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Fisetin and luteolin protect human retinal pigment epithelial cells from oxidative stress-induced cell death and regulate inflammation.

Hytti M, Piippo N, Korhonen E, Honkakoski P, Kaarniranta K, Kauppinen A - Sci Rep (2015)

Bottom Line: Here, we assess the ability of fisetin and luteolin, to protect ARPE-19 cells from oxidative stress-induced cell death and to decrease intracellular inflammation.The decrease in inflammation was associated with reduced activation of MAPKs and CREB, but was not linked to NF- κB or SIRT1.The ability of fisetin and luteolin to protect and repair stressed RPE cells even after the oxidative insult make them attractive in the search for treatments for AMD.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O.B. 1627, FI-70211, Kuopio, Finland.

ABSTRACT
Degeneration of retinal pigment epithelial (RPE) cells is a clinical hallmark of age-related macular degeneration (AMD), the leading cause of blindness among aged people in the Western world. Both inflammation and oxidative stress are known to play vital roles in the development of this disease. Here, we assess the ability of fisetin and luteolin, to protect ARPE-19 cells from oxidative stress-induced cell death and to decrease intracellular inflammation. We also compare the growth and reactivity of human ARPE-19 cells in serum-free and serum-containing conditions. The absence of serum in the culture medium did not prevent ARPE-19 cells from reaching full confluency but caused an increased sensitivity to oxidative stress-induced cell death. Both fisetin and luteolin protected ARPE-19 cells from oxidative stress-induced cell death. They also significantly decreased the release of pro-inflammatory cytokines into the culture medium. The decrease in inflammation was associated with reduced activation of MAPKs and CREB, but was not linked to NF- κB or SIRT1. The ability of fisetin and luteolin to protect and repair stressed RPE cells even after the oxidative insult make them attractive in the search for treatments for AMD.

No MeSH data available.


Related in: MedlinePlus

Effects of fisetin and luteolin on SIRT1 levels in HNE-treated serum-starved ARPE-19 cells and efficacy of SIRT1 siRNA in ARPE-19 cells.Both fisetin and luteolin decreased intracellular SIRT1 levels when compared to untreated controls or cells treated with HNE and the solvent DMSO alone (a). Results were obtained using SIRT1 specific ELISA. Also SIRT1 siRNA effectively decreased protein levels in ARPE-19 as measured by both Western Blot (b,c) or ELISA methods (d). Bands are from one representative Western blot (whole blots in Suppl. Fig. 5) and quantitative analysis represents a combination from 3 independent experiments with 2 parallel determinations per group/experiment. Data in graphs is shown as scatterplots with median. *denotes p < 0.05, **denotes p < 0.01, ns denotes not statistically significant, Mann–Whitney U-test.
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f7: Effects of fisetin and luteolin on SIRT1 levels in HNE-treated serum-starved ARPE-19 cells and efficacy of SIRT1 siRNA in ARPE-19 cells.Both fisetin and luteolin decreased intracellular SIRT1 levels when compared to untreated controls or cells treated with HNE and the solvent DMSO alone (a). Results were obtained using SIRT1 specific ELISA. Also SIRT1 siRNA effectively decreased protein levels in ARPE-19 as measured by both Western Blot (b,c) or ELISA methods (d). Bands are from one representative Western blot (whole blots in Suppl. Fig. 5) and quantitative analysis represents a combination from 3 independent experiments with 2 parallel determinations per group/experiment. Data in graphs is shown as scatterplots with median. *denotes p < 0.05, **denotes p < 0.01, ns denotes not statistically significant, Mann–Whitney U-test.

Mentions: Fisetin and luteolin have been associated with the activation of SIRT1, the human analogue of the yeast longevity gene Sir2 that, in addition to being involved in longevity, has also putative effects on the oxidative stress response and inflammatory signaling4243. To establish whether SIRT1 would be involved in the anti-inflammatory effects of fisetin and luteolin, we measured intracellular SIRT1 levels from cell lysates. Surprisingly, we detected a decline in SIRT1 levels in HNE-treated ARPE-19 cells 24 h after co-treatment with either fisetin or luteolin (Fig. 7a). Treatment with HNE and the solvent DMSO alone did not significantly decrease SIRT1 levels (p = 0.3939). These results suggest that down-regulation of SIRT1 might be involved in the anti-inflammatory effects of fisetin and luteolin in HNE-treated ARPE-19 cells, despite descriptions that SIRT1 is an anti-inflammatory protein in other cell lines. However, since the activation of SIRT1 is facilitated by allosteric activators, SIRT1 protein levels do not necessarily correlate with its activity4445. We therefore determined whether the downregulation we observed was linked to the anti-inflammatory effects of fisetin and luteolin by transfecting ARPE-19 cells either with specific SIRT1 siRNA or a noncoding control siRNA. Western blotting and ELISA measurements confirmed a significant decrease in the SIRT1 protein levels in ARPE-19 cells 48 h after the transfection (Fig. 7b–d, p = 0.0022 in both ELISA and western blot analysis). Subsequently, siRNA-transfected cells were treated with HNE 48 h post-transfection and then, 1 h later exposed to either fisetin or luteolin, or their solvent DMSO. After 24 h of the polyphenol exposure, the levels of secreted IL-6 and IL-8 were analyzed and no differences were found between the control and SIRT1 siRNA-transfected groups (Fig. 8). This suggests that SIRT1 downregulation is not involved in mediating the anti-inflammatory activities exerted by fisetin and luteolin in HNE-treated ARPE-19 cells.


