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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 NCAM in RA differentiated C6 (a) and IMR-32 (d) cells, respectively.RT-PCR results for NCAM mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented by histograms. The expression of NCAM in C6 (c) and IMR-32 (f) cells was analysed by immunostaining. “*” 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-g004: Representative Western blots and their densitometry analysis for NCAM in RA differentiated C6 (a) and IMR-32 (d) cells, respectively.RT-PCR results for NCAM mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented by histograms. The expression of NCAM in C6 (c) and IMR-32 (f) cells was analysed by immunostaining. “*” 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: NCAM is a glycoprotein of immunoglobulin (Ig) superfamily, expressed on the surface of neurons and glia cells. It has a role in cell–cell adhesion, neurite outgrowth, synaptic plasticity, neuroprotection and learning and memory. We examined NCAM expression in control and treated groups and found that ASH-WEX treatment caused a minor increase in NCAM expression both in C6 and IMR-32 cells (Fig. 4 a, d). The low dose treatment of glutamate (0.5 mM) led to upregulation of NCAM expression (p<0.05) which was further increased in the high dose glutamate (1 mM) treated cells as seen on the Western blots. ASH-WEX (0.1%) pre-treatment led to normalization of NCAM expression in the low dose glutamate group but its expression remained significantly higher (around 45%) in the high dose treatment group (Fig. 4a,d). These changes were also apparent at mRNA level. Lower dose of glutamate (0.5 mM) exposure led to increase in NCAM mRNA level that was normalized by ASH-WEX in C6 cells (Fig. 4b). On the other hand high dose treatment group did not show normalization of NCAM mRNA in C6 cells when pretreated with ASH-WEX. Furthermore, ASH-WEX did not cause any recovery in NCAM mRNA expression in both low and high dose glutamate groups of IMR-32 (Fig. 4e). Immunocytostaining for NCAM was enhanced upon glutamate exposure in case of C6 cells as well as in IMR-32 cells (Fig. 4 c, f). ASH-WEX pre-treatment induced normalization was evident by NCAM staining. Consistent with the protein and mRNA expression data, NCAM immunocytostaining in IMR-32 cells revealed that ASH-WEX was not able to recover cells from glutamate-induced changes.


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 NCAM in RA differentiated C6 (a) and IMR-32 (d) cells, respectively.RT-PCR results for NCAM mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented by histograms. The expression of NCAM in C6 (c) and IMR-32 (f) cells was analysed by immunostaining. “*” 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-g004: Representative Western blots and their densitometry analysis for NCAM in RA differentiated C6 (a) and IMR-32 (d) cells, respectively.RT-PCR results for NCAM mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented by histograms. The expression of NCAM in C6 (c) and IMR-32 (f) cells was analysed by immunostaining. “*” 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: NCAM is a glycoprotein of immunoglobulin (Ig) superfamily, expressed on the surface of neurons and glia cells. It has a role in cell–cell adhesion, neurite outgrowth, synaptic plasticity, neuroprotection and learning and memory. We examined NCAM expression in control and treated groups and found that ASH-WEX treatment caused a minor increase in NCAM expression both in C6 and IMR-32 cells (Fig. 4 a, d). The low dose treatment of glutamate (0.5 mM) led to upregulation of NCAM expression (p<0.05) which was further increased in the high dose glutamate (1 mM) treated cells as seen on the Western blots. ASH-WEX (0.1%) pre-treatment led to normalization of NCAM expression in the low dose glutamate group but its expression remained significantly higher (around 45%) in the high dose treatment group (Fig. 4a,d). These changes were also apparent at mRNA level. Lower dose of glutamate (0.5 mM) exposure led to increase in NCAM mRNA level that was normalized by ASH-WEX in C6 cells (Fig. 4b). On the other hand high dose treatment group did not show normalization of NCAM mRNA in C6 cells when pretreated with ASH-WEX. Furthermore, ASH-WEX did not cause any recovery in NCAM mRNA expression in both low and high dose glutamate groups of IMR-32 (Fig. 4e). Immunocytostaining for NCAM was enhanced upon glutamate exposure in case of C6 cells as well as in IMR-32 cells (Fig. 4 c, f). ASH-WEX pre-treatment induced normalization was evident by NCAM staining. Consistent with the protein and mRNA expression data, NCAM immunocytostaining in IMR-32 cells revealed that ASH-WEX was not able to recover cells from glutamate-induced changes.

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