Non-alcoholic fatty liver disease (NAFLD), one of the most common chronic liver diseases, is characterized by excessive triglyceride (TG) accumulation as lipid droplets in the cytoplasm of hepatocytes in the absence of excessive alcohol intake (Rada et al., 2020). NAFLD is a major risk factor for the development of cirrhosis and possible subsequent hepatocellular carcinoma (Bertot and Adams, 2019). NAFLD begins with excessive TG in the liver (steatosis), which can progress to non-alcoholic steatohepatitis (NASH) (Pierantonelli and Svegliati-Baroni, 2019). This condition is characterized by fat deposits, inflammation and damage to hepatocytes. The prevalence of NAFLD has increased rapidly worldwide because of changes in lifestyle and dietary habits (Yasutake et al., 2014). Obesity and the intake of foods high in sugar and fat are closely associated with the development and progression of NAFLD (Eng and Estall, 2021). Recent studies in humans and animals have shown that high dietary intake of fructose is an important cause of hepatic steatosis (Abdelmalek et al., 2010; Alwahsh et al., 2014; Bray and Popkin, 2014; Sellmann et al., 2015). High fructose intake causes excessive lipid accumulation in the liver via numerous pathways, among which increased lipid synthesis plays a pivotal role (Jegatheesan and De Bandt, 2017). Activation of hepatic de novo lipogenesis (DNL) can contribute to the production and accumulation of TG in the liver, and this activation is regulated by several transcription factors (Jegatheesan and De Bandt, 2017). The two major mediators of pathways involved in hepatic DNL are carbohydrate response element-binding protein (ChREBP), which is a glucose-sensing transcription factor, and sterol regulatory element binding protein-1c (SREBP-1c), which is an insulin-sensing transcription factor (Jegatheesan and De Bandt, 2017). These two mediators operate mainly by upregulating lipogenesis-related genes, and this upregulation has been observed in the liver of rats with NAFLD (Jegatheesan et al., 2015). The glycerol-3-phosphate (G3P) pathway is another important pathway for triglyceride (TGs) synthesis in the liver, and is under the control of two key enzymes, i.e. acyl-CoA:glycerol-sn-3-phosphate acyltransferase (GPAT-1) and diacylglycerol/acyl-CoA acyltransferase-2 (DGAT2) (Gluchowski et al., 2019; Karasawa et al., 2019). GPAT-1 catalyzes the first step in TG synthesis, and DGAT2 is a rate-limiting enzyme catalyzing the final step in TG synthesis. Previous studies have demonstrated that the suppression of GPAT-1 and DGAT-2 reverses diet-induced hepatic steatosis in experimental animals (Park et al., 2011; Zhao et al., 2015; Gluchowski et al., 2019). Increased hepatic lipid accumulation promotes inflammatory cytokine production and oxidative stress, which contribute to the progression of hepatic steatosis to NASH and fibrosis (Jegatheesan and De Bandt, 2017; Eng and Estall, 2021). Therefore, improving lipid metabolism and/or reducing hepatic oxidative stress and inflammation are considered an effective strategy to prevent NAFLD progression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a cytoprotective transcription factor that plays a major role in the elimination of ROS and subsequent reduction in the inflammatory response (Tu et al., 2019; Galicia-Moreno et al., 2020). It activates elements of the antioxidant response (ARE) and upregulates the expression of several antioxidant genes, including heme oxygenase-1 (HO-1) (Galicia-Moreno et al., 2020). The induction of HO-1, one subtype of the heme oxygenases, catalyzes the rate limiting step in the breakdown of heme to the antioxidant bilirubin (Origassa and Câmara, 2013). HO-1 and its metabolites have been shown to exert several protective biological activities, displaying antioxidant, anti-inflammatory and anti-apoptotic properties (Guo et al., 2018; Kuo et al., 2020). Recently, it has been demonstrated that an increased Nrf2/HO-1 signaling might play a significant role in preventing hepatic oxidative stress and inflammation induced by a high-fructose diet (Hu et al., 2017; Zhao et al., 2018).

