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Neuritogenic effect of standardized extract of Centella asiatica ECa233 on human neuroblastoma cells.

Wanakhachornkrai O, Pongrakhananon V, Chunhacha P, Wanasuntronwong A, Vattanajun A, Tantisira B, Chanvorachote P, Tantisira MH - BMC Complement Altern Med (2013)

Bottom Line: While ECa 233 had only limited effects on cell viability, it significantly enhanced neurite outgrowth of IMR-32 cells at the concentrations of 1-100 μg/ml.ERK1/2 and Akt signaling pathways seemed to account for the neurotrophic effect observed.In conjunction with in vivo neuroprotective effect of ECa 233 previously reported, the results obtained support further development of ECa 233 for clinical use in neuronal injury or neurodegenerative diseases.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: In order to gain insight into neuroprotective effects of ECa 233, a standardized extract of Centella asiatica, previously demonstrated in animal models of memory impairment induced by transient global ischemia or intracerebroventricular injection of β-amyloid, the effect of ECa 233 on neurite outgrowth of human IMR-32 neuroblastoma cell line was investigated.

Methods: Cells were seeded and incubated with various concentrations of ECa 233. Morphometric analysis was carried out by a measurement of the longest neurite growth of cells at 24 and 48 h. Contributing signaling pathways possibly involved were subsequently elucidated by western blot analysis.

Results: While ECa 233 had only limited effects on cell viability, it significantly enhanced neurite outgrowth of IMR-32 cells at the concentrations of 1-100 μg/ml. Western blot analysis revealed that ECa 233 significantly upregulated the level of activated ERK1/2 and Akt of the treated cells suggesting their involvement in the neuritogenic effect observed, which was subsequently verified by the finding that an addition of their respective inhibitors could reverse the effect of ECa 233 on these cells.

Conclusions: The present study clearly demonstrated neurite outgrowth promoting activity of ECa 233. ERK1/2 and Akt signaling pathways seemed to account for the neurotrophic effect observed. In conjunction with in vivo neuroprotective effect of ECa 233 previously reported, the results obtained support further development of ECa 233 for clinical use in neuronal injury or neurodegenerative diseases.

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Representative photomicrograph of IMR-32 cells.(A) after pre-treated with LY294002 or PD098059 in the presence or absence of ECa 233 (1, 10 and 100 μg/ml) (scale bar = 50 μm). (B) Morphometric analysis of the cell treated with LY294002 or PD098059 on the increment of neurite outgrowth induced by ECa 233. Neurites from 60 living cells per treated condition were evaluated, (n = 3). Data are presented as mean ± S.E., ** = p < 0.001 vs non-treated control. ## = p < 0.001 vs ECa 233 treated groups.
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Figure 5: Representative photomicrograph of IMR-32 cells.(A) after pre-treated with LY294002 or PD098059 in the presence or absence of ECa 233 (1, 10 and 100 μg/ml) (scale bar = 50 μm). (B) Morphometric analysis of the cell treated with LY294002 or PD098059 on the increment of neurite outgrowth induced by ECa 233. Neurites from 60 living cells per treated condition were evaluated, (n = 3). Data are presented as mean ± S.E., ** = p < 0.001 vs non-treated control. ## = p < 0.001 vs ECa 233 treated groups.

Mentions: Subsequently, the involvement of ERK and Akt signaling pathways in the neurite outgrowth promoting activity of ECa 233 was confirmed by treatment of specific inhibitor of MEK (PD098059) or PI3K (LY294002) prior to the exposure to ECa 233. In comparison to neurite lengths of non-treated group which was 23.39 ± 0.54 μm (Figure 5A), ECa 233 at the concentrations of 1, 10 and 100 μg/ml significantly enhanced the neurite outgrowth into 36.12 ± 1.53, 36.79 ± 1.60 and 36.69 ± 1.98 μm respectively. Pretreatment of the cell with PD098059 significantly decreased the neurite outgrowth of the respective concentrations of ECa 233 into 22.45 ± 0.65, 20.76 ± 0.532 and 21.83 ± 0.53 μm. Similar results were demonstrated by LY294002 which significantly decreased the ECa 233-induced neurite outgrowth into 20.83 ± 0.81, 20.03 ± 0.49 and 20.76 ± 0.53 μm, respectively (Figure 5B). As expected, PD098059 (5 μM) or LY294002 (7.5 μM) in the concentration that exhibited no significant effect on neurite outgrowth (23.52 ± 0.84 and 20.43 ± 0.54 μm, respectively) completely abolished neurite stimulating effect of ECa 233.


