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The flavonoid, fisetin, inhibits UV radiation-induced oxidative stress and the activation of NF-kappaB and MAPK signaling in human lens epithelial cells.

Yao K, Zhang L, Zhang Y, Ye P, Zhu N - Mol. Vis. (2008)

Bottom Line: The effect of fisetin on the generation of reactive oxygen species (ROS) of SRA01/04 cells was determined by flow cytometry.Treatment of SRA01/04 cells with fisetin inhibited UVB-induced cell death and the generation of ROS.Fisetin inhibited UVB-induced activation and translocation of NF-kappaB/p65, which was mediated through an inhibition of the degradation and activation of IkappaB.

View Article: PubMed Central - PubMed

Affiliation: Eye Center, Affiliated Second Hospital, College of Medicine, Zhejiang University, Hangzhou, China. xlren@zju.edu.cn

ABSTRACT

Purpose: Ultraviolet (UV) radiation-induced oxidative stress plays a significant role in the progression of cataracts. This study investigated the photoprotective effect of fisetin on UV radiation-induced oxidative stress in human lens epithelial cells and the possible molecular mechanism involved.

Methods: SRA01/04 cells exposed to different doses of ultraviolet B (UVB) were cultured with various concentrations of fisetin and subsequently monitored for cell viability by the 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of fisetin on the generation of reactive oxygen species (ROS) of SRA01/04 cells was determined by flow cytometry. Translocation of nuclear factor kappa-B (NF-kappaB) was examined by immunocytochemistry. Expression of NF-kappaB/P65, inhibiter kappa B (IkappaB), and mitogen activated protein kinase (MAPK) proteins were measured by western blot.

Results: Treatment of SRA01/04 cells with fisetin inhibited UVB-induced cell death and the generation of ROS. Fisetin inhibited UVB-induced activation and translocation of NF-kappaB/p65, which was mediated through an inhibition of the degradation and activation of IkappaB. Fisetin also inhibited UVB-induced phosphorylation of the p38 and c-Jun N-terminal kinase (JNK) proteins of the MAPK family at various time points studied.

Conclusions: The flavonoid, fisetin, could be useful in attenuation of UV radiation-induced oxidative stress and the activation of NF-kappaB and MAPK signaling in human lens epithelial cells, which suggests that fisetin has a potential protective effect against cataractogenesis.

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Effect of fisetin on UVB-induced activation of NF-кB and degradation of IкB in HLE cells. A: SRA01/04 cells were exposed to UVB (30 mJ/cm2) with or without pretreatment with fisetin (25 μg/ml) for 1 h. Cells were harvested at 3 h and 6 h time points after UVB exposure, and cell lysates were prepared to determine the activation of NF-кB or degradation of IкB using western blot analysis. The graph represents the quantification results normalized to β-actin levels. Data represent the mean±SD of three individual experiments. The asterisk indicates p<0.05. B: Immunocytochemical analysis of NF-кB p65 localization is visualized. Cultured cells were incubated with anti-p65 antibody overnight at 4 °C as described in Methods. p65 is stained red, and the nuclei are stained blue. Representative fluorescent images were taken under fluorescence microscopy. Magnification, 100X.
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f3: Effect of fisetin on UVB-induced activation of NF-кB and degradation of IкB in HLE cells. A: SRA01/04 cells were exposed to UVB (30 mJ/cm2) with or without pretreatment with fisetin (25 μg/ml) for 1 h. Cells were harvested at 3 h and 6 h time points after UVB exposure, and cell lysates were prepared to determine the activation of NF-кB or degradation of IкB using western blot analysis. The graph represents the quantification results normalized to β-actin levels. Data represent the mean±SD of three individual experiments. The asterisk indicates p<0.05. B: Immunocytochemical analysis of NF-кB p65 localization is visualized. Cultured cells were incubated with anti-p65 antibody overnight at 4 °C as described in Methods. p65 is stained red, and the nuclei are stained blue. Representative fluorescent images were taken under fluorescence microscopy. Magnification, 100X.

Mentions: Activation of NF-кB was based on the detection of its translocation into cell nuclei from its initial location in the cytoplasm where it exists in an inactive form. Cells exposed to UVB exhibited an enhancement of nuclear NF-кB/p65 and a reduction of cytosolic NF-кB /p65 3 h after irradiation, and this became more evident 6 h after irradiation (Figure 3A). Western blot indicated that treatment with fisetin before UVB irradiation markedly abrogated UVB-induced activation of NF-кB/p65 in a time-dependent manner. Similarly, immunocytochemical studies showed NF-кB/p65 primarily resided in the cytoplasm and translocated into nuclei 3 h after exposure. In contrast, NF-кB/p65 mostly remained in the cytoplasm with scant translocation found in the cells treated with fisetin (Figure 3B). We determined whether UVB-induced degradation of IкB is inhibited by fisetin treatment, which inhibits the activation and translocation of NF-кB. Western blot indicated that exposure to UVB radiation resulted in a degradation of the IкB protein at each time point studied compared to non-UVB-exposed cells (Figure 3B). The UVB-induced degradation of IкB was inhibited after 3 h and almost completely inhibited 6 h after exposure in the cells pretreated with fisetin (Figure 3A).


