Amelioration by Idesia polycarpa Maxim. var. vestita Diels. of Oleic Acid-Induced Nonalcoholic Fatty Liver in HepG2 Cells through Antioxidant and Modulation of Lipid Metabolism

Idesia polycarpa Maxim. var. vestita Diels (I. polycarpa) is well known as an edible oil plant which contains abundant linoleic acid and polyphenols. The objective of this study was to maximize the by-product of defatted fruit of I. polycarpa. We found that the fraction D of ethyl acetate extract (EF-D) contained more polyphenols, which contribute to its strong antioxidant activity by antioxidant assays (DPPH, ABTS, and FRAP). Meanwhile, EF-D showed a significant lipid-lowering effect on oleic acid- (OA-) induced hepatic steatosis in HepG2 cells through enhancing antioxidant activity, reducing liver damage, and regulating lipid metabolism, antioxidant, and inflammation-related gene expression. The SOD and T-AOC levels significantly increased, but the levels of MDA, AST, and ALT decreased obviously when treated with EF-D. In general, EF-D improved the antioxidant enzyme activities and decreased the hepatic injury activities. Besides, treatment with EF-D for NAFLD influenced lipid metabolism and inflammation by activating PPARα which was associated with the increased expression of CPT1 and decreased expression of SCD, NF-κB, and IL-1. Moreover, EF-D improved the oxidative stress system through activation of the Nrf2 antioxidant signal pathways and upregulated its target genes of HO-1, NQO1, and GSTA2. The results highlighted the EF-D from the defatted fruit of I. polycarpa regarding lipid-lowering, proving it to be a potential drug resource of natural products for treating the nonalcoholic fatty liver disease (NAFLD).


Introduction
Nonalcoholic fatty liver disease (NAFLD) encompassed a broad-spectrum pathology from simple triglyceride (TG) deposition in hepatocytes to nonalcoholic steatohepatitis (NASH), liver cirrhosis, and even hepatocellular carcinoma (HCC) [1]. With the change of life, a more calorie diet and less exercise have caused a rise among the general population in recent years [2]. It has been proven that oxidative stress involved in the etiology of NASH caused an imbalance between prooxidant and antioxidant chemical species that led to oxidative damage of cellular macromolecules [3][4][5][6][7].
Related reports showed that the damage of the membrane system will lead to the increased oxygen pressure in NAFLD and can initiate an oxidative stress response. Furthermore, multiple metabolic pathways of NAFLD can be mediated by PPARs to regulate lipid metabolism, glucose metabolism, and immune pathways. At present, there is no specific drug for the effective treatment of NAFLD due to its unclear pathogenesis and "crosstalk" between multiple metabolic pathways [8,9]. Based on the understanding of the current situation of NAFLD, it is urgent to develop new drugs and explore new therapeutic ways.
Idesia polycarpa Maxim. var. vestita Diels (I. polycarpa), a member of the Salicaceae family and the only species of monotypic genus Idesia, is a common oil woody and edible plant distributed widely in East Asia, such as China, Japan, and Korea. Research concerning the use of I. polycarpa as a natural source of edible oil and dietary supplement increased gradually due to its various unsaturated fatty acids, such as linoleic acid and linolenic acid [10,11]. The relative report has been confirmed that the oil of its fruits is nontoxic and has anticarcinogenic and antidiabetic effects and antihypertensive properties [12,13]. Moreover, flavonoid and phenolic glycosides of I. polycarpa exhibited various biological activities, such as antioxidant, skin whitening, inhibition of platelet aggregation, and antiadipogenesis [14]. Previous researches demonstrated that the ethyl acetate extracts of I. polycarpa exhibited strong antioxidant [14], anti-inflammatory, and whitening activities in vitro and in vivo [15,16].
Taken together, these researches showed that I. polycarpa could be regarded as having potential medicinal value as a natural product for its antioxidant and anti-inflammatory activity. In our study, we isolated the EF-D from the defatted fruit of I. polycarpa by high-speed counter-current chromatography (HSCCC). Besides, we investigated the treatment effect and mechanism of EF-D-alleviated OA-induced NAFLD in HepG2 cells by antioxidant activities, related enzyme activities, and related gene expression. Our research has achieved the utilization by-product of the defatted fruit of I. polycarpa and demonstrated its potential medical value for alleviating NAFLD. After being placed in the shade to dry up at a constant weight at room temperature, the fruits were ground and sieved with a 40-mesh sieve. Before analysis, the oil of sam-ples was removed by n-hexane extraction 3 times and the solution was pooled. After vacuum filtration, the filtrate was combined and dried under vacuum at 37°C. The defatted powder was stockpiled at -20°C. Approximately 100 g of the defatted powder was weighed and extracted with 75% ethanol (1 : 40 g/mL) at 65-75°C for 4 h. The extracts were filtered, and the filtrates were concentrated by using a vacuum rotary evaporator. Before use, the primary 75% ethanol extract (EE) was suspended in distilled water. The solutions were sequentially extracted by ethyl acetate for 4 times. Lastly, the ethyl acetate extracts (EAE) were evaporated to dryness at 50°C.

