Regulation of UCP2 in nonalcoholic fatty liver disease: From mechanisms to natural product

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease associated with lipid deposition in liver cells and/or subsequent inflammation, excluding other known causes. NAFLD is a subset of metabolic syndrome that ranges from simple steatohepatitis (NASH), fibrosis to cirrhosis and hepatocellular carcinoma (HCC). At present, the pathogenesis of NAFLD remains unclear. Among the many factors that shape these transitions, uncoupling protein 2 (UCP2) may be involved in every stage of the disease. UCP2 is a carrier protein that responds to fatty acids (FAs) in mitochondrial intima and has a wide tissue distribution. However, the biological function of UCP2 has not been fully elucidated, and most of our current knowledge comes from cell and animal experiments. These data suggest that UCP2 plays a role in lipid metabolism, oxidative stress, apoptosis, and even cancer. In this review, we summarize the structure, distribution, and biological function of UCP2 and its role in the progression of NAFLD, as well as natural products targeting UCP2 to improve NAFLD.


| INTRODUCTION
The incidence of nonalcoholic fatty liver disease (NAFLD) has risen dramatically, affecting about a quarter of the global population (Younossi et al., 2018).NAFLD is an umbrella term for a range of diseases in which steatosis is present in more than 5% of liver cells with metabolic risk factors (especially obesity and type 2 diabetes), not including excessive alcohol consumption or other chronic liver diseases (Guo et al., 2022).NAFLD can be divided into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) (Leoni et al., 2018).NAFL is characterized by steatosis with or without mild lobular inflammation, and NASH is characterized by liver injury (hepatocellular balloon-like degeneration, diffuse lobular inflammation, and fibrosis) (Bessone et al., 2019).Simple steatosis can progress to fibrosis, cirrhosis to hepatocellular carcinoma (HCC), so NAFLD has become a world public health problem that cannot be ignored (Bessone et al., 2019).
NAFLD is often associated with features of metabolic syndrome, such as dyslipidemia and obesity (Dietrich & Hellerbrand, 2014).However, the pathogenesis of NAFLD is not well understood, which has become an obstacle to NAFLD treatment.Previous studies have shown that liver steatosis, caused by excess fatty acids (FAs), is the "first hit".Liver cells will eventually cause pathological changes such as injury, inflammation, and fibrosis due to oxidative stress and lipid peroxidation, resulting in a "second hit" (Basaranoglu et al., 2013).Nowadays, it is widely believed that the "multiple hit" theory is based on the "second hit" theory, which includes a variety of factors such as oxidative stress and lipotoxicity (Buzzetti et al., 2016).Collectively, the evidence of the past decade also supports the reliability of the multiple parallel hits hypothesis.However, large randomized controlled trials specifically targeting inflammatory pathways are still lacking (Tilg et al., 2021).
With no Food and Drug Administration-approved drugs, the current treatment options include dietary restrictions and lifestyle changes (Negi et al., 2022).However, there are a number of drug treatments designed to alleviate NAFLD-NASH that are currently being examined in various stages of clinical trials and are considered good candidates for the treatment of NAFLD/NASH (Negi et al., 2022).However, people with NAFLD often have difficulty maintaining an improved lifestyle.Therefore, it is of great practical significance to strengthen the research on the pathogenesis of NAFLD and find safe and effective drugs to prevent and treat NAFLD (Guo et al., 2022).Many studies have shown that compared with traditional therapies, natural products have obvious advantages in terms of low toxicity, fewer side effects, and light economic burden (Guo et al., 2022).This review will focus on the potential role of uncoupling protein 2 (UCP2), a member of the mitochondrial inner membrane transporter family, in NAFLD (Luby & Alves-Guerra, 2022).UCP2 is an attractive target, but the specific function of UCP2 is unclear.Nevertheless, UCP2 may play a role in various stages of NAFLD.Therefore, it makes sense to take a look at current knowledge about UCP2 and outline potential directions for further research.In this article, we also review some natural products that may have therapeutic potential for NAFLD.

