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Artemisinin protects PC12 cells against β -amyloid-induced apoptosis through activation of the ERK1/2 signaling pathway

View Article: PubMed Central - PubMed

ABSTRACT

Accumulating evidence displays that an abnormal deposition of amyloid beta-peptide (Aβ) is the primary cause of the pathogenesis of Alzheimer's disease (AD). And therefore the elimination of Aβ is regarded as an important strategy for AD treatment. The discovery of drug candidates using culture neuronal cells against Aβ peptide toxicity is believed to be an effective approach to develop drug for the treatment of AD patients. We have previously showed that artemisinin, a FDA-approved anti-malaria drug, has neuroprotective effects recently. In the present study, we aimed to investigate the effects and potential mechanism of artemisinin in protecting neuronal PC12 cells from toxicity of β amyloid peptide. Our studies revealed that artemisinin, in clinical relevant concentration, protected and rescued PC12 cells from Aβ25–35-induced cell death. Further study showed that artemisinin significantly ameliorated cell death due to Aβ25–35 insult by restoring abnormal changes in nuclear morphology, lactate dehydrogenase, intracellular ROS, mitochondrial membrane potential and activity of apoptotic caspase. Western blotting analysis demonstrated that artemisinin activated extracellular regulated kinase ERK1/2 but not Akt survival signaling. Consistent with the role of ERK1/2, preincubation of cells with ERK1/2 pathway inhibitor PD98059 blocked the effect of artemisinin while PI3K inhibitor LY294002 has no effect. Moreover, Aβ1-42 also caused cells death of PC12 cells while artemisinin suppressed Aβ1-42 cytotoxicity in PC12 cells. Taken together, these results, at the first time, suggest that artemisinin is a potential protectant against β amyloid insult through activation of the ERK1/2 pathway. Our finding provides a potential application of artemisinin in prevention and treatment of AD.

No MeSH data available.


ERK 1/2 pathway mediated the protect effects of artemisinin in PC12 cells. PC12 cells were pre-treated with10 μM PD98059 or 10 μM LY294002 (A), or 5, 10, 20 μM PD98059 (B) for 30 min, and treated with 25 μM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35 for a further 24 h, and the cell viability was determined by MTT assay (N=3). PC12 cells were pre-treated with the ERK1/2 inhibitor PD98059 (20 μM) or PI3K inhibitor LY294002 (20 μM) for 30 min, and afterward treated with 25 μM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35 for a further 24 h; the apoptosis cells was detected by staining with Hoechst 33342 and visualized by fluorescence microscopy (C). The number of apoptotic nuclei with condensed chromatin was counted from the photomicrographs and presented as a percentage of the total number of nuclei (N=3) (D). PC12 cells were pre-treated with the ERK1/2 inhibitor PD98059 (20 μM) or PI3K inhibitor LY294002 (20 μM) for 30 min, and afterward treated with 25uM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35, The expression of phosphorylated ERK1/2, total ERK1/2; and GAPDH were detected by Western blotting with specific antibodies (N=3). ###P<0.005 versus control group; **P<0.01, ***P<0.005 versus the artemisinin or artemisinin plus Aβ25-35-treated group as indicated were considered significantly different.
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f0030: ERK 1/2 pathway mediated the protect effects of artemisinin in PC12 cells. PC12 cells were pre-treated with10 μM PD98059 or 10 μM LY294002 (A), or 5, 10, 20 μM PD98059 (B) for 30 min, and treated with 25 μM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35 for a further 24 h, and the cell viability was determined by MTT assay (N=3). PC12 cells were pre-treated with the ERK1/2 inhibitor PD98059 (20 μM) or PI3K inhibitor LY294002 (20 μM) for 30 min, and afterward treated with 25 μM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35 for a further 24 h; the apoptosis cells was detected by staining with Hoechst 33342 and visualized by fluorescence microscopy (C). The number of apoptotic nuclei with condensed chromatin was counted from the photomicrographs and presented as a percentage of the total number of nuclei (N=3) (D). PC12 cells were pre-treated with the ERK1/2 inhibitor PD98059 (20 μM) or PI3K inhibitor LY294002 (20 μM) for 30 min, and afterward treated with 25uM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35, The expression of phosphorylated ERK1/2, total ERK1/2; and GAPDH were detected by Western blotting with specific antibodies (N=3). ###P<0.005 versus control group; **P<0.01, ***P<0.005 versus the artemisinin or artemisinin plus Aβ25-35-treated group as indicated were considered significantly different.

