Peng Zhi-Yong, Hamiel Christine R, Banerjee Anirban, Wischmeyer Paul E, Friese Randall S, Wischmeyer Paul
Department of Anesthesiology, University of Colorado Health Science Center, Denver, 80262, USA.
JPEN J Parenter Enteral Nutr. 2006 Sep-Oct;30(5):400-6; discussion 406-7. doi: 10.1177/0148607106030005400.
Glutamine (GLN) has been shown to improve outcome after experimental and clinical models of critical illness. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. The heat shock response has been shown to inhibit inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production. This study tested the hypothesis that GLN-mediated activation of the HSP pathway is responsible for improved survival and attenuation of iNOS expression after an inflammatory cytokine-induced injury.
Heat shock factor-1 (HSF-1) wild-type and knockout mouse embryonic fibroblasts (HSF-1+/+ and HSF-1-/-) were used in all experiments. Cells were treated with 0 mmol/L or 8 mmol/L GLN and cytomix (tumor necrosis factor-alpha, lipopolysaccharide, and interferon-gamma) in a concurrent treatment model once they had reached confluence. Cell viability was assayed with MTS/PMS mixture. Apoptosis and necrosis were assayed via immunohistochemistry. iNOS and HSP-70 expression were detected via Western blotting. NO production was measured using the Griess reagent.
GLN treatment significantly attenuated inflammatory cytokine-induced cell death and apoptosis in HSF-1+/+ cells vs 0 mmol/L GLN treatment; however, GLN's cellular protection was lost in HSF-1-/- cells. GLN supplementation attenuated cytomix-induced iNOS expression and NO production only in HSF-1+/+ cells. Further, GLN induced HSP-70 expression only in HSF-1+/+ cells.
This is the first demonstration that GLN-mediated cellular protection after inflammatory cytokine injury is due to HSF-1 expression and cellular capacity to activate an HSP response.
谷氨酰胺（GLN）已被证明可改善危重病实验模型和临床模型的预后。热休克蛋白（HSP）表达增强被认为是这种保护作用的原因。热休克反应已被证明可抑制诱导型一氧化氮合酶（iNOS）基因表达和一氧化氮（NO）生成。本研究检验了以下假设：炎症细胞因子诱导损伤后，GLN介导的HSP途径激活是生存率提高和iNOS表达减弱的原因。
所有实验均使用热休克因子-1（HSF-1）野生型和基因敲除小鼠胚胎成纤维细胞（HSF-1+/+和HSF-1-/-）。细胞汇合后，在同步处理模型中用0 mmol/L或8 mmol/L GLN和细胞混合液（肿瘤坏死因子-α、脂多糖和干扰素-γ）处理。用MTS/PMS混合液检测细胞活力。通过免疫组织化学检测细胞凋亡和坏死情况。通过蛋白质免疫印迹法检测iNOS和HSP-70的表达。使用格里斯试剂测量NO生成量。
与0 mmol/L GLN处理相比，GLN处理显著减轻了炎症细胞因子诱导的HSF-1+/+细胞死亡和凋亡；然而，GLN的细胞保护作用在HSF-1-/-细胞中丧失。补充GLN仅在HSF-1+/+细胞中减轻了细胞混合液诱导的iNOS表达和NO生成。此外，GLN仅在HSF-1+/+细胞中诱导HSP-70表达。
这是首次证明炎症细胞因子损伤后GLN介导的细胞保护作用是由于HSF-1表达和细胞激活HSP反应的能力。