| UCP2
UCP is located in the inner mitochondrial membrane and belongs to the mitochondrial transporter family SLC25 (Palmieri, 2013).The UCP family consists of five members (UCP1-UCP5) (Table 1).UCP1 is mainly expressed in brown adipose tissue.UCP1 allows mitochondrial membrane potential to be transduced to heat (Li et al., 2019).UCP2 was discovered in 1997, and the amino acid sequence of UCP2 has 59% homology with that of UCP1 (Cadenas, 2018).Compared with UCP1, UCP2 tissues are widely distributed, but UCP2 is usually only expressed in Kupffer cells in the liver.However, UCP2 expression in liver cells is very low and sometimes even undetectable (Brauner et al., 2001).The human UCP2 gene is located in 11q13 and consists of 8 exons and 7 introns, with 6 distal exons encoding UCP2 protein (Lentes et al., 1999).UCP2 is an antioxidant that inhibits the production of reactive oxygen species (ROS) in mitochondria (Cadenas, 2018).UCP2 also plays a role in uncoupling the transmembrane proton gradient (Vallejo et al., 2021).UCP3 is mainly present in skeletal muscle and is involved in the regulation of skeletal muscle respiration (Boss et al., 1997).UCP4 and UCP5 are located primarily in the brain (Zheng et al., 2015), which play an important role in energy balance and neuroprotection (Ramsden et al., 2012).

| UCP-2 and fatty acids
The liver showed increased liver fat, liver triglyceride accumulation and insulin resistance, and the sensitivity of liver cells to internal and external injuries increased during "the first hit" (Fang et al., 2018).Multiple studies have shown that increased plasma FAs levels lead to increased UCP2 expression (Reilly & Thompson, 2000).Previous studies have shown that UCP2 levels in white adipose tissue of ob/ob mice with leptin deficiency or leptin receptor deficiency are significantly increased after being fed a high-fat diet (HFD) (Fleury et al., 1997).In addition, many animal experiments have also shown that the basic expression of UCP2 is low or even undetectable in normal liver cells, but the expression of UCP2 is significantly increased in NAFLD and NASH (Baffy et al., 2002;El-Derany & El-Demerdash, 2020;Jiang et al., 2008;Nii et al., 2014;Yu et al., 2018).The upregulation effect of different FAs on UCP2 expression was also different.In liver cells, polyunsaturated FAs stimulated liver UCP2 mRNA level more than monounsaturated FAs, while saturated palmitic acid had little effect (Armstrong & Towle, 2001).This difference in FAs might be caused by transcription factors regulated by FAs and their interaction with UCP2.

| Regulation of peroxisome proliferator-activated receptors
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factor belonging to the nuclear receptor superfamily.The PPAR family consists of three isoforms, including PPARα, PPARγ, and PPARβ/δ, all of which can be activated by free FAs and their derivatives.Various unsaturated FAs can be used as ligands or activators of PPAR to induce UCP2 expression (Kersten et al., 2000).PPARα activated genes involved in the oxidation of various FAs, including carnitine palmitoyltransferase 1 (CPT1) and UCP2 (Wang et al., 2020), improving the treatment of NAFLD (Shin et al., 2020).PPARγ expression was low in healthy liver, but its expression was increased in animal models of steatosis (Rahimian et al., 2001), and PPARγ ligands induced UCP2 expression in fatty liver (Memon et al., 2000).However, the role of UCP2 in NAFLD is unclear.Studies have found that upregulation of UCP2 in human and rat NASH liver induces mitochondrial uncoupling reduces redox pressure in the mitochondrial respiratory chain, and acts as a protective mechanism against damage progression.But it also impairs the liver's ability to respond to additional acute energy demands such as ischemia-reperfusion (Serviddio et al., 2008).On the one hand, FAs enter the matrix in the form of acyl-CoA, and insufficient NADH reoxidation may result in more acyl-CoA being cleaved into non-esterified fatty acids (NEFA) (Eaton, 2002;Hunt & Alexson, 2002).NEFA cannot be further metabolized in the matrix or penetrate the inner membrane, and will then be transported to the cytoplasm (Hunt & Alexson, 2002), where this free fatty acids (FFA) accumulation is more unfavorable than the accumulation of triglycerides (Listenberger et al., 2003).On the other hand, a large number of FAs may enter the mitochondrial matrix and undergo βoxidation to produce excess NADH, which needs to be re-oxidized by mitochondrial respiration.In the case of excess energy, mitochondrial respiration may be limited by high proton levels (Nobes et al., 1990), and UCP2-mediated proton leakage reduces mitochondrial membrane potential and supports continuous FAs oxidation.Therefore, UCP2 may prevent the accumulation of FFA and its harmful effects in the mitochondrial matrix.

