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. 2017 Mar 6;7(1):64.
doi: 10.1038/s41598-017-00141-8.

AMD3100 inhibits the migration and differentiation of neural stem cells after spinal cord injury

Jia-Ming Liu  1 , Kai Zhao  1   2 , Liu-Xue Du  1 , Yang Zhou  1 , Xin-Hua Long  1 , Xuan-Yin Chen  1 , Zhi-Li Liu  3
Affiliations

AMD3100 inhibits the migration and differentiation of neural stem cells after spinal cord injury

Jia-Ming Liu et al. Sci Rep. .

Abstract

It was reported that CXCR4 signaling played an important role in the migration and differentiation of endogenous neural stem cells after spinal cord injury (SCI). However, the molecular mechanism of it is still unclear. Here, we established a model of SCI in rats and AMD3100 was used to treat them. The rats were then sacrificed and the injured spinal cord specimens were harvested. Additionally, the neural stem cells (NSCs) line was culture and treated with AMD3100 in vitro. Results showed the locomotor function of SCI rats was worse after treated with AMD3100. And the expression levels of Nestion in neural stem cells and β-tubulin in neuron cells were significantly increased in the injured spinal cord, which can be inhibited by the CXCR4 antagonist of AMD3100. Additionally, the expression of β-catenin and phosphorylase β-catenin protein was significantly down regulated by AMD3100. In vitro, the NSCs proliferation ability was inhibited and the migration was decreased after treated with AMD3100. Also, the expression of Nestion, β-tubulin, β-catenin and phosphorylase β-catenin protein was significantly decreased in AMD3100 group comparing with untreated group. Taken together, this study suggested that AMD3100 could inhibit the migration and differentiation of endogenous neural stem cells in rats with SCI. The mechanism of it maybe that AMD3100 could down regulate of SDF-1/CXCR4 by targeting β-catenin signaling pathway.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
The protein expression of SDF-1 up-regulated in neural stem cells of the lesion site on the 7th day after surgery. (A) Representative image of IHC staining of SDF-1 in sham group (×400). (B and C) Representative images of IHC staining of SDF-1 in untreated and AMD3100 groups (×400). The expression of SDF-1 protein increased in the neural stem cells after SCI. (D) The expression of SDF-1 was significant higher in untreated and AMD3100 groups than those in sham group. (*P < 0.05: untreated group vs. sham group, AMD3100 group vs. sham group).
Figure 2
Figure 2
The BBB scores of the rats during the 28 days period after surgery in the three groups. It showed that the scores were significantly lower in AMD3100 group than those in untreated group on the 7th, 14th and 28th day after SCI. (*P < 0.05).
Figure 3
Figure 3
The proliferation of neural stem cells treated by AMD3100 in vitro. As the time going on, the inhabitation rates of neural stem cells proliferation were gradually increased.
Figure 4
Figure 4
The expression of Nestin protein in neural stem cell after treated with AMD3100. (A) Representative images of IHC staining of Nestin in sham and untreated groups of rats (×40). It showed that the expression of Nestin protein increased at the injured site after SCI in rats comparing with those without SCI, especially in the region around central canal of spinal cord. (B) Representative images of IHC staining of Nestin in the three groups of rats (×400). It showed that the expression of Nestin increased in both of AMD3100 and untreated groups after SCI. (C) The expression of Nestin protein was measured in the three groups of rats. It revealed that the Nestin protein was inhibited by AMD3100. (D) The expression of Nestin protein in RASNF-01001 cells was significantly decreased in AMD3100 group. (F) The wound healing assay showed the migration ability of RASNF-01001 cells was reduced by AMD3100. (*P < 0.05: untreated group vs. sham group, AMD3100 group vs. sham group; # P < 0.05: AMD3100 group vs. untreated group).
Figure 5
Figure 5
The expression of β-tubulin protein in neural stem cell after treated with AMD3100. (A) Representative images of IHC staining of β-tubulin in the three groups of rats (×400). It showed that the expression of β-tubulin increased on the 7th day and then decreased from the 7th to 28th day after surgery. (B) The expression of β-tubulin protein was lower in AMD3100 group of rats than that in untreated group after surgery. (C) The expression of β-tubulin protein in RASNF-01001 cells was significantly lower than that of control group after treated with AMD3100. (*P < 0.05: untreated group vs. sham group, AMD3100 group vs. sham group; # P < 0.05: AMD3100 group vs. untreated group).
Figure 6
Figure 6
The expression of Wnt protein in the three groups of rats after surgery. (A) Representative images of IHC staining of Wnt in the three groups on the 7th day after surgery (×40). (B) Representative images of IHC staining of Wnt in the three groups (×400). It showed that the expression of Wnt protein in AMD3100 and untreated groups increased after surgery. (C) The expression level of Wnt protein was significantly higher in AMD3100 and untreated groups than those in sham group on the 7th, 14th and 28th day after surgery. However, no different was found for Wnt between AMD3100 and untreated groups at the different time. (*P < 0.05: untreated group vs. sham group, AMD3100 group vs. sham group).
Figure 7
Figure 7
The expression of β-catenin in the three groups of rats after surgery. (A) Representative images of IHC staining of β-catenin in the three groups (×400). It showed that the expression of β-catenin increased in both of AMD3100 and untreated groups. (B) The expression of β-catenin protein was measured and the expression of it was significantly lower in AMD3100 group comparing with that in untreated group at the different time after surgery. (*P < 0.05: untreated group vs. sham group, AMD3100 group vs. sham group; # P < 0.05: AMD3100 group vs. untreated group).
Figure 8
Figure 8
The expression of phosphorylase β-catenin protein in the three groups of rats after surgery. (A) Representative images of IHC staining of phosphorylase β-catenin in the three groups (×400). It showed that the expression of phosphorylase β-catenin also increased in both of AMD3100 and untreated groups. (B) The expression level of phosphorylase β-catenin was significantly higher in AMD3100 group than that in untreated group on the 7th, 14th and 28th day after surgery. (*P < 0.05: untreated group vs. sham group, AMD3100 group vs. sham group; # P < 0.05: AMD3100 group vs. untreated group).
Figure 9
Figure 9
The protein expression of Wnt, β-catenin and the phosphorylase of β-catenin in RASNF-01001 cells after treated with AMD3100. The expression levels of β-catenin and the phosphorylase of β-catenin were decreased in AMD3100 group, but the expression Wnt was not significantly changed after treated with AMD3100.
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