Anti-Inflammatory and Antioxidant Properties of Squalene in Copper Sulfate-Induced Inflammation in Zebrafish (Danio rerio)

Long-term or excessive oxidative stress can cause serious damage to fish. Squalene can be added to feed as an antioxidant to improve the body constitution of fish. In this study, the antioxidant activity was detected by 2,2-diphenyl-1-acrylhydrazyl (DPPH) test and fluorescent probe (dichloro-dihydro-fluorescein diacetate). Transgenic Tg (lyz: DsRed2) zebrafish were used to evaluate the effect of squalene on CuSO4-induced inflammatory response. Quantitative real-time reverse transcription polymerase chain reaction was used to examine the expression of immune-related genes. The DPPH assay demonstrated that the highest free radical scavenging exerted by squalene was 32%. The fluorescence intensity of reactive oxygen species (ROS) decreased significantly after 0.7% or 1% squalene treatment, and squalene could exert an antioxidative effect in vivo. The number of migratory neutrophils in vivo was significantly reduced after treatment with different doses of squalene. Moreover, compared with CuSO4 treatment alone, treatment with 1% squalene upregulated the expression of sod by 2.5-foldand gpx4b by 1.3-fold to protect zebrafish larvae against CuSO4-induced oxidative damage. Moreover, treatment with 1% squalene significantly downregulated the expression of tnfa and cox2. This study showed that squalene has potential as an aquafeed additive to provide both anti-inflammatory and antioxidative properties.


Introduction
Reactive oxygen species (ROS) and free radicals are involved in several diseases, including metabolic, and infectious diseases [1,2]. ROS can affect apoptosis and the immune system, and enhance the inflammatory response. Therefore, ROS are believed to cause many diseases and are a major factor in aging [3]. An imbalance between ROS production and the counteracting antioxidant system allows ROS to interact with and cause damage to proteins, lipids, and DNA. This imbalance is referred to as oxidative stress [2]. Inflammation and oxidative stress are interdependent mechanisms [4]. Moreover, oxidative stress might provoke inflammatory responses that can further enhance oxidative stress [5].
Zebrafish is a widely used animal model and has a variety of transgenic or mutant fish lines that can study human and aquatic animal diseases [6]. At the same time, zebrafish has a variety of inflammatory models [6]. When fish are stressed, the production of oxygen free radicals in tissues or cells increases [7,8]. This leads to a large amount of ROS in tissues or cells, causing oxidative damage [9]. Long-term or excessive oxidative stress can cause damage to fish, such as slow growth and development, decreased immune function, and increased disease incidence [8]. Oxidative stress damage to fish can reduce the quality of aquatic products and the feed conversion rate, causing serious economic

2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Activity of Squalene
The in vitro antioxidant activity of squalene was determined by measuring its DPPH radical scavenging activity. Compared with that of the 0% group, the results of each experimental group were significantly different (Figure 2). At the range of concentrations used in the experiment, the scavenging activity of squalene increased in a dose-dependent manner. DPPH radical scavenging activity of 1% squalene was the highest (32%). The results showed that squalene had certain DPPH free radical scavenging ability. DPPH radical scavenging activity of squalene. Each bar represents the mean ± S.D. for three different experiments performed in triplicate, * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the 0% group (control group). DPPH-2,2-Diphenyl-1-picrylhydrazyl.

In Vivo Antioxidant Effect of Squalene
The fluorescent probe dichloro-dihydro-fluorescein diacetate (H2DCFDA) was used as a ROS indicator to evaluate the effect of squalene on ROS production in zebrafish ( Figure 3A). Compared with that in the control group, the fluorescence intensity (FI) of  Copper chelating activity of squalene. Each bar represents the mean ± S.D. for three different experiments performed in triplicate, ns, not significant, * p < 0.05, *** p < 0.001 compared with the 0% group (control group).

2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Activity of Squalene
The in vitro antioxidant activity of squalene was determined by measuring its DPPH radical scavenging activity. Compared with that of the 0% group, the results of each experimental group were significantly different ( Figure 2). At the range of concentrations used in the experiment, the scavenging activity of squalene increased in a dose-dependent manner. DPPH radical scavenging activity of 1% squalene was the highest (32%). The results showed that squalene had certain DPPH free radical scavenging ability. Figure 2. DPPH radical scavenging activity of squalene. Each bar represents the mean ± S.D. for three different experiments performed in triplicate, * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the 0% group (control group). DPPH-2,2-Diphenyl-1-picrylhydrazyl.

