Fig 1.
Survival rates of zebrafish larvae exposed to varying concentrations of Elaeagnus angustifolia (EA) at 24, 48, and 72 hours post-fertilization (hpf).
Larvae were treated with EA at concentrations of 0.5, 0.75, 1.0, 2.0, 2.5, 3.0, 4.0, and 5.0 mg/mL, along with a positive control (20 µg/mL zinc oxide) and a negative control (E3 medium). Survival was assessed by manually counting live embryos under a standard microscope and expressing the result as a percentage of the initial number of embryos. Data are presented as mean ± standard deviation (% survival; n = 30 embryos per group, experiment performed in triplicate). Statistical analysis was performed using one-way ANOVA followed by Šídák’s multiple comparisons test. Significant differences relative to the negative control are indicated as follows: p < 0.05, **p < 0.001.
Fig 2.
Hatching rate of zebrafish embryos at 72 hpf following exposure to varying concentrations of Elaeagnus angustifolia extract (EA).
Hatching rate was determined as the proportion of embryos successfully emerging from their chorions after exposure to EA concentrations of 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 mg/mL, alongside a negative control (E3M) and a positive control (20 µg/mL Zinc Oxide). Data are presented as mean ± standard deviation (% hatching) for n = 30 embryos per group, with experiments performed in triplicate. Statistical analysis was conducted using one-way ANOVA followed by Šídák’s multiple comparisons test. Significant differences relative to the negative control group (E3M) are indicated as follows: ****p < 0.0001.
Fig 3.
Cardiac parameter assessment in the dorsal aorta of zebrafish larvae at 72 hours post-fertilization (hpf).
Zebrafish larvae were exposed to varying concentrations of Elaeagnus angustifolia (EA) extract (0.5, 0.75, 1.0, 1.5, 2.0 mg/mL), along with a positive control (20 µg/mL Zinc Oxide) and a negative control (E3M). Cardiac function was evaluated by measuring: (A) dorsal aortic blood flow velocity (µm/s), (B) vessel diameter (µm), (C) shear stress (dynes/cm²), (D) heart rate (beats per minute, bpm), and (E) flow rate (nL/min). Data are presented as mean ± standard error of the mean (SEM) (n = 5 per group). Statistical significance was determined using one-way ANOVA followed by Šídák’s multiple comparisons test. Significant differences relative to the negative control group (E3M) are indicated by asterisks: *p < 0.05, **p < 0.01.
Fig 4.
Behavioral and locomotion assay of zebrafish larvae at 96 hpf.
Zebrafish larvae were exposed to varying concentrations of Elaeagnus angustifolia (EA) (0.5, 0.75, 1.0, 1.5, and 2.0 mg/mL), as well as to a positive control (20 µg/mL Zinc Oxide) and a negative control (E3M). At 96 hpf, individual larvae were transferred to 96-well plates placed within the Viewpoint ZebraLab chamber and subjected to alternating 10-minute light and dark cycles for a total of 60 minutes. Locomotor activity was recorded to quantify both (A) the average distance traveled every 5 minutes during the assay and (B) the total distance traveled at the end of the recording period. Data are expressed as mean ± standard error of the mean (SEM) (n = 10 larvae per group; experiments performed in duplicate). Statistical analysis was conducted using one-way ANOVA followed by Šídák’s multiple comparisons test. Significant differences relative to the negative control (E3M) are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 5.
Oil Red O staining for lipid accumulation in zebrafish livers at 5 days post-fertilization (dpf).
Larvae were exposed to varying concentrations of Elaeagnus angustifolia (EA) (0.5, 0.75, 1.0, 1.5, and 2.0 mg/mL), and Oil Red O staining intensity was measured to quantify neutral lipid deposition in the liver. Data are presented as mean ± standard error of the mean (SEM) (n = 3 embryos per group; experiment performed in duplicate). Statistical significance was determined using one-way ANOVA followed by Šídák’s multiple comparisons test. Significant differences from the negative control group (E3M) are indicated by ****p < 0.0001.
Fig 6.
Fluorescence imaging of cancer-free and xenograft zebrafish larvae injected with triple-negative breast cancer (TNBC) cells (MDA-MB-231).
(A) At 48 hpf, zebrafish larvae in the xenograft group were injected with red fluorescent protein (RFP)-labeled MDA-MB-231 cells, while uninjected larvae served as negative controls. Images were acquired at 4 × magnification to visualize RFP fluorescence in the head and tail regions at 1 and 2 days post-injection (dpi). The left panels depict cancer-free larvae (negative control), while the right panels show xenografted larvae (positive control). For each group, representative images are presented for two individual embryos (Embryo 1 and Embryo 2), with the head region shown in the left column and the tail region in the right column. Scale bars = 700 µm. (B) Data are presented as mean ± standard error of the mean (SEM) (n = 6 embryos per group; experiment performed in duplicate). Statistical significance was determined using two-way ANOVA followed by Šídák’s multiple comparisons test, with differences relative to the untreated xenograft control group indicated as **p < 0.01 and ****p < 0.0001.
Fig 7.
Zebrafish xenograft model injected with triple-negative breast cancer (TNBC; MDA-MB-231) cells at 48 hours post-fertilization (hpf).
Images show xenografted larvae containing red fluorescent protein (RFP)-labeled TNBC cells at 1 and 2 days post-injection (dpi) at different concentrations (0.50 mg/ml and 0.75 mg/ml EA). (A) Representative fluorescence images of head and tail regions from xenografted larvae treated with 0.5 mg/mL EA (top) or 0.75 mg/mL EA (bottom) at both time points. (B) Quantitative analysis of RFP fluorescence intensity, reflecting tumor burden, in EA-treated xenografts compared to untreated xenograft controls. Data are expressed as mean ± standard error of the mean (SEM) (n = 6 embryos per group; experiment performed in duplicate). Statistical significance was determined by two-way ANOVA with Šídák’s multiple comparisons test, with differences compared to untreated xenograft controls denoted as **p < 0.01 and ***p < 0.001.