Attenuation of UV-B exposure-induced inflammation by abalone hypobranchial gland and gill extracts

Abstract

Exposure to solar ultraviolet B (UV-B) is a known causative factor for many skin complications such as wrinkles, black spots, shedding and inflammation. Within the wavelengths 280‑320 nm, UV-B can penetrate to the epidermal level. This investigation aimed to test whether extracts from the tropical abalone [Haliotis asinina (H. asinina)] mucus-secreting tissues, the hypobranchial gland (HBG) and gills, were able to attenuate the inflammatory process, using the human keratinocyte HaCaT cell line. Cytotoxicity of abalone tissue extracts was determined using an AlamarBlue viability assay. Results showed that HaCaT cells could survive when incubated in crude HBG and gill extracts at concentrations between <11.8 and <16.9 µg/ml, respectively. Subsequently, cell viability was compared between cultured HaCaT cells exposed to serial doses of UV-B from 1 to 11 (x10) mJ/cm2 and containing 4 different concentrations of abalone extract from both the HBG and gill (0, 0.1, 2.5, 5 µg/ml). A significant increase in cell viability was observed (P<0.001) following treatment with 2.5 and 5 µg/ml extract. Without extract, cell viability was significantly reduced upon exposure to UV-B at 4 mJ/cm2. Three morphological changes were observed in HaCaT cells following UV-B exposure, including i) condensation of cytoplasm; ii) shrunken cells and plasma membrane bubbling; and iii) condensation of chromatin material. A calcein AM‑propidium iodide live‑dead assay showed that cells could survive cytoplasmic condensation, yet cell death occurred when damage also included membrane bubbling and chromatin changes. Western blot analysis of HaCaT cell COX‑2, p38, phospho‑p38, SPK/JNK and phospho‑SPK/JNK following exposure to >2.5 µg/ml extract showed a significant decrease in intensity for COX‑2, phospho‑p38 and phospho‑SPK/JNK. The present study demonstrated that abalone extracts from the HGB and gill can attenuate inflammatory proteins triggered by UV-B. Hence, the contents of abalone extract, including cellmetabolites and peptides, may provide new agents for skin anti‑inflammation, preventing damage due to UV-B.

Figure 1

Analysis of the effective dose of abalone HBG and gill extracts on cell viability. Cytotoxicity test from abalone (A) HBG extract and (B) gill extract, using an AlamarBlue cell viability assay. Red arrows show the maximal concentrations of extract which allows for cell survival of >50%. (C and D) Live-dead staining (calcein AM-PI) of HaCaT cells treated with 12.5 μg/ml HBG and gill extract, respectively. HBG, hypobranchial gland; PI, propidium iodide.

Figure 2

Analysis of effective doses for abalone HBG and gill extract under UVB and HaCaT cell viability. (A) Graph comparing HaCaT cell viability among the four groups. Significant difference in cell viability when compared to the control group is shown at ^****P<0.001, ^**P<0.01 and ^*P<0.05. (B-I) Representative images of HaCaT cells treated with UV-B with and without extract, showing live-dead staining as indicated by green and red, respectively. HBG, hypobranchial gland.

Figure 3

Protective effect of abalone extracts following UV-B exposure, based on cell survival and morphology. Bright field (left) and fluorescence (right) images showing HaCaT cells: (A and B) after exposure to 6×10 mJ/cm² UV-B; (C and D) pre-treated with 2.5 μg/ml abalone extract and exposed to 6×10 mJ/cm² UV-B; (E and F) after exposure to 8×10 mJ/cm² UV-B; (G and H) pre-treated with 2.5 μg abalone extracts and exposed to 8×10 mJ/cm² UV-B. (I and J) Controls with no UV-B exposure or to extract. Red staining represents dead cells and green staining represents live cells. Three morphological cell types are observed, shown as (A, E and G) membrane bubbling, (A and E) condensation of cytoplasm and (A, E and G) chromatin.

Figure 4

Western blot analysis of inflammatory-related proteins present in HaCaT cells after UV-B exposure and with abalone extract. (A) Representative western blots, and (B) graphs showing relative protein levels for each protein. Densities of bands (quantified using densitometry UN-SCAN-IT software) were compared to the control group to identify relative change. −, no significant difference; +, significant difference at P<0.05; ++, significant difference at P<0.01; +++, significant difference at P<0.001; and ++++, significant difference at P<0.0001.