β-Sitosterol, which is used extensively in pharmaceuticals, nutraceuticals, and cosmetics, has high nutritional value along with immunomodulatory and anti-inflammatory properties. In this study, we investigated the antioxidant and anti-inflammatory effects of β-sitosterol in zebrafish and explored the associated molecular mechanisms. In an in vivo antioxidant experiment, zebrafish (Danio rerio) larvae were treated with different concentrations of β-sitosterol and then exposed to a nonlethal concentration of CuSO₄ to induce oxidative stress. Treatment with β-sitosterol at 70 or 100 μg/mL significantly reduced CuSO₄-induced oxidative stress in the zebrafish, demonstrating the strong antioxidant activity of β-sitosterol. Treatment with β-sitosterol protected zebrafish larvae against oxidative damage from CuSO₄ by upregulating the expressions of sod and gpx4b. In a zebrafish model of inflammation, pretreatment with β-sitosterol before CuSO4 exposure inhibited neutrophil recruitment and damage to lateral line neuromasts, indicating a potent anti-inflammatory effect derived from reductions in the expressions of il-8 and myd88. The results demonstrate the antioxidative and anti-inflammatory activities of β-sitosterol and suggest that β-sitosterol may be useful for the treatment of various inflammatory diseases.

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.

An 8-week feeding trial with high-carbohydrate- and 100, 200 and 300 mg/kg mulberry leaf flavonoids (MLF)-supplemented diets (HCF1, HCF2 and HCF3, respectively) was conducted to evaluate the protective effect of MLF on oxidized high-carbohydrate-induced glucose metabolism disorder, liver oxidative damage and intestinal microbiota disturbance in Monopterus albus. The results showed that HC diets had significant negative effects on growth, glucose metabolism, liver antioxidant and immunity, as well as intestinal microbiota, in comparison to CON diets. However, WGR and SR in the HCF3 group dramatically increased compared to the HC group. With the increase of MLF in the HC diet, the activities of glycolysis and antioxidant enzymes in the liver tended to increase, while the changes of gluconeogenesis-related enzyme activities showed the opposite trend and significantly changed in the HCF3 group. Additionally, MLF supplementation dramatically increased the mRNA expression involved in glycolysis, antioxidative enzymes and anti-inflammatory cytokines in comparison with the HC group. Furthermore, gluconeogenesis and pro-inflammatory cytokine genes’ expression dramatically decreased. Furthermore, the proportion of Clostridium and Rhodobacter in the HC group dramatically declined, and the proportion of Lactococcus dramatically increased, compared to the HC group. In addition, 300 mg/kg MLF supplementation significantly improved the species composition and homeostasis of intestinal microbiota. These results indicate that MLF can alleviate the negative effects of low growth performance, glucose metabolism disorder, liver oxidative damage and intestinal microbiota disturbance caused by HC diets, and the relief of MLF is dose-related.

Largemouth bass ranavirus (LMBV) infects largemouth bass, leading to significant mortality and economic losses. There are no safe and effective drugs against this disease. Oral vaccines that directly target the intestinal mucosal immune system play an important role in resisting pathogens. Herein, the B subunit of Escherichia coli heat-labile enterotoxin (LTB, a mucosal immune adjuvant) and the LMBV main capsid protein (MCP) were expressed using Saccharomyces cerevisiae surface display technology. The yeast-prepared oral vaccines were named EBY100-OMCP and EBY100-LTB-OMCP. The candidate vaccines could resist the acidic intestinal environment. After 7 days of continuous oral immunization, indicators of innate and adaptive immunity were measured on days 1, 7, 14, 21, 28, 35, and 42. High activities of immune enzymes (T-SOD, AKP, ACP, and LZM) in serum and intestinal mucus were detected. IgM in the head kidney was significantly upregulated (EBY100-OMCP group: 3.8-fold; BY100-LTB-OMCP group: 4.3-fold). IgT was upregulated in the intestines (EBY100-OMCP group: 5.6-fold; EBY100-LTB-OMCP group: 6.7-fold). Serum neutralizing antibody titers of the two groups reached 1:85. Oral vaccination protected against LMBV infection. The relative percent survival was 52.1% (EBY100-OMCP) and 66.7% (EBY100-LTB-OMCP). Thus, EBY100-OMCP and EBY100-LTB-OMCP are promising and effective candidate vaccines against LMBV infection.