The Study Of β-1,4-GalT Ⅰ In Schwann Cells During Inflammation

ObjectiveTo study the expression changed of β-1,4-Galactosyltransferase Ⅰ (β-1,4-GalT Ⅰ) in the Shwann cells stimulated with tumor necrosis factor-alpha (TNF-α), then explore its function and mechanism in Shwann cells’autocrine, proliferation and apoptosis.Methods1. To investigate the role of β-1,4-GalT Ⅰ in TNF-α autocrine, primary Schwann cells were stimulated with TNF-α, we use reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) to detect the secretion of TNF-α, and the expression of β-1,4-GalT Ⅰ were analysis by RT-PCR and Western blot, we also found the activation of ERK, JNK and p38by Western blot, then we transfected Schwann cells with the β-1,4-GalT Ⅰ siRNA and overexpression vector, after that analysing the secretion of TNF-α and the activation of ERK, JNK and p38. Meanwhile we used the TNFR1/2antibodies individually, then the expression of β-1,4-GalT Ⅰ and the secretion of TNF-α were dected.2. To analyses the effect of β-1,4-GalT Ⅰ in Schwann cells proliferation and apoptosis caused by TNF-α, different concentrations (0,0.001,0.01,0.1,1,10,100ng/mL) of recombined rat TNF-α were used, MTT shown the proliferation and TUNEL analysis the apoptosis of Schwann cells, RT-PCR and Western blot shown the expression of β-1,4-GalT Ⅰand TNFR1/2. Then overexpression β-1,4-GalT Ⅰ, detecting the activation of extracellular signal-regulated kinase (ERK1/2), when knocking down β-1,4-GalT Ⅰ expression, analying the activation of p38and JNK. Meanwile we used anti-TNFR1/2antibody individually to detecte the effect of TNFR1/2on Schwann cells proliferation and apoptosis.Results1. Shawnn cells treated with tumor necrosis factor-alpha (TNF-α) could induced the increasing expression of TNF-α mRNA and secretion, we also found the up-regulated expression of the mRNA and protein levels of β-1,4-GalT Ⅰ as well as the activation of ERK, JNK and p38pathways. When knockdown β-1,4-GalT Ⅰ expression suppressed TNF-a secretion stimulated by TNF-α and overexpression β-1,4-GalT Ⅰ could promoted TNF-α production in Shawnn cells. Meanwhile, anti-TNFR1antibody suppressed the expression of β-1,4-GalT Ⅰ and TNF-α autocrine. In Schwann cells with knocking down β-1,4-GalT Ⅰ could suppressed the activation of ERK, JNK and p38pathways induced by TNF-α. And in β-1,4-GalT Ⅰ overexpression Schwann cells the inhibitors of ERK, JNK and p38could prevent the increased of TNF-α autocine.2. Different concentration of TNF-α induced Shawnn cells proliferation and apoptosis, meanwile the expression of β-1,4-GalT Ⅰ and TNFR1/2were changed. When β-1,4-GalT Ⅰ overexpression, low concentration of TNF-α induced Shawnn cells proliferation was partially repressed. Concurrently, the activity of ERK1/2was decreased. While knocking down β-1,4-GalT Ⅰ expression, high concentration of TNF-α-induced Schwann cells apoptosis was partially rescued. Consistent with this, the activity of p38and JNK were decreased. We also found anti-TNFR2antibody suppressed low concentration of TNF-α-induced Shawnn cells proliferation, while anti-TNFR1antibody inhibited high concentration of TNF-α-induced Shawnn cells apoptosis.Conclusions1.β-1,4-GalT Ⅰ increased TNF-α-induced TNF-α secretion in Shawnn cells. β-1,4-GalT Ⅰ conferred its effect by promoting TNFR1and ERK, JNK and p38MAP kinase signal pathways activation induced by TNF-α which led to the induction of TNF-α secretion2.β-1,4-GalT Ⅰ may play an important role in regulating the Shawnn cells proliferation and apoptosis induced by different concentration of TNF-α. Overexpression of β-1,4-GalT Ⅰ repressed Shawnn cells proliferation induced by low concentration of TNF-α via ERK1/2signal pathway and TNFR2, while knocking down β-1,4-GalT Ⅰ expression inhibited Shawnn cells apoptosis induced by high concentration of TNF-α via p38, JNK signal pathways and TNFR1.