High-Speed Counter-Current Chromatography (HSCCC)
Separation of EAE. The EAE were separated by HSCCC. The peak fractions were collected manually according to the elution profile and evaporated under reduced pressure. Optimal two-phase solvent systems are evaluated with a partition coefficient (0:2 ≤ K ≤ 5). The two-phase solvent systems ethyl acetate-n-butyl alcohol-water = 4 : 1 : 5, 2 : 3 : 5, and 3 : 2 : 5 and n-hexane-ethyl acetate-methanol-water = 2 : 5 : 2 : 5, 3 : 2 : 1 : 5, 3 : 5 : 3 : 5, and 4 : 1 : 1 : 5, v/v/v/v, were screened as follows: the two-phase solvent system was prepared by adding each solvent to a separatory funnel. After shaking and thoroughly equilibrating at room temperature, the upper phase and lower phase were then separated and degassed ultrasonically for 30 min. The upper phase was used as the stationary phase while the lower phase as the mobile phase. The sample solution for HSCCC separation was prepared by dissolving 5 g of EAE in 20 mL of the lower phase. The multilayer coil column was entirely filled with the stationary phase at a flow rate of 30.0 mL/min. The apparatus was rotated at 800 rpm, then the mobile phase was pumped into the column at a flow rate of 4 mL/min in the head totail elution mode. The mobile phase eluting at the tail outlet indicated that hydrodynamic equilibrium had been reached. Subsequently, 20 mL of sample solution was injected into the column. The detection wavelength was set at 280 nm. Oxidative Medicine and Cellular Longevity was based on the TFC method reported elsewhere with proper modification. Briefly, 10 μL extracts were mixed with 100 μL FC reagent, and 90 μL 10% sodium carbonate solution was added 5 min later. The mixture was incubated at 25°C for 40 min with constant oscillation. The observance was gauged at a 765 nm wavelength. Gallic acid was used as a standard (0.002-0.025 mg/mL). The content of phenolics was calculated from a regression equation (y = 3:381x + 0:038, R 2 = 0:9995) and expressed as mg gallic acid equivalent/g of defatted fruit (mg GAE/g). The total flavonoid content was determined based on a colorimetric method with aluminium chloride [17,18]. Briefly, the 20 μL extracts (0.1 mg/mL) or rutin (0-0.1 mg/mL) was diluted in 60% ethanol solution and mixed with 30 μL of NaNO 2 (5%). After 6 min, 50 μL of 10% AlCl 3 was added to the above mixture; subsequently, 100 μL of NaOH (1 M) was added. After 15 min, the absorbance values were measured at 510 nm and compared with 50% ethanol as a blank control. The total flavonoid content was calculated from a regression equation (y = 0:4196x + 0:002, R 2 = 0:9995) and expressed as rutin equivalents (RE) per g of dry extract. 2.10.3. Measurement of SOD. SOD activity was measured by using a commercial test kit with reference to the manufacturer's instructions. The absorbance values were measured at 450 nm by using a microplate reader and the activity of SOD was expressed as U/mg prot.

