPX4 axis can trigger ferroptosis.
Serum ferritin (SF) levels are significantly increased in patients with NAFLD, which is considered to be an independent risk factor for the progression of NAFLD to NASH and fibrosis (Kowdley et al., 2012). Iron overload exacerbates insulin resistance associated with NASH (Fargion et al., 2005). In the methionine/choline-deficient diet (MCD) model, RSL-3 (a GPX4 inhibitor) aggravated liver steatosis, and inflammation in NASH, while deferoxamine mesylate nullified this effect through chelating iron. Treatment with GPX4 activator inhibited hepatic lipid peroxidation, while reducing the severity of NASH (Qi et al., 2020). Ferroptosis inhibition exhibits cytoprotection and alleviates inflammation in NASH (Tsurusaki et al., 2019). Iron accumulation in HSCs facilitates the progression of NASH to fibrosis via inducing fibrogenic activation, which is dependent on ROS (Gao et al., 2022b). As mentioned previously, targeting ferroptosis has significant potential for the prevention and treatment of NASH.
8 Stem cell-based therapy in NASH
At present, cell transplantation for the treatment of metabolic diseases emerging from obesity has become a research hotspot. Mesenchymal stem cells (MSCs) are promising in the treatment of NASH, with extensive sources and rapid proliferation. The transplantation of MSC-derived human hepatocyte-like cells can restrain NASH progression by augmenting liver regeneration, inhibiting inflammation, and regulating lipid metabolism (Winkler et al., 2014). Intravenous infusion of adipose-derived mesenchymal stem cells (ASCs) improves insulin resistance through the hepatic AMPK signaling pathway and suppresses liver inflammation with decreasing TNFα (Cao et al., 2015; Xie et al., 2017). A recent study demonstrated that the infusion of c-kit-positive liver sinusoidal endothelial cells (LSECs) improves homeostasis in the pericentral liver endothelium and diet-induced NASH in mice by impacting LSEC–macrophage–neutrophil crosstalk mediated by repressing C-X-C motif chemokine receptor 4 (CXCR4) transcription by CCAAT enhancer-binding protein α (CEBPA) (Duan et al., 2022). In addition to cell implantation, EVs from pan-PPAR agonist-stimulated-induced mesenchymal stem cell (pan PPAR-iMSC-EVs) can facilitate NASH progression, contributing to ameliorating hepatic steatosis, ER stress, oxidative stress, and apoptosis (Kim et al., 2021a). MSC-derived apoptotic vesicles have significant potential in the treatment of NASH, considering macrophage regulatory effects and efferocytosis mediated by calreticulin (Zheng et al., 2021). Exosomes from human umbilical cord mesenchymal stem cells (hUC-MSCs) alleviated inflammation and reversed PPARα expression, thereby improving NASH in MCD-induced mice (Shi et al., 2022). MSC-based treatments regulate the expression of PPARα and PPARγ to reduce hepatic fat accumulation and mitigate NASH inflammation, among which MSC-derived brown adipocytes are more effective than MSCs and MSC lysate (Lee et al., 2017). Given that the safety, survival rate, and colonization rate of stem-cell transplantation are difficult to control, stem-cell derivatives may be more promising (Figure 2).
FIGURE 2. Therapeutic strategies of stem cells and their derivatives in NASH. Implantation of stem cells or stem-cell-differentiated cells is used to treat NASH. Additionally, stem cell derivatives are also promising for NASH treatment.
9 Targeted therapeutic agent delivery based on exosomes in NASH
Exosomes as a type of EVs are lipid-bilayer airtight vesicle with diameters ranging from 40 to 160 nm that are generated in a process that intraluminal vesicles (ILVs) are released to extracellular space upon fusion of intracellular multivesicular bodies (MVBs) containing ILVs and the plasma membrane (Gould and Raposo, 2013; Kalluri and LeBleu, 2020). Exosomes are naturally occurring targeted drug delivery tools in vivo, carrying proteins, lipids, nucleic acids, and synthetic drugs (Keerthikumar et al., 2016; Barile and Vassalli, 2017). Exosomes have exhibited multiple advantages in targeted drug delivery: 1) some exosomes can have a long retention time in circulation because CD47 protects exosomes from phagocytosis by macrophages and monocytes (Kamerkar et al., 2017); 2) there are abundant sources of exosomes; 3) exosomes are well-tolerated, less immunogenic, less antigenic, and less toxic (Kordelas et al., 2014; Zhu et al., 2017); 4) exosomes are able to migrate across biological barriers such as the blood–brain barrier with no modification (Yuan et al., 2017); 5) exosomes protect and maintain the biological activity of cargo, such as protecting exosomal RNA from degradation by ribonucleases present in blood (Cheng et al., 2014); and 6) surface modification of exosomes at the cellular level or after isolation can enhance targeting efficiency (Hood, 2016; Antimisiaris et al., 2018). In brief, exosome-mediated targeted therapy has fewer side effects, while regulating the targets more accurately to treat diseases.
The role of macrophages in NASH is so important that targeting macrophages is considered a promising therapeutic pathway (Huang et al., 2010; Kazankov et al., 2019). M2 macrophage polarization activated by a retinoic-acid-related orphan receptor α (RORα) contributes to the release of IL-10, which promotes M1 macrophage apoptosis, thus ameliorating NASH via inhibiting hepatic steatosis, inflammation, and apoptosis (Wan et al., 2014; Han et al., 2017). In the human body, exosomes from adipose-derived stem cells polarize M2 macrophages through the transactivation of arginase-1 by exosome-borne active STAT3 (Zhao et al., 2018). Further experimental evidence for exosome-mediated targeted drug delivery in NASH results from a study in which exosomes loaded with RBP-J decoy oligodeoxynucleotides block Notch signaling in macrophages and ameliorate liver inflammation (He et al., 2022). Exosomes are dominantly captured by macrophages in vivo, and exosomes of macrophages-targeted drug delivery do not appear to require a plethora of modifications (Imai et al., 2015).