Naringin (4′,5,7-trihydroxyflavone 7-rhamnoglucoside) is found in grapefruit and related citrus species; its colonic metabolite is naringenin (Arafah et al., 2020). Both naringin and naringenin have been reported to display a number of pharmacological activities, including antioxidant, anti-inflammatory, hypoglycemic, hypolipidemic, cardioprotective and hepatoprotective effects (Ortiz-Andrade et al., 2008; Jain and Parmar, 2011; Alam et al., 2013; Hermenean et al., 2014; Ahmed et al., 2017; Rashmi et al., 2018; Chung et al., 2019; Pengnet et al., 2019; Yu et al., 2019). Previous studies have reported that naringin can ameliorate high-fat-diet-induced hepatic steatosis by decreasing oxidative stress, promoting fatty acid oxidation, inhibiting fatty acid synthesis in the liver (Pu et al., 2012; Mu et al., 2020), and modulating intestinal gut microbiota (Mu et al., 2020). In addition, our previous study indicated that daily administration of naringin at 100 mg/kg/day for 4 weeks can lower serum levels of TG, TC, and LDL-C in high fructose solution-fed rats. This finding also demonstrated the vascular-protective properties of naringin via decreasing vascular oxidative stress (Malakul et al., 2018). Growing evidence suggests that the lipid-lowering properties of natural products may be associated with the amelioration of NAFLD (Malakul et al., 2022; Prommaouan et al., 2022). However, the efficacy of naringin in preventing NAFLD progression in fructose-induced NAFLD has not yet been evaluated. Therefore, the purpose of the current study was to determine whether naringin could ameliorate NAFLD progression induced by fructose feeding, and to evaluate the hypothesis that naringin inhibits the progression of NAFLD via regulating the DNL and G3P pathways, enhancing the Nrf2/HO-1 pathway and decreasing inflammatory cytokine production.

The present results clearly demonstrate that treatment of rats with naringin, a major bioflavonoid in citrus fruits, was highly effective in preventing fructose-induced fatty liver disease. This could be due to its effect on decreasing the protein levels of ChREBP, SREBP-1c, nSREBP-1c and their target proteins, ACC and FAS, with a prominent decrease in GPAT-1 and DGAT2 expression. In addition, our study is the first to demonstrate that naringin can prevent the progression of NAFLD by suppressing the NF-kB/TNF-α pathway and increasing the Nrf2/HO-1 pathway in the liver of fructose-induced NAFLD rats.

Fructose is commonly used in large amounts throughout the world as a sweetener in food and beverages. After fructose consumption, it is metabolized rapidly in the liver and provides large amounts of hepatic triose-phosphate as a substrate for hepatic DNL (Federico et al., 2021). This process converts carbohydrates into fatty acids by the key enzymes ACC and FAS; these fatty acids are esterified into TG, which are then exported from the liver as very-low-density lipoprotein (VLDL) (Alves-Bezerra and Cohen, 2017). TG may accumulate as lipid droplets in the cytoplasm of hepatocytes due to increased DNL; the consequence of this phenomenon is fatty liver (Alves-Bezerra and Cohen, 2017; Gerges et al., 2021). Several studies suggest that overconsumption of dietary fructose causes lipid deposition in the liver and leads to the development of NAFLD (Alwahsh et al., 2014; Sellmann et al., 2015; Li et al., 2018; Zhao et al., 2018).

