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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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Carvacrol inhibits Fe2+-induced apoptotic cell death in SH-SY5Y cells. Fe2+(200 μmol/L, 24 h) caused a significant increase in the Bax/Bcl-2 ratio (A) and cleaved caspase-3 expression (B) in SH-SY5Y cells, which was significantly attenuated by carvacrol. (C) Representative images of DAPI staining and TUNEL assays used to analyze apoptotic cells (200×). (D) Apoptotic status was also determined using an Annexin V-FITC binding assay. (E) The rates of apoptosis. CAR, carvacrol. Mean±SD (n=3). cP<0.01 vs control; eP<0.05, fP<0.01 vs the Fe2+-treated group.
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fig2: Carvacrol inhibits Fe2+-induced apoptotic cell death in SH-SY5Y cells. Fe2+(200 μmol/L, 24 h) caused a significant increase in the Bax/Bcl-2 ratio (A) and cleaved caspase-3 expression (B) in SH-SY5Y cells, which was significantly attenuated by carvacrol. (C) Representative images of DAPI staining and TUNEL assays used to analyze apoptotic cells (200×). (D) Apoptotic status was also determined using an Annexin V-FITC binding assay. (E) The rates of apoptosis. CAR, carvacrol. Mean±SD (n=3). cP<0.01 vs control; eP<0.05, fP<0.01 vs the Fe2+-treated group.

Mentions: Because the intrinsic apoptotic pathway is mainly regulated by proteins that belong to the Bcl-2 and caspase families, changes in the expression of either pro-apoptotic or antiapoptotic Bcl-2 and caspase family members can affect the induction of apoptosis. To determine whether carvacrol acts by modulating the abundance of apoptotic proteins, the protein levels of Bax, Bcl-2 and caspase-3 were measured using Western blot analysis. SH-SY5Y cells were pretreated with carvacrol (333 μmol/L) and then exposed to Fe2+ for another 24 h. As shown in Figure 2A, exposure to Fe2+ at 200 μmol/L for 24 h significantly increased Bax expression and decreased Bcl-2 expression, while pretreatment with carvacrol inhibited the upregulation of Bax and the downregulation of Bcl-2. Consistent with these results, exposure to Fe2+ at 200 μmol/L for 24 h significantly increased cleaved caspase-3 protein levels compared to control conditions, indicating the involvement of caspase-3 in Fe2+-induced cell death in SH-SY5Y cells (Figure 2B), whereas pretreatment with carvacrol (333 μmol/L) for 2 h significantly downregulated the elevated protein expression of cleaved caspase-3.


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)

Carvacrol inhibits Fe2+-induced apoptotic cell death in SH-SY5Y cells. Fe2+(200 μmol/L, 24 h) caused a significant increase in the Bax/Bcl-2 ratio (A) and cleaved caspase-3 expression (B) in SH-SY5Y cells, which was significantly attenuated by carvacrol. (C) Representative images of DAPI staining and TUNEL assays used to analyze apoptotic cells (200×). (D) Apoptotic status was also determined using an Annexin V-FITC binding assay. (E) The rates of apoptosis. CAR, carvacrol. Mean±SD (n=3). cP<0.01 vs control; eP<0.05, fP<0.01 vs the Fe2+-treated group.
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fig2: Carvacrol inhibits Fe2+-induced apoptotic cell death in SH-SY5Y cells. Fe2+(200 μmol/L, 24 h) caused a significant increase in the Bax/Bcl-2 ratio (A) and cleaved caspase-3 expression (B) in SH-SY5Y cells, which was significantly attenuated by carvacrol. (C) Representative images of DAPI staining and TUNEL assays used to analyze apoptotic cells (200×). (D) Apoptotic status was also determined using an Annexin V-FITC binding assay. (E) The rates of apoptosis. CAR, carvacrol. Mean±SD (n=3). cP<0.01 vs control; eP<0.05, fP<0.01 vs the Fe2+-treated group.
Mentions: Because the intrinsic apoptotic pathway is mainly regulated by proteins that belong to the Bcl-2 and caspase families, changes in the expression of either pro-apoptotic or antiapoptotic Bcl-2 and caspase family members can affect the induction of apoptosis. To determine whether carvacrol acts by modulating the abundance of apoptotic proteins, the protein levels of Bax, Bcl-2 and caspase-3 were measured using Western blot analysis. SH-SY5Y cells were pretreated with carvacrol (333 μmol/L) and then exposed to Fe2+ for another 24 h. As shown in Figure 2A, exposure to Fe2+ at 200 μmol/L for 24 h significantly increased Bax expression and decreased Bcl-2 expression, while pretreatment with carvacrol inhibited the upregulation of Bax and the downregulation of Bcl-2. Consistent with these results, exposure to Fe2+ at 200 μmol/L for 24 h significantly increased cleaved caspase-3 protein levels compared to control conditions, indicating the involvement of caspase-3 in Fe2+-induced cell death in SH-SY5Y cells (Figure 2B), whereas pretreatment with carvacrol (333 μmol/L) for 2 h significantly downregulated the elevated protein expression of cleaved caspase-3.

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