β-sitosterol reduces the content of triglyceride and cholesterol in zebrafish. (A) Oil red O staining results, scale: 1 mm. (B) Filipin staining results, scale: 1 mm. (C) Quantitative results of oil red O staining, n = 6. (D) Quantitative results of Filipin staining, n = 6. Results are expressed as mean ± SE of the three repeated samples. ^##p < 0.05, ^###p < 0.01 compared with the negative control group; * p < 0.05, ** p < 0.01 compared with the HFD group.

β-sitosterol reduces the content of triglyceride and cholesterol in zebrafish. (A) Oil red O staining results, scale: 1 mm. (B) Filipin staining results, scale: 1 mm. (C) Quantitative results of oil red O staining, n = 6. (D) Quantitative results of Filipin staining, n = 6. Results are expressed as mean ± SE of the three repeated samples. ^##p < 0.05, ^###p < 0.01 compared with the negative control group; * p < 0.05, ** p < 0.01 compared with the HFD group.

Changes in microvessels in the zebrafish vitreous vascular system. (A) Confocal scan of the outer vasculature surrounding the ocular globe in Tg (fli1a: EGFP) zebrafish larvae. Through the translucent lens, transparent blood vessels (yellow arrows in (A)) can be seen from the outside, and red arrows indicate the branches between the vascular arcs. Scale: 10 μm. (B) Quantitative analysis of the vascular diameter in confocal scanning images, n = 6. The unit of length is in pixels (px). ^###p < 0.01 compared with the negative control group; * p < 0.05 compared with HFD group; ns, no significant difference.

β-Sitosterol reduces the contents of triglyceride and cholesterol in the liver of zebrafish. (A) Triglyceride content in the liver of zebrafish. (B) Cholesterol content in the liver of adult zebrafish. Results are expressed as mean ± SE of the three repeated samples. ^####p < 0.01 compared with the control group; ** p < 0.01 compared with the HFD group.

Lipid metabolome analysis of adult zebrafish liver. (A) Correlation analysis between samples in control, HFD, and β-sitosterol groups. (B) PCA analysis results in the positive POS ion mode. The X-axis represents the first principal component, and the Y-axis represents the second principal component. (C) PCA analysis results in NEG ion mode. (D) Lipid classification of the top 10 metabolites. B, β-sitosterol group; BC, blank control group; HFD, HFD group; PCA, principal component analysis; POS, positive; NEG, negative.

Lipid metabolome analysis of adult zebrafish liver. (A) Correlation analysis between samples in control, HFD, and β-sitosterol groups. (B) PCA analysis results in the positive POS ion mode. The X-axis represents the first principal component, and the Y-axis represents the second principal component. (C) PCA analysis results in NEG ion mode. (D) Lipid classification of the top 10 metabolites. B, β-sitosterol group; BC, blank control group; HFD, HFD group; PCA, principal component analysis; POS, positive; NEG, negative.

Orthogonal projections to latent structures discriminant analysis (OPLS-DA). (A) OPLS-DA score diagram. The x-axis (t1) represents the prediction component (inter-group difference component), and the y-axis (to1) represents the orthogonal component (intra-group difference component). (B) OPLS-DA model replacement test diagram. The x-axis represents the correlation between the permutation group and the original model group, the y-axis represents the value of R2Y or Q2Y (where R2Y and Q2Y of 1 in the x-axis are the values of the original model), and the two dashed lines are the regression lines fitted by R2Y and Q2Y.

Orthogonal projections to latent structures discriminant analysis (OPLS-DA). (A) OPLS-DA score diagram. The x-axis (t1) represents the prediction component (inter-group difference component), and the y-axis (to1) represents the orthogonal component (intra-group difference component). (B) OPLS-DA model replacement test diagram. The x-axis represents the correlation between the permutation group and the original model group, the y-axis represents the value of R2Y or Q2Y (where R2Y and Q2Y of 1 in the x-axis are the values of the original model), and the two dashed lines are the regression lines fitted by R2Y and Q2Y.

Analysis of differentially expressed lipid metabolites (DELMs) between HFD and β-sitosterol groups. (A) Volcano plot results; the x-axis represents the difference in multiple changes in the group compared with each substance (log₂), and the y-axis represents the p-value (log₁₀). The size of the scatter represents the VIP value of the OPLS-DA model. The larger the scatter point, the larger the VIP value, and the more reliable the DELMs screened. The blue dots in the figure represent downregulated DELMs, the red dots represent upregulated DELMs, and the gray represents the detected non-significant lipids. (B) Top 10 upregulated and downregulated lipid metabolites based on log₂FC. The x-axis represents the change in the difference multiples of each substance in the group (taking log₂ as the bottom) based on the upregulation and downregulation; upregulation is represented by the red color, downregulation is indicated by the blue color, and logFC is represented by the column length.

Differential lipid KEGG functional annotation and enrichment analysis results. (A) The annotation results of the TOP20 differential lipid KEGG enrichment. The entries shown in the same box indicate the hierarchical classification annotation of the KEGG pathway, and the column length shows the number of lipids annotated to each pathway. (B) Differential lipid KEGG annotation results for the TOP20 enrichment point map. (C) Differential lipid KEGG annotation results from TOP20 enrichment network map. (D) KEGG functional annotation and enrichment analysis results of the upregulated metabolites in differential lipids. (E) KEGG functional annotation and enrichment analysis results of the downregulated metabolites in differential lipids.

Differential lipid KEGG functional annotation and enrichment analysis results. (A) The annotation results of the TOP20 differential lipid KEGG enrichment. The entries shown in the same box indicate the hierarchical classification annotation of the KEGG pathway, and the column length shows the number of lipids annotated to each pathway. (B) Differential lipid KEGG annotation results for the TOP20 enrichment point map. (C) Differential lipid KEGG annotation results from TOP20 enrichment network map. (D) KEGG functional annotation and enrichment analysis results of the upregulated metabolites in differential lipids. (E) KEGG functional annotation and enrichment analysis results of the downregulated metabolites in differential lipids.

Differential lipid KEGG functional annotation and enrichment analysis results. (A) The annotation results of the TOP20 differential lipid KEGG enrichment. The entries shown in the same box indicate the hierarchical classification annotation of the KEGG pathway, and the column length shows the number of lipids annotated to each pathway. (B) Differential lipid KEGG annotation results for the TOP20 enrichment point map. (C) Differential lipid KEGG annotation results from TOP20 enrichment network map. (D) KEGG functional annotation and enrichment analysis results of the upregulated metabolites in differential lipids. (E) KEGG functional annotation and enrichment analysis results of the downregulated metabolites in differential lipids.

Effects of β-sitosterol on the expression of Ppar-γ and Rxr-α in zebrafish. (A) The mRNA level of ppar-γ in the liver. (B) The mRNA level of Ppar-γ in the liver. (C) The mRNA level of rxr-α in the liver. (D) The mRNA level of Rxr-α in the liver. Data are presented as the mean ± SE of the three repeated samples. ## p < 0.05, ### p < 0.01, #### p < 0.001 compared with the control group. * p < 0.05, *** p < 0.001 compared with HFD group.