Natural compounds found in fruits and vegetables, such as flavonoids, have been shown to ameliorate hepatic lipid accumulation, thus providing excellent therapeutic potential for the treatment of NAFLD (Braud et al., 2017). Several studies have demonstrated that naringin possesses great health benefits, and prevents various metabolic diseases, such as hyperlipidemia, cardiovascular dysfunction and obesity (Pu et al., 2012; Alam et al., 2013; Pengnet et al., 2019). We previously reported that the beneficial effects of oral administration of naringin on serum lipid levels in fructose-fed rats (Malakul et al., 2018); therefore, we hypothesized that naringin could prevent fructose-induced NAFLD because of its anti-hyperlipidemic properties. Previous study has reported that naringin is able to ameliorate NAFLD induced by high-fat diets in experimental animals by modulating the intestinal bacterial composition (Mu et al., 2020). However, the mechanisms by which naringin acts on hepatic lipid accumulation has not been elucidated clearly. In the present study, we further investigated the influences of naringin administration on lipid accumulation in the liver and related mechanisms in fructose-fed rats. Consistent with other findings (Li et al., 2018; Yang et al., 2019), we found that a 10% solution of fructose in the drinking water promoted hepatic steatosis, as demonstrated by an increase in liver weight, the liver/body weight ratio, Oil Red O staining area of the liver and hepatic TG and TC contents. In addition, the activities of serum AST and ALT, as indicators of liver function and liver injury, were increased in the high-fructose group. These results suggest that high fructose intake induced liver steatosis and damage, resulting in the development NAFLD. In this study, naringin treatment effectively reversed the fructose-induced alterations in these parameters, indicating that naringin improved liver steatosis and damage induced by fructose feeding.

Accumulating evidence has demonstrated that the principal route for fructose feeding-induced hepatic steatosis is the increase in DNL, and a rate-limiting enzyme for this pathway is ACC and FAS (Silva et al., 2019; Federico et al., 2021). Both ACC and FAS are known to be regulated by the transcription factors SREBP-1c and ChREBP (Alves-Bezerra and Cohen, 2017). SREBP-1c is a lipogenic transcriptional factor that is translocated into the nucleus for stimulating the DNL in response to a high-fat or high-carbohydrate diet, and may act in synergy with ChREBP, a glucose-mediated transcription factor, strongly regulates the gene transcription in DNL in response to dietary carbohydrates (Li et al., 2011; Alves-Bezerra and Cohen, 2017; Lane et al., 2019; Ortega-Prieto and Postic, 2019). Consistent with other studies (Erion et al., 2013; Kim et al., 2019), we found increased ChREBP, SREBP-1c and nSREBP-1c expression as well as upregulated levels of their downstream targets, FAS and ACC, in the liver of fructose-fed rats. These results indicate that fructose consumption induced the synergistic action of ChREBP and SREBP-1c on hepatic DNL, which creates fatty acids that are assimilated into TG. In addition to the DNL pathway, the G3P pathway is a crucial source for TG synthesis, which contributes to over 90% of total TG synthesis; a rate-limiting enzyme for this pathway in the liver is GPAT-1 and DGAT2 (Gluchowski et al., 2019; Karasawa et al., 2019). GPAT-1 provides glycerol-3-phosphate as a substrate for diacylglycerol, which serves as a precursor molecule for TG synthesis (Karasawa et al., 2019). DGAT2 catalyzes the acylation of diacylglycerol, resulting in enhanced TG synthesis (Zhao et al., 2015).