Neuritogenic effect of standardized extract of Centella asiatica ECa233 on human neuroblastoma cells.

Wanakhachornkrai O, Pongrakhananon V, Chunhacha P, Wanasuntronwong A, Vattanajun A, Tantisira B, Chanvorachote P, Tantisira MH - BMC Complement Altern Med (2013)

Representative photomicrograph of IMR-32 cells.(A) after pre-treated with LY294002 or PD098059 in the presence or absence of ECa 233 (1, 10 and 100 μg/ml) (scale bar = 50 μm). (B) Morphometric analysis of the cell treated with LY294002 or PD098059 on the increment of neurite outgrowth induced by ECa 233. Neurites from 60 living cells per treated condition were evaluated, (n = 3). Data are presented as mean ± S.E., ** = p < 0.001 vs non-treated control. ## = p < 0.001 vs ECa 233 treated groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750251&req=5

Figure 5: Representative photomicrograph of IMR-32 cells.(A) after pre-treated with LY294002 or PD098059 in the presence or absence of ECa 233 (1, 10 and 100 μg/ml) (scale bar = 50 μm). (B) Morphometric analysis of the cell treated with LY294002 or PD098059 on the increment of neurite outgrowth induced by ECa 233. Neurites from 60 living cells per treated condition were evaluated, (n = 3). Data are presented as mean ± S.E., ** = p < 0.001 vs non-treated control. ## = p < 0.001 vs ECa 233 treated groups.
Mentions: Subsequently, the involvement of ERK and Akt signaling pathways in the neurite outgrowth promoting activity of ECa 233 was confirmed by treatment of specific inhibitor of MEK (PD098059) or PI3K (LY294002) prior to the exposure to ECa 233. In comparison to neurite lengths of non-treated group which was 23.39 ± 0.54 μm (Figure 5A), ECa 233 at the concentrations of 1, 10 and 100 μg/ml significantly enhanced the neurite outgrowth into 36.12 ± 1.53, 36.79 ± 1.60 and 36.69 ± 1.98 μm respectively. Pretreatment of the cell with PD098059 significantly decreased the neurite outgrowth of the respective concentrations of ECa 233 into 22.45 ± 0.65, 20.76 ± 0.532 and 21.83 ± 0.53 μm. Similar results were demonstrated by LY294002 which significantly decreased the ECa 233-induced neurite outgrowth into 20.83 ± 0.81, 20.03 ± 0.49 and 20.76 ± 0.53 μm, respectively (Figure 5B). As expected, PD098059 (5 μM) or LY294002 (7.5 μM) in the concentration that exhibited no significant effect on neurite outgrowth (23.52 ± 0.84 and 20.43 ± 0.54 μm, respectively) completely abolished neurite stimulating effect of ECa 233.

Bottom Line: While ECa 233 had only limited effects on cell viability, it significantly enhanced neurite outgrowth of IMR-32 cells at the concentrations of 1-100 μg/ml.ERK1/2 and Akt signaling pathways seemed to account for the neurotrophic effect observed.In conjunction with in vivo neuroprotective effect of ECa 233 previously reported, the results obtained support further development of ECa 233 for clinical use in neuronal injury or neurodegenerative diseases.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: In order to gain insight into neuroprotective effects of ECa 233, a standardized extract of Centella asiatica, previously demonstrated in animal models of memory impairment induced by transient global ischemia or intracerebroventricular injection of β-amyloid, the effect of ECa 233 on neurite outgrowth of human IMR-32 neuroblastoma cell line was investigated.

Methods: Cells were seeded and incubated with various concentrations of ECa 233. Morphometric analysis was carried out by a measurement of the longest neurite growth of cells at 24 and 48 h. Contributing signaling pathways possibly involved were subsequently elucidated by western blot analysis.

Results: While ECa 233 had only limited effects on cell viability, it significantly enhanced neurite outgrowth of IMR-32 cells at the concentrations of 1-100 μg/ml. Western blot analysis revealed that ECa 233 significantly upregulated the level of activated ERK1/2 and Akt of the treated cells suggesting their involvement in the neuritogenic effect observed, which was subsequently verified by the finding that an addition of their respective inhibitors could reverse the effect of ECa 233 on these cells.

Conclusions: The present study clearly demonstrated neurite outgrowth promoting activity of ECa 233. ERK1/2 and Akt signaling pathways seemed to account for the neurotrophic effect observed. In conjunction with in vivo neuroprotective effect of ECa 233 previously reported, the results obtained support further development of ECa 233 for clinical use in neuronal injury or neurodegenerative diseases.

Show MeSH
Related in: MedlinePlus