Fig. 1. Thymosin synergizes with vinorelbine to inhibit human lung cancer A549 cells in the xenotransplanted zebrafish model. (A) Schematic diagram and grouping of our experiments. CM-DiI-labeled human lung cancer A549 cells are injected into the yolk sac of 2 dpf zebrafish to establish the xenotransplanted zebrafish model. The experimental groupings and their corresponding color markers are shown in the figure. (B) Representative images of xenotransplanted zebrafish in different groups. The dotted box indicates the yolk sac of the zebrafish. Scale bar, 100 µm. (C) Fluorescence intensity of CM-DiI-labeled A549 cells after treatment of vinorelbine and thymosin for 18 h. (D) The synergistic anti-cancer effect of thymosin on vinorelbine. Data are expressed as mean ± SEM. n = 10. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations: dpf: days post fertilization; MTC: maximum tolerable concentration; T: thymosin; V: vinorelbine.

Fig. 2. Thymosin alleviates vinorelbine-induced apoptosis of muscle cells in the xenotransplanted zebrafish model. (A) Representative images of xenotransplanted zebrafish in different groups. The acridine orange, a nucleic acid intercalating dye that emits green fluorescence when bound to dsDNA and selectively stains apoptotic cells, is utilized here. Red arrows indicate apoptotic muscle cells. Scale bar, 50 µm. (B) The number of apoptotic muscle cells after treatment with vinorelbine and thymosin for 18 h. (C) The transcription levels of apoptosis-promoting factor, Bax, and (D) apoptosis inhibitor, Bcl2, after treatment of vinorelbine and thymosin for 18 h. The mRNA level of the model control group was set at 1 in the correlation analysis of qRT-PCR results. Data are expressed as mean ± SEM. n = 10. ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 compared to the vinorelbine treatment group; #P < 0.05, ##P < 0.01, ###P < 0.001, comparison between the model control group and the vinorelbine treatment group. Abbreviations: T: thymosin; V: vinorelbine.

Fig. 3. Thymosin inhibits vinorelbine-induced macrophage reduction in the xenotransplanted zebrafish model (Tg:zlyz-EGFP). (A) Representative images of xenotransplanted zebrafish in different groups. Scale bar, 100 µm. (B) Fluorescence intensity of macrophages after treatment of vinorelbine and thymosin for 18 h. Data are expressed as mean ± SEM. n = 10. ∗∗∗P < 0.001 compared to the vinorelbine treatment group; ####P < 0.001, comparison between the model control group and the vinorelbine treatment group. Abbreviations: T: thymosin; V: vinorelbine.

Fig. 4. Thymosin suppresses vinorelbine-induced T cell decrease in the xenotransplanted zebrafish model (Tg:rag2-DsRed). (A) Representative images of xenotransplanted zebrafish in different groups. The dotted box indicates the thymus where T cells gather. Scale bar, 100 µm. (B) Fluorescence intensity of T cells after treatment of vinorelbine and thymosin for 18 h. Data are expressed as mean ± SEM. n = 10. ∗P < 0.005 compared to the vinorelbine treatment group; #P < 0.05, comparison between the model control group and the vinorelbine treatment group. Abbreviations: T: thymosin; V: vinorelbine.

Fig. 5. Thymosin mitigates vinorelbine-induced immunosuppression. (A) The transcription levels of immune-related factors after treatment of vinorelbine and thymosin for 18 h. The mRNA level of the model control group was set at 1 in the correlation analysis of qRT-PCR results. Data are expressed as mean ± SEM. n = 10. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 compared to the vinorelbine treatment group; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001, comparison between the model control group and the vinorelbine treatment group. Abbreviations: T: thymosin; V: vinorelbine.