Anthocyanins from black rice (Oryza sativa) promote immune responses in leukemia through enhancing phagocytosis of macrophages in vivo

Abstract

Rice is a staple food in numerous countries around the world. Anthocyanins found in black rice have been reported to reduce the risk of certain diseases, but the effects of crude extract of anthocyanins from Asia University-selected purple glutinous indica rice (AUPGA) on immune responses have not yet been demonstrated. The current study aimed to investigate whether AUPGA treatment could affect immune responses in murine leukemia cells in vivo. Murine acute myelomonocytic leukemia WEHI-3 cells were intraperitoneally injected into normal BALB/c mice to generate leukemia mice. A total of 50 mice were randomly divided into five groups (n=10 in each group) and were fed a diet supplemented with AUPGA at 0, 20, 50 or 100 mg/kg for three weeks. All mice were weighed and the blood, liver and spleen were collected for further experiments. The results indicated that AUPGA did not significantly affect animal body weight, but significantly increased spleen weight (P<0.05) and decreased liver weight (P<0.05) when compared with the control group. AUPGA significantly increased the T cell (CD3) population at treatments of 20 and 100 mg/kg (P<0.05). However, it only significantly increased the B cell (CD19) population at a treatment of 20 mg/kg (P<0.05). Furthermore, AUPGA at 50 and 100 mg/kg significantly increased the monocyte (CD11b) population and the level of macrophages (Mac-3; P<0.05 for both). AUPGA at 50 and 100 mg/kg significantly promoted macrophage phagocytosis in peripheral blood mononuclear cells (P<0.05), and all doses of AUPGA treatment significantly promoted macrophage phagocytotic activity in the peritoneum (P<0.05). AUPGA treatment significantly decreased natural killer cell activity from splenocytes (P<0.05). Finally, AUPGA treatment at 20 mg/kg treatment significantly promoted T cell proliferation (P<0.05), and treatment at 50 and 100 mg/kg significantly decreased B cell proliferation compared with the control group (P<0.05).

Keywords: WEHI-3 cells; crude extract of anthocyanins from black rice; macrophage; natural killer cell; phagocytosis.

Figure 1.

Effect of AUPGA treatment on the body, liver and spleen weights of leukemia BALB/c mice. All animals were treated for 22 days. (A) Representative images of the mouse bodies at the end of the treatment period. (B) Total body weight throughout the treatment period (measured every three days). (C) Representative images of the mouse livers at the end of the treatment period. (D) Weights of mouse livers at the end of the treatment period. *P<0.05 vs. AUPGA-untreated leukemia mice group. (E) Representative images of the mouse spleens at the end of the treatment period. (F) Weights of mouse spleens at the end of the treatment period. *P<0.05 vs. AUPGA-untreated leukemia mice group. AUPGA, Asia University selected purple glutinous indica rice.

Figure 1.

Effect of AUPGA treatment on the body, liver and spleen weights of leukemia BALB/c mice. All animals were treated for 22 days. (A) Representative images of the mouse bodies at the end of the treatment period. (B) Total body weight throughout the treatment period (measured every three days). (C) Representative images of the mouse livers at the end of the treatment period. (D) Weights of mouse livers at the end of the treatment period. *P<0.05 vs. AUPGA-untreated leukemia mice group. (E) Representative images of the mouse spleens at the end of the treatment period. (F) Weights of mouse spleens at the end of the treatment period. *P<0.05 vs. AUPGA-untreated leukemia mice group. AUPGA, Asia University selected purple glutinous indica rice.

Figure 1.

Effect of AUPGA treatment on the body, liver and spleen weights of leukemia BALB/c mice. All animals were treated for 22 days. (A) Representative images of the mouse bodies at the end of the treatment period. (B) Total body weight throughout the treatment period (measured every three days). (C) Representative images of the mouse livers at the end of the treatment period. (D) Weights of mouse livers at the end of the treatment period. *P<0.05 vs. AUPGA-untreated leukemia mice group. (E) Representative images of the mouse spleens at the end of the treatment period. (F) Weights of mouse spleens at the end of the treatment period. *P<0.05 vs. AUPGA-untreated leukemia mice group. AUPGA, Asia University selected purple glutinous indica rice.

Figure 2.

Effects of AUPGA treatment on cell markers of white blood cells from leukemia BALB/c mice. Blood was collected from mice and analyzed for cell markers (A) CD3, (B) CD19, (C) CD11b and (D) Mac-3 by flow cytometry. *P<0.05 vs. AUPGA-untreated leukemia mice group. AUPGA, Asia University selected purple glutinous indica rice.

Figure 3.

Effect of AUPGA treatment on macrophage phagocytosis in the PBMC and peritoneum of leukemia BALB/c mice. Blood samples were collected, then macrophages were isolated from the (A) PBMC and (B) peritoneum of each mouse. Macrophage phagocytosis activity was assayed by flow cytometry and quantified using CellQuest software. *P<0.05 vs. AUPGA-untreated leukemia mice group. AUPGA, Asia University selected purple glutinous indica rice; PBMC, peripheral blood mononuclear cell.

Figure 4.

Effect of AUPGA treatment on the cytotoxic activity of NK cells from leukemia BALB/c mice. Splenocytes were cultured in 96-well plates and target YAC-1 cells were added. NK cell cytotoxic activity was measured using flow cytometry. *P<0.05 vs. AUPGA-untreated leukemia mice group. AUPGA, Asia University selected purple glutinous indica rice; NK, natural killer.

Figure 5.

Effect of AUPGA treatment on T and B cell proliferation in leukemia BALB/c mice. (A) T cells were pretreated with or without Con A, then cell proliferation was analyzed by flow cytometry. (B) B cells were pretreated with or without LPS, then cell proliferation was analyzed by flow cytometry. *P<0.05 vs. AUPGA-untreated leukemia mice group. AUPGA, Asia University selected purple glutinous indica rice; LPS, lipopolysaccharide; Con A, concanavalin A.