Journal of Developmental Medicine(Electronic Version)  2022, Vol. 10 Issue (4): 250-260 DOI: 10.3969/j.issn.2095-5340.2022.04.002 |
|
|
|
|
|
|
| Effect of bone morphogenetic proteins/Smad1/5/8 pathway on the proliferation of mouse neural stem cell under inositol deficiency |
| Zhang Yan, Yang Aiyun, Li Shen, et al
|
1. Beijing Key Laboratory of Environment and Viral Oncology, Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; 2. Translational Medicine Laboratory, Beijing Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics,Beijing 100020, China
|
|
|
|
Abstract 【Abstract】 Objective To explore the effect of bone morphogenetic protein (BMP) / homologues of
the drosophila protein, mothers against decapentaplegic and the caenorhabditis elegans protein (Smad)
1/5/8 pathway on the proliferation of mouse neural stem cell (mNSC) under inositol deficiency, for further
clarifying the molecular mechanism of embryonic neurodevelopmental abnormalities. Method NE-
4C cell line was selected, divided into control group and 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 150
and 200 mmol/L inositol treatment groups. The cells were treated with lithium carbonate, an inhibitor of
inositol monophosphatase that interfered with inositol synthesis, which were divided into control group and
0.75, 1, 1.25, 1.5, 1.75, 2 and 2.5 mmol/L lithium carbonate treatment groups. The recovery experiment
was carried out with BMP signaling pathway inhibitor LDN-193189, and the cells were divided into control
group and 0.05, 0.1, 0.5, 1, 2, 4 and 8 μmol/L LDN-193189 treatment groups, and divided into control
group, lithium carbonate group, inositol free group, lithium carbonate+LDN-193189 group and LDN-
193189 group respectively, which were cultured for 24 h, the cell activity of each group was detected by
3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) method. Reverse transcriptionpolymerasechain reaction (RT-PCR) was used to detect the expressions of key genes Bmp2, Bmp4, Smad1,Smad5, Smad8, and downstream target gene Runx family transcription factor 2 (Runx2) of BMP signalingpathway. The expressions and localization of key genes of BMP/Smad1/5/8 pathway were detected byWestern blotting and immunofluorescence. Statistical methods performed by analysis of Variance andDunnett's t test. Result ① The cell activities in 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 150, 200mmol/L inositol treatment groups and control group were as follows: (119.1±1.5)%, (101.7±2.6)%,(98.9±1.4)%, (101.5±1.2)%, (97.9±1.5)%, (97.5±1.0)%, (96.5±1.2)%, (97.8±1.1)%, (94.4±1.2)%,(92.9±1.9)%, (82.9±1.5)%, (63.6±1.7)% and (147.8±2.4)%, the cell proliferation ability decreased gradually with the increase of inositol concentration in the culture medium (all P<0.01). The cell activitiesin 1, 1.25, 1.5, 1.75, 2, 2.5 mmol/L lithium carbonate treatment groups and control group were as follows:(119.8±1.6)%, (128.1±1.8)%, (138.5±2.2)%, (114.4±2.3)%, (111.0±1.9)%, (106.3±1.7)% and
(99.5±1.6)%, lithium carbonate significantly promoted cell proliferation (all P<0.05), and the effect on
cell proliferation was the most obvious at 1.5 mmol/L. ② The mRNA levels in lithium carbonate group,
inositol free group and control group were as follows: Bmp2 (2.17±0.26, 19.81±0.92 and 1.00±0.13),
Bmp4 (1.97±0.18, 4.23±0.31 and 1.00±0.27), Smad1 (1.91±0.15, 4.10±0.25 and 1.00±0.37), Smad5
(1.94±0.20, 2.88±0.27 and 1.00±0.28), Smad8 (1.62±0.14, 3.20±0.18 and 1.00±0.13) and Runx2
(1.79±0.13, 5.31±0.49 and 1.00±0.21), which were enhanced in both lithium carbonate group and inositol
free group (all P<0.01). ③ The cell activities in 0.05, 0.1, 0.5, 1, 2, 4, 8 μmol/L LDN-193189 treatment
groups and control group were as follows: (96.5±0.7)%, (80.4±1.6)%, (63.8±1.1)%, (56.9±1.8)%,
(29.3±2.4)%, (4.6±0.2)%, (2.6±0.2)% and (99.4±1.0)%, LDN-193189 inhibited cell proliferation in a dosedependentmanner (all P<0.001). The cell activities in lithium carbonate group, inositol free group, lithium carbonate+LDN-193189 group, LDN-193189 treatment groups and control group were as follows: (122.1±2.0)%,(144.7±2.7)%, (82.6±2.3)%, (66.6±2.0)%, (57.3±2.1)% and (101.2±2.2)%, LDN-193189 could reversethe cell proliferation induced by lithium carbonate or inositol free (all P<0.001). ④ The relative expressions ofphospho-Smad1/5/8 in lithium carbonate group, inositol free group, lithium carbonate+LDN-193189 group, LDN-193189 treatment groups and control group were as follows: 1.30±0.10, 1.62±0.10, 0.59±0.15, 0.11±0.08and 1.00±0.18, which indicated that LDN-193189 could reverse the up-regulation of phospho-Smad1/5/8 proteinexpression under inositol deficiency (P<0.01). ⑤ The results of the immunofluorescence showed that the redfluorescent labeled Runx2 protein was localized in the nucleus. Compared with control group, the expressionsof Runx2 were increased in both lithium carbonate group and inositol free group, almost no expression in LDN-193189 group, and decreased in lithium carbonate+LDN-193189 group. LDN-193189 could reverse the upregulationof Runx2 expression under inositol deficiency. The relative expressions of Runx2 protein in lithiumcarbonate group, inositol free group, lithium carbonate+LDN-193189 group, LDN-193189 treatment groups andcontrol group were as follows: 2.00±0.08, 2.09±0.11, 1.75±0.05, 1.76±0.13 and 1.04±0.10, which indicatedthat inositol deficiency promoted up-regulation of Runx2 expression (P<0.01), and the effect that LDN-193189
reversed the up-regulation of Runx2 expression induced by lithium carbonate was not significant, but P valuewas close to 0.05 (P=0.051). Conclusion Inositol deficiency leads to abnormal activation of BMP/Smad1/5/8pathway, regulates downstream target gene Runx2, and promots abnormal proliferation of neural stem cell.
|
|
Received: 07 March 2022
Published: 29 July 2022
|
|
|
|
|
|
