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. 2023 Mar 24;24(7):6120.
doi: 10.3390/ijms24076120.

Photoprotective Effects of Dendrobium nobile Lindl. Polysaccharides against UVB-Induced Oxidative Stress and Apoptosis in HaCaT Cells

Yunluan Long  1   2   3 , Wuji Wang  1   2   3 , Yanyan Zhang  1   2   3 , Fanpan Du  1   2   3 , Shiqian Zhang  1   2   3 , Zheng Li  1   2   3 , Jiang Deng  1   2   3 , Jingjie Li  1   2   3
Affiliations

Photoprotective Effects of Dendrobium nobile Lindl. Polysaccharides against UVB-Induced Oxidative Stress and Apoptosis in HaCaT Cells

Yunluan Long et al. Int J Mol Sci. .

Abstract

Acute ultraviolet (UV)-B radiation is the major external factor causing photodamage. In this study, we aimed to determine the effects of Dendrobium nobile Lindl. polysaccharides (DNPs) on photodamage in HaCaT keratinocytes after UVB irradiation and the underlying mechanisms. We found that DNPs significantly attenuated the decline in the viability and proliferation of HaCaT cells after UVB irradiation. Moreover, DNPs scavenged reactive oxygen species (ROS), improved the activities of endogenous antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase, and reduced the levels of malondialdehyde, while partially attenuating cell cycle arrest, suggesting their antioxidant and anti-apoptotic properties. The mitogen-activated protein kinase (MAPK) pathway was found to be important for the attenuation of UVB-induced photodamage in the HaCaT cells. Furthermore, DNPs exerted cytoprotective effects by downregulating UVB-induced ROS-mediated phosphorylation of MAPKs, including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase, and by inhibiting p53 expression as well as the apoptotic cascade response. Therefore, DNPs ameliorated UVB-induced oxidative damage and apoptosis in HaCaT cells via the regulation of MAPKs. Our findings thus highlight the Dendrobium nobile Lindl polysaccharides as promising therapeutic candidates for UVB-induced photodamage.

Keywords: Dendrobium nobile Lindl polysaccharides; HaCaT keratinocytes; MAPK; anti-apoptosis; antioxidant; ultraviolet B (UVB).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Protective effects of Dendrobium nobile Lindl. polysaccharides (DNPs) on the viability of ultraviolet (UV)-B-irradiated HaCaT cells. (A) Effects of various UVB radiation doses on the viability in HaCaT cells for 24 h. (B) Cytotoxicity of DNPs on HaCaT cells. (C) Effects of DNPs on UVB-irradiated HaCaT cells. # p < 0.05 and *** p < 0.001 vs. control group; ** p < 0.01 vs. UVB group.
Figure 2
Figure 2
DNPs inhibit UVB-induced intracellular ROS generation. (A) Images of DCFH-DA fluorescence observed via microscopy. Scale bar = 100 μm. (B) Quantification of fluorescence intensity. ### p < 0.001 vs. control group; *** p < 0.001 vs. UVB group.
Figure 3
Figure 3
DNPs alleviate UVB-induced oxidative damage in HaCaT cells. Activities of (A) SOD, (B) CAT, and (C) GSH-Px. (D) Malondialdehyde (MDA) levels. # p < 0.05, and ### p < 0.001 vs. control group; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. UVB group.
Figure 4
Figure 4
DNP pre-treatment rescues UVB-induced cell cycle arrest. (A) Cell cycle analysis of HaCaT cells subjected to different treatments using flow cytometry. (B) Statistical analysis of cells in different phases of the cell cycle.
Figure 5
Figure 5
DNPs exert protective effects by regulating MAPK and p53 pathways. (A) Protein expression levels of MAPKs and p53. Statistical analysis of the phosphorylation levels of p38 (B), JNK (C), ERK (D), and p53 (E). # p < 0.05 vs. control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. UVB group.
Figure 6
Figure 6
DNPs promote HaCaT cell proliferation. (A) Representative images of EdU staining. Proliferating cells are stained red, and cell nuclei are stained blue. Scale bar = 200 μm. (B) Statistical analysis of EdU-positive cells. ### p < 0.001 vs. control group; *** p < 0.001 vs. UVB group.
Figure 7
Figure 7
DNPs inhibit UVB-induced apoptosis of cells. (A) Representative images of Hoechst 33258 staining. The blue region indicates cell nuclei, and white arrows indicate the apoptotic cells with distinct features, such as intense fluorescence and presence of apoptotic bodies. Scale bar = 50 μm. (B) Quantification of apoptosis. ### p < 0.001 vs. control group; * p < 0.05 vs. UVB group.
Figure 8
Figure 8
DNPs inhibit UVB-induced apoptosis-related proteins expression in HaCaT cells. (A) Representative fluorescence images showing cleaved caspase-9 staining and (B) cleaved caspase-3 staining. Scale bar = 100 μm (A)/20 μm (B); Cleaved caspase-9 and cleaved caspase-3 positive cells were counted (C,D). ### p < 0.001 vs. control; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. UVB.
Figure 8
Figure 8
DNPs inhibit UVB-induced apoptosis-related proteins expression in HaCaT cells. (A) Representative fluorescence images showing cleaved caspase-9 staining and (B) cleaved caspase-3 staining. Scale bar = 100 μm (A)/20 μm (B); Cleaved caspase-9 and cleaved caspase-3 positive cells were counted (C,D). ### p < 0.001 vs. control; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. UVB.
Figure 9
Figure 9
Potential mechanism of DNP in ameliorating the UVB-induced photodamage of HaCaT cells.
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