Phloretin inhibits glucose transport and reduces inflammation in human retinal pigment epithelial cells

Abstract

During age-related macular degeneration (AMD), chronic inflammatory processes, possibly fueled by high glucose levels, cause a breakdown of the retinal pigment epithelium (RPE), leading to vision loss. Phloretin, a natural dihydroxychalcone found in apples, targets several anti-inflammatory signaling pathways and effectively inhibits transporter-mediated glucose uptake. It could potentially prevent inflammation and cell death of RPE cells through either direct regulation of inflammatory signaling pathways or through amelioration of high glucose levels. To test this hypothesis, ARPE-19 cells were incubated with or without phloretin for 1 h before exposure to lipopolysaccharide (LPS). Cell viability and the release of pro-inflammatory cytokines interleukin 6 (IL-6), IL-8 and vascular endothelial growth factor (VEGF) were measured. Glucose uptake was studied using isotope uptake studies. The nuclear levels of nuclear factor erythroid 2-related factor 2 (Nrf2) were determined alongside the phosphorylation levels of mitogen-activated protein kinases. Phloretin pretreatment reduced the LPS-induced release of IL-6 and IL-8 as well as VEGF. Phloretin increased intracellular levels of reactive oxygen species and nuclear translocation of Nrf2. It also inhibited glucose uptake into ARPE-19 cells and the phosphorylation of Jun-activated kinase (JNK). Subsequent studies revealed that Nrf2, but not the inhibition of glucose uptake or JNK phosphorylation, was the main pathway of phloretin's anti-inflammatory activities. Phloretin was robustly anti-inflammatory in RPE cells and reduced IL-8 secretion via activation of Nrf2 but the evaluation of its potential in the treatment or prevention of AMD requires further studies.

Keywords: Glucose transport; Inflammation; Nuclear factor erythroid 2-related factor 2 (Nrf2); Phloretin; Retinal degeneration.

Fig. 1

The effect of phloretin on cell viability. a Phloretin does not decrease RPE cell viability at the studied concentration of 100 µM. b Lipopolysaccharide (LPS) exposure with or without a 1 h pretreatment with 100 µM phloretin (PHL) does not reduce cell viability. c Brightfield microscopy images of cells exposed to vehicle or 10 µg/ml LPS with or without 1 h pretreatment with 100 µM phloretin reveal no changes to cellular morphology. Data are combined from three to nine independent experiments with four parallel samples per experiment and are represented as mean ± SEM. Representative microscopy images are shown. PHL—100 µM phloretin, LPS—10 µg/ml LPS, ns not statistically significant, ****p < 0.0001, Mann–Whitney U-test

Fig. 2

The effect of phloretin on pro-inflammatory cytokine secretion. A 1 h pretreatment with 100 µM phloretin (PHL) greatly reduces 10 mg/ml LPS (LPS)-induced secretion of IL-6 (a), IL-8 (b) or VEGF (c) in ARPE-19 cells. Data are combined from three independent experiments with three or four parallel samples per experiment and are represented as mean ± SEM. ns not statistically significant, **p < 0.01, ****p < 0.0001, Mann–Whitney U-test

Fig. 3

The uptake of [14C]-Glucose and [14C(U)]-Methyl α-D-glucopyranoside ([14C]-α-MDG) by ARPE-19 cells. a/b The uptake rate of 0.25 μCi/ml [14C]-Glucose (86.2 µM) (a) or of 0.5 μCi/ml [14C]-α-MDG (2.0 µM) (b) measured over 5 or 10 min, respectively, in the absence or presence of 100 µM phloretin (PHL) or 10 µM phloridzin (PHZ). Phloretin, but not phloridzin significantly reduces the glucose uptake rate. (c/d) The uptake rate of 0.25 μCi/ml [14C]-Glucose (86.2 µM) measured for 5 min after 1 h pre-incubation with PHL and LPS, or 24 h incubation with LPS. Glucose uptake was measured in the absence (c) or presence of PHL (d). Removal of phloretin from culture medium prior to glucose uptake rate measurement leads to an increased uptake of [14C]-Glucose. Data are presented as the mean ± SEM of data from three to five independent experiments. ns not statistically significant, *p < 0.05, Mann–Whitney U-test

Fig. 4

The effect of glucose content in cell-culture medium on cell viability and inflammation. a Cellular viability of untreated control cells in no glucose medium (noG) was significantly lowered when compared to high-glucose medium (HG) or normal medium conditions (DF12). b 10 µg/ml LPS or 100 µM phloretin (PHL) exposure had little effect on cell viability in the different culture media. c–e A 1 h pretreatment with 100 µM phloretin (PHL) significantly reduced the 10 µg/ml LPS-induced secretion of VEGF (c), IL-6 (d) or IL-8 (e) in all studied media. Data are combined from three independent experiments with three or four parallels per experiment and are represented as mean ± SEM. ns not statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001, Mann–Whitney U-test

Fig. 5

The effect of phloretin and LPS exposure on cellular signaling pathways in ARPE-19 cells. a Intracellular ROS levels are significantly increased in cells exposed to 100 µM phloretin (PHL) for 1 h and slightly increased in cells exposed to 10 µg/ml LPS (LPS) for 5 min. b–f Following a 1 h pretreatment with PHL and a 2 h exposure to LPS, the DNA-binding activity of NF-κB subunit p65 (b) or the intracellular levels of phosphorylated CREB (c), ERK1/2 (d), and p38 (e) remained unaffected. The phosphorylation of JNK (f) was reduced by cotreatment with 100 µM phloretin and 10 µg/ml LPS. g/h Phloretin increased the nuclear levels of Nrf2 as determined by Western Blotting (g, quantified h). Data are combined from three or four independent experiments, with two to four parallel samples per experiment and are represented as mean ± SEM. ns not statistically significant, *p < 0.05, ****p < 0.0001, Mann–Whitney U-test

Fig. 6

Effect of JNK inhibition or Nrf2 activation and knockdown on LPS-stimulated ARPE-19 cells. a/d A 1 h pretreatment with the JNK inhibitor SP600215 (SP, 10 µM) increased IL-6 (a) and slightly decreased IL-8 (d) secretion in 10 µg/ml LPS (LPS)-stimulated ARPE-19 cells. b/e A 1 h pretreatment with the Nrf2 activator sulforaphane (SUL, 20 µM) significantly decreased the secretion of IL-6 (b) or IL-8 (e) from LPS-treated RPE cells with or without phloretin (PHL). c/f Nrf2 knockdown by siRNA transfection before exposure to phloretin (PHL) and LPS increased IL-6 (c) or IL-8 (f) secretion when compared to phloretin and LPS stimulation alone, but only changes for IL-8 were statistically significantly different to the effect of scrambled control siRNA. Results are combined from three or four independent experiments with three or four parallel samples per experiment and are represented as mean ± SEM. ns not statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Mann–Whitney U-test