Int J Stem Cells.  2022 May;15(2):144-154. 10.15283/ijsc21093.

BMP‑6 Attenuates Oxygen and Glucose Deprivation-Induced Apoptosis in Human Neural Stem Cells through Inhibiting p38 MAPK Signaling Pathway

• Wang L
• Chen Y
• Wei L
• He J

• KMID: 2529786
• DOI: http://doi.org/10.15283/ijsc21093

Abstract

Background and Objectives
Neural stem cells (NSCs) remain in the mammalian brain throughout life and provide a novel therapeutic strategy for central nervous system (CNS) injury. Bone morphogenetic protein-6 (BMP-6) had shown a protective effect in different types of cells. However, the role of BMP-6 in NSCs is largely unclear. The present study was aimed to investigate whether BMP-6 could protect human NSCs (hNSCs) against the oxygen and glucose deprivation (OGD)-induced cell death.
Methods and Results
Upon challenge with OGD treatment, cell viability was significantly decreased in a time-dependent manner, as indicated by the CCK-8 assay. BMP-6 could attenuate the OGD-induced cell injury in a dose-dependent manner and decrease the number of TUNEL-positive cells. Moreover, BMP-6 markedly weakened the OGD-induced alterations in the expression of procaspase-8/9/3 and reversed the expression of cleaved-caspase-3. Interestingly, noggin protein (the BMP-6 inhibitor) attenuated the neuroprotective effect of BMP-6 in cultured hNSCs. Furthermore, the p38 MAPK signaling pathway was activated by OGD treatment and BMP-6 markedly inhibited the phosphorylation of p38 in a concentration-dependent manner. Pretreatment with noggin abolished the effect of BMP-6 on p38 activation. SB239063, a selective p38 inhibitor, exerted similar effects with BMP-6 in protecting hNSCs against the OGD-induced apoptosis. These results indicated that blocking the phosphorylation of p38 might contribute to the neuroprotective effect of BMP-6 against the OGD-induced injury in hNSCs.
Conclusions
These findings suggested that BMP-6 might be a therapeutic target in the OGD-induced cell death, which provides a novel therapeutic strategy for enhancing host and graft NSCs survival in hypoxic-ischemic brain injury.

Keyword

Human neural stem cells; BMP-6; Oxygen and glucose deprivation; Neuroprotection; p38 MAPK pathway

Figure

• 

  Fig. 1 Culture of hNSCs and establishment of OGD model. Human neural stem cells (hNSCs) were isolated from human fetal cortex and cultured in vitro. Neurospheres, 80∼120 μm in size, formed after 3∼5 d of culture (A) and majority of the cells expressed nestin (B). (C) hNSCs were identified by immunofluorescence double staining for nestin/SOX2. (D) After 5 days of culture in normal differentiation medium, Tuj1‑ and GFAP‑positive cells were detected by immunostaining. (E) hNSCs were exposed to oxygen and glucose deprivation (OGD) for different time (15, 30, 60, 120, or 180 mins) and cell viability was detected using CCK-8 assay. **p<0.01, ***p<0.001 versus normal control (0 min). (F, G) Following 60 min of the OGD treatment, the hypoxic cell was identified by HIF-1α immunostaining, and the number of HIF-1α-positive cells were significantly increased in the OGD group. ***p<0.001 versus control. Scale bars: A∼D and F=50 μm.

• 

  Fig. 2 BMP-6 protects hNSCs against OGD-induced cell death. (A) NSCs were pretreated with a serial concentration of BMP-6 (0, 1, 5, 10, 50, 100 or 200 ng/ml), and CCK-8 assay were performed to determine the cell viability. **p<0.01, ***p<0.001 versus OGD＋0 ng/ml group. (B) Adherent hNSCs were pretreated with BMP‑6 (100 ng/ml), noggin (3 μg/ml) or noggin (3 μg/ml)＋BMP‑6 (100 ng/ml). After OGD treatment, apoptosis was detected by TUNEL staining. Scale bars=100 μm. (C) Quantitative data from three independent experiments (n=3) were shown as the percentage of TUNEL-positive cells in total DAPI-stained cells. **p<0.01, ***p<0.001 versus normal control (ctrl); #p<0.05 versus OGD group, &p<0.05 versus OGD＋BMP-6 group.

• 

  Fig. 3 BMP-6 pretreatment inhibits the expression of apoptotic signal proteins. NSCs were pretreated with BMP‑6 (100 ng/ml), noggin (3 μg/ml) or noggin (3 μg/ml)＋BMP‑6 (100 ng/ml). After exposure to OGD, the apoptotic signal proteins were determined by Western blotting (WB). (A) The representative WB bands were illustrating the expression of cleaved-caspase-3 and procaspase-8/9/3, and β-Actin was used as a reference protein. (B∼E) The ratio of cleaved-caspase-3 to procaspase-3 expression were quantified and expression levels of procaspase-8/9/3 were normalized to β‑actin. Data represents as the mean±standard deviation of at least three independent experiments. *p<0.05, **p<0.01 versus OGD group.

• 

  Fig. 4 BMP-6 suppresses the phosphorylation of p38 MAPK in hNSCs. (A) hNSCs were pretreated with different concentrations of BMP‑6, then cells were subjected to the OGD treatment. Western blotting (WB) was used to determine the expression levels of p‑p38 and p38. β‑actin was used as an internal reference. (B) hNSCs were pretreated with BMP‑6 (100 ng/ml), noggin (3 μg/ml) or noggin (3 μg/ml)＋BMP‑6 (100 ng/ml). After the OGD treatment, p‑p38 and p38 were detected by WB. (C, D) Alterations in the ratio of p‑p38/p38 was quantified. Data are presented as the mean±standard deviation of at least three independent experi-ments. *p<0.05, **p<0.01 and ***p<0.001 versus normal control group (ctrl). #p<0.05, ##p<0.01 versus OGD＋0 ng/ml group.

• 

  Fig. 5 Neuroprotective effect of BMP‑6 is involved in the inactiva-tion of p38 MAPK pathway. hNSCs were pretreated with BMP‑6 (100 ng/ml) or SB239063 (10 μM). After exposure to OGD, the apoptotic cells and apoptotic signal proteins were detected by TUNEL staining and Western blotting, respectively. (A) Representative Western blot bands were illustrating the expression of cleaved-caspase-3 and procaspase-3. (B, C) The ratio of cleaved-caspase-3 to procaspase-3 expression were quantified and expression level of procaspase-3 was normalized to β‑ actin. Data are presented as the means±standard deviation of at least three independent experiments. **p<0.01, ***p<0.001 versus normal control group (ctrl). #p<0.05, ##p<0.01 versus OGD group. (D, E) Representative images and quantitative analysis for TUNEL staining. Data from three independent experiments (n=3) represents the percentage of TUNEL-positive cells from total DAPI-stained cells. Scale bar=50 μm. **p<0.01, ***p<0.001 versus normal control group (ctrl). ##p<0.01 versus OGD group.

Reference

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