Protective Effects of Emodin on Oxidized Fish Oil-Induced Metabolic Disorder and Oxidative Stress through Notch-Nrf2 Crosstalk in the Liver of Teleost Megalobrama amblycephala

Dietary oxidized lipids are key perpetrator to accumulate excessive reactive oxygen species (ROS) that induce oxidative stress for animals. Immoderate oxidative stress dysregulates cell fate, perturbs cellular homeostasis, thereby interrupts metabolism and normal growth. Therefore, a 12-week feeding trial with fish oil (FO, control group), oxidized fish oil (OF), and emodin-supplemented (OF+E) diets was conducted to evaluate the therapeutic mechanism of emodin on metabolic and oxidative resistance in Megalobrama amblycephala liver. Morphologically, emodin remits oxidized fish oil-induced cellular constituents damage, evidenced by lipid droplets enlargement and accumulation, mitochondria rupture, and nucleus aggregation, which were functionally related to oxidative stress, metabolism, and cell fate determination. Consecutively, glucose, lipid, and amino acid metabolism were retained under emodin stimulation. Specifically, fatty acid metabolic genes optimized fatty acid utilization and metabolism, featured as total saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) alternation. Physiologically, inflammation, autophagy, apoptosis, as well as antioxidant capacity were alleviated by emodin. Interactively, fatty acid metabolism was correlated with antioxidant capacity; while the crosstalk and dynamic equilibrium between apoptosis and autophagy determine the cell fate under oxidative stress amelioration. Synergistically, Nrf2 and Notch signaling were active to antioxidant defense. In particular, oxidative stress blocked the crosstalk between Notch and Nrf2 signaling, while emodin rescued Notch-Nrf2 interaction to ameliorate oxidative stress. In conclusion, these results suggest that elevated ROS levels by oxidative stress activates Notch and Nrf2 signaling but intercepts Notch-Nrf2 crosstalk to stimulate cell fate and antioxidant program; dietary emodin alleviates oxidative stress and returns overall ROS levels to a moderate state to maintain homeostatic balance. The crosstalk between Notch and Nrf2 signaling might be the potential therapeutic target for emodin to ameliorate oxidative stress and metabolic disorder in M. amblycephala liver.


Introduction
Internal homeostasis is an important precondition for animal physiological health, growth, and reproduction maintenance. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated during cellular metabolism, which play important roles in internal homeostasis, cell signal transduction, cell proliferation and differentiation [1]. Generally, animals are exposed to internal or external adverse environments that could dysregulate ROS or RNS homeostasis, which trigger oxidative stress consequently. Oxidative stress is the most extensive and harmful stress among all stresses. When oxidative stress exceeds the repair capacity of the cell, oxidative damage will lead to physiological disorder,

Experimental Diets
According to our previous research, oxidized fish oil in the diet has been shown to induce oxidative stress [18], and emodin was confirmed to improve the antioxidant capacity in M. amblycephala [19]. Therefore, this experiment applied oxidized fish oil with a peroxide value (POV) of 375.33 mmol/kg to induce oxidative stress with the fish model of M. amblycephala. Specifically, isonitrogen and isoenergy (33.11% crude protein and 14.68 kJ/g energy) diets were formulated, including basal diets containing 6% fish oil (control, 6F), oxidized lipid enriched diets containing 6% oxidized fish oil (6OF), and emodin enriched diets containing 30 mg/kg emodin (6OF+E) ( Table 1). All ingredients were prepared and the diets were pelleted according to the established methods [19].