Measurement of T-AOC.
T-AOC was measured by using a commercial test kit concerning the manufacturer's instructions. The absorbance values were measured at 593 nm by using a microplate reader and T-AOC was expressed as μM/mg prot.
2.11. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The total RNA of cells was isolated using a TRIzol lysis reagent (Qiagen Sciences, Germantown, MD), according to the instructions of the manufacturer. The mRNA was reverse-transcribed into cDNA with a reverse-transcription kit (Takara Bio Inc), employing a SuperScript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA). RT-PCR products were run with a loading star (Dyne Bio, South Korea) on a 1% agarose gel. Relative expression levels were calculated using the 2 -ΔΔCt method and were normalized to GAPDH levels. The primer sequences are listed in Table 1. 2.12. Statistical Analysis. The data were expressed as means ± standard deviation (SD) of three replications per experiment. Analysis of variance (ANOVA) (SPSS15.0, SPSS Inc., Chicago, IL, USA) and GraphPad Prism5 software (Graph-Pad Software, USA) were used. Values of p < 0:05, p < 0:01 or p < 0:001 were considered statistically significant.

Selection of Active
Parts. The compounds of EE and EAE from the defatted fruit of I. polycarpa were detected by UPLC (Figure 1(a)). We found that the activities of EAE were relatively stronger compared with EE which was consistent with previous reports. Base on this analysis, we establish a valid way to isolate and purify EAE for its utilization.
Our study confirmed the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3 : 2 : 1 : 5, v/v/v/v) as optimal for its appropriate K values and the retention of the stationary phase (43.7%). In the HSCCC separation, the fractions were separated in 100 min with a total mobile elution volume of 300 mL (Figure 1(b)). Four fractions were collected and detected by UPLC (Figure 1(c)). Fractions A of ethyl acetate extract (EF-A) and C (EF-C) were low in content and complex in composition. Therefore, we selected fractions B of ethyl acetate extract (EF-B), D (EF-D), EE, and EAE for further antioxidant assays.
The TPC and TFC of four different extracts are shown in Table 2. Among the four different extracts, EF-D showed the highest total flavonoid content, followed by EAE, EE, and EF-B. The phenolic contents of EE and EF-D are higher than others. In the DPPH scavenging assay (Figure 2(a)), the IC 50 value (Table 2) (the concentration required scavenge 50% of radical) showed EF-D was the strongest activity than others. In the ABTS assay (Figure 2(b)), the activity was dosedependent in low concentrations, and the IC 50 value was VC > EE > EF − D > EF − B > EAE. FRAP was associated with the reduction of ferric ions to ferrous ions. At the concentrations of 5-100 μg/mL, the phenomenon of concentration dependence was obvious, and EF-D showed the highlight activity by evaluating the value of A 0:5 (Figure 2(c)). Comprehensively, EF-D was the strongest fraction in antioxidant activity compared to others and then was selected for lipidlowering study in vitro.

OA Induced Hepatic Steatosis and Cell Viability. The
HepG2 cells were treated with 0.1-2 mM concentration of OA for 24 h to induce hepatic steatosis. Compared to the control, it did not cause cytotoxicity to the cells when treated with 0.1-2 mM OA (Figure 3(a)). Cell viability reached a peak when treated with 1 mM OA. The number of lipid droplets in HepG2 cells was increased with the concentrations of OA (Figure 3(b)). Comprehensively, 1 mM OA was selected as a final concentration for inducing accumulation of lipid.
No evidence of toxicity of EF-D (10-200 μg/mL) was found in the HepG2 cells after treating for 24 h. Cell toxicity of EF-D (100-400 μg/mL) was obvious when treated for 48 h (Figure 3(c)). In addition, the viability was higher than the cells that were not treated with EF-D in a concentration-dependent manner (Figure 3(d)). Taken together, 10, 20, 40, and 80 μg/mL of EF-D were chosen for further study.