In Vivo Antioxidant Effect of Squalene
The fluorescent probe dichloro-dihydro-fluorescein diacetate (H2DCFDA) was used as a ROS indicator to evaluate the effect of squalene on ROS production in zebrafish ( Figure 3A). Compared with that in the control group, the fluorescence intensity (FI) of Figure 2. DPPH radical scavenging activity of squalene. Each bar represents the mean ± S.D. for three different experiments performed in triplicate, * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the 0% group (control group). DPPH-2,2-Diphenyl-1-picrylhydrazyl.

In Vivo Antioxidant Effect of Squalene
The fluorescent probe dichloro-dihydro-fluorescein diacetate (H 2 DCFDA) was used as a ROS indicator to evaluate the effect of squalene on ROS production in zebrafish ( Figure 3A). Compared with that in the control group, the fluorescence intensity (FI) of the CuSO 4 treatment group increased ( Figure 3B,C). This indicated that CuSO 4 induced more ROS in zebrafish, and the experimental model was established. The FI of the 100 µM quercetin group was significantly decreased compared with that in the CuSO 4 group ( Figure 3B,C). Similar to quercetin, the mean FI of ROS was decreased in the 0.2%, 0.5%, 0.7%, and 1% squalene-treated groups ( Figure 3B,C). No significant difference was found between the 0.7% and 1% squalene-treated groups. After treatment with squalene, the FI decreased with the increase in squalene concentration. However, the effect of 0.05% squalene treatment on the production of reactive oxygen was not significant. In summary, squalene inhibited ROS generation in a dose dependent manner. At the lowest concentration (0.05% squalene), the effect of treatment was not significant.
( Figure 3B,C). Similar to quercetin, the mean FI of ROS was decreased in the 0.2%, 0.5%, 0.7%, and 1% squalene-treated groups ( Figure 3B,C). No significant difference was found between the 0.7% and 1% squalene-treated groups. After treatment with squalene, the FI decreased with the increase in squalene concentration. However, the effect of 0.05% squalene treatment on the production of reactive oxygen was not significant. In summary, squalene inhibited ROS generation in a dose dependent manner. At the lowest concentration (0.05% squalene), the effect of treatment was not significant.  Fluorescence intensity of juvenile zebrafish compared with that of the group using CuSO4 alone, the data were presented as medians for fou different larvae. #### p < 0.001, compared with the control group. ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the CuSO4 group. H2DCFDA-dichlorodihydrofluorescein diacetate.

Effect of Squalene on the Expression of Antioxidant Genes sod and gpx4b
The expression levels of sod (encoding superoxide dismutase) and gpx4b (encoding glutathione peroxidase 4b) were assessed using RT-qPCR. Compared with those in the control group, the expression levels of sod and gpx4b in the CuSO4 alone treatment group were significantly reduced ( Figure 4A,B). After combined treatment with squalene for 24 (C) Fluorescence intensity of juvenile zebrafish compared with that of the group using CuSO 4 alone, the data were presented as medians for four different larvae. #### p < 0.001, compared with the control group. ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the CuSO 4 group. H 2 DCFDA-dichlorodihydrofluorescein diacetate.

Effect of Squalene on the Expression of Antioxidant Genes sod and gpx4b
The expression levels of sod (encoding superoxide dismutase) and gpx4b (encoding glutathione peroxidase 4b) were assessed using RT-qPCR. Compared with those in the control group, the expression levels of sod and gpx4b in the CuSO 4 alone treatment group were significantly reduced ( Figure 4A,B). After combined treatment with squalene for 24 h, the expression of sod in the 1% squalene treatment group was upregulated by 5-fold. The sod expression levels in the remaining experimental groups were increased by 2-fold ( Figure 4A). The difference between all the experimental groups and the CuSO 4 alone treatment group was statistically significant. The effect of squalene on the expression of gpx4b was dose-dependent ( Figure 4B). The expression of gpx4b gene relative to β-actin, the data were expressed as the mean ± SD, three biological replicates, ## p < 0.05, ### p < 0.01, compared with the control group. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with CuSO4 alone treatment group. sod-superoxide dismutase; gprx4bglutathione peroxidase 4b.