Naringin has been reported to reduce lipid synthesis by reducing the hepatic expression of several proteins involved in the DNL pathway, such as SREBP-1, ACC and FAS in mice fed a high-fat diet (Pu et al., 2012), diabetic rats fed a high-fat diet (Kumar Sharma et al., 2011), alcoholic fatty liver disease in rats (Zhou et al., 2019) and in obese C57BL/6J mice (Mu et al., 2020). Our data showed that treatment with naringin reversed the upregulated hepatic expression of ChREBP, SREBP-1c, nSREBP-1c, FAS and ACC in fructose-fed rats. Another interesting observation in the current study was that naringin treatment in fructose-fed rats also reduced the protein levels of GPAT-1 and DGAT2. These results suggest that downregulating the expression of ChREBP, SREBP-1c, nSREBP-1c, FAS and ACC, as well as the downregulating GPAT-1 and DGAT2 expression, are critical mechanisms involved in the naringin-induced inhibition of TG synthesis in fructose-fed rats. This finding was consistent with our results illustrated in the liver morphology and hepatic lipid contents. Therefore, the reduced expression of DNL and G3P pathway-related proteins may contribute to the anti-steatotic effect of naringin. According to the two-hit hypothesis of NAFLD pathogenesis, oxidative stress and inflammation play important roles in the second-hit action, wherein simple steatosis can progress to NASH. During NAFLD, there is increased lipid accumulation in hepatocytes and oxidative stress is promoted. These alterations result in the release of local inflammatory cytokines that further exacerbate NAFLD (Rada et al., 2020). The nuclear transcription factor Nrf2, which is redox-sensitive, plays a crucial role in the modulation of proteins and enzymes against oxidative stress and inflammation, by activating the expression of several antioxidant enzymes (Park et al., 2011). During exposure to oxidative stress or inflammation, Nrf2 dissociates from its cytoplasmic inhibitor protein and subsequently translocates to the nucleus to activate the downstream transcription of various antioxidant enzymes, including HO-1 (Park et al., 2011). HO-1 is regarded as one of the most important intracellular antioxidant mechanisms; it converts heme to carbon monoxide (CO), ferrous iron and biliverdin (Duvigneau et al., 2019). Both CO and biliverdin exert potent cellular protective effects via their antioxidant and anti-inflammatory activities (Duvigneau et al., 2019). A number of studies have demonstrated that high fructose intake induces oxidative stress and inflammation in various tissues, including the liver, which may activate the Nrf2/HO-1 signaling pathway, a cellular antioxidant defense (Hu et al., 2017; Sharma et al., 2017; Zhao et al., 2018). Therefore, the pharmacological activation of Nrf2 expression has the potential to inhibit the progression of NAFLD. Consistent with previous findings (Hu et al., 2017; Sharma et al., 2017), we demonstrated increased ROS generation and down-regulated expression of Nrf2 and HO-1 in the liver of fructose-fed rats. The results indicate that hepatic oxidative stress can be induced in fructose-fed rat.

Previous studies demonstrated that naringin reduced oxidative stress through the activation of antioxidative pathways (Gopinath and Sudhandiran, 2012; Lv et al., 2018). Thus, we further investigated whether the Nrf2/HO-1 signaling pathway is involved in the mechanisms of the protective effect of naringin in high fructose-induced NAFLD. The results reveal that naringin administration in rats fed with a high-fructose solution up-regulated Nrf2 and HO-1 expression in the liver. As predicted, positive DHE staining, as a marker of ROS generation, was decreased in the liver section by naringin administration. This effect may have been attributable to the activation of the Nrf2/HO-1 signaling pathway.

Chronic oxidative stress, generated through the oxidation of cytotoxic free fatty acids, is closely linked to the inflammatory response in the liver. NF-κB is a critical transcription factor that regulates the production of inflammatory cytokines involved in the pathogenesis of inflammatory diseases and has been implicated in the development of the chronic inflammatory state in NAFLD (Chen et al., 2014). Experimental studies have shown that overconsumption of fructose activates NF-κB, which leads to the release of pro-inflammatory cytokines that develop into inflammatory responses (Bai et al., 2020; Heeba et al., 2021; Li et al., 2022). These findings are supported by our results, which revealed higher levels of NF-κB and pro-inflammatory cytokines (TNF-α) in the fructose-fed rats compared with the control rats. In this study, treatment with naringin reversed the upregulated hepatic expression of NF-κB and TNF-α in fructose-fed rats. Taken together, the findings of this study indicate that naringin can activate the Nrf2/HO-1 signaling pathway in the liver, and that these effects may contribute to the reduction of oxidative stress, inflammation and NAFLD progression.

The present findings demonstrate the effectiveness of treatment with naringin on hepatic steatosis in fructose-induced NAFLD in rats. Our study found that naringin reduced the hepatic DNL pathway by downregulating the protein levels of ChREBP, SREBP-1c, nSREBP-1c and their target proteins, ACC and FAS, and decreased the G3P pathway by downregulating the hepatic expression of GPAT-1 and DGAT2 in the liver. Thus, naringin protects against fructose-induced hepatic lipid accumulation. In addition, naringin protects against fructose-induced liver injury and inflammation in rats, in part by activating the Nrf2/HO-1 pathway and modulating the NF-κB pathway (Figure 6). Therefore, we have demonstrated that naringin has beneficial effects that prevent and improve fructose-induced NAFLD pathogenesis.