Experimental Animals and Rearing Conditions
The experimental fish M. amblycephala was generated from our research institute Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences. The experiment was conducted in fiberglass tanks (300 L each) of indoor fresh water circulation system to support equal supplemental aeration and water flow (3 L/min). Prior the feeding trail, fish were acclimated in the tank fed with the control feed for 14 days. After acclimation, fish with similar body weight (intimal average weight 5.20 ± 0.01 g) were randomly assigned to nine tanks (3 tanks for each group, 25 individuals for each tank, 225 fish in total). During the 12-week rearing experiment, fish were fed with the respective diets to near satiation four times a day (8:00, 11:00, 14:00, and 17:00). During the experiment, one-third of the water in the tank was replaced weekly, the water temperature was kept as 26 ± 1 • C, and the water quality maintained as: pH 7.6-7.8, DO > 6 mg/L, NH 3 < 0.01 mg/L.

Sample Collection
After a 12-week feeding experiment, the fish were starved for 24 h to evacuate the digestive tract contents before sampling. Nine fish in each group were taken (three fish from each tank were randomly selected) and anesthetized with tricaine mesylate (MS-222, 100 mg/L) for sampling. Blood samples were obtained from caudal vein and stored in heparin coated tubes, and then centrifuged at 4500 rpm, 4 • C for 10 min to obtain the plasma. The anesthetized fish were then dissected to remove the liver tissues on ice, immediately isolated for TEM analysis or frozen in liquid nitrogen and stored at −80 • C for subsequent analysis.

Liver Histological Ultrastructure
According to our previously established method [18], TEM was used to detect histological ultrastructure. In detail, the livers of M. amblycephala (3 replicates per group) were collected immediately and fixed in 2.5% glutaraldehyde for 24 h, post-fixed in 1% osmium tetroxide (OsO4) for 1 h, and stored at 4 • C till sectioning. The sections were embedded in epoxy resin Epon812, cut into thin slices (70 µm thick) with RMC PowerTome XL microtome, and stained with uranyl acetate and lead citrate. A Hitachi HT7700 transmission electron microscope (Hitachi, Tokyo, Japan) was used to observe the ultrastructure morphology.

Fatty Acid and Amino Acid Analysis
For fatty acid analysis, whole fish samples were hydrolyzed with BHT for 1.5 h, neutralized with ddH 2 O and n-hexane, and centrifuged at 1000 rpm for 10 min. The supernatant Antioxidants 2022, 11, 1179 5 of 18 was isolated to analyze fatty acid composition by meteorological chromatography-mass spectrometer (GC-MS, Aglilent 7890B-5977A).
For amino acid analysis, whole fish samples were hydrolyzed with 6 mol/L HCl and filled with nitrogen for 24 h. The samples were applied to determine the amino acid content by liquid chromatography analyzer (Agilent-1100).

Correlation Analysis
Pearson's correlation test was performed to analyze the correlations between parameters or key genes. The significance threshold was set at a p-value < 0.05. The heatmap was created in R with the pheatmap package.

RNA Extraction and RT-PCR Analysis
Total RNA from nine livers in each group was extracted with RNAiso Plus reagent (Takara Co. Ltd., Dalian, China), and the total RNA was incubated with RNase-free DNase (Takara Co. Ltd., Dalian, China) to remove contaminated genomic DNA. Absorbance under OD 260/280 and electrophoresis (1.5% agarose) was applied to evaluate the quantity and quality of RNAs. Primers for RT-PCR was designed with primer Premier 5.0 according to the sequence we obtained with RNA-seq and synthesized by Shanghai Generay Biotechnology Co., Ltd., China (primers were shown in Table 2). RT-PCR analysis was performed using SYBR ® Primix Ex TaqTM II (TliRNase Plus) kit according to the manufacturer's protocol with ABI 7500 real-time PCR system. β-actin was used as the housekeeping gene, and the relative expression was calculated using the 2 −∆∆CT method.
Note: The mRNA sequences for each gene were obtained from M. amblycephala transcriptome sequencing database. Primers for RT-PCR were designed using primer premier 5.0.

Statistical Analysis
To determine the variances for each parameter among different groups, data were all validated for normality and homogeneity, followed by independent samples t-test with . Results were expressed as mean ± standard error of the mean (mean ± SEM).

