谷氨酰胺增强蛋白O-GlcNAc修饰减轻LPS诱导心肌细胞损伤.docVIP

谷氨酰胺增强蛋白O-GlcNAc修饰减轻LPS诱导心肌细胞损伤 龚俊松，景亮 东南大学附属中大医院 210009 【摘要】目的：本研究观察谷氨酰胺（Gln）诱导热休克蛋白70（HSP70）表达对脂多糖（LPS）刺激乳鼠心肌细胞的保护作用及O位-N-乙酰葡萄糖胺(O-GlcNAc)修饰在Gln诱导HSP70表达信号转导通路中的作用。方法：原代培养SD乳鼠心肌细胞后依据培养基内加入成分分五组：对照组（C组）给予双蒸水；LPS组给予LPS 4ug/ml；GL组给予Gln 5mM +LPS 4ug/ml；GLA组给予Gln 5mM+LPS 4ug/ml+Alloxan 1mM（Alloxan为O位-N-乙酰氨基葡萄糖转移酶(OGT)抑制剂，能减少O-GlcNAc修饰水平）；GLP组给予Gln 5mM+LPS 4ug/ml+PUGNAc 100uM（PUGNAc为O位-N-乙酰氨基葡萄糖苷酶(OGA)抑制剂，能增加O-GlcNAc修饰水平）。上述5组心肌细胞孵育6h后，以台盼蓝拒染法测定细胞生存率；比色法测定细胞培养液LDH水平(反映心肌细胞损伤情况)；Western Blotting分析心肌细胞HSP70蛋白水平、胞核热休克因子-1（HSF-1）蛋白水平和O-GlcNAc修饰水平；电泳迁徙率变动实验（electrophoretic mobility shift assay，EMSA）测定心肌细胞核HSF-1转录活性。计量资料以均值(标准差(±s)表示，组间数据采用单因素方差分析，以P＜0.05有统计学意义。结果：各组细胞生存率无显著性变化。与C组相比，LPS组细胞损伤显著增加 (P.05)，但在Gln+LPS组这种心肌细胞损伤得到明显恢复(P.05)。与C组相比，LPS组的心肌细胞O-GlcNAc修饰水平、HSP70蛋白表达、胞核HSF-1蛋白表达及转录活性明显增加(P.05)；Gln+LPS组的O-GlcNAc修饰水平、HSP70蛋白表达、胞核HSF-1蛋白表达及转录活性与LPS组相比更进一步增加(P.05)。Gln在LPS刺激心肌细胞的上述作用可为Alloxan完全抑制（GLA组），也可为PUGNAc所逆转(GLP组)。结论：LPS刺激乳鼠心肌细胞给予Gln治疗，可通过增加O-GlcNAc修饰水平，增加核内转录因子HSF-1水平及转录活性，进而促进HSP70的蛋白表达，减轻细胞损伤。调控O-GlcNAc修饰关键酶的活性可能是脓毒血症治疗的潜在靶点。 【关键词】谷氨酰胺；O位-N-乙酰葡萄糖胺修饰；热休克蛋白70；热休克因子-1 【Abstract】AIMS: To investigate the protective effect of glutamine-induced heat shock proteins(HSP) expression in LPS-treated rat cardiocytes and whether its possible molecular mechanisms are relative to enzyme activities of O-linked β-N-acetylglucosamine (O-GlcNAc) modification pathway. METHODS:Primary cultures of neonatal rat cardiocytes were divided into control (treated with double distilled water), LPS (lipopolysaccharide, 4μg/ml), Gln+LPS (Gln 5mM+LPS 4μg/ml), Gln+LPS+Alloxan(an inhibitor of O-linked β-N-acetyl glucosamine transferase/OGT, 1mM), and Gln+LPS+PUGNAc (an inhibitor of O-linked β-N-acetyl glucosaminidase/OGA, 1μM ) groups. After 6-hour incubation, cell viability was measured by Trypan blue exclusion method and cell necrosis was assessed by detecting the release of lactate dehydrogenase(LDH) in culture medium in all groups, respectively. The levels of HSP70 expression, endonuclear heat