| Regulation of AMPK
UCP2 is also associated with AMP-activated protein kinase (AMPK), an evolutionarily conserved stress-sensitive protein kinase that is activated by detecting ATP reduction to maintain cellular metabolic homeostasis (Zhao et al., 2023), UCP2 activation may be related to pathways that depend on its upstream AMPK-mediated antioxidant response (Yu et al., 2019).In the liver, AMPK promotes FAs oxidation and ketogenesis and inhibits cholesterol and triglyceride formation (Carling, 2004).Therefore, AMPK can improve insulin resistance and prevent fat accumulation in the liver (Li, Li, Yang, et al., 2022).Activation of AMPK is part of adiponectin insulin sensitization (Yamauchi et al., 2002).Adiponectin is a fat-derived bioactive protein that promotes UCP2 expression in nonparenchymal cells by stimulating the production of mitochondrial superoxides (Zhou et al., 2012).UCP2 plays a key role in mediating the beneficial effects of adiponectin on mitochondrial function.The effect of adiponectin on endotoxin-induced liver injury was significantly diminished in UCP2-deficient mice, suggesting that increased UCP2 expression may be required for adiponectin to activate its liver protective function (M.Zhou et al., 2008).Further studies have shown that the expression of UCP2 was parallel to AMPK activity (Shin et al., 2020).Previous studies have shown that UCP2 deficiency leads to reduced liver utilization and clearance of FFA.Interestingly, studies have shown that downregulation of UCP2 can promote the recovery of hepatic steatosis (Evans et al., 2008).In addition, another study has shown that increased UCP2 expression in the livers of genetically or dietologically obese mice has neither a protective nor detrimental effect on the severity of fatty liver disease (Baffy et al., 2002).These seemingly contradictory results have led to the discussion of the relationship between UCP2 and NAFLD.

| UCP-2 and the production of reactive oxygen species
During the "the second hit" of NAFLD, many factors including inflammatory cytokines, adipokines, mitochondrial dysfunction, and oxidative stress lead to necrotizing inflammation and fibrosis that can develop and eventually lead to cirrhosis (Fang et al., 2018).In NAFLD, damaged fatty acid oxidation (FAO) in hepatocyte mitochondria leads to lipid accumulation and oxidative damage (Zeng et al., 2022).Due to its uncoupling effect, UCP-2 can affect the mitochondrial oxidative respiratory chain, inhibit the production of mitochondrial ROS, etc. (Hu et al., 2019) It was found that ROS production and TNF-α expression were increased, UCP2 expression was decreased, mitochondrial content and biomarkers were decreased in NAFLD (Rafiei et al., 2017).UCP2 can disperse the mitochondrial intima potential and pH gradient to reduce ROS production (Sreedhar & Zhao, 2017).Chronic oxidative stress leads to the accumulation of advanced glycation end products (AGEs), and oxidative stress associated with mitochondrial dysfunction enhances liver protein glycosylation, thereby exacerbating inflammatoryinduced liver injury.Studies have shown that oxidative stress and liver glycosylation are increased in UCP2 −/− mice (Kuhla et al., 2010).It was found that early liver injury decreased the expression of UCP2 in liver tissue, significant steatosis of liver cells, increased production of oxygen free radicals in cells, and promoted lipid peroxidation (Wang, Cheng, et al., 2021).Studies have shown that UCP2 deficiency enhances the AGE/RAGE interaction.Aging is accompanied by increased oxidative stress.In addition, oxidative stress leads to increased MG-AGE and accumulation of highly modified AGE, accompanied by upregulation of RAGE expression.UCP2 deficiency was associated with significantly elevated levels of oxidative stress in all age groups (Kuhla et al., 2011).Therefore, UCP2 played a protective role in NAFLD by improving oxidative stress (Chang et al., 2011;Ma et al., 2012).However, some studies have also shown that UCP2 expression in the liver of mice with steatosis liver is decreased, oxidative stress is reduced, and mitochondrial function is improved (Evans et al., 2009).Although studies linking ROS to UCP2 activation have shown that UCP2 in NAFLD is not only highly expressed but also functional, there is no good evidence that UCP2 contributes to reducing ROS production.We speculate that the increased amount of UCP2 is not sufficient to inhibit ROS production in hepatocytes.UCP2 expression in normal liver was found in Kupffer cells, but was very low in hepatocytes (Jin et al., 2013).Interestingly, this state seems to change under certain metabolic conditions.UCP2 expression decreased and mitochondrial ROS increased in peritoneal macrophages of ob/ob mice (Lee et al., 1999).These results suggested that the increase of UCP2 expression in hepatocytes in fatty liver was associated with downregulation of UCP2 in macrophages, a change that may be inferred to Kupffer cells.Notably, in most studies, UCP2 expression was evaluated in whole liver mitochondria (Yang et al., 2000).In addition, the expression of UCP2 in endotoxin-induced UCP2 −/− mouse hepatocytes and Kupffer cells was different in UCP2 mice that received macrophages from UCP2 +/+ mice via bone marrow transplantation (Alves-Guerra et al., 2003).Therefore, the effect of UCP2 on ROS in NASH was likely related to its decreased expression in liver macrophages.The details of negative transcriptional regulation of UCP2 in macrophage responses to endotoxins and obesity remain to be elucidated.Although the increase in mitochondrial ROS production in steatohepatitis may be due to the downregulation of UCP2 in liver macrophages, studies have also shown that the effect of UCP2 ablation on fatty liver cells is considerable, where UCP2 deficiency appears to reduce ATP storage (Fülöp et al., 2006).If alterations in UCP2 expression have the potential to alter steatohepatitis, cell specificity should be considered for any intervention.