As gatekeepers of liver homeostasis, LSECs make a significant contribution to the course of NAFLD/NASH (Hammoutene and Rautou, 2019). LSEC capillarization occurs at the early stage of NAFLD, defenestration, also called the loss of fenestrae aggravated in the cirrhotic phase (Miyao et al., 2015). In line with the aforementioned description, LSEC dysfunction occurs early in NAFLD, associated with augmented vascular resistance and steatosis (Pasarín et al., 2012; Nasiri-Ansari et al., 2022). LSECs without the physiological function promote pathologic angiogenesis, NASH progression, and fibrosis (Hammoutene and Rautou, 2019). Therapeutic approaches targeting LSECs are auspicious strategies for NASH treatment. The expression of Krüppel-like factor 2 (KLF2) activated by resveratrol or statins in LSECs repairs endothelial dysfunction while deactivating HSCs (Gracia-Sancho et al., 2010; Marrone et al., 2013). Furthermore, the blockade of endothelial Notch reverses LSEC dedifferentiation and improves LSEC angiocrine and NASH progression in an eNOS-dependent pathway (Duan et al., 2018; Fang et al., 2022). These are therapeutic targets for LSECs. How to treat NASH with targeting LSECs has been shown in a study that RUNX1 siRNA immunonano-lipocarrier tagged with VEGFR3 antibodies (specific marker of LSECs), specifically silenced the RUNX1 gene in LSECs and improved NASH inflammation (Ding et al., 2010; Tripathi et al., 2021). Due to hyaluronic acid (HA) that is a naturally LSEC-targeted ligand, HA-coated nanocapsules efficiently deliver bioactive agents to LSECs (Fraser et al., 1985; Kren et al., 2009; Toriyabe et al., 2011). Moreover, the uptake efficiency of LSECs is affected by the density of ligands at the surface and the diameter of nanoparticles (Kamps et al., 1997; Akhter et al., 2014). Avoiding phagocytosis of macrophages is also the key to targeting LSEC by exosomes. CD47-enriched exosomes are possible to address this problem (Kamerkar et al., 2017; Belhadj et al., 2020). Otherwise, macrophage depletion by injection with clodronate-containing liposomes also delays the phagocytosis of exosomes by macrophages (Imai et al., 2015). As previously stated, exosomes of LSEC-targeted drug delivery are full of potential for NASH treatment.
Apart from macrophages and LSECs, other cells in the liver may be therapeutic targets in the treatment of NASH. However, exosomes targeting other cells in therapeutic agent delivery are even more difficult based on hepatic histological characteristics (Figure 3).
FIGURE 3. Engineered exosomes of targeted drug delivery against NASH. Preparation of engineered exosomes: drugs are to be loaded in the exosomes following exosomal surface modification. The targeting ability of engineered exosomes is enhanced by surface modification. Exosomes targeting cells other than macrophages require CD47 modification. Engineered exosomes taken up by liver sinusoid: infusion of engineered exosomes is used to treat NASH by delivering drugs to targets. Targeting cells other than LSECs and macrophages in therapeutic agents delivery is difficult due to hepatic histological characteristics and LSEC capillarization occurred at the early stage of NAFLD.
10 Conclusion and perspective
Despite several drugs in late-stage clinical development, there is no existing therapy approved to treat NAFLD. Lifestyle intervention (diet, physical activity, and exercise) continues to be the mainstay of treatment (Romero-Gómez et al., 2017; Powell et al., 2021). NASH is a dynamic liver inflammatory response involving multiple metabolic disorders. In this review, the focus is placed on the summary of the role of metabolic regulation, the gut microbiome, antioxidant, miRNA, inhibition of apoptosis, ferroptosis resistance, and stem cell-based therapy in the management of NASH. As the therapeutic effect of single-targeted therapy was limited, combined therapy for distinct pathogenesis and targets and different periods exhibits better efficacy (Rajamoorthi et al., 2017; Puengel et al., 2022). This may increase the side effects of drugs. Precise targeted drug delivery such as engineered exosomes can use the aforementioned mechanisms and combat these problems. Exosome-mediated targeted drug delivery can be used to perform precise drug administration to selective targets with few adverse effects, according to the stage of development and pathological characteristics of NASH.
Author contributions
ZY wrote the whole review. LW supervised the whole study.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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Keywords: non-alcoholic fatty liver disease, lipid peroxidation, non-alcoholic steatohepatitis, metabolic homeostasis, ferroptosis, targeted therapeutics, the gut microbiome
Citation: Yang Z and Wang L (2023) Current, emerging, and potential therapies for non-alcoholic steatohepatitis. Front. Pharmacol. 14:1152042. doi: 10.3389/fphar.2023.1152042
Received: 31 January 2023; Accepted: 20 March 2023;
Published: 30 March 2023.
Edited by:
Runping Liu, Beijing University of Chinese Medicine, ChinaReviewed by:
David Sacerdoti, University of Verona, ItalyPrasanna K. Santhekadur, JSS Academy of Higher Education and Research, India
Copyright © 2023 Yang and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Lin Wang, fierywang@163.com