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. 2023 Mar 3;13(3):438.
doi: 10.3390/brainsci13030438.

The Inhibition Effects of Sodium Nitroprusside on the Survival of Differentiated Neural Stem Cells through the p38 Pathway

Lingling Jiao  1 , Tongying Xu  1 , Xixun Du  1 , Xi Chen  1 , Qian Jiao  1 , Hong Jiang  1   2
Affiliations

The Inhibition Effects of Sodium Nitroprusside on the Survival of Differentiated Neural Stem Cells through the p38 Pathway

Lingling Jiao et al. Brain Sci. .

Abstract

Nitric oxide (NO) is a crucial factor in regulating neuronal development. However, certain effects of NO are complex under different physiological conditions. In this study, we used differentiated neural stem cells (NSCs), which contained neural progenitor cells, neurons, astrocytes, and oligodendrocytes, to observe the physiological effects of sodium nitroprusside (SNP) on the early developmental stage of the nervous system. After SNP treatment for 24 h, the results showed that SNP at 100 μM, 200 μM, 300 μM, and 400 μM concentrations resulted in reduced cell viability and increased cleaved caspase 3 levels, while no significant changes were found at 50 μM. There were no effects on neuronal differentiation in the SNP-treated groups. The phosphorylation of p38 was also significantly upregulated with SNP concentrations of 100 μM, 200 μM, 300 μM, and 400 μM, with no changes for 50 μM concentration in comparison with the control. We also observed that the levels of phosphorylation increased with the increasing concentration of SNP. To further explore the possible role of p38 in SNP-regulated survival of differentiated NSCs, SB202190, the antagonist of p38 mitogen-activated protein kinase, at a concentration of 10 mM, was pretreated for 30 min, and the ratio of phosphorylated p38 was found to be decreased after treatment with SNP. Survival and cell viability increased in the SB202190 and SNP co-treated group. Taken together, our results suggested that p38 is involved in the cell survival of NSCs, regulated by NO.

Keywords: differentiation; neural stem cells; sodium nitroprusside; survival.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Culture and identification of NSCs. (A) Cells in spheres were nestin positive. (B) The β-tubulin III positive cells and GFAP positive cells were observed after 7 days of culture in the differentiation medium. Scale bars = 20 μm.
Figure 2
Figure 2
The effects of SNP on the survival of differentiated NSCs. (A) The survival and morphological changes of differentiated NSCs after SNP treatment. Scale bar = 100 μm. (B) Cell survival of differentiated NSCs after SNP treatment. (C) Expressions of caspase 3 and cleaved caspase 3 in differentiated NSCs. (D) Data analysis of the ratio of cleaved caspase 3/total caspase 3. The values of the ratio of cleaved caspase 3/total caspase 3 in SNP-treated groups were normalized by the control. All data were obtained from three independent experiments and more than three replicates for every experiment. The values are the mean ± SD and were analyzed via a one-way ANOVA test. Compared with control * p < 0.05, ** p < 0.01, *** p < 0.001; compared with the SNP 50 μM group # p < 0.05, ### p < 0.001; compared with the SNP 100 μM group ^ p < 0.05, ^^ p < 0.01; compared with the SNP 200 μM group; compared with the SNP 300 μM group, n = 5.
Figure 3
Figure 3
Effects of p38 on the survival of differentiated NSCs induced by SNP. (A,B) The expression of p38 and p-p38 of differentiated NSCs after SNP treatment. All data were obtained from three independent experiments and more than three replicates for every experiment. The values are the mean ± SD and were analyzed via a one-way ANOVA test. Compared with control * p < 0.05, *** p < 0.001; compared with the SNP 50 μM group # p < 0.05, ### p < 0.001, n = 3. (C,D) Expressions of p38 and p-p38 were detected after SNP and SB202190 treatment. (E) The survival and morphological changes of differentiated NSCs after SNP and SB202190 treatment. Scale bar = 100 μm. (F) Cell survival of differentiated NSCs after SNP and SB202190 treatment. All data of D and F were obtained from three independent experiments and more than three replicates for every experiment. The values are the mean ± SD and were analyzed via t-test. Compared with control *** p < 0.001; compared with the SNP 200 μM group ### p < 0.001; compared with the SB202190 group ^^^ p < 0.001, n = 5.
Figure 4
Figure 4
The effects of SNP on the differentiation of NSCs. (A) Immunocytochemistry staining of β-tubulin III positive cells. Scale bar = 50 μm. (B) Data analysis of the percentage of β-tubulin III positive cells, n = 5. (C) The expression of β-tubulin III was tested by Western blots. (D) Data analysis of the ratio of β-tubulin III/β-actin. All data were obtained from three independent experiments and three replicates for every experiment. The values are the mean ± SD and were analyzed via a one-way ANOVA test, n = 3.
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