Mentions: We have previously reported that ERK1/2 pathway rather than PI3K pathway mediated the neuroprotective effects of artemisinin in cells against H2O2 or NO insult [18], [28]. In order to examine whether the up regulation of ERK1/2 phosphorylation is involved in the survival promoting effect of artemisinin on cell apoptosis induced by Aβ25-35, we investigated the effects of specific inhibitors for the ERK1/2 and PI3K pathways (the ERK1/2 pathway inhibitor PD98059 and PI3K inhibitor LY294002) and we found that the ERK1/2 pathway inhibitor blocked the effects of artemisinin while LY294002, a PI3K kinase inhibitor had no effect (Fig. 6A). To further confirm the effects of ERK/1/2 pathway inhibitor PD98059 in more detail, the cultures preincubated with different concentrations of the ERK inhibitor PD98059 (5, 10, 20 μM) for 30 min were treated with artemisinin (25 μM) for 1 h, and the viability of cells was determined by the MTT assay after another 24 h. As shown in Fig. 6B, the protective effect of artemisinin was blocked in the presence of appropriate concentration of ERK inhibitor PD98059. Nuclei condensation was observed in PC12 cells treated with Aβ25-35 along in Hoechst 33342 staining assay, while pretreatment of cells with artemisinin (25 μM) for 1 h reversed the effect of Aβ25-35 (Fig. 6C). However, a pretreatment of PD98059 definitely abolished the effect of artemisinin (Fig. 6D). Western blot in Fig. 6E showed that Aβ25-35 was able to significantly down regulation the phosphorylation of ERK1/2, and artemisinin reversed decreased phosphorylation of ERK1/2, while PD98059 pretreatment blocked the reversed effects of artemisinin. The concentrations of inhibitors themselves used in the current experiments, had no effect on the cell death as shown. Thus these data put together suggested that the ERK1/2 pathway rather than the Akt pathway is mediating the protective effect of artemisinin.


