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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 densometery analysis for PSA-NCAM in RA differentiated C6 (a) and IMR-32 (d) cells, respectively.RT-PCR results for PST mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented by histograms. The expression of PSA-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-g005: Representative Western blots and their densometery analysis for PSA-NCAM in RA differentiated C6 (a) and IMR-32 (d) cells, respectively.RT-PCR results for PST mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented by histograms. The expression of PSA-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: The polysialylated neuronal cell adhesion molecule (PSA-NCAM) is considered as a marker of developing and migrating neurons and of synaptogenesis in the immature vertebrate nervous system. However, it persists in the mature normal brain in some regions which retain a capability for morphofunctional reorganization throughout life. We examined PSA-NCAM in control and treated groups and found that glutamate exposure led to an increase in the PSA-NCAM expression by about 25% at low glutamate dose both in C6 and IMR-32 cells which was further enhanced in ASH-WEX pre-treatment group in the IMR-32 cells (Fig. 5 a,d). The PSA-NCAM was around 15% (p<0.05) higher at high dose glutamate treatment group in C6 cells as compared to control. ASH-WEX pretreated group did not show any significant change (Fig. 5a). In contrast, there was a dose dependent increase in PSA-NCAM expression in the IMR-32 cells from 15–45% that was further enhanced in the ASH-WEX pretreatment (Fig. 5d). The expression of polysialyltransferase (PST) mRNA was examined by RT-PCR and was found to be significantly increased both in glutamate and ASH-WEX treatment groups as compared to control (Fig. 5 b,e). Immunocytostaining revealed that PSA-NCAM expression was enriched along the projections of the differentiated cells in the control group that was further enhanced by low glutamate treatment both in the C6 and IMR-32 cells. High dose glutamate led to disruption of surface expression of PSA-NCAM both in C6 and IMR-32 cells (Fig. 5 c,f).


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 densometery analysis for PSA-NCAM in RA differentiated C6 (a) and IMR-32 (d) cells, respectively.RT-PCR results for PST mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented by histograms. The expression of PSA-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-g005: Representative Western blots and their densometery analysis for PSA-NCAM in RA differentiated C6 (a) and IMR-32 (d) cells, respectively.RT-PCR results for PST mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented by histograms. The expression of PSA-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: The polysialylated neuronal cell adhesion molecule (PSA-NCAM) is considered as a marker of developing and migrating neurons and of synaptogenesis in the immature vertebrate nervous system. However, it persists in the mature normal brain in some regions which retain a capability for morphofunctional reorganization throughout life. We examined PSA-NCAM in control and treated groups and found that glutamate exposure led to an increase in the PSA-NCAM expression by about 25% at low glutamate dose both in C6 and IMR-32 cells which was further enhanced in ASH-WEX pre-treatment group in the IMR-32 cells (Fig. 5 a,d). The PSA-NCAM was around 15% (p<0.05) higher at high dose glutamate treatment group in C6 cells as compared to control. ASH-WEX pretreated group did not show any significant change (Fig. 5a). In contrast, there was a dose dependent increase in PSA-NCAM expression in the IMR-32 cells from 15–45% that was further enhanced in the ASH-WEX pretreatment (Fig. 5d). The expression of polysialyltransferase (PST) mRNA was examined by RT-PCR and was found to be significantly increased both in glutamate and ASH-WEX treatment groups as compared to control (Fig. 5 b,e). Immunocytostaining revealed that PSA-NCAM expression was enriched along the projections of the differentiated cells in the control group that was further enhanced by low glutamate treatment both in the C6 and IMR-32 cells. High dose glutamate led to disruption of surface expression of PSA-NCAM both in C6 and IMR-32 cells (Fig. 5 c,f).

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