In Vivo Neutrophil Recruitment Assay
The CuSO4 inflammation model was constructed using transgenic zebrafish Tg (lyz: DsRED2) to verify the effect of squalene on neutrophil migration after the zebrafish were stimulated by Cu 2+ (Figure 5A). In the control group, neutrophils were localized to the The expression of gpx4b gene relative to β-actin, the data were expressed as the mean ± SD, three biological replicates, ## p < 0.05, ### p < 0.01, compared with the control group. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with CuSO 4 alone treatment group. sod-superoxide dismutase; gprx4b-glutathione peroxidase 4b.

In Vivo Neutrophil Recruitment Assay
The CuSO 4 inflammation model was constructed using transgenic zebrafish Tg (lyz: DsRED2) to verify the effect of squalene on neutrophil migration after the zebrafish were stimulated by Cu 2+ ( Figure 5A). In the control group, neutrophils were localized to the caudal hematopoietic tissue in the ventral trunk and tail ( Figure 5B(I)). In contrast, in fish treated with CuSO 4 , neutrophils migrated to the horizontal midline and formed clusters near the lateral line neuromasts ( Figure 5B(II)). However, pretreatment with squalene solution (0.05%, 0.2%, 0.5%, 0.7%, and 1%) significantly inhibited neutrophil migration to inflammatory sites ( Figure 5C). The results for all squalene pretreatment groups were significantly different when compared with the CuSO 4 alone treatment group ( Figure 5C). There was no significant difference between 0.2%, 0.5%, 0.7%, and 1% groups.

Effect of Squalene on Expression of tnfa and cox-2
The expression levels of tnfa (encoding tumor necrosis factor alpha) and cox-2 (encoding cyclooxygenase 2) were significantly upregulated in zebrafish after CuSO4 only stimulation, by more than 2.5 times ( Figure 6A,B). Compared with the CuSO4 alone treatment group, the expression levels of tnfa and cox-2 were downregulated after treatment with squalene for 24 h, in a dose-dependent manner ( Figure 6A,B). The most marked inhibition was achieved using 1% squalene. (C) Quantification of the number of neutrophils aggregated to the lateral line after CuSO 4 treatment, the data are presented as medians for four different larvae. Data are expressed as the mean ± SD. #### p < 0.001, compared with the control group. ns, not significant. ** p < 0.01, *** p < 0.001, compared with the CuSO 4 alone treatment group, n = 4. dpf-days post-fertilization; DMSO-dimethyl sulfoxide.

Effect of Squalene on Expression of tnfa and cox-2
The expression levels of tnfa (encoding tumor necrosis factor alpha) and cox-2 (encoding cyclooxygenase 2) were significantly upregulated in zebrafish after CuSO 4 only stimulation, by more than 2.5 times ( Figure 6A,B). Compared with the CuSO 4 alone treatment group, the expression levels of tnfa and cox-2 were downregulated after treatment with squalene for 24 h, in a dose-dependent manner ( Figure 6A,B). The most marked inhibition was achieved using 1% squalene. Figure 6. The expression of immune response-related genes relative to β-actin in zebrafish larv after CuSO4 stimulation and squalene treatment. (A) The expression of tnfa gene relative to β-ac (B) The expression of cox-2 gene relative to β-actin. The data were expressed as the mean ± SD, thr biological replicates, #### p < 0.001, compared with the control group. * p < 0.05, ** p < 0.01 compar with CuSO4 alone treatment group. tnfa-tumor necrosis factor alpha; cox-2-cyclooxygenase 2.

Discussion
The CuSO4-induced inflammation model can be established by simply adding t compound CuSO4 into the zebrafish larvae culture medium. The accumulation neutrophils in the neuromasts is one of the most frequently used indicators for the lev of inflammation in CuSO4-induced inflammation model and has been applied to asse the effect of known anti-inflammatory drugs [6]. Neutrophils migrate to inflammato foci and can be observed in zebrafish [31,32]. Therefore, in this study, CuSO4 was select to stimulate oxidative stress and induce inflammation. The established zebrafi inflammation model was used to verify the anti-inflammatory and antioxidant effects The expression of cox-2 gene relative to β-actin. The data were expressed as the mean ± SD, three biological replicates, #### p < 0.001, compared with the control group. * p < 0.05, ** p < 0.01 compared with CuSO 4 alone treatment group. tnfa-tumor necrosis factor alpha; cox-2-cyclooxygenase 2.

