Resveratrol Inhibits Secretion of Interleukin 8 by Regulation of Autophagic Flux in Ultraviolet B-stimulated Keratinocytes
Authors: Chen Hong-Ying , Chen Xu , Li Li , Gu Heng 
Published: May 2021

Abstract

Objective:
The plant polyphenol resveratrol (3,4′,5-trihydroxystilbene) (RSV) has been proposed for use because of its protective effect on ultraviolet (UV)-induced skin disorders. In UVB-induced skin damage, cell autophagy and apoptosis have been approved to prevent the damage and to contribute to the cytoprotective role of RSV; however, the detailed mechanism remains unknown. So, we conducted this study to investigate the cytoprotective effects of RSV on UVB-irradiated human epidermal keratinocytes (HEKs) and its undergoing mechanisms.

Methods:
Secretion of thirty-six inflammatory cytokines of HEKs induced by 50 mJ/cm2 UVB at 0, 12, 24, and 48 hours were detected by a human cytokine assay and the interleukin (IL)-8 protein level in the culture media were determined by ELISA. Next, HEKs were treated with or without 100 μmol/L RSV in the presence or absence of 50 mJ/cm2 UVB, and activator protein 1 and NF-κB-related proteins were measured by Western blot. Furthermore, cells exposed to UVB radiation were treated with apoptosis activators procaspase-activating compound 1 (PAC-1), apoptosis activator 2 (AA2) or RSV to investigate the effect of RSV on the percentage of apoptotic cells by flow cytometry. Then cells were treated with autophagy inhibitors LY294002, 3-methyladenine (3-MA) or RSV in the presence of UVB and chloroquine (CQ) to investigate the effect of RSV on autophagy through detecting microtubule-associated protein 1 light chain 3 (LC3) expression by western blot. Finally, the effect of LY294002, 3-MA, ATG5 siRNA, PAC-1, and AA2 on RSV-mediated reduction of IL-8 expression was determined by ELISA assay.

Results:
RSV treatment decreased the secretion of IL-8 (UVB vs. UVB + RSV: [1454.05 pg/mL ± 52.95 pg/mL] vs. [553.68 pg/mL ± 206.03 pg/mL], P < 0.001), and downregulated the protein level of c-Fos in UVB-irradiated HEKs (UVB vs. UVB + RSV: [0.103 ± 0.009] vs. [0.048 ± 0.015], P < 0.01). In UVB-irradiated HEKs, the result of western blot showed that LY294002 and 3-MA inhibited RSV-induced LC3 II accumulation (UVB + CQ + RSV vs. UVB + CQ + 3-MA + RSV vs. UVB + CQ + LY294002 + RSV: [1.15 ± 0.03] vs. [0.77 ± 0.13] vs. [0.67 ± 0.13], P < 0.01), and the result of flow cytometry showed that PAC-1 and AA2 prevented RSV from reducing cell apoptosis (UVB + RSV vs. UVB + PAC-1 + RSV vs. UVB + AA2 + RSV: [19.56% ± 0.62%] vs. [94.33% ± 0.15%] vs. [94.97% ± 1.91%], P < 0.001). The data of ELSA assay showed that LY294002, 3-MA, and ATG5 siRNA reversed the RSV-mediated inhibition of IL-8 protein secretion by UVB-irradiated HEKs (UVB + LY294002 vs. UVB + LY294002 + RSV: [3283.00 pg/mL ± 444.05 pg/mL] vs. [1608.58 pg/mL ± 128.42 pg/mL], P < 0.05; UVB + 3-MA vs. UVB + 3-MA + RSV: [2941.88 pg/mL ± 103.80 pg/mL] vs. [1867.51 pg/mL ± 153.84 pg/mL], P < 0.01; UVB + siATG5 vs. UVB + siATG5 + RSV: [2530.11 pg/mL ± 685.34 pg/mL] vs. [3011.42 pg/mL ± 435.69 pg/mL], P > 0.05), whereas neither PAC-1 nor AA2 exerted similar effects.

Conclusion:
RSV may regulate autophagic flux to inhibit IL-8 expression in UVB-challenged keratinocytes.