| UCP2 AND LIVER CANCER
4.1 | UCP-2 and reactive oxygen species NAFLD may progress to cirrhosis and HCC (Estes et al., 2018).ROS plays an important role in cancer, and one feature that distinguishes cancer cells from normal cells is their ability to produce a higher number of reactive oxygen species and to rely more heavily on antioxidant defense systems (Prasad et al., 2017).ROS regulates a variety of cell signaling pathways and is involved in cell proliferation, invasion, angiogenesis, and metastasis (Prasad et al., 2017).UCP2 is an antioxidant that inhibits the production of ROS in mitochondria (Deng et al., 2012).It has been shown that lipid and protein oxidation are increased in HepG2 cells exposed to ROS, while this increase is significantly reduced the expression of UCP2 under the same conditions.UCP2 can limit the oxidative damage of HepG2 cells under oxidative stress, thereby improving cell function and antiapoptotic ability (Collins et al., 2005).

| UCP-2 and apoptosis
In addition, most cancers were caused by an imbalance between cell proliferation and cell death.Mitochondria play an important role in the regulation of apoptosis (Zhou et al., 2019).While intrinsic apoptotic pathways are primarily responsive to metabolic perturbations and intracellular oxidative stress, extrinsic pathways are activated through cell surface death receptors.There is evidence of crosstalk between these two pathways, with mitochondria involved in both processes (Al-Aamri et al., 2021).Based on its assumed biological function, UCP2 may regulate apoptosis.UCP2 overexpression induced G1 cell cycle arrest and non-apoptotic cell death in Hepa 1-6 cells, and UCP2 overexpression inhibited cell proliferation and led to loss of cell viability through non-apoptotic cell death (Cai et al., 2022;Palanisamy et al., 2014).Studies have shown that overexpression of UCP2 in liver cancer cells contributes to autophagy and anti-apoptosis after FAs injury.During palmitic acid, UCP2 upregulates, and hepatocyte autophagy increases.Overexpression of UCP2 in HCC cells contributes to autophagy, which played a protective role in hepatocyte lipid apoptosis (Lou et al., 2014).