Discussion
The CuSO 4 -induced inflammation model can be established by simply adding the compound CuSO 4 into the zebrafish larvae culture medium. The accumulation of neutrophils in the neuromasts is one of the most frequently used indicators for the level of inflammation in CuSO 4 -induced inflammation model and has been applied to assess the effect of known anti-inflammatory drugs [6]. Neutrophils migrate to inflammatory foci and can be observed in zebrafish [31,32]. Therefore, in this study, CuSO 4 was selected to stimulate oxidative 9 of 16 stress and induce inflammation. The established zebrafish inflammation model was used to verify the anti-inflammatory and antioxidant effects of squalene on zebrafish. This study has shown that inflammation occurs after zebrafish are stimulated, including increased ROS content and neutrophil migration to the neuromast. In this study, the use of squalene reduced inflammation and enhanced the anti-inflammatory and antioxidant capacities of zebrafish.

Antioxidant Effect of Squalene on Zebrafish
To assess the antioxidant activity of squalene, we first used an in vitro DPPH assay. The DPPH radical scavenging assay is widely used to determine the antioxidant activity of natural compounds because of its simplicity, sensitivity, comparable nature, and reproducibility [33][34][35]. The extract of Prunus mume (squalene-rich) had a good DPPH free radical scavenging ability, and its ethanol extract had the highest antioxidant activity (96.08-97.71%), whereas its water extract had the lowest antioxidant activity (76.09%) [36]. In our study, the free radical scavenging rate of the 1% squalene treatment group was 32% ( Figure 2). With the increase in squalene concentration, the scavenging rate of DPPH free radical increased. This indicated that squalene can scavenge DPPH free radicals and squalene has certain antioxidant activity.
Although the determination of the antioxidant capacity by the DPPH method in vitro is simple and convenient, it cannot replace animal experiments in vivo [37,38]. Therefore, this study used zebrafish to establish an inflammatory model to test the antioxidant and anti-inflammatory effects of squalene. CuSO 4 stimulated the generation of ROS, and ROS production could induce an inflammatory reaction [39][40][41]. In addition, CuSO 4 could interfere with the expression of antioxidant genes, such as sod and gpx [42,43]. Therefore, this study mainly studied the effects of squalene treatment on ROS production and antioxidant gene expression in vivo. To eliminate the factors affecting the experimental results due to the complexation of Cu 2+ with squalene during incubation, first, we tested the CCA of squalene, which showed that the complexation rate of 1% squalene to Cu 2+ was 13.3% ( Figure 1). The CCA of squalene is very low and would not affect the subsequent experimental results. Squalene can reduce AAPH (2,2 -azobis-2-methyl-propanimidamide, dihydrochloride) and H 2 O 2 -induced oxidative stress in Vero cells and reduce UV-induced intracellular ROS levels [44]. Squalene-based PLGA NPs (ploy lactic-co-glycolic acid nanoparticles) effectively reduced ROS levels in normal mouse hepatocytes [45]. Then, an in vivo experimental model was established by soaking zebrafish in CuSO 4 to produce oxidative stress, permitting the verification of the antioxidant effect of squalene on zebrafish. ROS generation was then evaluated using the dye H 2 DCFDA [46][47][48]. The results showed that squalene treatment could effectively reduce ROS produced in zebrafish in a dosedependent manner ( Figure 3A,B). The 0.7% and 1% squalene treatment groups showed the best effect, with no significant differences between these two groups ( Figure 3B). This demonstrated that squalene had a significant antioxidant effect, which could reduce the production of ROS in zebrafish larvae and protect zebrafish from stress.
Antioxidant enzymes include glutathione peroxidase (GPX, GSH-Px) and superoxide dismutase (SOD) [49,50]. GSH-Px plays a crucial role against oxidative stress, turning toxic substances into innocuous products by scavenging their free radicals [51,52]. GPx converts H 2 O 2 and lipid peroxides into water and lipid alcohols to exert similar antioxidant effects [53]. Therefore, SOD and GSH-Px can reflect the body's ability to resist oxidative damage [54]. In the oxidative damage model of zebrafish embryos induced by cadmium (Cd), a tree peony seed protein hydrolysate could inhibit Cd-induced oxidation by upregulating the expression of sod and gpx mRNA [55]. Rats fed with 2% squalene showed a significant decrease in lipid peroxidation and a significant increase in SOD and CAT activity, indicating the antioxidant properties of squalene in experimental induction [56]. Administration of squalene in rats improved GPx activity and the GSH level in heart tissue and protected the heart from cyclophosphamide-induced oxidative stress [57]. In this study, we found that squalene could effectively upregulate the expression of sod (by 2.5-fold) and gpx4b (by 1.3-fold) in the CuSO 4 -induced inflammation model ( Figure 4A,B). Compared with the CuSO 4 treatment alone group, 1% squalene treatment significantly upregulated the expression of sod ( Figure 4A). Both 0.7% and 1% squalene significantly upregulated the expression of gpx4b ( Figure 4B). In summary, squalene can significantly upregulate the expression of antioxidant genes sod and gpx4b, and can effectively reduce the ROS produced in zebrafish. This reduced CuSO 4 -induced oxidative stress, indicating that squalene had an antioxidant effect on zebrafish.

