Camu-Camu Fruit Extract Inhibits Oxidative Stress and Inflammatory Responses by Regulating NFAT and Nrf2 Signaling Pathways in High Glucose-Induced Human Keratinocytes

Abstract

Myrciaria dubia (HBK) McVaugh (camu-camu) belongs to the family Myrtaceae. Although camu-camu has received a great deal of attention for its potential pharmacological activities, there is little information on the anti-oxidative stress and anti-inflammatory effects of camu-camu fruit in skin diseases. In the present study, we investigated the preventative effect of 70% ethanol camu-camu fruit extract against high glucose-induced human keratinocytes. High glucose-induced overproduction of reactive oxygen species (ROS) was inhibited by camu-camu fruit treatment. In response to ROS reduction, camu-camu fruit modulated the mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NFAT) signaling pathways related to inflammation by downregulating the expression of proinflammatory cytokines and chemokines. Furthermore, camu-camu fruit treatment activated the expression of nuclear factor E2-related factor 2 (Nrf2) and subsequently increased the NAD(P)H:quinone oxidoreductase1 (NQO1) expression to protect keratinocytes against high-glucose-induced oxidative stress. These results indicate that camu-camu fruit is a promising material for preventing oxidative stress and skin inflammation induced by high glucose level.

Keywords: NFAT; Nrf2; camu-camu fruit; high glucose; inflammation; keratinocytes; oxidative stress.

Figure 1

The HPLC (high-performance liquid chromatography) results of ellagic acid (peak 1) and quercetin standards (peak 2) (A,B) and the contents of ellagic acid and quercetin in the camu-camu fruit extract (C).

Figure 2

Antioxidative activities of the camu-camu fruit extract. DPPH radical-scavenging activity of the camu-camu fruit extract (A). Levels of ROS production in the HaCaT cells treated with 15 mM D-glucose for 6 h (B,C). The cells were stained with DCF-DA for 30 min; then, the number of cells was plotted versus the dichlorofluorescein detected by the FL-2 channel. All the data are shown as the means ± SD of three independent experiments. # Significant differences between the untreated group and the high glucose-induced group. (# p < 0.05, ### p < 0.001). * Significant differences between the high glucose-induced group and the other groups (* p < 0.05; ** p < 0.01; *** p < 0.001). Dex, dexamethasone.

Figure 3

Effect of the camu-camu fruit extract on cell viability and mRNA expression of proinflammatory cytokines and chemokines in high glucose-induced HaCaT cells. Effect of the camu-camu fruit on the cell viability of high glucose-induced HaCaT cells (A). Expression of mRNA of IL-8, MDC, RANTES, and TARC in HaCaT cells under a high glucose-treated condition was measured by RT-PCR analysis (B). Band intensities of IL-8 (C), MDC (D), RANTES (E), and TARC (F) were calculated by densitometry and compared to GAPDH. The data are shown as the means ± SD of three independent experiments. # Significant differences between the untreated group and the high glucose-induced group (# p < 0.05; ## p < 0.01; ### p < 0.001). * Significant differences between the high glucose-induced group and the other groups (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 4

Effects of the camu-camu fruit on the MAPK/AP-1 signaling pathways in high glucose-stimulated HaCaT cells. JNK, p38, ERK, and their phosphorylation were analyzed by Western blotting (A). Phosphorylation of c-Jun was assessed by the Western blot analysis (E). Band intensities for p-JNK (B), p-ERK (C), p-p38 (D), and p-c-Jun (F) were measured by densitometry and normalized; the percentage was calculated on the basis of the level of β-actin. The data are shown as the means ± SD of three independent experiments. # Significant differences between the untreated group and the high glucose-induced group (# p < 0.05; ### p < 0.001). * Significant differences between the high glucose-induced group and the other groups (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

Effects of the camu-camu fruit on the NF-κB signaling pathway in high glucose-stimulated HaCaT cells. (A) The expression of NF-κB, IKBα and phosphorylation of IKBα were performed by Western blotting. Band intensities of NF-κB and p-IKBα (B) were measured by densitometry and normalized; the percentage was calculated on the basis of the level of β-actin. Values are shown as the means ± SD of three independent experiments. # Significant differences between the untreated group and the high glucose-induced group (# p < 0.05; ### p < 0.001). * Significant differences between the high glucose-induced group and the other groups (* p < 0.05; ** p < 0.01).

Figure 6

Effects of the camu-camu fruit on the NFATc1 and COX-2 signaling pathways in high glucose-stimulated HaCaT cells. Phosphorylation of NFATc1 (A) and COX-2 (C) was evaluated by Western blot analysis. Band intensities for p-NFATc1 (B) and COX-2 (D) were quantified using densitometry and normalized; the percentage was calculated on the basis of the level of β-actin. The data are shown as the means ± SD of three independent experiments. # Significant differences between the untreated group and the high glucose-induced group (# p < 0.05). * Significant differences between the high glucose-induced group and the other groups (* p < 0.05; ** p < 0.01).

Figure 7

Regulatory effects of the camu-camu fruit extract on the Nrf2/ARE pathway in high glucose-stimulated HaCaT cells were assessed by Western blot analysis (A) and band intensities for Nrf2 and NQO-1 were quantified by densitometry and normalized; the percentage was calculated on the basis of the level of β-actin (B). The data are shown as the means ± SD of at least two independent experiments. # Significant differences between the untreated group and the high glucose-induced group (# p < 0.05) * Significant differences between the high glucose-induced group and the other groups (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 8

Anti-inflammatory effects of the camu-camu fruit on high-glucose-induced keratinocytes.