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Uridine from Pleurotus giganteus and Its Neurite Outgrowth Stimulatory Effects with Underlying Mechanism.

Phan CW, David P, Wong KH, Naidu M, Sabaratnam V - PLoS ONE (2015)

Bottom Line: An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated.Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1 ± 0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells.MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells.

View Article: PubMed Central - PubMed

Affiliation: Mushroom Research Centre, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT
Neurodegenerative diseases are linked to neuronal cell death and impairment of neurite outgrowth. An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated. The chemical constituents of P. giganteus (linoleic acid, oleic acid, cinnamic acid, caffeic acid, p-coumaric acid, succinic acid, benzoic acid, and uridine) were tested for neurite outgrowth activity. Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1 ± 0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells. Uridine which was present in P. giganteus (1.80 ± 0.03 g/100g mushroom extract) increased the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt). Further, phosphorylation of the mammalian target of rapamycin (mTOR) was also increased. MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells. This study demonstrated that P. giganteus may enhance neurite outgrowth and one of the key bioactive molecules responsible for neurite outgrowth is uridine.

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(a) The effects of different concentrations of uridine (1–500 μM) on neurite outgrowth in differentiating N2a cells. NGF (50 ng/mL) was used as positive control and medium only (vehicle) with no treatment was used as control. Aqueous and ethanol extracts (20 μg/mL) were compared to uridine. (b) The mean of the longest neurite length of N2a. Values are mean ± SD from three independent experiments. Different alphabets represent significant differences between samples (p < 0.01). Phase-contrast photomicrographs of (c) uridine (100μM) induced neurites as indicated by arrows and (d) negative control cells treated with only vehicle (serum-free medium). Scale bar represents 20 μm.
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pone.0143004.g004: (a) The effects of different concentrations of uridine (1–500 μM) on neurite outgrowth in differentiating N2a cells. NGF (50 ng/mL) was used as positive control and medium only (vehicle) with no treatment was used as control. Aqueous and ethanol extracts (20 μg/mL) were compared to uridine. (b) The mean of the longest neurite length of N2a. Values are mean ± SD from three independent experiments. Different alphabets represent significant differences between samples (p < 0.01). Phase-contrast photomicrographs of (c) uridine (100μM) induced neurites as indicated by arrows and (d) negative control cells treated with only vehicle (serum-free medium). Scale bar represents 20 μm.

Mentions: Taking into account that the uridine content is 1.6–1.8% (g/100 g), 20 μg/mL in ethanol and aqueous extracts would correspond to 0.32 and 0.36μg/mL of uridine; respectively. The molecular weight of uridine is 244g/mol and this would correspond to about 1.5 μM of uridine. To determine the optimum uridine concentration for neurite outgrowth activity, pure uridine at the range of 1–500 μM was tested. Treatment with uridine significantly (p < 0.01) increased neurite outgrowth in N2a cells in a dose dependent manner up to 100 μM (Fig 4A). One μM of uridine induced 19.4 ± 4% of neurite bearing cell which was 2 times higher than that of negative control (p < 0.01) and with no significant difference from the NGF control (p > 0.01). This means that the amount of uridine present in the mushroom extract (1.5 μM) would be sufficient to induce a significantly (p < 0.01) high percentage of neurite bearing cell. Uridine at 100 μM exhibited a significantly (p < 0.01) higher percentage in neurite-bearing cells (49.2 ± 1.6%) when compared to NGF- and mushroom extract-treated cells. Concentration of more than 100 μM did not cause additional neurite outgrowth stimulation. Therefore, the optimum uridine concentration used for the subsequent experiment was taken as 100 μM. The dose-response data was analysed to obtain the concentration of uridine that caused half-maximal neurite outgrowth stimulatory activity (EC50) and the value was found to be 10.5 μM.


Uridine from Pleurotus giganteus and Its Neurite Outgrowth Stimulatory Effects with Underlying Mechanism.

Phan CW, David P, Wong KH, Naidu M, Sabaratnam V - PLoS ONE (2015)

(a) The effects of different concentrations of uridine (1–500 μM) on neurite outgrowth in differentiating N2a cells. NGF (50 ng/mL) was used as positive control and medium only (vehicle) with no treatment was used as control. Aqueous and ethanol extracts (20 μg/mL) were compared to uridine. (b) The mean of the longest neurite length of N2a. Values are mean ± SD from three independent experiments. Different alphabets represent significant differences between samples (p < 0.01). Phase-contrast photomicrographs of (c) uridine (100μM) induced neurites as indicated by arrows and (d) negative control cells treated with only vehicle (serum-free medium). Scale bar represents 20 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643974&req=5

pone.0143004.g004: (a) The effects of different concentrations of uridine (1–500 μM) on neurite outgrowth in differentiating N2a cells. NGF (50 ng/mL) was used as positive control and medium only (vehicle) with no treatment was used as control. Aqueous and ethanol extracts (20 μg/mL) were compared to uridine. (b) The mean of the longest neurite length of N2a. Values are mean ± SD from three independent experiments. Different alphabets represent significant differences between samples (p < 0.01). Phase-contrast photomicrographs of (c) uridine (100μM) induced neurites as indicated by arrows and (d) negative control cells treated with only vehicle (serum-free medium). Scale bar represents 20 μm.
Mentions: Taking into account that the uridine content is 1.6–1.8% (g/100 g), 20 μg/mL in ethanol and aqueous extracts would correspond to 0.32 and 0.36μg/mL of uridine; respectively. The molecular weight of uridine is 244g/mol and this would correspond to about 1.5 μM of uridine. To determine the optimum uridine concentration for neurite outgrowth activity, pure uridine at the range of 1–500 μM was tested. Treatment with uridine significantly (p < 0.01) increased neurite outgrowth in N2a cells in a dose dependent manner up to 100 μM (Fig 4A). One μM of uridine induced 19.4 ± 4% of neurite bearing cell which was 2 times higher than that of negative control (p < 0.01) and with no significant difference from the NGF control (p > 0.01). This means that the amount of uridine present in the mushroom extract (1.5 μM) would be sufficient to induce a significantly (p < 0.01) high percentage of neurite bearing cell. Uridine at 100 μM exhibited a significantly (p < 0.01) higher percentage in neurite-bearing cells (49.2 ± 1.6%) when compared to NGF- and mushroom extract-treated cells. Concentration of more than 100 μM did not cause additional neurite outgrowth stimulation. Therefore, the optimum uridine concentration used for the subsequent experiment was taken as 100 μM. The dose-response data was analysed to obtain the concentration of uridine that caused half-maximal neurite outgrowth stimulatory activity (EC50) and the value was found to be 10.5 μM.

Bottom Line: An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated.Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1 ± 0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells.MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells.

View Article: PubMed Central - PubMed

Affiliation: Mushroom Research Centre, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT
Neurodegenerative diseases are linked to neuronal cell death and impairment of neurite outgrowth. An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated. The chemical constituents of P. giganteus (linoleic acid, oleic acid, cinnamic acid, caffeic acid, p-coumaric acid, succinic acid, benzoic acid, and uridine) were tested for neurite outgrowth activity. Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1 ± 0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells. Uridine which was present in P. giganteus (1.80 ± 0.03 g/100g mushroom extract) increased the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt). Further, phosphorylation of the mammalian target of rapamycin (mTOR) was also increased. MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells. This study demonstrated that P. giganteus may enhance neurite outgrowth and one of the key bioactive molecules responsible for neurite outgrowth is uridine.

Show MeSH
Related in: MedlinePlus