Target Proteins of Phloretin for Its Anti-Inflammatory and Antibacterial Activities Against Propionibacterium acnes-Induced Skin Infection

Abstract

Phloretin is a natural chalcone with antibacterial and anti-inflammatory effects. This study investigated the anti-acne activity of phloretin against Propionibacterium acnes-induced skin infection and the potential target proteins of its anti-inflammatory and antibacterial effects. Phloretin potently inhibited the growth of P. acnes and P. acnes-induced Toll-like receptor (TLR) 2-mediated inflammatory signaling in human keratinocytes. Secreted embryonic alkaline phosphatase assay confirmed that the anti-inflammatory activity of phloretin is associated with the P. acnes-stimulated TLR2-mediated NF-κB signaling pathway. Phloretin significantly decreased the level of phosphorylated c-Jun N-terminal kinase (JNK), showing a binding affinity of 1.184 × 10^-5 M^-1. We also found that phloretin binds with micromolar affinity to P. acnes β-ketoacyl acyl carrier protein (ACP) synthase III (KAS III), an enzyme involved in fatty acid synthesis. Conformation-sensitive native polyacrylamide gel electrophoresis showed that phloretin reduced KAS III-mediated 3-ketoacyl ACP production by over 66%. A docking study revealed that phloretin interacts with the active sites of JNK1 and KAS III, suggesting their involvement in P. acnes-induced inflammation and their potential as targets for the antibacterial activity of phloretin. These results demonstrate that phloretin may be useful in the prevention or treatment of P. acnes infection.

Keywords: Propionibacterium acnes; antimicrobial activity; inflammation; phloretin; plant natural product.

Figure 1

Chemical structure of phloretin (molecular weight: 274.27).

Figure 2

Effects of phloretin, CU-CPT22, benzoyl peroxide, and triclosan on viability of (A) HaCaT cells and (B) human HEK293 cells.

Figure 3

Dose-dependent reduction in secreted embryonic alkaline phosphatase (SEAP) activity reflecting suppression of NF-κB activation in (A) 200 ng/mL Pam₃CSK₄-stimulated HEK-Blue^® hTLR2 cells and (B) P. acnes-stimulated HEK-Blue^® hTLR2 cells. SEAP activities of culture supernatants containing different concentrations of phloretin or CU-CPT22 (0, 1, 2.5, 5, 10, 20, 40, 50 μM) were determined and compared with levels without phloretin or CU-CPT22. All experiments were performed three times independently. *** p < 0.001 vs. cells treated with Pam₃CSK₄ or P. acnes only; n.s, represents no significance.

Figure 4

Effects of phloretin on Propionibacterium acnes-induced inflammation. (A) Effects of phloretin on cytokine levels in P. acnes-stimulated HaCaT cells. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-12 levels in the culture medium of P. acnes-stimulated HaCaT cells were evaluated by enzyme-linked immunosorbent assay. (B) Expression of intracellular signaling molecules in P. acnes-stimulated HaCaT cells with or without phloretin treatment, as determined by Western blotting; β-actin was used as a loading control. (C) Indicated levels of phosphorylation of c-Jun N-terminal kinase (JNK) protein in P. acnes-stimulated HaCaT cells with or without phloretin treatment at 0, 10, 20, or 40 μM, quantified using ImageJ. ** p < 0.01, *** p < 0.001 vs. cells treated with P. acnes only. n.s, represents no significance.

Figure 5

Binding of phloretin to JNK1. (A) Fluorescence spectra of JNK1 in the presence of phloretin at the indicated concentrations (pH 7.0). Samples were excited at 290 nm, and emission was recorded at the indicated wavelengths. (B) Model of phloretin binding to JNK1. Hydrophobic residues are shown in white, and residues that participate in hydrogen bonding with phloretin are shown in yellow.

Figure 6

Inhibition of β-ketoacyl acyl carrier protein (ACP) synthase III (KAS III) activity and binding by phloretin. (A) Results of conformation-sensitive native polyacrylamide gel electrophoresis (PAGE). Upper and lower bands represent malonyl-ACP and 3-keto hexanoyl-ACP (reaction product), respectively. (B) Normalized fluorescence spectra of P. acnes KAS III in the presence of indicated concentrations of phloretin. (C) Docking pose of phloretin with P. acnes KAS III. Hydrophobic residues are shown in white, and residues that participate in hydrogen bonding with phloretin are shown in yellow.