The Anti-Inflammatory Effect of Squalene in the Zebrafish Model
Exposure to copper causes hair cell necrosis in zebrafish lateral line. In addition, it can promote neutrophil migration and accumulation in the inflammatory area, followed by an acute inflammatory response [43,58]. Treatment with Bacillus coagulans XY2 before CuSO 4 exposure significantly reduced neutrophil mobilization, thereby alleviating the acute inflammation induced by CuSO 4 [59]. Therefore, this study used transgenic Tg (lyz: DsRED2) zebrafish to establish an inflammatory model to study the effect of squalene on neutrophil migration. The results showed that squalene treatment could reduce the number of migratory neutrophils ( Figure 5A,B). Among the squalene concentrations, the 0.7% and 1% squalene treatment groups showed the best effects, with no significant difference between them ( Figure 5B). In the zebrafish inflammation model, squalene could effectively inhibit neutrophil migration and the inflammatory response, demonstrating a good antiinflammatory effect. It reduced the number of neutrophils in the inflammatory foci and alleviated the inflammatory symptoms to a certain extent.
Squalene has been shown to inhibit intracellular oxidative stress in LPS-stimulated mouse macrophages by inhibiting TNFα, inducible nitric oxide synthase (iNOS), COX-2, and NFE2 like BZIP transcription factor 2 (Nrf2) signaling pathways [44,60]. Similarly, squalene downregulated COX-2 and iNOS expression in a mouse acute colitis model [24,28]. There is abundant evidence that certain pro-inflammatory cytokines, such as IL-1β and TNFα, are involved in the process of pathological inflammation. In addition, COX-2 could be induced during tissue damage or inflammation in response to cytokines [61]. Pinostrobin, as a potential anti-inflammatory drug, could down-regulate Tnfa and Cox-2 expression in LPS-stimulated RAW 264.7 macrophages and zebrafish larvae by inhibiting gene expression [62]. Squalene dose-dependently reduced the increased levels of TNF-α and COX-2 in LPS-induced RAW cells [44]. Moreover, addition of squalene to human monocytes and neutrophils activated by incubation with lipopolysaccharide was found to suppress COX-2, IL1β, and TNF mRNA expression [60]. Therefore, in this study, the expression of tnfa and cox-2 was tested in 3 dpf zebrafish treated with 10 µM CuSO 4 for 24 h to verify the anti-inflammatory effect of squalene. Compared with the control, the expression of tnfa and cox-2 in zebrafish was significantly increased after stimulation with CuSO 4 (Figure 6A,B). This indicated the regulation of cells after an inflammatory response. However, the expression of tnfa and cox-2 was significantly downregulated after treatment with squalene in a dose-dependent manner. In addition, squalene had beneficial regulatory effect on the post-inflammatory response. In summary, squalene could inhibit the migration of neutrophils to the inflammatory site and inhibit the inflammatory response by downregulating the expression of tnfa and cox-2, which had an anti-inflammatory effect. These results provide the evidence that squalene exerts its anti-inflammatory activities via mechanisms targeting pro-inflammatory mediators (tnfα, cox-2) mediators and pathways in closely related phagocytic cells that cooperate during the onset, progression, and resolution of inflammation.