The flavonoid, fisetin, inhibits UV radiation-induced oxidative stress and the activation of NF-kappaB and MAPK signaling in human lens epithelial cells.

Yao K, Zhang L, Zhang Y, Ye P, Zhu N - Mol. Vis. (2008)

Effect of fisetin on UVB-induced activation of NF-кB and degradation of IкB in HLE cells. A: SRA01/04 cells were exposed to UVB (30 mJ/cm2) with or without pretreatment with fisetin (25 μg/ml) for 1 h. Cells were harvested at 3 h and 6 h time points after UVB exposure, and cell lysates were prepared to determine the activation of NF-кB or degradation of IкB using western blot analysis. The graph represents the quantification results normalized to β-actin levels. Data represent the mean±SD of three individual experiments. The asterisk indicates p<0.05. B: Immunocytochemical analysis of NF-кB p65 localization is visualized. Cultured cells were incubated with anti-p65 antibody overnight at 4 °C as described in Methods. p65 is stained red, and the nuclei are stained blue. Representative fluorescent images were taken under fluorescence microscopy. Magnification, 100X.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2571947&req=5

f3: Effect of fisetin on UVB-induced activation of NF-кB and degradation of IкB in HLE cells. A: SRA01/04 cells were exposed to UVB (30 mJ/cm2) with or without pretreatment with fisetin (25 μg/ml) for 1 h. Cells were harvested at 3 h and 6 h time points after UVB exposure, and cell lysates were prepared to determine the activation of NF-кB or degradation of IкB using western blot analysis. The graph represents the quantification results normalized to β-actin levels. Data represent the mean±SD of three individual experiments. The asterisk indicates p<0.05. B: Immunocytochemical analysis of NF-кB p65 localization is visualized. Cultured cells were incubated with anti-p65 antibody overnight at 4 °C as described in Methods. p65 is stained red, and the nuclei are stained blue. Representative fluorescent images were taken under fluorescence microscopy. Magnification, 100X.
Mentions: Activation of NF-кB was based on the detection of its translocation into cell nuclei from its initial location in the cytoplasm where it exists in an inactive form. Cells exposed to UVB exhibited an enhancement of nuclear NF-кB/p65 and a reduction of cytosolic NF-кB /p65 3 h after irradiation, and this became more evident 6 h after irradiation (Figure 3A). Western blot indicated that treatment with fisetin before UVB irradiation markedly abrogated UVB-induced activation of NF-кB/p65 in a time-dependent manner. Similarly, immunocytochemical studies showed NF-кB/p65 primarily resided in the cytoplasm and translocated into nuclei 3 h after exposure. In contrast, NF-кB/p65 mostly remained in the cytoplasm with scant translocation found in the cells treated with fisetin (Figure 3B). We determined whether UVB-induced degradation of IкB is inhibited by fisetin treatment, which inhibits the activation and translocation of NF-кB. Western blot indicated that exposure to UVB radiation resulted in a degradation of the IкB protein at each time point studied compared to non-UVB-exposed cells (Figure 3B). The UVB-induced degradation of IкB was inhibited after 3 h and almost completely inhibited 6 h after exposure in the cells pretreated with fisetin (Figure 3A).

Bottom Line: The effect of fisetin on the generation of reactive oxygen species (ROS) of SRA01/04 cells was determined by flow cytometry.Treatment of SRA01/04 cells with fisetin inhibited UVB-induced cell death and the generation of ROS.Fisetin inhibited UVB-induced activation and translocation of NF-kappaB/p65, which was mediated through an inhibition of the degradation and activation of IkappaB.

View Article: PubMed Central - PubMed

Affiliation: Eye Center, Affiliated Second Hospital, College of Medicine, Zhejiang University, Hangzhou, China. xlren@zju.edu.cn

ABSTRACT

Purpose: Ultraviolet (UV) radiation-induced oxidative stress plays a significant role in the progression of cataracts. This study investigated the photoprotective effect of fisetin on UV radiation-induced oxidative stress in human lens epithelial cells and the possible molecular mechanism involved.

Methods: SRA01/04 cells exposed to different doses of ultraviolet B (UVB) were cultured with various concentrations of fisetin and subsequently monitored for cell viability by the 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of fisetin on the generation of reactive oxygen species (ROS) of SRA01/04 cells was determined by flow cytometry. Translocation of nuclear factor kappa-B (NF-kappaB) was examined by immunocytochemistry. Expression of NF-kappaB/P65, inhibiter kappa B (IkappaB), and mitogen activated protein kinase (MAPK) proteins were measured by western blot.

Results: Treatment of SRA01/04 cells with fisetin inhibited UVB-induced cell death and the generation of ROS. Fisetin inhibited UVB-induced activation and translocation of NF-kappaB/p65, which was mediated through an inhibition of the degradation and activation of IkappaB. Fisetin also inhibited UVB-induced phosphorylation of the p38 and c-Jun N-terminal kinase (JNK) proteins of the MAPK family at various time points studied.

Conclusions: The flavonoid, fisetin, could be useful in attenuation of UV radiation-induced oxidative stress and the activation of NF-kappaB and MAPK signaling in human lens epithelial cells, which suggests that fisetin has a potential protective effect against cataractogenesis.

Show MeSH
Related in: MedlinePlus