Emodin Alleviates Oxidized Fish
Oil Induced Morphological Impairment in the Liver of M. amblycephala TEM was applied to reveal the histological alternation of the liver tissue induced by dietary oxidized fish oil and emodin. In comparison with the control group (6F, Figure 1A), lipid droplets (LD) were enlarged in size and accumulated in quantity ( Figure 1B, arrows in white color), mitochondria (MT) were ruptured gradually ( Figure 1B, arrows in red color), and nucleolus (NC) was aggregated ( Figure 1B, arrows in blue color) under oxidized fish oil (6OF). These morphological impairments indicate 6OF-induced oxidative stress, metabolic disorder, and cell fate dysregulation in the liver. Heartily, we found the structure of LD, MT, and NC was visibly rescued under emodin stimulation (6OF+E), indicating the ameliorative effects of emodin on oxidative stress, cellular metabolism, and cell fate determination ( Figure 1C).

Statistical Analysis
To determine the variances for each parameter among different groups, data were all validated for normality and homogeneity, followed by independent samples t-test with SPSS 25.0 (IBM, Chicago, USA). Results were expressed as mean ± standard error of the mean (mean ± SEM).

Emodin Alleviates Oxidized Fish Oil Induced Morphological Impairment in the Liver of M. amblycephala
TEM was applied to reveal the histological alternation of the liver tissue induced by dietary oxidized fish oil and emodin. In comparison with the control group (6F, Figure  1A), lipid droplets (LD) were enlarged in size and accumulated in quantity ( Figure 1B, arrows in white color), mitochondria (MT) were ruptured gradually ( Figure 1B, arrows in red color), and nucleolus (NC) was aggregated ( Figure 1B, arrows in blue color) under oxidized fish oil (6OF). These morphological impairments indicate 6OF-induced oxidative stress, metabolic disorder, and cell fate dysregulation in the liver. Heartily, we found the structure of LD, MT, and NC was visibly rescued under emodin stimulation (6OF+E), indicating the ameliorative effects of emodin on oxidative stress, cellular metabolism, and cell fate determination ( Figure 1C).

Emodin Alleviates Metabolic Disorder Induced by Oxidative Stress in the Liver of M. amblycephala
To reveal the metabolic alterations retrieved form the mitochondria histology, glucose, lipid, and protein metabolic-related indexes were detected with the liver tissue. Glucometabolic-related glucose content ( Figure 2A) and amylase activity ( Figure 2B) were inhibited by 6OF; protein metabolic-related alkaline phosphatase (ALP) activity ( Figure 2C) was also inhibited by 6OF; lipid and fatty acid metabolic related lipase ( Figure 2D), lipoprotein lipase ( Figure 2E), triglyceride ( Figure 2F), total cholesterol (TC, Figure 2G), and fatty acid synthetase ( Figure 2H) were all inhibited by 6OF. However, the aberrant expression of these genes was rescued by emodin (6OF+E), and exhibited no significant difference with that in 6F (p ≥ 0.05) (Figure 2A-H).

Emodin Rescues Fatty Acid Metabolism under Oxidative Stress in the Liver of M. amblycephala
To further explore the potential alteration of fatty acid metabolism that is regulated by OF, fatty acid composition, fatty acid catabolism, and anabolism were analyzed. Fatty acid composition of the diet reveal saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) were significantly increased (p < 0.05), while the polyunsaturated fatty acids (PUFA) were significantly decreased (p < 0.05) after the oxidization of fish oil ( Figure  3A-C, Table 3). However, the fatty acid composition from the whole fish reveal SFA exhibited no significant difference between 6F, 6OF, and 6OF+E (p > 0.05); MUFA in 6OF was increased, but restored to the 6F level under emodin (6OF+E); PUFA in 6OF and 6OF+E was significantly decreased (p < 0.05), and exhibited the same variation as that in the diet ( Figure 3A-C, Table 3). These results indicate M. amblycephala presents positive adaptability to fatty acid metabolism under fatty acid composition alteration in the diet induced by oxidized fish oil, especially the SFA and MUFA utilization. This interesting finding inspired us to explore the fatty acid catabolic and anabolic regulation.