Fish Husbandry
Transgenic Tg (lyz: DsRED2) and wild-type (AB strain) zebrafish (Danio rerio) were purchased from the Institute of Hydrobiology of the Chinese Academy of Sciences, China Zebrafish Resource Center (CZRC, Wuhan, China). Zebrafish were maintained and handled according to the Zebrafish Book (zfin.org, 1 June 2022). The zebrafish were fed twice a day. The night before spawning, male and female fish in a 1:1 ratio were transferred to a darkened mating cage. Mating and spawning occurred within 1 h after turning on the lights in the morning. Selected healthy embryos were washed and examined under a microscope. Embryos were housed at 28 • C in embryo-rearing media (E3, see Supplementary Material) (Nanjing EzeRinka Biotechnology Co., Ltd., Nanjing, China). At 3 days post-fertilization (dpf), the larvae were used for the experiments. The components of E3 were NaCl (29.4 g/100 mL), KCl (1.27 g/100 mL), CaCl 2 ·2H 2 O (4.85 g/100 mL), and MgSO 4 ·7H 2 O (8.13 g/100 mL) [63,64].

Preparation of Test Samples
Squalene (Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) exists as an oily liquid at room temperature [17]; therefore, the concentration of squalene is expressed by the volume fraction. Dimethyl sulfoxide (DMSO) (Sangon Biotech Co., Ltd., Shanghai, China) was used as the solvent. The squalene samples were dissolved in 6% DMSO and 90% E3 to form a 4% stock solution. The stock solution was diluted to different concentrations with E3 before use, such that the concentrations of the test sample were 0, 0.05%, 0.2%, 0.5%, 0.7%, and 1%. H 2 DCFDA (Merck Co., Ltd., Shanghai, China) was prepared as a stock of 20 µg/mL in DMSO and frozen at −20 • C. Its working solution was prepared on the day of the experiment.

The Metal Chelating Ability
The CCA was determined using PV (Merck Co., Ltd.) [65]. A total of 30 µL of different concentrations of squalene solution (0.05%, 0.2%, 0.5%, 0.7%, and 1%) or water (control) were mixed with 200 µL 50 mmol/L sodium acetate buffer. Each group had 3 repetitions. Thereafter, 30 µL of 100 mg/L CuSO 4 ·5H 2 O solution was added to each group and oscillated for 2 min. After 2 min, 8.5 µL of 2 mmol/L catechol violet solution was added to start the reaction. All the reaction mixtures were shaken at 25 • C for 10 min, and then the absorbance was measured at 632 nm using an ultraviolet-visible (UV-Vis) spectrophotometer [37]. The CCA of squalene was calculated as Cu 2+ chelating ability of squalene (%) = (Abs control − Abs sample) × 100/Abs control where Abs control is the absorbance of the control and Abs sample is the absorbance of the sample.

Antioxidant Capacity In Vitro
DPPH (Sangon Biotech Co., Ltd.) antioxidant capacity was determined according to a previously published protocol [34,37]. A total of 20 µL of squalene solution at different concentrations (0.05%, 0.2%, 0.5%, 0.7%, and 1%) was added to 180 µL of a methanol solution of 0.1 mM DPPH (the ratio was 1:9). Each group had 3 repetitions. Methanol, E3, and DMSO were used as controls, with quercetin as the positive control. After mixing, the reactions were placed in dark at room temperature for 15 min. Then, the absorbance was measured at 517 nm using an UV-Vis spectrophotometer. The calculation formula was Scavenging Activity (%) = (Abs control − Abs sample) × 100/(Abs control)

