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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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The protective effects of carvacrol against Fe2+-induced cytotoxicity in SH-SY5Y cells. (A) SH-SY5Y cells were treated with different concentrations of Fe2+ (10, 50, 100, 200 or 300 μmol/L) for 24 h, and cell viability was estimated using CCK-8 assays. (B) SH-SY5Y cells were treated with different concentrations of carvacrol (33, 67, 164, 333 or 666 μmol/L) for 24 h. Cell viability was estimated using CCK-8 assays. (C) Cells were pretreated with 67, 164 or 333 μmol/L carvacrol for 2 h and then incubated with 200 μmol/L Fe2+ for 24 h. (D) Cells were pretreated with 164 or 333 μmol/L carvacrol for 2 h prior to treatment with Fe2+ (200 μmol/L) for 24 h, and then morphological changes were analyzed (200×). CAR, carvacrol. Mean±SD (n=3). cP<0.01 vs the untreated control; fP<0.01 vs the Fe2+-treated group.
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fig1: The protective effects of carvacrol against Fe2+-induced cytotoxicity in SH-SY5Y cells. (A) SH-SY5Y cells were treated with different concentrations of Fe2+ (10, 50, 100, 200 or 300 μmol/L) for 24 h, and cell viability was estimated using CCK-8 assays. (B) SH-SY5Y cells were treated with different concentrations of carvacrol (33, 67, 164, 333 or 666 μmol/L) for 24 h. Cell viability was estimated using CCK-8 assays. (C) Cells were pretreated with 67, 164 or 333 μmol/L carvacrol for 2 h and then incubated with 200 μmol/L Fe2+ for 24 h. (D) Cells were pretreated with 164 or 333 μmol/L carvacrol for 2 h prior to treatment with Fe2+ (200 μmol/L) for 24 h, and then morphological changes were analyzed (200×). CAR, carvacrol. Mean±SD (n=3). cP<0.01 vs the untreated control; fP<0.01 vs the Fe2+-treated group.

Mentions: To investigate the toxicity of ferrous iron and carvacrol in SH-SY5Y cells and to determine the influence of carvacrol on Fe2+ toxicity in these cells, the SH-SY5Y cells were treated with 10, 50, 100, 200 or 300 μmol/L of Fe2+ and 33, 66, 164, 333, or 666 μmol/L of carvacrol for 24 h. CCK-8 assays were performed to assess cell viability. The viability of cells incubated with Fe2+ at 10, 50, 100, 200 and 300 μmol/L for 24 h was 93.2%±7.5%, 88.0%±2.0%, 79.0%±5.3%, 65.0%±2.5%, and 53.0%±2.2% of the control values, respectively (Figure 1A). The results showed that Fe2+ at 10–300 μmol/L gradually decreased cell viability, with significant decreases at 200 and 300 μmol/L of Fe2+, compared to the untreated control cells. Fe2+ at 200 μmol/L was chosen for the following experiments. The presence of up to 333 μmol/L carvacrol did not affect the viability of SH-SY5Y cells (Figure 1B), suggesting that the carvacrol concentration used in this study was not cytotoxic. The decrease in viability of cells that was caused by 200 μmol/L of Fe2+ was significantly prevented by pretreatment with 164 and 333 μmol/L of carvacrol (Figure 1C). Likewise, as shown in Figure 1D, exposure to Fe2+ at 200 μmol/L caused clear morphological alterations that were typical of neuronal damage, while pretreatment with carvacrol for 2 h before Fe2+ exposure partially attenuated iron-induced cytotoxicity and cell damage.


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)

The protective effects of carvacrol against Fe2+-induced cytotoxicity in SH-SY5Y cells. (A) SH-SY5Y cells were treated with different concentrations of Fe2+ (10, 50, 100, 200 or 300 μmol/L) for 24 h, and cell viability was estimated using CCK-8 assays. (B) SH-SY5Y cells were treated with different concentrations of carvacrol (33, 67, 164, 333 or 666 μmol/L) for 24 h. Cell viability was estimated using CCK-8 assays. (C) Cells were pretreated with 67, 164 or 333 μmol/L carvacrol for 2 h and then incubated with 200 μmol/L Fe2+ for 24 h. (D) Cells were pretreated with 164 or 333 μmol/L carvacrol for 2 h prior to treatment with Fe2+ (200 μmol/L) for 24 h, and then morphological changes were analyzed (200×). CAR, carvacrol. Mean±SD (n=3). cP<0.01 vs the untreated control; fP<0.01 vs the Fe2+-treated group.
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fig1: The protective effects of carvacrol against Fe2+-induced cytotoxicity in SH-SY5Y cells. (A) SH-SY5Y cells were treated with different concentrations of Fe2+ (10, 50, 100, 200 or 300 μmol/L) for 24 h, and cell viability was estimated using CCK-8 assays. (B) SH-SY5Y cells were treated with different concentrations of carvacrol (33, 67, 164, 333 or 666 μmol/L) for 24 h. Cell viability was estimated using CCK-8 assays. (C) Cells were pretreated with 67, 164 or 333 μmol/L carvacrol for 2 h and then incubated with 200 μmol/L Fe2+ for 24 h. (D) Cells were pretreated with 164 or 333 μmol/L carvacrol for 2 h prior to treatment with Fe2+ (200 μmol/L) for 24 h, and then morphological changes were analyzed (200×). CAR, carvacrol. Mean±SD (n=3). cP<0.01 vs the untreated control; fP<0.01 vs the Fe2+-treated group.
Mentions: To investigate the toxicity of ferrous iron and carvacrol in SH-SY5Y cells and to determine the influence of carvacrol on Fe2+ toxicity in these cells, the SH-SY5Y cells were treated with 10, 50, 100, 200 or 300 μmol/L of Fe2+ and 33, 66, 164, 333, or 666 μmol/L of carvacrol for 24 h. CCK-8 assays were performed to assess cell viability. The viability of cells incubated with Fe2+ at 10, 50, 100, 200 and 300 μmol/L for 24 h was 93.2%±7.5%, 88.0%±2.0%, 79.0%±5.3%, 65.0%±2.5%, and 53.0%±2.2% of the control values, respectively (Figure 1A). The results showed that Fe2+ at 10–300 μmol/L gradually decreased cell viability, with significant decreases at 200 and 300 μmol/L of Fe2+, compared to the untreated control cells. Fe2+ at 200 μmol/L was chosen for the following experiments. The presence of up to 333 μmol/L carvacrol did not affect the viability of SH-SY5Y cells (Figure 1B), suggesting that the carvacrol concentration used in this study was not cytotoxic. The decrease in viability of cells that was caused by 200 μmol/L of Fe2+ was significantly prevented by pretreatment with 164 and 333 μmol/L of carvacrol (Figure 1C). Likewise, as shown in Figure 1D, exposure to Fe2+ at 200 μmol/L caused clear morphological alterations that were typical of neuronal damage, while pretreatment with carvacrol for 2 h before Fe2+ exposure partially attenuated iron-induced cytotoxicity and cell damage.

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