Artemisinin protects PC12 cells against β -amyloid-induced apoptosis through activation of the ERK1/2 signaling pathway
ERK 1/2 pathway mediated the protect effects of artemisinin in PC12 cells. PC12 cells were pre-treated with10 μM PD98059 or 10 μM LY294002 (A), or 5, 10, 20 μM PD98059 (B) for 30 min, and treated with 25 μM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35 for a further 24 h, and the cell viability was determined by MTT assay (N=3). PC12 cells were pre-treated with the ERK1/2 inhibitor PD98059 (20 μM) or PI3K inhibitor LY294002 (20 μM) for 30 min, and afterward treated with 25 μM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35 for a further 24 h; the apoptosis cells was detected by staining with Hoechst 33342 and visualized by fluorescence microscopy (C). The number of apoptotic nuclei with condensed chromatin was counted from the photomicrographs and presented as a percentage of the total number of nuclei (N=3) (D). PC12 cells were pre-treated with the ERK1/2 inhibitor PD98059 (20 μM) or PI3K inhibitor LY294002 (20 μM) for 30 min, and afterward treated with 25uM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35, The expression of phosphorylated ERK1/2, total ERK1/2; and GAPDH were detected by Western blotting with specific antibodies (N=3). ###P<0.005 versus control group; **P<0.01, ***P<0.005 versus the artemisinin or artemisinin plus Aβ25-35-treated group as indicated were considered significantly different.
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f0030: ERK 1/2 pathway mediated the protect effects of artemisinin in PC12 cells. PC12 cells were pre-treated with10 μM PD98059 or 10 μM LY294002 (A), or 5, 10, 20 μM PD98059 (B) for 30 min, and treated with 25 μM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35 for a further 24 h, and the cell viability was determined by MTT assay (N=3). PC12 cells were pre-treated with the ERK1/2 inhibitor PD98059 (20 μM) or PI3K inhibitor LY294002 (20 μM) for 30 min, and afterward treated with 25 μM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35 for a further 24 h; the apoptosis cells was detected by staining with Hoechst 33342 and visualized by fluorescence microscopy (C). The number of apoptotic nuclei with condensed chromatin was counted from the photomicrographs and presented as a percentage of the total number of nuclei (N=3) (D). PC12 cells were pre-treated with the ERK1/2 inhibitor PD98059 (20 μM) or PI3K inhibitor LY294002 (20 μM) for 30 min, and afterward treated with 25uM artemisinin for 1 h, then incubated with or without 0.3 μM Aβ25-35, The expression of phosphorylated ERK1/2, total ERK1/2; and GAPDH were detected by Western blotting with specific antibodies (N=3). ###P<0.005 versus control group; **P<0.01, ***P<0.005 versus the artemisinin or artemisinin plus Aβ25-35-treated group as indicated were considered significantly different.
Mentions: We have previously reported that ERK1/2 pathway rather than PI3K pathway mediated the neuroprotective effects of artemisinin in cells against H2O2 or NO insult [18], [28]. In order to examine whether the up regulation of ERK1/2 phosphorylation is involved in the survival promoting effect of artemisinin on cell apoptosis induced by Aβ25-35, we investigated the effects of specific inhibitors for the ERK1/2 and PI3K pathways (the ERK1/2 pathway inhibitor PD98059 and PI3K inhibitor LY294002) and we found that the ERK1/2 pathway inhibitor blocked the effects of artemisinin while LY294002, a PI3K kinase inhibitor had no effect (Fig. 6A). To further confirm the effects of ERK/1/2 pathway inhibitor PD98059 in more detail, the cultures preincubated with different concentrations of the ERK inhibitor PD98059 (5, 10, 20 μM) for 30 min were treated with artemisinin (25 μM) for 1 h, and the viability of cells was determined by the MTT assay after another 24 h. As shown in Fig. 6B, the protective effect of artemisinin was blocked in the presence of appropriate concentration of ERK inhibitor PD98059. Nuclei condensation was observed in PC12 cells treated with Aβ25-35 along in Hoechst 33342 staining assay, while pretreatment of cells with artemisinin (25 μM) for 1 h reversed the effect of Aβ25-35 (Fig. 6C). However, a pretreatment of PD98059 definitely abolished the effect of artemisinin (Fig. 6D). Western blot in Fig. 6E showed that Aβ25-35 was able to significantly down regulation the phosphorylation of ERK1/2, and artemisinin reversed decreased phosphorylation of ERK1/2, while PD98059 pretreatment blocked the reversed effects of artemisinin. The concentrations of inhibitors themselves used in the current experiments, had no effect on the cell death as shown. Thus these data put together suggested that the ERK1/2 pathway rather than the Akt pathway is mediating the protective effect of artemisinin.

View Article: PubMed Central - PubMed

ABSTRACT

Accumulating evidence displays that an abnormal deposition of amyloid beta-peptide (A&beta;) is the primary cause of the pathogenesis of Alzheimer's disease (AD). And therefore the elimination of A&beta; is regarded as an important strategy for AD treatment. The discovery of drug candidates using culture neuronal cells against A&beta; peptide toxicity is believed to be an effective approach to develop drug for the treatment of AD patients. We have previously showed that artemisinin, a FDA-approved anti-malaria drug, has neuroprotective effects recently. In the present study, we aimed to investigate the effects and potential mechanism of artemisinin in protecting neuronal PC12 cells from toxicity of &beta; amyloid peptide. Our studies revealed that artemisinin, in clinical relevant concentration, protected and rescued PC12 cells from A&beta;25&ndash;35-induced cell death. Further study showed that artemisinin significantly ameliorated cell death due to A&beta;25&ndash;35 insult by restoring abnormal changes in nuclear morphology, lactate dehydrogenase, intracellular ROS, mitochondrial membrane potential and activity of apoptotic caspase. Western blotting analysis demonstrated that artemisinin activated extracellular regulated kinase ERK1/2 but not Akt survival signaling. Consistent with the role of ERK1/2, preincubation of cells with ERK1/2 pathway inhibitor PD98059 blocked the effect of artemisinin while PI3K inhibitor LY294002 has no effect. Moreover, A&beta;1-42 also caused cells death of PC12 cells while artemisinin suppressed A&beta;1-42 cytotoxicity in PC12 cells. Taken together, these results, at the first time, suggest that artemisinin is a potential protectant against &beta; amyloid insult through activation of the ERK1/2 pathway. Our finding provides a potential application of artemisinin in prevention and treatment of AD.

No MeSH data available.