Antioxidant Capacity In Vivo
Before the formal experiment, no death or deformity was found in zebrafish soaked with CuSO 4 or squalene in the experimental concentration range. Quantification of ROS in zebrafish larvae was determined using the cell-permeable fluorescent probe H 2 DCFDA [37]. Zebrafish larvae of at 3 dpf were transferred to a 6-well plate, 10 larvae per well. The zebrafish larvae were treated with 2 mL squalene (0, 0.05%, 0.2%, 0.5%, 0.7%, or 1%) or 100 µM quercetin (positive control) for 1 h. Each group had 4 repetitions. Then, oxidative stress was induced in the zebrafish using CuSO 4 at a final concentration of 10 µM. After 20 min of exposure, the larvae were washed three times with fresh E3 medium. The larvae were then incubated with H 2 DCFDA solution (20 µg/mL) for 1 h in the dark at 28 • C. The larvae were then washed three times with fresh E3 medium. Zebrafish larvae were immobilized in 3% methylcellulose (Merck Co., Ltd.). The larvae were observed under a fluorescence microscope (Olympus, Tokyo, Japan) and photographed. FI quantification was performed in individual confocal slices using ImageJ software (version 1.6, NIH, Bethesda, MD, USA). Larvae treated with CuSO 4 alone were used as the negative control, and larvae treated with quercetin were used as positive controls.

In Vivo Neutrophil Recruitment Assay
The Tg (lyz: DsRED2) zebrafish transgenic line was used for the neutrophil recruitment assay in vivo [66]. The 3 dpf zebrafish larvae were treated with 2 mL squalene (0, 0.05%, 0.2%, 0.5%, 0.7%, or 1%) for 1 h (10 larvae per well, and each group had 4 repetitions.). Next, the larvae were treated with 20 µM CuSO 4 for 1 h. Neutrophil migration was monitored using images taken using a fluorescence microscope (Olympus). Neutrophil migration was quantified by calculating the number of labeled cells detected in the lateral line region. Larvae treated with DMSO alone were used as a control, and larvae treated with CuSO 4 alone were used as a negative control.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis
The expression of selected genes (sod (encoding superoxide dismutase), gpx4b (encoding glutathione peroxidase 4b), tnfa (encoding tumor necrosis factor alpha), and cox2 (encoding cyclooxygenase 2) was analyzed using RT-qPCR (the primers are shown in Table 1, see Supplementary Material) [37]. The 3 dpf zebrafish larvae were transplanted into a 6-well plate, at 30 larvae per well. The 30 hatched larvae in each group were treated with different doses of 2 mL squalene (0, 0.05%, 0.2%, 0.5%, 0.7%, or 1%) for 1 h. Each group had 3 repetitions. The larvae were then treated with 10 µM CuSO 4 (a concentration that induced oxidative stress and inflammation in zebrafish without resulting in fish death up to 24 h [14]) for 24 h. The larvae were collected after 24 h and stored at −80 • C for RNA extraction. The total RNA extraction was carried out using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) [67]. RNA integrity was determined by electrophoresis through 1% agarose gels [68]. The RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit (Trans Gen Biotech, Shanghai, China). The cDNA was then used as the template for a real-time quantitative polymerase chain reaction using the following cycling conditions: 95 • C for 10 min, followed by 40 cycles of 95 • C for 30 s, and 60 • C for 30 s. The results were calculated using the 2 −∆∆ct method [69] with the actb gene (encoding beta-actin) as the control.

Statistical Analyses
Normality of the data and homogeneity of the variances were confirmed using Shapiro-Wilk and Levene tests. The data were subjected to two-way analysis of variance (ANOVA) with squalene and copper exposure as the factors. A probability level of 5% (p < 0.05) was considered as significant. Kruskal-Wallis non-parametric one-way ANOVA was used when the normality test failed and differences between groups were determined by the Mann-Whitney test. When non-parametric statistics were used, the data are presented using Tukey's boxplot (the bottom and top of the box are the minimum and maximum numbers, respectively, for each boxplot).

Conclusions
This study found that squalene could reduce the production of ROS in a CuSO 4induced zebrafish inflammation model, and upregulated the expression of sod and gpx4b to inhibit oxidative stress. Squalene could also inhibit the migration of neutrophils to inflammatory sites and downregulated the expression of pro-inflammatory cytokine genes tnfa and cox2 to inhibit inflammation. The anti-inflammatory and antioxidant effects of squalene on fish were studied from the aspects of aquatic animal models and gene expression changes. The results provide scientific support for squalene as a feed additive to improve the anti-inflammatory and antioxidant capacity of aquatic products.