Emodin Rescues Fatty Acid Metabolism under Oxidative Stress in the Liver of M. amblycephala
To further explore the potential alteration of fatty acid metabolism that is regulated by OF, fatty acid composition, fatty acid catabolism, and anabolism were analyzed. Fatty acid composition of the diet reveal saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) were significantly increased (p < 0.05), while the polyunsaturated fatty acids (PUFA) were significantly decreased (p < 0.05) after the oxidization of fish oil ( Figure 3A-C, Table 3). However, the fatty acid composition from the whole fish reveal SFA exhibited no significant difference between 6F, 6OF, and 6OF+E (p > 0.05); MUFA in 6OF was increased, but restored to the 6F level under emodin (6OF+E); PUFA in 6OF and 6OF+E was significantly decreased (p < 0.05), and exhibited the same variation as that in the diet ( Figure 3A-C, Table 3). These results indicate M. amblycephala presents positive adaptability to fatty acid metabolism under fatty acid composition alteration in the diet induced by oxidized fish oil, especially the SFA and MUFA utilization. This interesting finding inspired us to explore the fatty acid catabolic and anabolic regulation.
In the fatty acid catabolism process, we found the transcriptional expression of lipoprotein lipase (LPL, Figure 3D), adipose triglyceride lipase (ATGL, Figure 3E), carnitine palmitoyltransferase I (CPT1, Figure 3F), carnitine palmitoyltransferase II (CPT2, Figure 3G), and cyclooxygenase 2 (COX2, Figure 3H) were all inhibited by 6OF; while uncoupling protein 2 (UCP2, Figure 3I) was activated by 6OF. Meanwhile, in fatty acid anabolic process, transcriptional expression of fatty acid synthase (FAS, Figure 3J) and sterol regulatory element-binding protein 1 (SREBP1, Figure 3K) were also down-regulated under 6OF. However, results also show the expression of these key genes were restored to the control level under 6OF+E stimulation ( Figure 3D-K). Note: letters "a, b, and c" represent the difference in the feed, "A, B, and C" represent the difference in the M. amblycephala.   Note: letters "a, b, and c" represent the difference in the feed, "A, B, and C" represent the difference in the M. amblycephala.

Emodin Alleviates Antioxidant Capacity under Oxidative Stress in the Liver of M. amblycephala
To evaluate whether dysregulated metabolism impacts antioxidant capacity, we next evaluated the antioxidant-related parameters in the liver. 6OF significantly increased the content or activity of reactive oxygen species (ROS, Figure 4A), total superoxide dismutase (T-SOD, Figure 4B), inducible nitric oxide synthase (iNOS, Figure 4C), glutathione peroxidase (GPx, Figure 4D), anti-superoxide anion (ASAFR, Figure 4E), and malondialdehyde (MDA, Figure 4F); and significantly decreased the content of nitric oxide (NO, Figure 4G) and reduced glutathione (GSH, Figure 4H). Analogously, emodin (6OF+E) also alleviates the increased or decreased activity or content to the control level after 12-weeks stimulation (p > 0.05, Figure 4A-H).