Effects of EF-D on TG Accumulation in OA-Induced
Hepatic Steatosis. To examine the lipid-lowering role of EF-D, we measured the TG accumulation in HepG2 cells. TG accumulation was significantly increased in the OA group compared with the control group, while EF-D inhibited TG   5 Oxidative Medicine and Cellular Longevity accumulation in a concentration-dependent manner when compared with the OA group. Rosiglitazone (ROSI) was recognized as the most effective and widely used medicine for NAFLD at present [27]. The inhibition of the lipid accumulation effect of EF-D was more sensitive to ROSI (Figure 4(a)). As shown in Figures 4(b) and 4(c), OA increased the intracellular lipid content when compared to the control. The number and size of lipid droplets were significantly reduced when EF-D was added compared with the OA group. Additionally, lipid deposits decreased by 10.2, 17.5, 24.7, 38.7, and 42.5% with 10 μM (3.57 μg/mL) ROSI and 10, 20, 40, and 80 μg/mL EF-D treatments. These   Oxidative Medicine and Cellular Longevity data initially implied that EF-D had a regulatory lipid-lowing effect on OA-induced hepatic steatosis.

Effects of EF-D on Key Markers in Responses to Oxidative
Stress. In this study, the activities of AST and ALT in the OA-induced HepG2 cells were higher than those of the corresponding control group, while treatment with EF-D observably suppressed the increase of ALT and AST in a concentration-dependent manner compared with the OA group (Figures 5(a) and 5(b)). The results demonstrated that EF-D reduced lipid accumulation by lowering liver injuryrelated enzyme activities. MDA content is commonly known as a marker of oxidative stress, and its level can directly influence the peroxidation of membrane. The levels of SOD and T-AOC can reflect the capacity of the antioxidant.
As the results showed, EF-D dose-dependently decreased the content of MDA ( Figure 5(c)) and elevated the levels of SOD ( Figure 5(d)) and T-AOC ( Figure 5(e)) in comparison to the OA group which further confirmed that EF-D alleviated lipid accumulation by decreasing enzyme activities associated with liver injury and enhancing antioxidase activities.

The Effects of EF-D on the Expression of Antioxidant
Genes Related to Hepatic Steatosis. Nuclear factor erythroid-2-related factor 2 (Nrf2), a key transcription factor in oxidative stress, can activate the expression of downstream related genes [28]. Our results demonstrated that EF-D attenuated lipid accumulation through increasing mRNA expression of Nrf2 and upregulating mRNA levels of its downstream oxidative response genes including heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase, quinone 1 (NQO1), and glutathione S-transferase alpha 2 (GSTA2) (Figure 6(a)).

The Effects of EF-D on the Expression of Lipogenic Genes and Inflammatory Cytokine Genes Related to Hepatic
Steatosis. Stearoyl coenzyme A desaturase (SCD) and carnitine palmitoyltransferase-1 (CPT1) are key genes for the lipid synthesis pathway and lipid catabolism pathway. Our results showed that EF-D downregulated SCD and upregulated CPT1 mRNA expression when compared with the OA group. Peroxisomal proliferator-activated receptor α (PPARα) is closely related to several important metabolic pathways. Our results showed that EF-D upregulated the mRNA expression  Oxidative Medicine and Cellular Longevity of PPARα which indicated EF-D regulated lipid metabolism by activating PPARα. (Figure 6(b)). Nuclear factor kappa-B (NF-κB), tumor necrosis factor (TNF), and interleukin (IL) are the most critical factors associated with inflammation in NAFLD [29,30]. These findings indicated that EF-D distinctly downregulated the mRNA expression of NF-κB and IL-1 compared with the OA group ( Figure 6(c)). These results implied that EF-D may play a potential role in preventing the process of inflammatory cytokines.

