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Carvacrol protects neuroblastoma SH-SY5Y cells against Fe(2+)-induced apoptosis by suppressing activation of MAPK/JNK-NF-κB signaling pathway.

Cui ZW, Xie ZX, Wang BF, Zhong ZH, Chen XY, Sun YH, Sun QF, Yang GY, Bian LG - Acta Pharmacol. Sin. (2015)

Bottom Line: Treatment of SH-SY5Y cells with Fe(2+) (50-200 μmol/L) dose-dependently decreased the cell viability, which was significantly attenuated by pretreatment with carvacrol (164 and 333 μmol/L).Treatment with Fe(2+) increased the Bax level and caspase-3 activity, and decreased the Bcl-2 level, resulting in cell apoptosis.Pretreatment with carvacrol significantly inhibited Fe(2+)-induced activation of NF-κB, expression of the pro-inflammatory cytokines, and cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT

Aim: Carvacrol (2-methyl-5-isopropylphenol), a phenolic monoterpene in the essential oils of the genera Origanum and Thymus, has been shown to exert a variety of therapeutic effects. Here we examined whether carvacrol protected neuroblastoma SH-SY5Y cells against Fe(2+)-induced apoptosis and explored the underlying mechanisms.

Methods: Neuroblastoma SH-SY5Y cells were incubated with Fe(2+) for 24 h, and the cell viability was assessed with CCK-8 assay. TUNEL assay and flow cytometric analysis were performed to evaluate cell apoptosis. The mRNA levels of pro-inflammatory cytokines and NF-κB p65 were determined using qPCR. The expression of relevant proteins was determined using Western blot analysis or immunofluorescence staining.

Results: Treatment of SH-SY5Y cells with Fe(2+) (50-200 μmol/L) dose-dependently decreased the cell viability, which was significantly attenuated by pretreatment with carvacrol (164 and 333 μmol/L). Treatment with Fe(2+) increased the Bax level and caspase-3 activity, and decreased the Bcl-2 level, resulting in cell apoptosis. Furthermore, treatment with Fe(2+) significantly increased the gene expression of IL-1β, IL-6 and TNF-α, and induced the nuclear translocation of NF-κB. Treatment with Fe(2+) also significantly increased the phosphorylation of p38, ERK, JNK and IKK in the cells. Pretreatment with carvacrol significantly inhibited Fe(2+)-induced activation of NF-κB, expression of the pro-inflammatory cytokines, and cell apoptosis. Moreover, pretreatment with carvacrol inhibited Fe(2+)-induced phosphorylation of JNK and IKK, but not p38 and ERK in the cells.

Conclusion: Carvacrol protects neuroblastoma SH-SY5Y cells against Fe(2+)-induced apoptosis, which may result from suppressing the MAPK/JNK-NF-κB signaling pathways.

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Effect of Fe2+ on the expression of NF-κB in SH-SY5Y cells. (A) The mRNA expression of NF-κB p65 in SH-SY5Y cells exposed to different concentrations of Fe2+ for 24 h. (B) The mRNA expression of NF-κB p65 in SH-SY5Y cells exposed to 200 μmol/L Fe2+ at different time-points. (C) Fe2+ increased the protein expression of NF-κB p65 in SH-SY5Y cells in a concentration-dependent manner. (D) Fe2+ increased the protein expression of NF-κB p65 in SH-SY5Y cells in a time-dependent manner. The data are presented as the mean±SD (n=3). bP<0.05 and cP<0.01, relative to the untreated control.
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fig4: Effect of Fe2+ on the expression of NF-κB in SH-SY5Y cells. (A) The mRNA expression of NF-κB p65 in SH-SY5Y cells exposed to different concentrations of Fe2+ for 24 h. (B) The mRNA expression of NF-κB p65 in SH-SY5Y cells exposed to 200 μmol/L Fe2+ at different time-points. (C) Fe2+ increased the protein expression of NF-κB p65 in SH-SY5Y cells in a concentration-dependent manner. (D) Fe2+ increased the protein expression of NF-κB p65 in SH-SY5Y cells in a time-dependent manner. The data are presented as the mean±SD (n=3). bP<0.05 and cP<0.01, relative to the untreated control.

Mentions: Previous studies have shown that NF-κB activation is necessary for the induction of proinflammatory mediators13,35. Next, we wanted to investigate whether NF-κB was modulated by Fe2+. SH-SY5Y cells were treated with 10, 50, 100, 200 or 300 μmol/L of Fe2+ for 24 h and for 1, 3, 6, 12, or 24 h with 200 μmol/L Fe2+, and then qPCR and Western blot analyses were used to determine the expression of NF-κB mRNA and protein. Both the mRNA and protein levels of NF-κB increased in concentration- and time-dependent manners after Fe2+ exposure (Figure 4). NF-κB mRNA levels increased significantly following the addition of 50 μmol/L Fe2+ and after 3-h Fe2+ exposure compared to control cells (Figure 4A and 4B). Similar behavior was observed for NF-κB protein expression levels, which increased significantly following the exposure to 100 μmol/L Fe2+ and after 6 h of exposure to Fe2+ compared to control cells (Figure 4C and 4D).


