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Luteolin and Apigenin Attenuate 4-Hydroxy-2-Nonenal-Mediated Cell Death through Modulation of UPR, Nrf2-ARE and MAPK Pathways in PC12 Cells.

Wu PS, Yen JH, Kou MC, Wu MJ - PLoS ONE (2015)

Bottom Line: Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin.In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity.Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan, 717, Taiwan.

ABSTRACT
Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.

No MeSH data available.


Related in: MedlinePlus

Luteolin and apigenin enhance 4-HNE-mediated unfolded protein response.(A) Western blot analysis of ATF4 and eIF2α phosphorylation in lysates obtained from PC12 cells incubated in the presence of vehicle only (V), or presence of 25 μM HNE plus vehicle (Veh), or co-treatment with 20 μM luteolin or apigenin for 2 h. (B) Effects of flavones on XBP1 splicing. PC12 cells were treated with luteolin and apigenin (20 μM) 30 min prior to 4-HNE (25 μM) treatment for 4 h at 37°. RNA was prepared and semi-quantitative RT-PCR was used for the analysis of mRNA levels of XBP1s and XBP1u, as described in Materials and Methods. (C) Western blot analysis of GRP78 in cell lysates prepared from PC12 cells, which were pretreated with luteolin or apigenin (20 μM) for 30 min followed by 4-HNE for 6 h. Immunoblots and polyacrylamide gels were then analyzed using Phoretix Gel Analysis Software as described under Materials and methods. Data represent the mean ± SD of three independent experiments. **, p<0.01 represents significant differences compared with vehicle control (without oxidant). ##, p<0.01 represents significant differences compared with the 4-HNE-treated vehicle group.
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pone.0130599.g003: Luteolin and apigenin enhance 4-HNE-mediated unfolded protein response.(A) Western blot analysis of ATF4 and eIF2α phosphorylation in lysates obtained from PC12 cells incubated in the presence of vehicle only (V), or presence of 25 μM HNE plus vehicle (Veh), or co-treatment with 20 μM luteolin or apigenin for 2 h. (B) Effects of flavones on XBP1 splicing. PC12 cells were treated with luteolin and apigenin (20 μM) 30 min prior to 4-HNE (25 μM) treatment for 4 h at 37°. RNA was prepared and semi-quantitative RT-PCR was used for the analysis of mRNA levels of XBP1s and XBP1u, as described in Materials and Methods. (C) Western blot analysis of GRP78 in cell lysates prepared from PC12 cells, which were pretreated with luteolin or apigenin (20 μM) for 30 min followed by 4-HNE for 6 h. Immunoblots and polyacrylamide gels were then analyzed using Phoretix Gel Analysis Software as described under Materials and methods. Data represent the mean ± SD of three independent experiments. **, p<0.01 represents significant differences compared with vehicle control (without oxidant). ##, p<0.01 represents significant differences compared with the 4-HNE-treated vehicle group.

Mentions: We reported previously that 4-HNE triggers ER stress, activates three canonical arms of the unfolded protein responses, and induces pro-apoptotic CHOP expression in PC12 cells [12]. To investigate how luteolin and apigenin affect 4-HNE-mediated UPR in PC12 cells, we started with analyzing PERK-eIF2α pathway, which is thought to be the most important stress response pathway leading to attenuation of global protein translation. Some selected proteins with internal ribosomal entry sites (IRES), such as ATF4 and GRP78/BiP, are translated more efficiently [48]. Fig 3A shows that PC12 cells treated with 25 μM 4-HNE for 2 h led to increases in eIF2α phosphorylation and ATF4 protein expression. Co-treatment of PC12 cells with 4-HNE (25 μM) and luteolin or apigenin (20 μM) did not change 4-HNE-mediated eIF2α phosphorylation, but significantly enhanced ATF4 protein expression.


Luteolin and Apigenin Attenuate 4-Hydroxy-2-Nonenal-Mediated Cell Death through Modulation of UPR, Nrf2-ARE and MAPK Pathways in PC12 Cells.

Wu PS, Yen JH, Kou MC, Wu MJ - PLoS ONE (2015)

Luteolin and apigenin enhance 4-HNE-mediated unfolded protein response.(A) Western blot analysis of ATF4 and eIF2α phosphorylation in lysates obtained from PC12 cells incubated in the presence of vehicle only (V), or presence of 25 μM HNE plus vehicle (Veh), or co-treatment with 20 μM luteolin or apigenin for 2 h. (B) Effects of flavones on XBP1 splicing. PC12 cells were treated with luteolin and apigenin (20 μM) 30 min prior to 4-HNE (25 μM) treatment for 4 h at 37°. RNA was prepared and semi-quantitative RT-PCR was used for the analysis of mRNA levels of XBP1s and XBP1u, as described in Materials and Methods. (C) Western blot analysis of GRP78 in cell lysates prepared from PC12 cells, which were pretreated with luteolin or apigenin (20 μM) for 30 min followed by 4-HNE for 6 h. Immunoblots and polyacrylamide gels were then analyzed using Phoretix Gel Analysis Software as described under Materials and methods. Data represent the mean ± SD of three independent experiments. **, p<0.01 represents significant differences compared with vehicle control (without oxidant). ##, p<0.01 represents significant differences compared with the 4-HNE-treated vehicle group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472230&req=5

pone.0130599.g003: Luteolin and apigenin enhance 4-HNE-mediated unfolded protein response.(A) Western blot analysis of ATF4 and eIF2α phosphorylation in lysates obtained from PC12 cells incubated in the presence of vehicle only (V), or presence of 25 μM HNE plus vehicle (Veh), or co-treatment with 20 μM luteolin or apigenin for 2 h. (B) Effects of flavones on XBP1 splicing. PC12 cells were treated with luteolin and apigenin (20 μM) 30 min prior to 4-HNE (25 μM) treatment for 4 h at 37°. RNA was prepared and semi-quantitative RT-PCR was used for the analysis of mRNA levels of XBP1s and XBP1u, as described in Materials and Methods. (C) Western blot analysis of GRP78 in cell lysates prepared from PC12 cells, which were pretreated with luteolin or apigenin (20 μM) for 30 min followed by 4-HNE for 6 h. Immunoblots and polyacrylamide gels were then analyzed using Phoretix Gel Analysis Software as described under Materials and methods. Data represent the mean ± SD of three independent experiments. **, p<0.01 represents significant differences compared with vehicle control (without oxidant). ##, p<0.01 represents significant differences compared with the 4-HNE-treated vehicle group.
Mentions: We reported previously that 4-HNE triggers ER stress, activates three canonical arms of the unfolded protein responses, and induces pro-apoptotic CHOP expression in PC12 cells [12]. To investigate how luteolin and apigenin affect 4-HNE-mediated UPR in PC12 cells, we started with analyzing PERK-eIF2α pathway, which is thought to be the most important stress response pathway leading to attenuation of global protein translation. Some selected proteins with internal ribosomal entry sites (IRES), such as ATF4 and GRP78/BiP, are translated more efficiently [48]. Fig 3A shows that PC12 cells treated with 25 μM 4-HNE for 2 h led to increases in eIF2α phosphorylation and ATF4 protein expression. Co-treatment of PC12 cells with 4-HNE (25 μM) and luteolin or apigenin (20 μM) did not change 4-HNE-mediated eIF2α phosphorylation, but significantly enhanced ATF4 protein expression.

Bottom Line: Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin.In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity.Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan, 717, Taiwan.

ABSTRACT
Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.

No MeSH data available.


Related in: MedlinePlus