Aquaporin-8 promotes human dermal fibroblasts to counteract hydrogen peroxide-induced oxidative damage: A novel target for management of skin aging

Abstract

The skin is subjected to various external factors that contribute to aging including oxidative stress from hydrogen peroxide (H₂O₂). This study investigated the distribution of aquaporin-8 (AQP8), a protein that transports H₂O₂ across biological membranes, in skin cells, and its effects in mitigating H₂O₂-induced oxidative damage. Human dermal fibroblasts were treated with increasing concentrations of H₂O₂ to evaluate oxidative damage. Cell viability, reactive oxygen species (ROS) generation, and the expression of specific genes associated with skin aging (IL-10, FPR2, COL1A1, KRT19, and Aggrecan) were evaluated and AQP8 expression was assessed via quantitative polymerase chain reaction and western blotting. Small-interfering RNA was used to silence the AQP8 gene and evaluate its significance. The results show that H₂O₂ treatment reduces cell viability and increases ROS generation, leading to oxidative damage that affects the expression of target molecules. Interestingly, H₂O₂-treated cells exhibit high levels of AQP8 expression and gene silencing of AQP8 reverses high levels of ROS and low levels of COL1A1, KRT19, and Aggrecan expression in stressed cells, indicating that AQP8 plays a vital role in preventing oxidative damage and consequent aging. In conclusion, AQP8 is upregulated in human dermal fibroblasts during H₂O₂-induced oxidative stress and may help prevent oxidative damage and aging. These findings suggest that AQP8 could be a potential therapeutic target for skin aging. Further research is necessary to explore the feasibility of using AQP8 as a preventive or therapeutic strategy for maintaining skin health.

Keywords: aging; aquaporin-8; dermal fibroblasts; hydrogen peroxide; oxidative stress.

Figure 1

H₂O₂ treatment induces cytotoxicity in human dermal fibroblasts. (a and b) The cells were exposed to increasing concentrations of H₂O₂ (100, 200, 400, 800, and 1,200 μM) for 24 h. (a) The cell morphology was observed under a light microscopy (10×). (b) The cell viability was determined using an MTT assay. (c) The cells were exposed to H₂O₂ (800 μ M) for different time intervals. The cell viability was determined using an MTT assay. *p  <  0.05, as compared to the control. All experiments were performed independently at least three times.

Figure 2

H₂O₂ treatment induces oxidative stress in human dermal fibroblasts. The cells were exposed to increasing concentrations of H₂O₂ (100, 200, 400, 800, and 1,200 μM) for 3 h. Intracellular ROS generation was detected using CellROX™ Green Reagent. (a) Representative fluorescent microscopy images of ROS detection. Green foci represent CellROX dye upon oxidation by ROS and binding to DNA (blue). (b) Quantitative analysis of (a). *p  <  0.05, as compared to the control. All experiments were performed independently at least three times.

Figure 3

Analysis of gene expression in human dermal fibroblasts. The cells were exposed to increasing concentrations of H₂O₂ (100, 200, 400, 800, and 1,200 μM) for 3 h. The expression level of target gene was identified by qPCR. *p  <  0.05, **p  <  0.01, ***p  <  0.001, as compared to the control. All experiments were performed independently at least three times. Abbreviations: AQP8, aquaporin-8; IL-10, interleukin-10; FPR2, N-formyl peptide receptor 2; COL1A1, collagen type I1; KRT19, keratin 19.

Figure 4

Analysis of protein expression in human dermal fibroblasts. The cells were exposed to increasing concentrations of H₂O₂ (100, 200, 400, 800, and 1,200 μM) for 3 h. The protein expression of target molecule was determined by Western blot analysis. (a) Representative immunoblotting images of protein expression. (b) Quantitative analysis of (a). *p  <  0.05, as compared to the control. All experiments were performed independently at least three times. Abbreviations: AQP8, aquaporin-8; Cola1, collagen type I α1; KRT19, keratin 19.

Figure 5

AQP8 promotes human dermal fibroblasts to counteract H₂O₂-induced oxidative damage. The cells were transfected with siRNA of AQP8 and then exposed to increasing concentrations of H₂O₂ (100, 200, 400, 800, and 1,200 μM) for 3 h. (a and b) The effectiveness of siRNA in silencing AQP8 gene expression was validated by Western blot analysis. (c) Intracellular ROS generation was detected using CellROX^TM Green Reagent. (d) The gene expression of target gene was identified by qPCR. *p  <  0.05, **p  <  0.01, ***p  <  0.001, as compared to the control. All experiments were performed independently at least three times. Abbreviations: AQP8, aquaporin-8; COL1A1, collagen type I1; KRT19, keratin 19.