Effect of Syncytin-1 overexpression in skeletal muscle cells on co-culture model of spinal cord anterior horn motor neurons, Schwann cells, and skeletal muscle cells / 中华神经医学杂志

ABSTRACT

Objective:To explore the effect of Syncytin-1 overexpression in the skeletal muscle cells on changes of sodium-dependent neutral amino acid transporter 1 (ASCT1), inflammatory factors and neuroprotective factors in co-culture model of spinal cord anterior horn motor neurons, Schwann cells, and skeletal muscle cells.Methods:(1) Spinal cord anterior horn motor neurons, skeletal muscle cells, and Schwann cells were primarily cultured in vitro; the expressions of choline acetyltransferase (ChAT), α-smooth muscle actin (α-SMA) and calcium-binding protein B (S100B) in the neurons, muscle cells, and Schwann cells were detected by immunofluorescence staining, respectively. (2) Plasmids containing Syncytin-1 or control plasmids were transfected into the skeletal muscle cells, respectively; 24 h after that, these transfected skeletal muscle cells were co-cultured with spinal cord anterior horn motor neurons and Schwann cells, respectively (Syncytin-1 recombinant plasmid transfection group or control plasmid transfection group); changes of morphology and junction of co-culture cells were observed under inverted microscope. Forty-eighty h after co-culture, enzyme-linked immunosorbent assay (ELISA) was used to detect the tumor necrosis factor α (TNF-α), inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) concentrations in the supernatant of co-culture cells; real-time quantitative (qRT)-PCR and Western blotting were used to detect the Syncytin-1, ASCT1, TNF-α, iNOS, and VEGF mRNA and protein expressions in the co-culture cells Results:(1) Immunofluorescent staining showed that more than 95% spinal cord anterior horn motor neurons were with positive CHAT expression, more than 95% skeletal muscle cells were with positive α-SMA expression, and more than 95% Schwann cells were with positive S100B expression; all of which were localized in the cytoplasm. (2) There were no obvious differences in number or morphology of co-culture cells between the Syncytin-1 recombinant plasmid transfection group and control plasmid transfection group. As compared with the control plasmid transfection group, Syncytin-1 recombinant plasmid transfection group had significantly increased concentrations of TNF-α, iNOS and VEGF in the supernatant of co-cultured cells, and statistically increased mRNA and protein expressions of TNF-α, iNOS, syncytin-1 and VEGF, and significantly decreased ASCT1 mRNA and protein expressions ( P<0.05). Conclusion:Syncytin-1 overexpression in the skeletal muscle cells may decrease the ASCT1 expression, induce the inflammatory factor release, and increase the neuroprotective factor VEGF expression.