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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 HSP70 in RA differentiated C6 (a) and IMR-32 (e) cells, respectively.RT-PCR results for HSP70 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry analysis was represented by histograms. The expression of HSP70 in C6 (c) and 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-g003: Representative Western blots and their densitometry analysis for HSP70 in RA differentiated C6 (a) and IMR-32 (e) cells, respectively.RT-PCR results for HSP70 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry analysis was represented by histograms. The expression of HSP70 in C6 (c) and 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: HSP70 is a member of heat shock protein family and serves as a housekeeper in the cell, assisting in the correct folding, trafficking, and degradation of many proteins during normal and stressed conditions. We examined HSP70 expression in control and ASH-WEX pretreated cells that were challenged with glutamate. As shown in Fig. 3a and e, HSP70 Western blots revealed significant increase after exposure to glutamate in a dose dependent manner in both C6 and IMR-32 cells suggesting that glutamate treatment evoked stress response in these cells. ASH-WEX treatment both in C6 and IMR-32 cells resulted in a moderate induction of HSP70 expression and led to normalization of increase in HSP70 induced by low dose of glutamate (Fig. 3a, e). Upregulation in HSP70 expression (about 60% increase in C6 cells and 40% in IMR-32 cells) induced by higher dose of glutamate was not recovered significantly upon ASH-WEX pre-treatment (Fig. 3a,e). The RT-PCR analysis showed increase (p<0.05) in HSP70 mRNA levels at low dose glutamate treatment group in both cell types; the high dose glutamate did not cause higher induction of HSP70 mRNA (Fig. 3b,f). Furthermore, the immunocytostaining for HSP70 showed enhanced intensity in glutamate treated groups as compared to control (Fig. 3c,g). ASH-WEX pre-treatment resulted in downregulation of HSP70 in low dose glutamate group in both the cell lines; high dose glutamate groups remained unaffected (Fig. 3d,h).


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 HSP70 in RA differentiated C6 (a) and IMR-32 (e) cells, respectively.RT-PCR results for HSP70 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry analysis was represented by histograms. The expression of HSP70 in C6 (c) and 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351387&req=5

pone-0037080-g003: Representative Western blots and their densitometry analysis for HSP70 in RA differentiated C6 (a) and IMR-32 (e) cells, respectively.RT-PCR results for HSP70 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry analysis was represented by histograms. The expression of HSP70 in C6 (c) and 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: HSP70 is a member of heat shock protein family and serves as a housekeeper in the cell, assisting in the correct folding, trafficking, and degradation of many proteins during normal and stressed conditions. We examined HSP70 expression in control and ASH-WEX pretreated cells that were challenged with glutamate. As shown in Fig. 3a and e, HSP70 Western blots revealed significant increase after exposure to glutamate in a dose dependent manner in both C6 and IMR-32 cells suggesting that glutamate treatment evoked stress response in these cells. ASH-WEX treatment both in C6 and IMR-32 cells resulted in a moderate induction of HSP70 expression and led to normalization of increase in HSP70 induced by low dose of glutamate (Fig. 3a, e). Upregulation in HSP70 expression (about 60% increase in C6 cells and 40% in IMR-32 cells) induced by higher dose of glutamate was not recovered significantly upon ASH-WEX pre-treatment (Fig. 3a,e). The RT-PCR analysis showed increase (p<0.05) in HSP70 mRNA levels at low dose glutamate treatment group in both cell types; the high dose glutamate did not cause higher induction of HSP70 mRNA (Fig. 3b,f). Furthermore, the immunocytostaining for HSP70 showed enhanced intensity in glutamate treated groups as compared to control (Fig. 3c,g). ASH-WEX pre-treatment resulted in downregulation of HSP70 in low dose glutamate group in both the cell lines; high dose glutamate groups remained unaffected (Fig. 3d,h).

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