Enzymatically synthesized glycogen prevents ultraviolet B-induced cell damage in normal human epidermal keratinocytes

Abstract

Enzymatically synthesized glycogen is a product from starch. Enzymatically synthesized glycogen has been reported to possess various health beneficial effects such as anti-oxidative and anti-inflammatory effects. In this study, we investigated the effect of enzymatically synthesized glycogen on ultraviolet B-induced oxidative stress and apoptosis in normal human epidermal keratinocytes. Treatment with enzymatically synthesized glycogen suppressed ultraviolet B-induced reactive oxygen species, caspase-3 activity, and DNA fragmentation in normal human epidermal keratinocytes. Furthermore, enzymatically synthesized glycogen increased in the expression level of heme oxygenase-1, NAD(P)H: quinone oxidoreductase 1, and NF-E2-related factor 2, a transcriptional factor for heme oxygenase-1 and NAD(P)H: quinone oxidoreductase 1. Although enzymatically synthesized glycogen did not increase in its mRNA expression level of NF-E2-related factor 2, enzymatically synthesized glycogen retained its protein degradation. Knockdown of heme oxygenase-1 and NAD(P)H: quinone oxidoreductase 1 canceled enzymatically synthesized glycogen-suppressed reactive oxygen species accumulation in normal human epidermal keratinocytes. It is, therefore, concluded that enzymatically synthesized glycogen inhibited ultraviolet B-induced oxidative stress through increasing the expression level of heme oxygenase-1 and NAD(P)H: quinone oxidoreductase 1 through the NF-E2-related factor 2 pathway in normal human epidermal keratinocytes.

Keywords: anti-oxidative protein; enzymatically synthesized glycogen; normal human epidermal keratinocytes; reactive oxygen species; ultraviolet B.

Fig. 1

ESG and RG inhibited UVB-induced ROS accumulation in NHEK. UVB-induced ROS accumulation was measured with or without ESG, RG, the residue, and OG in NHEK. NHEK was treated with ESG, RG, the residue, and OG for 24 h. The cells were exposed by UVB irradiation (20 mJ/cm²), followed by incubation with 20 µM DCFH-DA and 1 µg/ml DAPI for 30 min. The fluorescence intensity of DCF and DAPI was measured at 485/535 nm and 355 and 460 nm, respectively. The fluorescence intensity of DCF was normalized by that of DAPI. Data are presented as the means ± SD (n = 4). Different letters indicate significant differences (p<0.05; Tukey-Kramer test).

Fig. 2

ESG and RG increased in the protein expression level of HO-1, NQO1, and Nrf2 in NHEK. (A) NHEK was treated with ESG, RG, and OG for 24 h and subject to Western blotting for detection of HO-1, NQO1, and Nrf2. (B) NHEK was treated with ESG, RG, and OG for 24 h and subjected to real time PCR for detection of Nrf2 gene (NFE2L2). Data are presented as the means ± SD (n = 4). Different letters indicate significant differences (p<0.05; Tukey-Kramer test).

Fig. 3

Effect of ESG and RG on Nrf2 stability in NHEK. (A) For time-course analysis of Nrf2 protein expression, NHEK treated with 600 µg/ml ESG, RG, and OG in the presence of 10 µg/ml cycloheximide (CHX) for indicated periods and subject to Western blotting for detection of Nrf2. (B) For detection of serine phosphorylation of Nrf2, Nrf2 protein was immunoprecipitated (IP) in NHEK treated with 600 µg/ml ESG, RG, and OG for 1 h, followed by Western blot analysis using anti-pSer antibody. Data are presented as the means ± SD (n = 4). Different letters indicate significant differences (p<0.05; Tukey-Kramer test).

Fig. 4

Knockdown of HO-1 and NQO-1 canceled the function of ESG against UVB-induced ROS accumulation in NHEK UVB-induced ROS accumulation was measured in HO-1- and/or NQO1-knocked NHEK as the same procedure as described in Fig. 1. Data are presented as the means ± SD (n = 4). Different letters indicate significant differences (p<0.05; Tukey-Kramer test).

Fig. 5

ESG and RG inhibited UVB-induced caspases activity and cleavage in NHEK. UVB-induced apoptosis was evaluated with or without ESG, RG, and OG in NHEK. NHEK was treated with ESG, RG, and OG for 24 h and irradiated with UVB (20 mJ/cm²). (A) The activity of caspase-3, -8 and -9 was measured using corresponding peptide substrate by monitoring the released fluorescence intensity of AMC at 380/460 nm in cell lysate. (B) The cleavages of caspase proteins were detected in cell lysate by Western blotting. Data are presented as the means ± SD (n = 4). Different letters indicate significant differences (p<0.05; Tukey-Kramer test).

Fig. 6

ESG and RG inhibited UVB-induced cytotoxicity and DNA fragmentation in NHEK. NHEK was treated with ESG, RG, and OG for 24 h and irradiated with UVB (20 mJ/cm²). (A) Cytotoxicity was measurement by MTT assay. (B) After DNA was extracted, DNA fragmentation was measured by agarose gel electrophoresis. Data are presented as the means ± SD (n = 4). Different letters indicate significant differences (p<0.05; Tukey-Kramer test).