Emodin Alleviates Antioxidant Capacity under Oxidative Stress in the Liver of M. amblycephala
To evaluate whether dysregulated metabolism impacts antioxidant capacity, we next evaluated the antioxidant-related parameters in the liver. 6OF significantly increased the content or activity of reactive oxygen species (ROS, Figure 4A), total superoxide dismutase (T-SOD, Figure 4B), inducible nitric oxide synthase (iNOS, Figure 4C), glutathione peroxidase (GPx, Figure 4D), anti-superoxide anion (ASAFR, Figure 4E), and malondialdehyde (MDA, Figure 4F); and significantly decreased the content of nitric oxide (NO, Figure 4G) and reduced glutathione (GSH, Figure 4H). Analogously, emodin (6OF+E) also alleviates the increased or decreased activity or content to the control level after 12-weeks stimulation (p > 0.05, Figure 4A-H). To reveal the relationship between fatty acid metabolism and antioxidant capacity, a correlation analysis was conducted with Pearson analysis ( Figure S1). Under oxidative stress (6OF), fatty acid metabolism-related indexes were synergistically correlated with antioxidant enzyme activities, but the correlation indexes were different with that in 6F. To reveal the relationship between fatty acid metabolism and antioxidant capacity, a correlation analysis was conducted with Pearson analysis ( Figure S1). Under oxidative stress (6OF), fatty acid metabolism-related indexes were synergistically correlated with antioxidant enzyme activities, but the correlation indexes were different with that in 6F. However, after emodin stimulation (6OF+E), the synergy was reduced with the only correlation of LPL and NO, indicating the regulation between fatty acid metabolism and antioxidant capacity were closely related to emodin stimulation.

Hypothetical Regulation of Notch-Nrf2 Crosstalk on Oxidative Stress Amelioration
Based on the above results, we raise the hypothetical regulation schematic of Notch-Nrf2 crosstalk under oxidative stress amelioration (Figure 7). Oxidative stress-induced ROS activates Notch1b signaling and subsequently activates Nrf2 signaling, evidenced by the crosstalk between Notch ligands and down-stream modulators with Nrf2 key elements. Activated crosstalk of Notch-Nrf2 increased apoptosis and autophagy in the liver, which facilitates the cells to resist oxidative stress. Exhilaratingly, dietary emodin inhibited Notch-Nrf2 crosstalk to ameliorate apoptosis and autophagy, thereby alleviating oxidative stress.
represents p < 0.05, and ** represents p < 0.01. Data were analyzed by Students' t-test, results were indicated as mean ± SEM, n = 9.

Hypothetical Regulation of Notch-Nrf2 Crosstalk on Oxidative Stress Amelioration
Based on the above results, we raise the hypothetical regulation schematic of Notch-Nrf2 crosstalk under oxidative stress amelioration (Figure 7). Oxidative stress-induced ROS activates Notch1b signaling and subsequently activates Nrf2 signaling, evidenced by the crosstalk between Notch ligands and down-stream modulators with Nrf2 key elements. Activated crosstalk of Notch-Nrf2 increased apoptosis and autophagy in the liver, which facilitates the cells to resist oxidative stress. Exhilaratingly, dietary emodin inhibited Notch-Nrf2 crosstalk to ameliorate apoptosis and autophagy, thereby alleviating oxidative stress. Figure 7. Hypothetical regulation of Notch-Nrf2 crosstalk on oxidative stress amelioration. Hypothetical regulation of Notch-Nrf2 crosstalk was raised based on the results in this study. Arrows in red color represent the regulation by oxidized fish oil, in blue color represent the regulation by emodin under oxidized fish oil. "×" represents oxidized fish oil blocks the co-regulation between Notch and Nrf2 signaling.

Discussion
Fish oil is enriched with HUFAs that could dominantly promote the growth of fish. However, fish oil is prone to oxidative rancidity, and oxidated diet is one of the most important exogenous factors leading to oxidative stress on fish [19]. Oxidative stress control and prevention have become a critical issue for aquatic animals [8]. In aquatic animals, unavoidable oxidation of dietary lipids could induce lipid peroxidation, which increases ROS production during cellular metabolism [19]. Our previous study indicates dietary oxidized lipids-induced ROS production exceeded the ability to quench the reactive species in the intestine, thereby impaired the cellular structural integrity, suppressed the immunity and antioxidant capacity of the intestine, and eventually inhibited the growth performance of M. amblycephala [1,17,19]. However, as the crucial organ for metabolism, Figure 7. Hypothetical regulation of Notch-Nrf2 crosstalk on oxidative stress amelioration. Hypothetical regulation of Notch-Nrf2 crosstalk was raised based on the results in this study. Arrows in red color represent the regulation by oxidized fish oil, in blue color represent the regulation by emodin under oxidized fish oil. "×" represents oxidized fish oil blocks the co-regulation between Notch and Nrf2 signaling.

