Fig. 4. Effect of hesperetin on NF-κB protein and mRNA expressions in LPS-treated THP-1 macrophages under hyperglycemic conditions. NF-κB levels were evaluated by western blot analysis of cell lysates (A). The percentage of NF-κB (whole)/β-actin (B) and the percentage of NF-κB (nuclear)/TBP (C) were revealed by image J. Cells (1 × 10⁶ cells/mL) were treated with hesperetin for 48 h, and NF-κB mRNA levels were evaluated by qPCR (D). Immunofluorescence images show downregulation of NF-ĸB (E) and Ac-NF-ĸB (F) in LPS-treated THP-1 macrophages under hyperglycemic conditions following hesperetin treatment. Differentiated THP-1 cells were cultured in the presence of LPS (100 ng/mL) under normoglycemic or hyperglycemic conditions in the absence or presence of hesperetin (50 μM) for 48 h (400 × Magnification). Signal quantification was performed using ImageJ software. The data are representative of at least three independent experiments. Data were analyzed using the 2^−ΔΔCT method.

NF-κB, nuclear factor-κB; TBP, TATA-binding protein; NG, normoglycemic 5.5 mM/L glucose; HG, hyperglycemic 25 mM/L glucose; LPS, lipopolysaccharide; H0, LPS + hyperglycemic + hesperetin 0 μM; DAPI, 4′,6-diamidino-2-phenylindole.

^##P < 0.01 compared with NG; ^**P < 0.01 compared with H0.