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Propolis Inhibits UVA-Induced Apoptosis of Human Keratinocyte HaCaT Cells by Scavenging ROS

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Kim, Han Bit, Yoo, Byung Sun·2016·Toxicological Research
DOI 10.5487/tr.2016.32.4.345PMC PMC5080852
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Abstract

Propolis is a resinous material collected by honeybees from several plant sources. This research aimed at showing its protective effect against UVA-induced apoptosis of human keratinocyte HaCaT cells. Using Hoechst staining, it was demonstrated that propolis (5 and 10 μg/mL) significantly inhibited the apoptosis of HaCaT cells induced by UVA-irradiation. Propolis also showed the protective effect against loss of mitochondrial membrane potential induced by UVA-irradiaiton in HaCaT cells. Propolis also inhibited the expression of activated caspase-3 induced by UVA-irradiation. To investigate the role of ROS in UVA-induced apoptosis and protection by propolis, the generation of ROS was determined in cells. The results showed that the generation of ROS was markedly reduced in cells pretreated with propolis. Consequently, propolis protected human keratinocyte HaCaT cells against UVA-induced apoptosis, which might be related to the reduction of ROS generation by UVA-irradiation.

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Fig. 1 Propolis inhibited UVA-induced apoptosis in HaCaT cells. Cells were pretreated with or without propolis (5 and 10 μg/mL) for 1 hr and then irradiated with UVA (4 J/cm2). After incubation for 6 hr, cells were stained with Hoechst 33258. (A) Representative morphology visualized under a fluorescence microscope (BX70, Olympus, Tokyo, Japan). Cells with brightly fluorescent and fragmented nuclei were apoptotic. (B) Quantitative analysis of the percentage of apoptotic cells. The percentage of apoptotic cells = The numbers of apoptotic cells/(The numbers of apoptotic cells + The numbers of viable cells). *P < 0.05 when compared to control group.
Fig. 1 Propolis inhibited UVA-induced apoptosis in HaCaT cells. Cells were pretreated with or without propolis (5 and 10 μg/mL) for 1 hr and then irradiated with UVA (4 J/cm2). After incubation for 6 hr, cells were stained with Hoechst 33258. (A) Representative morphology visualized under a fluorescence microscope (BX70, Olympus, Tokyo, Japan). Cells with brightly fluorescent and fragmented nuclei were apoptotic. (B) Quantitative analysis of the percentage of apoptotic cells. The percentage of apoptotic cells = The numbers of apoptotic cells/(The numbers of apoptotic cells + The numbers of viable cells). *P < 0.05 when compared to control group.CC BY-NC · PubMed Central
Fig. 2 Propolis recovered the loss of UVA-induced mitochondrial membrane potential in HaCaT cells. HaCaT cells were pre-treated with or without propolis (5 and 10 μg/mL) for 1 hr and then irradiated with UVA (4 J/cm2) for 6 hr. Mitochondrial membrane potential was evaluated using Rhodamine 123 staining. The change of MMP were imaged on fluorescence microscope (×200).
Fig. 2 Propolis recovered the loss of UVA-induced mitochondrial membrane potential in HaCaT cells. HaCaT cells were pre-treated with or without propolis (5 and 10 μg/mL) for 1 hr and then irradiated with UVA (4 J/cm2) for 6 hr. Mitochondrial membrane potential was evaluated using Rhodamine 123 staining. The change of MMP were imaged on fluorescence microscope (×200).CC BY-NC · PubMed Central
Fig. 3 Effect of propolis on UVA-induced caspase-3 activation in HaCaT cells. HaCaT cells were pretreated with or without propolis (1~10 μg/mL) for 1 hr and then irradiated with UVA (4 J/cm2). Cells were harvested by centrifugation and protein was extracted. The extracts were analyzed for Western blot analysis of cleaved caspase-3 expression level. Densitometric analysis was performed to determine the intensity of cleaved caspase-3 bands. Values are normalized to β-actin band and expressed as the percentage of control group. Data are mean ± standard error of three independent experiments. *P < 0.05 when compared to control group.
Fig. 3 Effect of propolis on UVA-induced caspase-3 activation in HaCaT cells. HaCaT cells were pretreated with or without propolis (1~10 μg/mL) for 1 hr and then irradiated with UVA (4 J/cm2). Cells were harvested by centrifugation and protein was extracted. The extracts were analyzed for Western blot analysis of cleaved caspase-3 expression level. Densitometric analysis was performed to determine the intensity of cleaved caspase-3 bands. Values are normalized to β-actin band and expressed as the percentage of control group. Data are mean ± standard error of three independent experiments. *P < 0.05 when compared to control group.CC BY-NC · PubMed Central
Fig. 4 Effect of propolis on UVA-induced ROS generation in HaCaT cells. HaCaT cells were pretreated with or without propolis (5 and 10 μg/mL) for 1 hr and then irradiated with UVA (4 J/cm2). After incubation for 6 hr, cells were stained with DCFH-DA for 30 min and washed with PBS. The intracellular levels of ROS were imaged on fluorescence microscope.
Fig. 4 Effect of propolis on UVA-induced ROS generation in HaCaT cells. HaCaT cells were pretreated with or without propolis (5 and 10 μg/mL) for 1 hr and then irradiated with UVA (4 J/cm2). After incubation for 6 hr, cells were stained with DCFH-DA for 30 min and washed with PBS. The intracellular levels of ROS were imaged on fluorescence microscope.CC BY-NC · PubMed Central

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Metadata

Authors
Kim, H. B., & Yoo, B. S.
Affiliations
Kyonggi University (KR)
Corresponding author
Yoo, Byung Sun
Venue
Toxicological Research
Publisher
Springer Science and Business Media LLC
Published
2016-10-30
Volume
32
Issue
4
Pages
345-351
ISSN
  • 1976-8257 (ISSN-L)
  • 2234-2753
DOI
10.5487/tr.2016.32.4.345
PMC
PMC5080852
OpenAlex
W2531157978
Cited by
42
References
1
Citations by year
2026: 1 · 2025: 1 · 2024: 5 · 2023: 1 · 2022: 7 · 2021: 7 · 2020: 1 · 2019: 4 · 2018: 9 · 2017: 5
FWCI
6.51
Type
journal article
Language
en
Open access
Licence
Locations
  • Toxicological Research (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
Kyonggi University(KGU)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Insect Science › Bee Products Chemical Analysis
  • Health Sciences › Medicine › Dermatology › Skin Protection and Aging
  • Health Sciences › Medicine › Pharmacology (medical) › Healthcare and Venom Research
Keywords
propolis, HaCaT keratinocytes, reactive oxygen species, mitochondrial membrane potential, caspase-3, photoprotection, photodamaged skin

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