Discussion
Fish oil is enriched with HUFAs that could dominantly promote the growth of fish. However, fish oil is prone to oxidative rancidity, and oxidated diet is one of the most important exogenous factors leading to oxidative stress on fish [19]. Oxidative stress control and prevention have become a critical issue for aquatic animals [8]. In aquatic animals, unavoidable oxidation of dietary lipids could induce lipid peroxidation, which increases ROS production during cellular metabolism [19]. Our previous study indicates dietary oxidized lipids-induced ROS production exceeded the ability to quench the reactive species in the intestine, thereby impaired the cellular structural integrity, suppressed the immunity and antioxidant capacity of the intestine, and eventually inhibited the growth performance of M. amblycephala [1,17,19]. However, as the crucial organ for metabolism, immunity and antioxidant resistance, the role and underlying mechanism of liver in resisting dietary oxidized lipids remains unclear in M. amblycephala.
As aquatic animals contain high amounts of lipids with polyunsaturated fatty acid residues that is the substrate for oxidation, oxidative stress could induce lipid peroxidation thereby disrupting the lipid metabolism. Generally, due to the high damaging capacity and biological activity, the cellular metabolism of ROS is under fine control in quiescent conditions. However, when steady-state ROS concentration is transiently or chronically enhanced, excessive accumulation of ROS could damage cellular constituents, and disrupt metabolic and antioxidant capacity [20]. In the present study, elevated ROS reveal dietary oxidized fish oil-induced oxidative stress in M. amblycephala liver. Meanwhile, as the end product of lipid peroxidation, MDA concentration was increased under 6OF, indicating dietary oxidized fish oil-induced lipid peroxidation in the liver of M. amblycephala.
Morphologically, cellular integrity is vital for the function of liver cells [21]. Nucleus is the control center of the cell that contains most of the genomic DNA and all of the chromosome, it maintains the integrity of genes and controls the activities of the cell by regulating gene expression [22]. Mitochondrion is the dominant organelle to produce energy through respiration that is critical for any metabolism and cellular activity [23]. Lipid droplets are lipid-rich cellular organelles to regulate the storage and hydrolysis of neutral lipids, they play a very important role in the regulation of intracellular lipid storage and lipid metabolism. Meanwhile, lipid droplets include a close association to inflammatory responses [24], lipotoxicity protection [25], as well as a platform for protein binding and degradation [26]. In the present study, TEM reveals oxidative stress-induced nucleus aggression, thereby impairing the cell fate evidenced by apoptosis and autophagy-related gene expression, indicating the metabolic disorder and cell fate determinate dysregulation in the liver.
Glucose is regarded as the major substrate for animals, and amino acids and fatty acids are also intermediates of the metabolic pathway to drive energy production [27]. Meanwhile, evidence indicate that glycolytic metabolism closely interacts with fatty acid and amino acid metabolic profiles to maintain normal cellular function, homeostasis, as well as cell fate determination [27]. It is clear that long-term administration of oxidized fish oil could induce lipid deposition in the liver [28]. In this study, oxidized fish oil led to lipid deposition in the liver, as evidenced by lipid droplets enlargement and accumulation. Similar results were also found in channel catfish [29], yellow catfish [30], loach [31], and largemouth bass [32]. In the study of channel catfish, lipid deposition in the liver induced by oxidized fish oil was originated from activated lipid synthesis [29]. However, lipid synthesis and lipolysis were both inhibited under oxidative stress in our present study, which was supported by decreased lipase, LPL, TG, TC, and FAS activity, as well as decreased lipid metabolic related gene expression of LPL, ATGL, CPT1, CPT2, COX2, FAS, and SREBP1. This contrasting result reveals there might have different regulatory mechanisms among different fishes under oxidative stress, or the regulatory mechanism was specific to different oxidative stress levels. Therefore, we suppose the oxidative stress was too severe to maintain lipid synthesis and lipolysis, thereby results in lipid deposition and inhibited lipid metabolism in our study. However, the underlying mechanism needs further investigation. Additionally, glycolytic and amino acid metabolism were also inhibited under oxidized fish oil and restored by emodin, indicating that nutrient metabolisms are closely associated with each other, and the regulation of the metabolic pathway could be important in oxidative stress resistance.