| UCP-2 and chemotherapy resistance
Furthermore, chemotherapy resistance is one of the main causes of cancer treatment failure.Recent studies have found that UCP2 may regulate the sensitivity of cancer cells to anti-tumor drugs.It was found that UCP2 and miR-214 expression were negatively correlated.The expression of miR-214 was significantly down-regulated and UCP2 protein was highly expressed in HCC patients.UCP2 overexpression mediates gemcitabine (GEM) resistance in HCC patients (Yu et al., 2016).In HCC cell lines with low endogenous UCP2 expression, ectopic overexpression of UCP2 significantly reduces the induction of mitochondrial superoxide by t GEM.In contrast, in HCC cell lines with high endogenous UCP2 expression, silencing UCP2 mRNA by RNA interference significantly enhanced GEM-induced mitochondrial superoxide production and apoptosis.In conclusion, mitochondrial uncoupling plays a key role in the GEM resistance of HCC cell lines.Therefore, synergistic targeting of UCP2 in combination with other chemotherapy agents may be more effective in HCC patients (Yu et al., 2015).The results showed that different tissue differentiation, lymph node metastasis, TNM stage, survival status, and UCP2 level were independent risk factors for paclitaxel resistance, and UCP2 level was positively correlated with paclitaxel resistance.The paclitaxel resistance and poor survival of UCP2-positive liver cancer patients can be used as the basis for prognosis assessment (Zhao, 2020).These studies provided more support for the role of UCP2 in cancer cell adaptation (139).However, it is unclear whether long-term high expression of UCP2 in NAFLD In summary, it is an interesting possibility to consider UCP2 as a negative regulator of ROS production, as a regulator of apoptosis, and as a factor in the adaptation of cancer cells to oxidative stress.Further studies are needed to determine the role of UCP2 in cancer, particularly in NAFLD-associated HCC formation.

| Natural products targeting UCP2 in NAFLD
Natural products play an important role in improving fatty liver by targeting UCP2.We summarized natural products that have the effect of improving NAFLD.The development of natural products may be a broad direction for NAFLD treatment.

| Total flavonoids of hawthorn leaves
Total flavonoids of hawthorn leaves (TFHL) are derived from hawthorn leaves, which have anti-inflammatory, antioxidant activity, and neuroprotective property (Wang et al., 2018).In the NASH model group, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and the contents of triacylglycerols, cholesterol, malondialdehyde, and UCP2 in liver tissue were significantly higher than those in normal rats.TFHL alleviated hepatic inflammatory damage by inhibiting oxidative stress, lipid peroxidation, and UCP2 overexpression in liver tissue, thereby preventing the further development of NASH (Chen et al., 2009).

| Punicalagin-enriched pomegranate extract
Pomegranate (Punica granatum L.) has been used in traditional herbal medicine, which has antioxidant, antiinflammatory qualities, and also used in treatment and prevention of cancer and other diseases.Punicalaginenriched pomegranate extract (PE) significantly inhibited HFD-induced hyperlipidemia and hepatic lipid deposition.With PE supplementation, the expression of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL1), interleukin-4 (IL4), and interleukin-6 (IL6) was increased and oxidative stress in hepatocytes was enhanced, followed by normalization of Nrf2 activation.In addition, PE treatment reduced UCP2 expression, restored ATP content, inhibited mitochondrial protein oxidation, and improved the activity of liver mitochondrial complex.In contrast, although PGC1α was increased and mitochondrial βoxidation-related gene expression was elevated after PE treatment, mitochondrial content was not affected.Punicalagin is the main active component of PE, and both Punicalagin and PE-protected cells from palmitate-induced mitochondrial dysfunction and insulin resistance (Zou et al., 2014).

| Total flavonoids of astmgali radix
Total flavonoids of Astmgali Radix (TFA) are the main active ingredient of astragalus, which has anti-damage and anti-tumor effects (Cui et al., 2010;Tang et al., 2009).TTFA significantly increased PPARγ levels and increased UCP2 and farnesoid X receptor (FXR) levels, compared to the model group.TFA improved liver function, alleviated liver pathological changes, and reduced collagen deposition and formation of liver pseudolobule in cirrhotic rats.The anti-fibrotic effect of TFA was realized by regulating the PPARγ signaling pathway and interacting with FXR (Cheng et al., 2017).

| Bergamot polyphenolic fraction
Bergamot polyphenolic fraction (PF) is a natural mixture of Citrus flavonoids extracted from processed bergamot fruits (Janda et al., 2018).PF decreased intracellular lipid content and was associated with increased expression of βoxidation genes (acyl-CoA oxidase 1, PPARα, and UCP2) and lipid phagocytosis (ATG7), suggesting that PF may decrease intracellular lipid neutral fat by increasing intracellular pathways associated with βoxidation (Mirarchi et al., 2022).