Carvacrol protects neuroblastoma SH-SY5Y cells against Fe(2+)-induced apoptosis by suppressing activation of MAPK/JNK-NF-κB signaling pathway.

Cui ZW, Xie ZX, Wang BF, Zhong ZH, Chen XY, Sun YH, Sun QF, Yang GY, Bian LG - Acta Pharmacol. Sin. (2015)

Effect of Fe2+ on the expression of NF-κB in SH-SY5Y cells. (A) The mRNA expression of NF-κB p65 in SH-SY5Y cells exposed to different concentrations of Fe2+ for 24 h. (B) The mRNA expression of NF-κB p65 in SH-SY5Y cells exposed to 200 μmol/L Fe2+ at different time-points. (C) Fe2+ increased the protein expression of NF-κB p65 in SH-SY5Y cells in a concentration-dependent manner. (D) Fe2+ increased the protein expression of NF-κB p65 in SH-SY5Y cells in a time-dependent manner. The data are presented as the mean±SD (n=3). bP<0.05 and cP<0.01, relative to the untreated control.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4816236&req=5

fig4: Effect of Fe2+ on the expression of NF-κB in SH-SY5Y cells. (A) The mRNA expression of NF-κB p65 in SH-SY5Y cells exposed to different concentrations of Fe2+ for 24 h. (B) The mRNA expression of NF-κB p65 in SH-SY5Y cells exposed to 200 μmol/L Fe2+ at different time-points. (C) Fe2+ increased the protein expression of NF-κB p65 in SH-SY5Y cells in a concentration-dependent manner. (D) Fe2+ increased the protein expression of NF-κB p65 in SH-SY5Y cells in a time-dependent manner. The data are presented as the mean±SD (n=3). bP<0.05 and cP<0.01, relative to the untreated control.
Mentions: Previous studies have shown that NF-κB activation is necessary for the induction of proinflammatory mediators13,35. Next, we wanted to investigate whether NF-κB was modulated by Fe2+. SH-SY5Y cells were treated with 10, 50, 100, 200 or 300 μmol/L of Fe2+ for 24 h and for 1, 3, 6, 12, or 24 h with 200 μmol/L Fe2+, and then qPCR and Western blot analyses were used to determine the expression of NF-κB mRNA and protein. Both the mRNA and protein levels of NF-κB increased in concentration- and time-dependent manners after Fe2+ exposure (Figure 4). NF-κB mRNA levels increased significantly following the addition of 50 μmol/L Fe2+ and after 3-h Fe2+ exposure compared to control cells (Figure 4A and 4B). Similar behavior was observed for NF-κB protein expression levels, which increased significantly following the exposure to 100 μmol/L Fe2+ and after 6 h of exposure to Fe2+ compared to control cells (Figure 4C and 4D).

Bottom Line: Treatment of SH-SY5Y cells with Fe(2+) (50-200 μmol/L) dose-dependently decreased the cell viability, which was significantly attenuated by pretreatment with carvacrol (164 and 333 μmol/L).Treatment with Fe(2+) increased the Bax level and caspase-3 activity, and decreased the Bcl-2 level, resulting in cell apoptosis.Pretreatment with carvacrol significantly inhibited Fe(2+)-induced activation of NF-κB, expression of the pro-inflammatory cytokines, and cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT

Aim: Carvacrol (2-methyl-5-isopropylphenol), a phenolic monoterpene in the essential oils of the genera Origanum and Thymus, has been shown to exert a variety of therapeutic effects. Here we examined whether carvacrol protected neuroblastoma SH-SY5Y cells against Fe(2+)-induced apoptosis and explored the underlying mechanisms.

Methods: Neuroblastoma SH-SY5Y cells were incubated with Fe(2+) for 24 h, and the cell viability was assessed with CCK-8 assay. TUNEL assay and flow cytometric analysis were performed to evaluate cell apoptosis. The mRNA levels of pro-inflammatory cytokines and NF-κB p65 were determined using qPCR. The expression of relevant proteins was determined using Western blot analysis or immunofluorescence staining.

Results: Treatment of SH-SY5Y cells with Fe(2+) (50-200 μmol/L) dose-dependently decreased the cell viability, which was significantly attenuated by pretreatment with carvacrol (164 and 333 μmol/L). Treatment with Fe(2+) increased the Bax level and caspase-3 activity, and decreased the Bcl-2 level, resulting in cell apoptosis. Furthermore, treatment with Fe(2+) significantly increased the gene expression of IL-1β, IL-6 and TNF-α, and induced the nuclear translocation of NF-κB. Treatment with Fe(2+) also significantly increased the phosphorylation of p38, ERK, JNK and IKK in the cells. Pretreatment with carvacrol significantly inhibited Fe(2+)-induced activation of NF-κB, expression of the pro-inflammatory cytokines, and cell apoptosis. Moreover, pretreatment with carvacrol inhibited Fe(2+)-induced phosphorylation of JNK and IKK, but not p38 and ERK in the cells.

Conclusion: Carvacrol protects neuroblastoma SH-SY5Y cells against Fe(2+)-induced apoptosis, which may result from suppressing the MAPK/JNK-NF-κB signaling pathways.

Show MeSH
Related in: MedlinePlus