Fig. 2
CA2-2 increases the level of TNF-a, ROS, NO, iNOS, and some CD markers in macrophages. (A) chemical structure of CA2-2. (B) ELISA analysis of TNF-α secretion by macrophage RAW 264.7 cells. The culture supernatants were harvested after treatment with LPS (1 µg/mL) and IFN-γ (20 ng/mL) for M1 polarization; IL-4 (20 ng/mL) for M2 polarization, and none treatment (M0) for 24 h. (C) TNF-α secretion by macrophage RAW 264.7 cells. The culture supernatants were harvested after cell co-treatment with LPS (1 µg/mL) and IFN-γ (20 ng/mL) and CA2-2 (0.01 µM) for 24 h. (D) Effect of CA2-2 on the ROS content in mouse peritoneal macrophages. The incubation time with cells is 2 h. (E) Effect of CA2-2 on the NO content in RAW 264.7 cells. The incubation time with cells is 2 h. The concentration of LPS used as positive control is 0.5 µg/ml. (F) Effect of CA2-2 on the level of iNOS expression in peritoneal macrophages. The level of iNOS expression is normalized to the level of beta-actin expression. The concentration of CA2-2 is 0.01 µM, incubation time of cells with the glycoside: 0 min (control), 15 min, 30 min, 1 h, and 4 h. (G-I) CA2-2 increases CD86, CD80, and MHC CLASS II markers in RAW 264.7 macrophages. The meaning of different colors is: (G) blue – nonactivated macrophages M0; green – activated by LPS + IFN-γ macrophages M1; red – activated by CA2-2 macrophages M1; (H-I) black – nonactivated macrophages M0; pink – activated by CA2-2 macrophages M1; blue – activated by LPS (10 ng/mL) + IFN-γ macrophages M1; red – activated by LPS (1 µg/mL) + IFN-γ + CA2-2 macrophages M1; green – activated by activated by LPS + IFN-γ + CA2-2 macrophages. (J-K) CA2-2 increases CD86 and CD38 markers in mouse peritoneal macrophages. Data are presented as mean ± SE (n = 5). *p < 0.05 compared to control; *** p < 0.001 compared to M0 macrophages