| (−) -epicatechin
(−) -epicatechin (EC) is found in many chocolates (Theobroma cacao L. Sterculiaceae), which is known for its mitochondrial function, and antioxidant activity (Chun et al., 2022).EC decreased the activities of triglyceride, low-density lipids, ALT, and alkaline phosphatase (ALP), suggesting a reduction of liver injury.Compared to the untreated group, liver cells had less ballooning, lipid droplets, less collagen accumulation, and less inflammatory cell infiltration.EC decreased the expression of perilipin 2 (PLIN2) and CD36 and increased the expression of adiponectin and UCP2.In summary, EC improved biochemical characteristics, microscopic characteristics, and protein expression.Therefore, it might be a possible treatment for NASH (Hidalgo et al., 2020).

| Resveratrol
Resveratrol is a polyphenol found in a variety of vegetables and fruits.It has many properties, including antioxidative stress, anti-inflammation, and anti-cancer activity (Galiniak et al., 2019).Studies have shown that resveratrol inhibited the proteolytic cleavage of SREBPs-1 and SREBPs-2, inhibited the βoxidation of FFA and ROS production by CPT1 and UCP2.It also reduced pancreatic lipase activity in vivo and in vitro and prevented the occurrence and development of NAFLD (Khaleel et al., 2018).Increased mitochondrial numbers, specifically increased expression of the liver uncoupling protein UCP2, were associated with normalization of liver fat content resulting from resveratrol supplementation in a high-fat diet (Poulsen et al., 2012).Interestingly, it has been found that resveratrol treatment has no significant effect on UCP2 expression.This means that regardless of the positive or negative effects of UCP2 on steatosis, the defat effect of this phenolic compound is not mediated by this uncoupling protein (Jiang et al., 2008).

| Quercetin
Quercetin, a flavonoid, is widely distributed in fruits and vegetables, which is known for its antiinflammatory, antioxidant, and anti-viral properties (Di Petrillo et al., 2022).In the model group, UCP2 expression decreased, while quercetin treatment significantly upregulated UCP2 expression, suggesting that quercetin induced the upregulation of UCP2 and inhibited the production of excess mitochondrial ROS and inflammatory factor TNF-α.Thus, the expression of CPT1α was up-regulated, the function of mitochondrial fatty acid βoxidation was enhanced, and finally, the steatosis of hepatocytes was improved (Li, Li, Li, et al., 2022;Li, Li, Yang, et al., 2022).In addition, our previous studies also found that quercetin can improve lipid deposition in HepG2 cells by reducing the phosphorylation level of AKT (Li et al., 2023).

| Blue honeysuckle
Blue Honeysuckle (BH) (Lonicera caerulea L.) is a promising berry crop with a valuable chemical composition (Naugžemys et al., 2022).BH extract improved fat density and mass, adipocyte histopathology, liver enzyme activity, lipid metabolism and the expression of UCP2, adiponectin, and sterol regulatory element binding protein 1 (SREBP1) and other related genes in rat, improving the risk factors of NAFLD (Chun et al., 2018).

| Phytosterol esters
Phytosterols have attracted more and more attention because of their good cholesterol-lowering, antioxidant, anti-inflammatory, and cardiovascular protective activities (Wang, Xiao, et al., 2021).Treatment with phytosterol esters improved the liver histology of NAFLD, reduced the liver total lipids, and reduced the serum ALT and AST levels in rats.Low and medium doses of PSEs effectively prevented the occurrence and progression of NAFLD by reducing blood low-density lipoprotein cholesterol (LDL-C), improving oxidative stress, and regulating key cytokines.These effects may be related to upregulation of mRNA and protein expression of TNF-α and certain metabolism-related genes (PPARα, PPARγ, and UCP2) in the liver (Song et al., 2017).
In summary, there are few studies on natural product regulation of UCP2 in the treatment of NAFLD, and the specific role of UCP2 is still unclear, and the research is not deep enough.Many signaling pathways closely related to NAFLD should be associated with UCP2, such as AMPK, Wnt/β-catenin signaling pathways, etc., to increase the depth of treatment for NAFLD.In addition, most studies mainly focus on the cellular and animal level, which cannot fully simulate the pathological characteristics of human NAFLD, and its drug activity needs to be further studied and confirmed.At present, there is still a lack of specific drugs to treat NAFLD in clinical practice, resulting in no control drugs in animal, cell, and clinical experiments, resulting in a lack of unified and clear efficacy standards for natural products to treat NAFLD.In addition, there are currently no clinically approved drugs to treat NAFLD, resulting in no control drugs in animal experiments, cell experiments, and clinical trials, resulting in the lack of unified and clear efficacy standards for natural products to treat NAFLD.Current first-line treatment focuses on lifestyle changes through diet and exercise to treat NAFLD, but there are many drugs in development that are considered good candidates for the treatment of NAFLD/NASH (Negi et al., 2022).

