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  • 1\'-hydroxy Midazolam br Carotene concentrations in both

    2020-08-12


    3.6. β-Carotene concentrations in both extracts
    The levels of β-carotene, which is known as one of major car-otenoids in Chinese cabbage, were measured in TL and NL by HPLC. β-Carotene was found at 40.5 min retention time (RT), and regarding its concentration, TL extract contained 27.9 ± 2.1 μg/g, whereas β-car-otene was not detected in the NL extract. Generally, 1\'-hydroxy Midazolam can be observed via absorbance at 450 nm in the visible range. Besides β-carotene, four minor peaks were detected at 7–11 min RT. Those peaks detected at 7.4, 7.7, 9.2 and 10.5 mins RT were considered as lutein, zeaxanthin, β-apo-8′-carotenal and β-cryptoxanthin, respectively, compared with the previous report (Howe & Tanumihardjo, 2006) (see Fig. 8).
    4. Discussion
    Immunosuppression by abnormally recruited Tregs is one of me-chanisms underlying PC’s ability to avoid host immunity (Ene-Obong et al., 2013; Hiraoka et al., 2006; Ino et al., 2013; Tang et al., 2014). We found that the TL extract suppressed tumor growth (Fig. 3) and changed the immune-cell population in the tumor and spleen (Figs. 4 and 6). The alteration in the tumor notably decreased the numbers of Tregs among tumor cells, and this phenomenon was proved by mRNA and protein expression as well (Figs. 4 and 5). In case of the spleen, the number of effector T cells was increased by the TL extract and NL extract, but TL had a more powerful effect than NL (Fig. 6). This result was reflected in the findings about the cell population and might be caused by the
    Fig. 4. Immune-cell population in tumor tissue. Tumor tissue was excised from the mice, and the population of immune cells such as NK cells, macrophages, dendritic cells, CD4+ T cells, CD8+ T cells, B cells, and CD4+FOXP3+ (Treg) cells was analyzed by flow cytometry (A–G). Data are presented as the means ± SD from three independent experiments. Statistical significance was evaluated by Student’s t test (*p < 0.05 Cancer vs TL or NL) (Cancer: group inoculated with cancer; NL: NL treated group inoculated with cancer; TL: TL treated group inoculated with cancer).
    Fig. 5. Expression of genes specific for Tregs and genes involved their recruitment (Foxp3, CCR5, and CCL5) as well as genes of anti-inflammatory cytokines (IL-10 and TGF-β) in tumor tissue (A–E). Tumor tissue was excised from the mice with or without treatment with NL or TL; expression of genes was determined by PCR. Data are presented as the means ± SD from three independent experiments. Statistical significance was evaluated by Student’s t test (*p < 0.05 Cancer vs TL or NL) (Cancer: group in-oculated with cancer; NL: NL treated group inoculated with cancer; TL: TL treated group inoculated with cancer).
    Fig. 6. Immune-cell population, T-cell polarization, and the expression levels of Treg-related and anti-inflammatory cytokine genes in the spleen. The profile of immune cells such as dendritic cells, macrophages, NK cells, CD4+ cells, CD8+ cells, and B cells was determined (A–F). In addition, T-cell polarization was measured by flow cytometry via confirmation of the expression of cell adhesion proteins (G–L). Lastly, the genes associated with Treg and their recruitment and anti-inflammatory cytokines were analyzed by PCR (M–Q). Data are presented as the means ± SD from three independent experiments. Statistical significance was evaluated by Student’s t test (#p < 0.05 Non-induction vs Cancer, *p < 0.05 Cancer vs TL or NL) (Non-induction: group without inoculation with cancer; Cancer: group inoculated with cancer; NL: NL treated group inoculated with cancer; TL: TL treated group inoculated with cancer).
    Fig. 7. Protein levels of cytokines and expression of genes in splenocytes reactivated by anti-CD3 antibody stimulation. The anti-CD3 antibody was used to boost T-cell functions. The expression of genes associated with Tregs and their recruitment and genes of anti-inflammatory cytokines like FOXP3, IL-10, TGF-β, CCR5, and CCL5 was evaluated by PCR (A–E), and the cytokines such as IFN-γ, TNF-α, IL-4, IL-13, and IL-10 were analyzed by an ELISA (F–J). Data are presented as the means ± SD from three independent experiments. Statistical significance was evaluated by Student’s t test (#p < 0.05 Non-induction vs Cancer, *p < 0.05 Cancer vs TL or NL) (Non-induction: group without inoculation with cancer; Cancer: group inoculated with cancer; NL: NL treated group inoculated with cancer; TL: TL treated group inoculated with cancer).
    blocking of Treg cell recruitment according to the gene expression profile (Fig. 6). Additionally, the same trend was observed after re-boosting of splenocytes by the anti-CD3 antibody for 2 days. FOXP3 and CCL5 are known as genes involved in the weakening of immunity by accumulating Tregs. These genes were downregulated, and cytokines such as TNFα and IL-4 that enhance the immune system were upre-gulated by treatment with the TL extract (Fig. 7).