Fisetin and luteolin protect human retinal pigment epithelial cells from oxidative stress-induced cell death and regulate inflammation.

Hytti M, Piippo N, Korhonen E, Honkakoski P, Kaarniranta K, Kauppinen A - Sci Rep (2015)

Effects of fisetin and luteolin on SIRT1 levels in HNE-treated serum-starved ARPE-19 cells and efficacy of SIRT1 siRNA in ARPE-19 cells.Both fisetin and luteolin decreased intracellular SIRT1 levels when compared to untreated controls or cells treated with HNE and the solvent DMSO alone (a). Results were obtained using SIRT1 specific ELISA. Also SIRT1 siRNA effectively decreased protein levels in ARPE-19 as measured by both Western Blot (b,c) or ELISA methods (d). Bands are from one representative Western blot (whole blots in Suppl. Fig. 5) and quantitative analysis represents a combination from 3 independent experiments with 2 parallel determinations per group/experiment. Data in graphs is shown as scatterplots with median. *denotes p < 0.05, **denotes p < 0.01, ns denotes not statistically significant, Mann–Whitney U-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4664957&req=5

f7: Effects of fisetin and luteolin on SIRT1 levels in HNE-treated serum-starved ARPE-19 cells and efficacy of SIRT1 siRNA in ARPE-19 cells.Both fisetin and luteolin decreased intracellular SIRT1 levels when compared to untreated controls or cells treated with HNE and the solvent DMSO alone (a). Results were obtained using SIRT1 specific ELISA. Also SIRT1 siRNA effectively decreased protein levels in ARPE-19 as measured by both Western Blot (b,c) or ELISA methods (d). Bands are from one representative Western blot (whole blots in Suppl. Fig. 5) and quantitative analysis represents a combination from 3 independent experiments with 2 parallel determinations per group/experiment. Data in graphs is shown as scatterplots with median. *denotes p < 0.05, **denotes p < 0.01, ns denotes not statistically significant, Mann–Whitney U-test.
Mentions: Fisetin and luteolin have been associated with the activation of SIRT1, the human analogue of the yeast longevity gene Sir2 that, in addition to being involved in longevity, has also putative effects on the oxidative stress response and inflammatory signaling4243. To establish whether SIRT1 would be involved in the anti-inflammatory effects of fisetin and luteolin, we measured intracellular SIRT1 levels from cell lysates. Surprisingly, we detected a decline in SIRT1 levels in HNE-treated ARPE-19 cells 24 h after co-treatment with either fisetin or luteolin (Fig. 7a). Treatment with HNE and the solvent DMSO alone did not significantly decrease SIRT1 levels (p = 0.3939). These results suggest that down-regulation of SIRT1 might be involved in the anti-inflammatory effects of fisetin and luteolin in HNE-treated ARPE-19 cells, despite descriptions that SIRT1 is an anti-inflammatory protein in other cell lines. However, since the activation of SIRT1 is facilitated by allosteric activators, SIRT1 protein levels do not necessarily correlate with its activity4445. We therefore determined whether the downregulation we observed was linked to the anti-inflammatory effects of fisetin and luteolin by transfecting ARPE-19 cells either with specific SIRT1 siRNA or a noncoding control siRNA. Western blotting and ELISA measurements confirmed a significant decrease in the SIRT1 protein levels in ARPE-19 cells 48 h after the transfection (Fig. 7b–d, p = 0.0022 in both ELISA and western blot analysis). Subsequently, siRNA-transfected cells were treated with HNE 48 h post-transfection and then, 1 h later exposed to either fisetin or luteolin, or their solvent DMSO. After 24 h of the polyphenol exposure, the levels of secreted IL-6 and IL-8 were analyzed and no differences were found between the control and SIRT1 siRNA-transfected groups (Fig. 8). This suggests that SIRT1 downregulation is not involved in mediating the anti-inflammatory activities exerted by fisetin and luteolin in HNE-treated ARPE-19 cells.

Bottom Line: Here, we assess the ability of fisetin and luteolin, to protect ARPE-19 cells from oxidative stress-induced cell death and to decrease intracellular inflammation.The decrease in inflammation was associated with reduced activation of MAPKs and CREB, but was not linked to NF- κB or SIRT1.The ability of fisetin and luteolin to protect and repair stressed RPE cells even after the oxidative insult make them attractive in the search for treatments for AMD.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O.B. 1627, FI-70211, Kuopio, Finland.

ABSTRACT
Degeneration of retinal pigment epithelial (RPE) cells is a clinical hallmark of age-related macular degeneration (AMD), the leading cause of blindness among aged people in the Western world. Both inflammation and oxidative stress are known to play vital roles in the development of this disease. Here, we assess the ability of fisetin and luteolin, to protect ARPE-19 cells from oxidative stress-induced cell death and to decrease intracellular inflammation. We also compare the growth and reactivity of human ARPE-19 cells in serum-free and serum-containing conditions. The absence of serum in the culture medium did not prevent ARPE-19 cells from reaching full confluency but caused an increased sensitivity to oxidative stress-induced cell death. Both fisetin and luteolin protected ARPE-19 cells from oxidative stress-induced cell death. They also significantly decreased the release of pro-inflammatory cytokines into the culture medium. The decrease in inflammation was associated with reduced activation of MAPKs and CREB, but was not linked to NF- κB or SIRT1. The ability of fisetin and luteolin to protect and repair stressed RPE cells even after the oxidative insult make them attractive in the search for treatments for AMD.

No MeSH data available.


Related in: MedlinePlus