| CONCLUSIONS AND PERSPECTIVES
The incidence of NAFLD is increasing, but the pathogenesis is unknown and has been extensively studied.Currently, in the framework of the "two hit hypothesis", increasing evidence supports the importance of mitochondrial dysfunction in NAFLD, in which UCP plays an important role in the uncoupling of oxidative phosphorylation.The relationship between UCP2 and NAFLD is still controversial.Most importantly, the biological functions and regulatory mechanisms of UCP2 remain much debated.In addition, much of our knowledge about UCP2's role in liver disease comes from animal models and is not fully representative of NAFLD in humans.Despite these limitations, we were able to identify several areas where UCP2 plays a role in the pathogenesis of NAFLD associated with metabolic syndrome.In addition to promoting liver fat accumulation and insulin resistance, elevated plasma FAs levels and certain lipogenic bioactive factors increase UCP2 expression and activation in fatty liver.UCP2 might alter fat distribution and metabolism in the liver, but there is no convincing evidence that upregulation of UCP2 has an effect on the occurrence of steatosis.Consistent with the "two hit" theory of NAFLD progression, UCP2 promoting the transition from steatosis to steatohepatitis seems more feasible.UCP2 had the ability to influence hepatocyte bioenergetics in the face of other challenges.Oxidative stress is a major causative factor in steatohepatitis, and UCP2 has been shown to control ROS production at the primary source of the mitochondrial respiratory chain.Identifying specific regulation of UCP2 expression in hepatocytes allowed us to distinguish between these roles in parenchymal and nonparenchymal cells.From the perspective of NAFLD multiple parallel hits hypothesis (Tilg et al., 2021), the role of UCP2 in different links was studied.Finally, UCP2 might mitigate ROS-induced apoptosis and was a factor in the development of HCC associated with obesity and insulin resistance.In summary, UCP2 was closely related to the pathogenesis of NAFLD, and studying the role of UCP2 in NAFLD might provide new mechanisms and therapeutic targets.
By targeting UCP2, natural products may be important to overcome the limitations of NAFLD therapy.Importantly, the natural product targets UCP2, regulating oxidative stress, apoptosis, and autophagy.In addition, many physicians are attracted to using natural products to alleviate NAFLD because of their safety, mass availability, and low cost, which have been demonstrated in many recent studies.For example, in addition to pre-clinical studies, there are also some clinical studies.Recent lines of evidence from other randomized controlled trials (RCTS) and animal models have also found that natural products have a therapeutic effect on NAFLD (Tarantino et al., 2021).Moreover, our previous study found that in an open-label exploratory clinical trial to evaluate the effects of GLS (Coptidis Rhizoma-Evodiae Fructus 2:1) on fibroblast growth factor 21 in patients with NAFLD.GLS has a significant liver protective effect in patients with NAFLD.It can cause the secretion of FGF-21 to decrease in response to self-injury (Zhang et al., 2022).The multi-target and multi-pathway therapeutic effect of natural products on liver diseases is its advantage, but the intensity of the effect on the target is not enough.The current research depth is more limited to the study of all hepatocyte abnormalities, and more attention should be paid to the study of non-hepatocellular glycolysis in different liver diseases, such as fibroblasts, Kupffer cells, macrophages, and T cells.In addition, many natural product studies are still in the pre-clinical stage, and more clinical data is needed to support the safety and efficacy of natural products.In addition, the research of natural products for NAFLD should not only stay in the pre-clinical research stage, but should strive to be transformed into clinical drugs.