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Water extract from the leaves of Withania somnifera protect RA differentiated C6 and IMR-32 cells against glutamate-induced excitotoxicity.

Kataria H, Wadhwa R, Kaul SC, Kaur G - PLoS ONE (2012)

Bottom Line: We demonstrate that RA-differentiated C6 and IMR-32 cells, when exposed to glutamate, undergo loss of neural network and cell death that was accompanied by increase in the stress protein HSP70.ASH-WEX pre-treatment inhibited glutamate-induced cell death and was able to revert glutamate-induced changes in HSP70 to a large extent.Furthermore, the analysis on the neuronal plasticity marker NCAM (Neural cell adhesion molecule) and its polysialylated form, PSA-NCAM revealed that ASH-WEX has therapeutic potential for prevention of neurodegeneration associated with glutamate-induced excitotoxicty.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar, India.

ABSTRACT
Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative disorders. Search for herbal remedies that may possibly act as therapeutic agents is an active area of research to combat these diseases. The present study was designed to investigate the neuroprotective role of Withania somnifera (Ashwagandha), also known as Indian ginseng, against glutamate induced toxicity in the retinoic acid differentiated rat glioma (C6) and human neuroblastoma (IMR-32) cells. The neuroprotective activity of the Ashwagandha leaves derived water extract (ASH-WEX) was evaluated. Cell viability and the expression of glial and neuronal cell differentiation markers was examined in glutamate challenged differentiated cells with and without the presence of ASH-WEX. We demonstrate that RA-differentiated C6 and IMR-32 cells, when exposed to glutamate, undergo loss of neural network and cell death that was accompanied by increase in the stress protein HSP70. ASH-WEX pre-treatment inhibited glutamate-induced cell death and was able to revert glutamate-induced changes in HSP70 to a large extent. Furthermore, the analysis on the neuronal plasticity marker NCAM (Neural cell adhesion molecule) and its polysialylated form, PSA-NCAM revealed that ASH-WEX has therapeutic potential for prevention of neurodegeneration associated with glutamate-induced excitotoxicty.

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Representative Western blots and their densitometry analysis for GFAP (a) and NF200 (e) for RA differentiated C6 and IMR-32 cells, respectively.RT-PCR results for GFAP and NF200 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry analysis was represented by histograms. The expression of GFAP in C6 (c) and NF200 in IMR-32 (g) cells was analysed by immunocytostaining and relative intensity was plotted as histogram as analysed by Image pro-plus software. “*” represents the statistical significant difference between all the treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. “#” represents the statistical difference between “glutamate + ASH-WEX” treated groups with their respective “glutamate” treatment groups. “*” and “#” = p<0.05.
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pone-0037080-g002: Representative Western blots and their densitometry analysis for GFAP (a) and NF200 (e) for RA differentiated C6 and IMR-32 cells, respectively.RT-PCR results for GFAP and NF200 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry analysis was represented by histograms. The expression of GFAP in C6 (c) and NF200 in IMR-32 (g) cells was analysed by immunocytostaining and relative intensity was plotted as histogram as analysed by Image pro-plus software. “*” represents the statistical significant difference between all the treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. “#” represents the statistical difference between “glutamate + ASH-WEX” treated groups with their respective “glutamate” treatment groups. “*” and “#” = p<0.05.

Mentions: GFAP is an astrocyte-specific intermediate filament thought to provide structural support to normal astrocytes. Increase in GFAP production is a sign of astrogliosis, reactive injury, and neurodegeneration in the differentiated cells. Cells were exposed to 0.5 mM or 1 mM glutamate (24 h) after the pretreatment with 0.1% ASH-WEX (24 h). As shown in Fig. 2a, there was a significant increase (p<0.05) in GFAP expression in the C6 cells upon treatment with glutamate which was suppressed with ASH-WEX pre-treatment in the 0.5 mM glutamate treatment group. In the 1 mM glutamate treatment group 0.1% ASH-WEX was not able to normalize the GFAP expression level (Fig. 2a). We performed RT-PCR in the control and treated groups and found that there was a significant increase in GFAP mRNA in glutamate treated groups. ASH-WEX pre-treatment was able to suppress the upregulation in GFAP mRNA in both low and high dose glutamate groups (Fig. 2b). Single cell quantitative immunocytofluoroscence for GFAP in these groups further revealed dose dependent increase in GFAP expression in the glutamate-exposed as compared to the control cells (Fig. 2c and d). In cells pretreated with ASH-WEX, the expression level of GFAP was normalized in low dose glutamate group but there was no significant difference in expression of GFAP in high dose group (Fig. 2d).


