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Figure 6 shows the effect of withanolide-A on glutamate- induced changes in expression of JNK, p-38, ERK, and their phosporylated forms as observed in the respective immuno- blots (6A) while Fig. 6b—d represents ratios of densitometric analyses of these proteins. Glutamate exposure leads to sig- nificant increase in phospho-JNK, phospho-p-38, and phospho-ERK 1/2 expression levels in cells as observed by immunoblots (Fig. 6a). Densitometric analysis demonstrated significant increase in phosho-JNK in glutamate-treated cells (85.49 + 4.49%) while as pre-treatment with MK-801 (50.12 + 2.75%) and withanolide-A at concentrations 2.5, 5, 10, and 20 uM have shown marked decrease as

Figure 6 shows the effect of withanolide-A on glutamate- induced changes in expression of JNK, p-38, ERK, and their phosporylated forms as observed in the respective immuno- blots (6A) while Fig. 6b—d represents ratios of densitometric analyses of these proteins. Glutamate exposure leads to sig- nificant increase in phospho-JNK, phospho-p-38, and phospho-ERK 1/2 expression levels in cells as observed by immunoblots (Fig. 6a). Densitometric analysis demonstrated significant increase in phosho-JNK in glutamate-treated cells (85.49 + 4.49%) while as pre-treatment with MK-801 (50.12 + 2.75%) and withanolide-A at concentrations 2.5, 5, 10, and 20 uM have shown marked decrease as

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Fig. 1 Effect of withanolide-A on glutamate-induced toxicity (a). Percentage cell viability by MTT assay (b). Percent viable cells by Trypan blue staining. Data expressed are the mean + SD of three Figure la, b shows the results of cell death assays. MTT assay of withanolide-A in 2.5, 5, and revealed neuroprotective effects 10 uM groups when compared with glutamate a (Fig. la). However, with increase in concentratio 20 uM, no significant change was found on cell viability (d not shown). Additionally, similar results were observed Trypan blue assay (Fig. 1b). The percentage of cell viabi observed was 74.50 + 3.2% and for 2.5 uM 71.15 + 1.9 for 5 uM and 69.9 + 1.3% 70.8 + 1.4 for 20 uM withanolide-A in MTT assay. Trypan b assay revealed percent viable ce 68.4 + 2.0 for 2.5 uM, 70.5 + one group ns beyond ata for MK-801, 70.21 + 1.3 and for 10 uM, ue Is as 73.2 + 2.4% for MK-801, 8 for 5 uM, 69.1 + 2.5% or 10 uM, and 68.2 + 0.8 for 20 uM group. While normalizing the effects of withanolide-A wi h MK-801 with respect to
Fig. 1 Effect of withanolide-A on glutamate-induced toxicity (a). Percentage cell viability by MTT assay (b). Percent viable cells by Trypan blue staining. Data expressed are the mean + SD of three Figure la, b shows the results of cell death assays. MTT assay of withanolide-A in 2.5, 5, and revealed neuroprotective effects 10 uM groups when compared with glutamate a (Fig. la). However, with increase in concentratio 20 uM, no significant change was found on cell viability (d not shown). Additionally, similar results were observed Trypan blue assay (Fig. 1b). The percentage of cell viabi observed was 74.50 + 3.2% and for 2.5 uM 71.15 + 1.9 for 5 uM and 69.9 + 1.3% 70.8 + 1.4 for 20 uM withanolide-A in MTT assay. Trypan b assay revealed percent viable ce 68.4 + 2.0 for 2.5 uM, 70.5 + one group ns beyond ata for MK-801, 70.21 + 1.3 and for 10 uM, ue Is as 73.2 + 2.4% for MK-801, 8 for 5 uM, 69.1 + 2.5% or 10 uM, and 68.2 + 0.8 for 20 uM group. While normalizing the effects of withanolide-A wi h MK-801 with respect to
independent experiments with 6 wells in each treatment group. Asterisk indicates significance with respect to untreated, number sign indicates significance with respect to glutamate treatment. *"'p < 0.001, p< 0.01 Figure 3A shows the effect of withanolide-A on the ROS production as measured by confocal microscopy, Fig. 3B
independent experiments with 6 wells in each treatment group. Asterisk indicates significance with respect to untreated, number sign indicates significance with respect to glutamate treatment. *"'p < 0.001, p< 0.01 Figure 3A shows the effect of withanolide-A on the ROS production as measured by confocal microscopy, Fig. 3B
