谷氨酰胺在体内／外对巨噬细胞炎症因子分泌的影响

The different effects of glutamine on macrophage cytokines release in vivo and in vitro

目的观察谷氨酰胺（Gln）在体内／外条件下对巨噬细胞炎症反应及热休克蛋白（HsP）表达的影响，探讨G1n在脓毒症时抑制体内炎症反应的机制。方法实验1：将培养的腹腔巨噬细胞株RAw264．7分为Gln0、0．5和8mmol／I。3组，分别于内毒素脂多糖（LPS）刺激后0、1、4、12和24h收集细胞及上清液。实验2：45只昆明小鼠被随机均分为假手术（Sham）组、模型组、Gln组；采用盲肠结扎穿孔术（CLP）制备小鼠脓毒症模型，术后即刻从尾静脉注射Gln0．75g／kg（Gln组）或等量生理盐水（Sham组和模型组）。6h后采血，腹腔灌洗分离巨噬细胞。用酶联免疫吸附法（ELIsA）检测细胞上清液、血清、巨噬细胞裂解液中肿瘤坏死因子-a（TNF—a）、白细胞介素-6（IL-6）、IL-10浓度；用蛋白质免疫印迹法检测巨噬细胞HSP72蛋白表达。结果体外条件下G1n可显著促进RAw264．7细胞释放TNF—a、IL-6和IL-10，呈剂量和时间依赖性（P〈O．05或P〈0．01）；LPS刺激4h后，Gln8mmol／L组RAW264．7细胞HSP72表达显著增加（P均〈0．01）。体内条件下，Gln组腹腔巨噬细胞TNF—a、IL-6含量均明显低于模型组（P〈0．01和P〈0．05），3组间IL—10含量比较差异无统计学意义；Gln组血清TNF—a浓度明显低于模型组（P〈0．05），两组血清IL-6、IL-10浓度差异无统计学意义；GIn组巨噬细胞HSP72表达较模型组和Sham组均显著升高（P均〈0．01）。结论G1n体外作用时可显著促进巨噬细胞释放TNF—a和IL-6，且该作用不能被HSP72表达所抑制。体内作用条件下Gln抑制腹腔巨噬细胞合成TNF—a和IL-6。体外和体内条件下Gln都可诱导巨噬细胞表达HSP72，提示HSP72表达可能不是G1n在脓毒症时抑制机体炎症反应的主要机制。

Objective To evaluate the effects of glutamine （Gin） on macrophages cytokines release and heat shock protein 72 （HSP72） expression in vivo and in vitro, and explore the anti-inflammation mechanisms of Gin during sepsis. Methods In experiment one, mouse peritoneal macrophage cell line RAW264. 7 cells were divided into 0, 0. 5 and 8 mmol/L Gin groups, and cells and supernatants were harvested at 0, 1, 4, 12 and 24 hours after lipopolysaccharide （LPS） challenge. In experiment two, forty-five Kunming mice were randomized into sham-operation group （Sham）, sepsis model group, and Gin group. Sepsis was induced by cecal ligation and puncture （CLP）. Either Gin 0.75 g/kg （Gin group） or saline （Sham and model group） was administered immediately after CLP via tail-vein injection. Blood sample was collected at 6 hours, and macrophages were harvested by peritoneal lavage. Tumor necrosis factor-a （TNF-a）, interleukin-6 （IL-6） and IL-10 contents in supernatant, serum and cell lysate were analyzed with enzyme-linked immunosorbent assay （ELISA）, macrophages HSP72 expression was assessed with Western blotting. Results Gin promoted RAW264.7 cells to release TNF-a, IL-6 and IL-10 in a dose-dependent and time-dependent manner in vitro （P〈0.05 or P〈0.01）, and significantly increased HSP72 expression in 8 mmol/L Gin group at 4 hours after LPS stimulation （both P〈 0.01）. In vivo, in animals given Gin intracellular TNF-a and IL-6 levels were significantly lower than sepsis animals （P〈0. 01 and P〈0.05）, but there was no statistically significant difference in intracellular IL-10 levels among three groups. The serum levels of TNF-a in Gin group were significantly lower than in model group （P〈0.05）, while serum IL-6 and IL-10 levels were similar between two groups. Gln treatment led to significant HSP72 expression compared to model and Sham groups （both P〈0. 01）. Conclusion Gln can promote inflammatory cytokines release from macrophages in vitro, which cannot be attenuated by HSP72 expression induced by Gin in LPS challenged RAW264.7 macrophages. Gln treatment significantly decreases intracellular TNF-a and IL-6 levels in vivo during sepsis. HSP72 expression increases after Gln treatment both in vivo and in vitro. These data implicate that HSP72 may not play a major role in attenuating the inflammatory response after Gln administration in sepsis.