Water extract from the leaves of Withania somnifera protect RA differentiated C6 and IMR-32 cells against glutamate-induced excitotoxicity.

Kataria H, Wadhwa R, Kaul SC, Kaur G - PLoS ONE (2012)

Representative Western blots and their densitometry analysis for GFAP (a) and NF200 (e) for RA differentiated C6 and IMR-32 cells, respectively.RT-PCR results for GFAP and NF200 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry analysis was represented by histograms. The expression of GFAP in C6 (c) and NF200 in IMR-32 (g) cells was analysed by immunocytostaining and relative intensity was plotted as histogram as analysed by Image pro-plus software. “*” represents the statistical significant difference between all the treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. “#” represents the statistical difference between “glutamate + ASH-WEX” treated groups with their respective “glutamate” treatment groups. “*” and “#” = p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351387&req=5

pone-0037080-g002: Representative Western blots and their densitometry analysis for GFAP (a) and NF200 (e) for RA differentiated C6 and IMR-32 cells, respectively.RT-PCR results for GFAP and NF200 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry analysis was represented by histograms. The expression of GFAP in C6 (c) and NF200 in IMR-32 (g) cells was analysed by immunocytostaining and relative intensity was plotted as histogram as analysed by Image pro-plus software. “*” represents the statistical significant difference between all the treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. “#” represents the statistical difference between “glutamate + ASH-WEX” treated groups with their respective “glutamate” treatment groups. “*” and “#” = p<0.05.
Mentions: GFAP is an astrocyte-specific intermediate filament thought to provide structural support to normal astrocytes. Increase in GFAP production is a sign of astrogliosis, reactive injury, and neurodegeneration in the differentiated cells. Cells were exposed to 0.5 mM or 1 mM glutamate (24 h) after the pretreatment with 0.1% ASH-WEX (24 h). As shown in Fig. 2a, there was a significant increase (p<0.05) in GFAP expression in the C6 cells upon treatment with glutamate which was suppressed with ASH-WEX pre-treatment in the 0.5 mM glutamate treatment group. In the 1 mM glutamate treatment group 0.1% ASH-WEX was not able to normalize the GFAP expression level (Fig. 2a). We performed RT-PCR in the control and treated groups and found that there was a significant increase in GFAP mRNA in glutamate treated groups. ASH-WEX pre-treatment was able to suppress the upregulation in GFAP mRNA in both low and high dose glutamate groups (Fig. 2b). Single cell quantitative immunocytofluoroscence for GFAP in these groups further revealed dose dependent increase in GFAP expression in the glutamate-exposed as compared to the control cells (Fig. 2c and d). In cells pretreated with ASH-WEX, the expression level of GFAP was normalized in low dose glutamate group but there was no significant difference in expression of GFAP in high dose group (Fig. 2d).

Bottom Line: We demonstrate that RA-differentiated C6 and IMR-32 cells, when exposed to glutamate, undergo loss of neural network and cell death that was accompanied by increase in the stress protein HSP70.ASH-WEX pre-treatment inhibited glutamate-induced cell death and was able to revert glutamate-induced changes in HSP70 to a large extent.Furthermore, the analysis on the neuronal plasticity marker NCAM (Neural cell adhesion molecule) and its polysialylated form, PSA-NCAM revealed that ASH-WEX has therapeutic potential for prevention of neurodegeneration associated with glutamate-induced excitotoxicty.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar, India.

ABSTRACT
Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative disorders. Search for herbal remedies that may possibly act as therapeutic agents is an active area of research to combat these diseases. The present study was designed to investigate the neuroprotective role of Withania somnifera (Ashwagandha), also known as Indian ginseng, against glutamate induced toxicity in the retinoic acid differentiated rat glioma (C6) and human neuroblastoma (IMR-32) cells. The neuroprotective activity of the Ashwagandha leaves derived water extract (ASH-WEX) was evaluated. Cell viability and the expression of glial and neuronal cell differentiation markers was examined in glutamate challenged differentiated cells with and without the presence of ASH-WEX. We demonstrate that RA-differentiated C6 and IMR-32 cells, when exposed to glutamate, undergo loss of neural network and cell death that was accompanied by increase in the stress protein HSP70. ASH-WEX pre-treatment inhibited glutamate-induced cell death and was able to revert glutamate-induced changes in HSP70 to a large extent. Furthermore, the analysis on the neuronal plasticity marker NCAM (Neural cell adhesion molecule) and its polysialylated form, PSA-NCAM revealed that ASH-WEX has therapeutic potential for prevention of neurodegeneration associated with glutamate-induced excitotoxicty.

Show MeSH
Related in: MedlinePlus