Fig. 3 Effect of withanolide-A on generation of reactive oxygen species (ROS) in glutamate-induced toxicity. (A) ROS were captured via live cell imaging and then (B) estimated by fluorimetric analysis; a is untreated, b is glutamate only, ¢ is glutamate + MK-801, d is glutamate + withanolide-A 2.5 uM, e is glutamate + withanolide-A 5 uM, f is gluta- mate + withanolide-A 10 uM, g glutamate + withanolide-A 20 1M. Asterisk indicates significance with respect to untreated. Number sign indicates significance with respect to glutamate treatment. Data expressed are the mean (arbitrary units) + SD of three independent experiments with 7 wells in each experiment. “p< 0.001, “p< 0.001, **p < 0.01. (C) Quantification of ROS levels via flow cytometry measurements for effect of withanolide-A on the ROS produc- tion. There was a marked escalation in the generation of ROS shows corresponding histogram of relative florescence units (AU), and Fig. 3C demonstrates flow cytometric
Fig. 3 Effect of withanolide-A on generation of reactive oxygen species (ROS) in glutamate-induced toxicity. (A) ROS were captured via live cell imaging and then (B) estimated by fluorimetric analysis; a is untreated, b is glutamate only, ¢ is glutamate + MK-801, d is glutamate + withanolide-A 2.5 uM, e is glutamate + withanolide-A 5 uM, f is gluta- mate + withanolide-A 10 uM, g glutamate + withanolide-A 20 1M. Asterisk indicates significance with respect to untreated. Number sign indicates significance with respect to glutamate treatment. Data expressed are the mean (arbitrary units) + SD of three independent experiments with 7 wells in each experiment. “p< 0.001, “p< 0.001, **p < 0.01. (C) Quantification of ROS levels via flow cytometry measurements for effect of withanolide-A on the ROS produc- tion. There was a marked escalation in the generation of ROS shows corresponding histogram of relative florescence units (AU), and Fig. 3C demonstrates flow cytometric
relative floursence units. Asterisk indicates significance with respect to untreated. Number sign indicates significance with respect to glutamate treatment. Data expressed are the mean (arbitrary units) + SD of three independent experiments with 7 wells in each experiment. p< 0.001, HH < 0.001, *n < 0.01
relative floursence units. Asterisk indicates significance with respect to untreated. Number sign indicates significance with respect to glutamate treatment. Data expressed are the mean (arbitrary units) + SD of three independent experiments with 7 wells in each experiment. p< 0.001, HH < 0.001, *n < 0.01
withanolide-A. (-actin was used as a loading control. Asterisk indicates significance with respect to untreated. Number sign indicates significance qith respect to glutamate treatment. “'p < 0.001, “*p < 0.01, ED < 0.001
withanolide-A. (-actin was used as a loading control. Asterisk indicates significance with respect to untreated. Number sign indicates significance qith respect to glutamate treatment. “'p < 0.001, “*p < 0.01, ED < 0.001
MAPK family proteins (INK, ERK1/2, and p38), and inhibition of PI3K/ Akt pathway. However, withanolide-A treatment rescued the cells by abat- ing intracellular calcium levels, attenuating excessive generation of ROS, correcting mitochondrial machinery, and invoking activation of PI3K/Akt and inhibition of MAPK family proteins Fig. 7 A schematic diagram illustrating the proposed mechanism by which withanolide-A rescued RA differentiated Neuro2a cells against glutamate- induced toxicity. Glutamate-induced toxicity caused by the over-activation of glutamate receptors resulting in huge influx of Ca”* ions. This accumu: lation of Ca”* results in the generation of ROS, increased levels of pro- apoptotic and decreased levels of anti-apoptotic proteins, activation o!
MAPK family proteins (INK, ERK1/2, and p38), and inhibition of PI3K/ Akt pathway. However, withanolide-A treatment rescued the cells by abat- ing intracellular calcium levels, attenuating excessive generation of ROS, correcting mitochondrial machinery, and invoking activation of PI3K/Akt and inhibition of MAPK family proteins Fig. 7 A schematic diagram illustrating the proposed mechanism by which withanolide-A rescued RA differentiated Neuro2a cells against glutamate- induced toxicity. Glutamate-induced toxicity caused by the over-activation of glutamate receptors resulting in huge influx of Ca”* ions. This accumu: lation of Ca”* results in the generation of ROS, increased levels of pro- apoptotic and decreased levels of anti-apoptotic proteins, activation o!
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