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Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes.

Xu J, Hu H, Chen B, Yue R, Zhou Z, Liu Y, Zhang S, Xu L, Wang H, Yu Z - PLoS ONE (2015)

Bottom Line: Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear.Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes.Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, North Sichuan Medical College Affiliated Hospital, Nanchong 637000, Sichuan, China; Department of Anesthesiology, North Sichuan Medical College Affiliated Hospital, Nanchong 637000, Sichuan, China.

ABSTRACT
Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.

No MeSH data available.


Related in: MedlinePlus

Effects of lycopene on ER stress inducer (thapsigargin, TG)-induced changes in neonatal cardiomyocytes.Cell viability was detected by CCK-8 assay. The total proteins of cardiomyocytes were extracted and the protein expression levels of Bip/GRP78 and GADD153 were detected by western blot. (A) TG evoked a dose-dependent decrease in the viability of neonatal cardiomyocytes. Cells were pretreated with TG (0.25, 0.5, 0.75 and 1.0μM) for 10h, respectively. (B) TG-mediated loss of cell viability increased with treatment duration. Cells were treatment with 0.5μM TG for 2, 4, 8, 10 and 20h, respectively. (C) Lycopene reversed TG-caused loss of cell viability. Cells were pretreated with or without 0.5μM TG for 10h after the lycopene pretreatment. (D) The relative proportion of TUNEL-positive cells is shown in each group. Representative western blot image, quantitative analysis normalized to the β-actin and control respectively showed the expression of GRP78 (E) and GADD153 (F). These assays were repeated three times. Values are mean ± SEM. *P<0.05, **P<0.01 versus control; ##P <0.01 versus H/R group. (Lyc, Lycopene; H/R, hypoxia/ reoxygenation).
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pone.0136443.g006: Effects of lycopene on ER stress inducer (thapsigargin, TG)-induced changes in neonatal cardiomyocytes.Cell viability was detected by CCK-8 assay. The total proteins of cardiomyocytes were extracted and the protein expression levels of Bip/GRP78 and GADD153 were detected by western blot. (A) TG evoked a dose-dependent decrease in the viability of neonatal cardiomyocytes. Cells were pretreated with TG (0.25, 0.5, 0.75 and 1.0μM) for 10h, respectively. (B) TG-mediated loss of cell viability increased with treatment duration. Cells were treatment with 0.5μM TG for 2, 4, 8, 10 and 20h, respectively. (C) Lycopene reversed TG-caused loss of cell viability. Cells were pretreated with or without 0.5μM TG for 10h after the lycopene pretreatment. (D) The relative proportion of TUNEL-positive cells is shown in each group. Representative western blot image, quantitative analysis normalized to the β-actin and control respectively showed the expression of GRP78 (E) and GADD153 (F). These assays were repeated three times. Values are mean ± SEM. *P<0.05, **P<0.01 versus control; ##P <0.01 versus H/R group. (Lyc, Lycopene; H/R, hypoxia/ reoxygenation).

Mentions: To further confirm whether lycopene could alleviate ER stress and ER stress-induced injury, we used thapsigargin (TG), a commonly used agent known to rapidly elicit ER stress in cardiomyocytes. Cardiomyocytes were exposed to a range of TG concentrations from 0.25 to l.0μM for 10h to find an optimal dose for subsequent assays (Fig 6A). TG evoked a dose-dependent decrease in the cell viability of cardiomyocytes, among that treatment with 0.5μM TG reduced cell viability to 65.67% of control, while l.0μM TG decreased cell viability to 45.71% of control. As illustrated in Fig 6B, 10 and 20h treatment with 0.5μM TG markedly reduced cell viability to 65.83% and 40.34% of control, respectively, whereas 2 and 4h treatment had no significant effects. According to these results, treatment with 0.5μM TG for 10h was chosen for the following experiments. As shown in Fig 6C, compared with controls treatment, 5μM lycopene pretreatment decreased the TG-induced loss of cell viability. Similarly, exposure of cardiomyocytes to TG caused an increase in the rate of apoptosis, whereas lycopene significantly reversed the changes (Fig 6C and 6D). Compared with controls, the protein expression of GRP78 and CHOP/GADD153 have been significantly elevated with 0.5μM TG pretreatment, conversely, lycopene markedly alleviated TG-induced increase in GRP78 and CHOP/GADD153 protein expression of cardiomyocytes (Fig 6E and 6F).


Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes.

Xu J, Hu H, Chen B, Yue R, Zhou Z, Liu Y, Zhang S, Xu L, Wang H, Yu Z - PLoS ONE (2015)

Effects of lycopene on ER stress inducer (thapsigargin, TG)-induced changes in neonatal cardiomyocytes.Cell viability was detected by CCK-8 assay. The total proteins of cardiomyocytes were extracted and the protein expression levels of Bip/GRP78 and GADD153 were detected by western blot. (A) TG evoked a dose-dependent decrease in the viability of neonatal cardiomyocytes. Cells were pretreated with TG (0.25, 0.5, 0.75 and 1.0μM) for 10h, respectively. (B) TG-mediated loss of cell viability increased with treatment duration. Cells were treatment with 0.5μM TG for 2, 4, 8, 10 and 20h, respectively. (C) Lycopene reversed TG-caused loss of cell viability. Cells were pretreated with or without 0.5μM TG for 10h after the lycopene pretreatment. (D) The relative proportion of TUNEL-positive cells is shown in each group. Representative western blot image, quantitative analysis normalized to the β-actin and control respectively showed the expression of GRP78 (E) and GADD153 (F). These assays were repeated three times. Values are mean ± SEM. *P<0.05, **P<0.01 versus control; ##P <0.01 versus H/R group. (Lyc, Lycopene; H/R, hypoxia/ reoxygenation).
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pone.0136443.g006: Effects of lycopene on ER stress inducer (thapsigargin, TG)-induced changes in neonatal cardiomyocytes.Cell viability was detected by CCK-8 assay. The total proteins of cardiomyocytes were extracted and the protein expression levels of Bip/GRP78 and GADD153 were detected by western blot. (A) TG evoked a dose-dependent decrease in the viability of neonatal cardiomyocytes. Cells were pretreated with TG (0.25, 0.5, 0.75 and 1.0μM) for 10h, respectively. (B) TG-mediated loss of cell viability increased with treatment duration. Cells were treatment with 0.5μM TG for 2, 4, 8, 10 and 20h, respectively. (C) Lycopene reversed TG-caused loss of cell viability. Cells were pretreated with or without 0.5μM TG for 10h after the lycopene pretreatment. (D) The relative proportion of TUNEL-positive cells is shown in each group. Representative western blot image, quantitative analysis normalized to the β-actin and control respectively showed the expression of GRP78 (E) and GADD153 (F). These assays were repeated three times. Values are mean ± SEM. *P<0.05, **P<0.01 versus control; ##P <0.01 versus H/R group. (Lyc, Lycopene; H/R, hypoxia/ reoxygenation).
Mentions: To further confirm whether lycopene could alleviate ER stress and ER stress-induced injury, we used thapsigargin (TG), a commonly used agent known to rapidly elicit ER stress in cardiomyocytes. Cardiomyocytes were exposed to a range of TG concentrations from 0.25 to l.0μM for 10h to find an optimal dose for subsequent assays (Fig 6A). TG evoked a dose-dependent decrease in the cell viability of cardiomyocytes, among that treatment with 0.5μM TG reduced cell viability to 65.67% of control, while l.0μM TG decreased cell viability to 45.71% of control. As illustrated in Fig 6B, 10 and 20h treatment with 0.5μM TG markedly reduced cell viability to 65.83% and 40.34% of control, respectively, whereas 2 and 4h treatment had no significant effects. According to these results, treatment with 0.5μM TG for 10h was chosen for the following experiments. As shown in Fig 6C, compared with controls treatment, 5μM lycopene pretreatment decreased the TG-induced loss of cell viability. Similarly, exposure of cardiomyocytes to TG caused an increase in the rate of apoptosis, whereas lycopene significantly reversed the changes (Fig 6C and 6D). Compared with controls, the protein expression of GRP78 and CHOP/GADD153 have been significantly elevated with 0.5μM TG pretreatment, conversely, lycopene markedly alleviated TG-induced increase in GRP78 and CHOP/GADD153 protein expression of cardiomyocytes (Fig 6E and 6F).

Bottom Line: Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear.Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes.Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, North Sichuan Medical College Affiliated Hospital, Nanchong 637000, Sichuan, China; Department of Anesthesiology, North Sichuan Medical College Affiliated Hospital, Nanchong 637000, Sichuan, China.

ABSTRACT
Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.

No MeSH data available.


Related in: MedlinePlus