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

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.

Figure 1. The morphological changes in C6 (a) and IMR-32 (f) cells were studied using phase contrast images.

Cell viability and toxicity of various concentrations of glutamate was assayed by MTT and LDH assays in RA differentiated C6 (b,c) and IMR-32 (g,h) cells. (d) and (i) histograms represents the relative percentage viability of glutamate and ASH-WEX treated C6 and IMR-32 cells, respectively, as compared to the control cells. (e) and (j) histograms represents the relative LDH activity when the control and ASH-WEX pretreated cells were exposed to different glutamate concentrations. “*” represents the statistical significant difference between all the treatment groups (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.

Figure 2. 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.

Figure 3. 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.

Figure 4. 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.

Figure 5. 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.

Figure 6. Representative Gelatin Zymograms for MMP 2 & 9 from media obtained from different groups of C6 (a) and IMR-32 (b) cells.

The zymograms were analysed using spot –denso method in Alpha Ease software and data was represented as histograms. “*” 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.