Cholesterol is a sterol type of lipid that serves as an essential structural component of animal cell membranes and a precursor for the biosynthesis of steroid hormones, bile acid, and vitamin D. Early research identifies cholesterol itself as an antioxidant that protects cells from free radical damage [33]. Interestingly, cholesterol was significantly reduced under oxidative stress in our present study. Mechanically, cholesterol was mainly synthesized from dietary lipid with fatty acid metabolism [34]. From this point of view, if the dietary lipid was oxidized to induce peroxidation for the organism, the synthesis of cholesterol will be unavoidably dysregulated. Consistently, reports indicate that oxidative damage could deplete cholesterol and disrupt cell membrane function [35,36]. Meanwhile, peroxidationinduced cellular structural impairment might be another factor to reduce cholesterol level in the liver. Additionally, it is clear that emodin could reduce cholesterol level [37,38]. In our study, cholesterol in 6OF+E was reduced in comparison with 6F, indicating ameliorative effects of emodin on oxidative stress. Impaired lipid metabolism was reported to interact with immunity and antioxidant dysregulation [39]. iNOS-derived NO signaling plays a central role in inflammatory regulation and hepatic protection [40]. Meanwhile, interferon system was reported to induce iNOS via activating NF-κB [41]. In the present study, NF-κB, IL-1β, and IL-6 expression was activated and iNOS content was increased under oxidized fish oil stimulation, indicating the immune system was activated, which was in accordance with that in zebrafish [42]. Antioxidant system functions importantly to remove the excessive ROS and ASAFR formation, and SOD and GPx are vital to decrease hydrogen peroxide in this process [43,44]. The present study shows that oxidized fish oil markedly increased T-SOD, GPx, and ASAFR activity, as well as ROS and MDA levels, indicating antioxidant system was activated in the liver of M. amblycephala. Similar studies also confirmed that oxidized fish oil dysregulates antioxidant capacity in channel fish [29] and tilapia [45].
Recent advances in physiological relevance reveal ROS acts as an important signaling molecule as well as a critical factor in cell fate determination [27]. Previous evidence suggests that ROS acts as signaling mediators linking between metabolic alteration and cell fate, such as cell cycle progression, apoptosis, and autophagy [46]. In this study, autophagy and apoptosis-related genes were all activated under oxidized fish oil stimulation, indicating prolonged oxidative stress results in apoptosis and autophagy.
Our previous reports reveal cell fate determination-related apoptosis and autophagy were closely related to Nrf2 and Notch signaling [1,17], which inspired us to uncover the relationship between apoptosis-autophagy and Nrf2-Notch signaling. Activation of Nrf2-ARE pathway protects cells from oxidative stress-induced cell death [47]. Notch signaling plays an important role in the process of cell fate determination, including cell growth, cell proliferation, and programmed death [48]. Through Notch-Nrf2 crosstalk studies, it has been clearly shown that NICD can activate the Nrf2 pathway and Nrf2 can inversely activate the Notch1 pathway in the liver [11]. Recent studies also indicate ARE is the upstream of the Notch1 major transcription start site. Furthermore, as the binding site of NICD, Rbpjκ is conserved on the promoters of Nrf2 among animal species [49]. Notch1 is one of the transmembrane Notch family receptors that drive Notch signaling, together with the Rbpjκ transcription factor [50]. Therefore, the crosstalk between Nrf2 and Notch signaling has been shown to enhance cyto-protection and maintenance of cellular homeostasis under oxidative stress, rather than