G-CSF augments the neuroprotective effect of conditioned medium of dental pulp stem cells against hypoxic neural injury in SH-SY5Y cells

Abstract

Objectives: Dental pulp stem cells (DPSCs) can differentiate into functional neurons and have the potential for cell therapy in neurological diseases. Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein family shown neuroprotective effect in models of nerve damage.we evaluated the protective effects of G-CSF, conditioned media from DPSCs (DPSCs-CM) and conditioned media from transfected DPSCs with plasmid encoding G-CSF (DPSC-CMT) on SH-SY5Y exposed to CoCl₂ as a model of hypoxia-induced neural damage.

Materials and methods: SH-SY5Y exposed to CoCl₂ were treated with DPSCs-CM, G-CSF, simultaneous combination of DPSCs-CM and G-CSF and finally DPSC-CMT. Cell viability and apoptosis were determined by resazurin (or lactate dehydrogenase (LDH) assay alternatively) and propidium iodide (PI) staining. Western blot analysis was performed to detect changes in apoptotic protein levels. The interleukin-6 and interleukin-10 IL6/IL10 levels were measured with Enzyme-Linked Immunosorbent Assay (ELISA).

Results: DPSCs-CM and G-CSF were able to significantly protect SH-SY5Y against neural cell damage caused by CoCl₂ according to resazurin and LDH analysis. Also, the percentage of apoptotic cells decreased when SH-SY5Y were treated with DPSCs-CM and G-CSF simultaneously. After transfection of DPSCs with G-CSF plasmid, DPSC-CMT could significantly improve the protection. The amount of β-catenin, cleaved PARP and caspase-3 were significantly decreased and the expression of survivin was considerably increased when hypoxic SH-SY5Y treated with DPSCs-CM plus G-CSF according to Western blot. Decreased level of IL-6/IL-10, which exposed to CoCl₂, after treatment with DPSCs-CM indicated the suppression of inflammatory mediators.

Conclusion: Combination therapy of G-CSF and DPSCs-CM improved the protective activity.

Keywords: Cobaltous chloride; Granulocyte colony-stimulating factor; Hypoxia; Stem cells; Transfection.

Figure 1

Effect of CoCl₂ on SH-SY5Y cells viability and DPSCs-CM on SH-SY5Y exposed to CoCl₂. 1×10⁴ cells/well of SH-SY5Y cells were exposed to 0.6 and 1.2 mM conncentration of CoCl₂ for 6, 12, 24 and 48 hr. The cell viability was assesed by resazurin asssay. The value presented as mean ± SD (n=9). ***P<0.001

Figure 2

Protective effect of G-CSF and DPSCs-CM on SH-SY5Y exposed to CoCl₂. 1×10⁴ cells/well were exposed to hypoxic condition by 0.6 mM of CoCl₂ for 24 hr. Cells were treated with DPSCs-CM and different concentration of G-CSF (10, 100, 500 ng/ml) for 24 hr Value presented as mean±SD (n=9), ***P<0.001 in comparison with CoCl₂

Figure 3

Effect of CoCl₂ on secretion of IL-6 and IL-10 in SH-SY5Y cells. The value presented as mean ± SD (n=9), ***P<0.001 in comparison with CoCl₂

Figure 4

Western blot analysis of SH-SY5Y cells to detect the effect of DPSCs-CM and G-CSF on expression of β-catenin, cleaved caspase-3, cleaved PARP, and survivin on hypoxic condition. 1×10⁵ cells/well of SH-SY5Y were exposed to 0.6 mM of CoCl₂ then treated with DPSCs-CM and G-CSF (500 ng /ml) for 24 hr and the expression of Survivin, Cleaved PARP], β-catenin, and Cleaved Caspase-3 was determined by western blot. Value presented as mean ± SD (n=9), **P<0.01, ***P<0.001

Figure 5

a) Investigation of G-CSF-GFP expression in DPSCs transfected with lipofectamin using fluorescent microscopy. All scale bars represent 100 μm. b) LDH release and c) flow cytometery analysis of SH-SY5Y cells treated with DPSCs-CMT and G-CSF in hypoxic condition. 1×10⁵ cells/well of SH-SY5Y were treated with 0.6 mM of CoCl2, DPSCs-CM and G-CSF (10 and 500 ng /ml) for 24 hr and the amount of LDH release was determined. Value presented as mean ± SD (n=9), ***P< 0.001 in comparison with CoCl₂

Figure 6

Schematic representation of protective mechanism of DPSCs-CMT and G-CSF in SH-SY5Y cells exposed to hypoxic condition