Phloretin Modulates Human Th17/Treg Cell Differentiation In Vitro via AMPK Signaling

Figure 3

Phloretin influences Th17/Treg differentiation via glycolysis AMPK signaling. (a) The effect of phloretin on the expression of phosphorylated AMPK and mTOR was detected by western blot in activated CD4⁺ T cells. After naïve CD4⁺ T cells were activated, DMSO, 50 μM phloretin, 50 μM phloretin combined with 1 μM Com C, 1 μM Com C, 200 μM AICAR, and 200 μM AICAR combined with 1 μM Com C were added to the culture medium, respectively, and activated CD4⁺ T cells were continuously cultured for 6 h. Total AMPK and beta-actin were used as loading control, respectively. , , , and ns (no significant difference) versus the control group; ^##, ^####, and NS (no significant difference) versus the phloretin treatment group; ^Δ, ^ΔΔΔΔ, and NS (no significant difference) versus the Com C treatment group; ^θθθθ versus the AICAR treatment group (, ). (b) The frequency of Th17 cells generated under Th17 polarization conditions. After naïve CD4⁺ T cells were activated, DMSO, 50 μM phloretin, 50 μM phloretin combined with 1 μM Com C, 1 μM Com C, 200 μM AICAR, and 200 μM AICAR combined with 1 μM Com C were added to Th17-polarization culture medium, respectively, and cells were continuously cultured for 3 days. and ns versus the control group; ^# and ^#### versus the phloretin treatment group; ^ΔΔΔΔ and NS versus the Com C treatment group; ^θθθθ versus the AICAR treatment group (, ). (c) The frequency of Treg cells generated under Treg polarization conditions. After naïve CD4⁺ T cells were activated, DMSO, 50 μM phloretin, 50 μM phloretin combined with 1 μM Com C, 1 μM Com C, 200 μM AICAR, and 200 μM AICAR combined with 1 μM Com C were added to Treg polarization culture medium, respectively, and cells were continuously cultured for 3 days. , , and ns versus the control group; ^#### and NS versus the phloretin treatment group ;^ΔΔΔ and ^ΔΔΔΔ versus the Com C treatment group; ^θθθθ versus the AICAR treatment group (, ). (a–c) One-way ANOVA followed by Tukey’s multiple comparison test was used in each statistical analysis.

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