as a simple on-off switch [51]. In our present study, oxidized fish oil robustly activated Nrf2 and Notch signaling, and induced aberrant Nrf2-Notch crosstalk by molecular impairment of related key genes. Under emodin stimulation, Nrf2 and Notch signaling was ameliorated and the crosstalk was restored, indicating the inhibition of Nrf2 and Notch signaling, as well as the promotion of Nrf2-Notch crosstalk plays a key role in liver development and in maintenance of hepatic function under oxidative stress. In accordance with our finding, Notch1-Nrf2 crosstalk exerts cellular protection by reducing the formation of ROS [52], promoting apoptosis and aggravating tight joint or oxidative damage [53]. Additionally, Notch or Nrf2 inhibition has been well used as a therapeutic target in the treatment of oxidative stress with different bioactive compounds, such as emodin [17], quercetin [54], and chlorogenic acid isomers [55] on Nrf2; as well as emodin [1], dibenzazepine [12], melatonin [56], and niclosamide [57] on Notch. We speculate that the ROS-Notch-Nrf2 pathway is a conserved pathway that is designed to allow cells to respond to changes in environmental levels of ROS. However, there are limited reports illustrating the breakdown of Nrf2-Notch crosstalk under oxidative stress. Presumably, a system for downregulation of this crosstalk should exist for the homeostasis maintenance, perhaps target gene products of each signaling contribute to a negative feedback mechanism for Notch and/or Nrf2 signaling regulation; the detailed mechanism requires further investigation. Our findings reveal, to our knowledge, the first observation of ROS activation and Notch-Nrf2 signaling in response to oxidative stress amelioration of M. amblycephala. Additionally, our study is supported by the previous report of Nrf2-Notch interaction in total liver cells [58].
To combat the adverse effects of external stress, especially the oxidative stress that could induce cellular lipid peroxidation, homeostasis imbalance, immune or antioxidant breakdown and even death, dietary implementation with functional additives has become an effective approach for stress control and prevention in aquatic animals [59]. For the characterized advantages of minor side effects and drug resistance, the medical herb extract emodin has been widely used in clinical trial for humans and aquaculture [59]. In consistent with our previous results that emodin protects the intestine from oxidative stress impairment [1,17,18], we demonstrate emodin alleviates morphological impairment, fatty acid metabolic disorder, antioxidant disorder, and cell fate determination by targeting Notch-Nrf2 crosstalk in the liver of M. amblycephala. Similar functional effects of emodin was also reported in other animals [60][61][62][63].
Taken together, our study emphasizes that the loss of normal ROS-Notch-Nrf2 cellular homeostatic mechanism was associated with excessive autophagy, apoptosis, and metabolic disorder in the liver. Meanwhile, our data demonstrate a vital homeostatic mechanism that emodin prevents excessive stress injury of liver cells and allows them to respond to injury and cellular repair.

Conclusions
In conclusion, dietary oxidized lipid induced oxidative stress and dysregulated lipid metabolism in the liver of M. amblycephala. Singular activation of Nrf2 and Notch signaling, interrupted Nrf2/Notch1 crosstalk, as well as apoptosis and autophagy were involved in the regulation. Furthermore, the therapeutic Nrf2-Notch crosstalk targeting holds great promise for the treatment of oxidative stress in aquatic animals, which could promote the application of emodin from scientific research into aquaculture practice.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11061179/s1, Figure S1: Interaction analysis between fatty acid metabolism and antioxidant capacity; Figure S2: Interaction analysis among inflammation, apoptosis, and autophagy.  Institutional Review Board Statement: This study was approved by the Animal Care and Use Committee of Nanjing Agri-cultural University (Nanjing, China; protocol code: WXFC 2017-0006, approved 27 May 2017). All animal procedures were carried out in accordance with the China Laboratory Animal Care and Use Guidelines. The ethics in this experiment is the same as the ethics previously published [19].

Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.