Discussion
In the recent years, multiple natural phytochemicals, including polyphenols and carotenoid components from food, have been demonstrated to behave with beneficial effects on suppressing the development of NAFLD through improving hepatic glycolipid metabolism dysfunction [31,32]. However, there are not many reports on treating NAFLD with natural products. The purpose of the study was to explore the antioxidant and lipid-lowering effect of I. polycarpa in vitro. It has been reported that many natural products that are rich in polyphenols and glycosides can repress lipid accumulation [33,34]. Our study found that the fragment EF-D from the defatted fruit of I. polycarpa can effectively ameliorate OA-induced NAFLD in HepG2 cells. Interestingly, we found that EF-D mainly  (Figure 7). We identified the two compounds by comparing to the standard substances which were isolated from the defatted fruit of I. polycarpa and identified by mass spectrometry in our laboratory [36]. The antioxidant activities and cell cytotoxicity of two new monomers have been proven [36]. Our findings are consistent with the previous studies and demonstrate that the defatted fruit of I. polycarpa acts as a natural product for ameliorating NAFLD. Oxidative stress plays a key role in causing the NAFLD process. High activity molecules such as reactive oxygen (ROS) and reactive nitrogen (RNS) free radicals caused an imbalance between prooxidant and antioxidant chemical species that led to oxidative damage of cellular macromolecules [37,38]. A previous study has reported that polyphenols can reduce the production of ROS and RNS [39]. The MDA, SOD, and T-AOC are important enzymes of the endogenous antioxidant defense system. Our results showed that EF-D decreases the level of MDA and increases the activities for SOD and T-AOC. Thus, the antioxidant capacity of EF-D seems to have an important connection with treating NAFLD. It has been reported that polyphenols through activation of Nrf2-ARE signal pathways respond to oxidative  Figure 6: Effects of EF-D on the mRNA expression of oxidative stress and inflammatory cytokine genes: (a) Nrf2, HO-1, NQO1, and GSTA2; (b) SCD, CPT1, and PPARα; (c) NF-κB and IL-1 mRNA expression. Data are represented as mean ± SD (n = 3). * p < 0:05 and * * p < 0:01 OA versus control. ## p < 0:01 and ### p < 0:001 OA+ROSI and OA+EF-D versus OA. 10 Oxidative Medicine and Cellular Longevity stress for inhibiting lipid accumulation [40]. Furthermore, Nrf2 promotes the activation of downstream relative target genes, such as HO-1, NQO1, CAT, and GSTA2, leading to improved oxidative stress resistance [41]. The expression of genes can influence the generation of relative protease which further regulated related metabolic pathways. It has been demonstrated that genetic control of all characters is mediated through specific enzymes. More precisely, the message of the gene is ultimately carried out by the enzymes [42].
Our results indicated that EF-D could decrease OA-induced oxidative damage through activation of the Nrf2 signaling pathway. It has been reported that SCD can regulate lipid metabolism and further affect insulin sensitivity [43]. CPT1 is a key regulatory enzyme and rate-limiting enzyme for the betaoxidation of long-chain fatty acids in liver tissues and cells, which is closely related to the catabolism pathway of fatty acids. Earlier studies showed PPARs were target genes for remission in multiple pathways that are disrupted in NAFLD [44]. In particular, PPARα is located primarily in the liver, adipose tissue, kidney, heart, skeletal muscle, and large intestine where it is thought to regulate the fatty acid synthesis and oxidation, gluconeogenesis, ketogenesis, and lipoprotein assembly [45]. Besides, studies have demonstrated that inflammatory metabolism is closely related to oxidative injury and lipid metabolism [46]. In our experiments, EF-D decreased the mRNA expression of SCD, NF-κB, and IL-1 and increased the mRNA expression of CPT1 and PPARα. Taking the above results together, EF-D inhibited inflammation and improved lipid metabolism disorders through activating PPARα and Nrf2 antioxidant pathways.

Conclusion
In conclusion, EF-D can improve the disturbance of lipid metabolism and inflammation in OA-induced hepatic steatosis in HepG2 cells through strong antioxidant activity. Moreover, the mechanism was achieved through regulating relative metabolic enzyme activities and activating PPARα and Nrf2 antioxidant signal pathways. Evidences collected in this research suggested that EF-D is beneficial for lipidlowering and acts as a candidate medicine for ameliorating NAFLD.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare no conflict of interest.