谷氨酰胺对RAW264.7细胞HSP 72表达和TNF-α释放的影响

Effects of glutamine on HSP 72 expression and TNF-α production of RAW264.7 macrophages

目的探讨不同条件下谷氨酰胺(Gln)对小鼠腹腔巨噬细胞株RAW264.7热休克蛋白(HSP)72表达和肿瘤坏死因子(TNF)-α释放的影响。方法将RAW264.7细胞与含Gln(0、0.5和8 mmol/L)的培养液分别进行下列处理:①共同培养165 min后,加入脂多糖(LPS)刺激0、1、4、12和24 h;②共同培养的同时行热应激预处理45 min,37℃培养2 h后加入LPS刺激4 h;③共同培养的同时行DON预处理,165 min后加入LPS刺激4 h;④两者与含LPS的培养液共同培养1 h,改用含0.5 mmol/L Gln和LPS的培养液继续培养4 h。ELISA法检测细胞上清液TNF-α的浓度,Western blotting检测细胞HSP72蛋白表达。结果①LPS刺激后4 h,RAW264.7细胞均有HSP 72表达,经8 mmol/L Gln培养者较经0和0.5 mmol/L Gln培养者HSP 72表达显著增加(P<0.01),而24 h时三者HSP 72表达差异无统计学意义(P>0.05);Gln促进TNF-α释放,与Gln呈时间和浓度依赖关系。②热应激显著促进Gln诱导的HSP 72表达(P<0.01),抑制Gln诱导的TNF-α释放,但TNF-α释放仍随Gln浓度增加而增加。③DON预处理不影响HSP 72的表达,但显著抑制TNF-α释放(P<0.01)。④短时间不同浓度Gln处理,与0.5和8 mmol/L Gln共同培养者较与0 mmol/L Gln共同培养者HSP 72表达显著增加(P<0.01);TNF-α的释放随Gln浓度增加有升高趋势,但三者间差异无统计学意义(P>0.05)。结论Gln可诱导RAW264.7细胞表达HSP 72,但并不足以抑制其TNF-α释放。除诱导HSP 72表达外,Gln在脓毒症时调节机体炎症反应还可能存在其他机制。

Objective To investigate the effects of glutamine （Gln） on heat shock protein （HSP） 72 expression and tumor necrosis factor （TNF）-α production of RAW264. 7 macrophages under different conditions. Methods RAW264. 7 macrophages and culture fluid containing different concentrations of Gln （0, 0.5 and 8 mmol/L） were treated by the following four procedures respectively. ①After co-culture for 165 min and stimulation with LPS, cells and supernatants were harvested at different time points （0, 1, 4, 12 and 24 h）. ②After co-cuhure with sublethal heat stress for 45 min, incubation in 37 ℃ for 2 h and stimulation with LPS for 4 h, cells and supernatants were harvested. ③After co-culture with DON for 165 min and stimulation with LPS for 4 h, cells and supernatants were harvested. ④After culture of RAW264.7 macrophages with culture fluid containing Gln（0, 0.5 and 8 mmol/L） and LPS for 1 h and that containing Gln （0.5 mmol/L） and LPS for another 4 h, cells and supernatants were harvested. TNF-α production in supernatants was determined by ELISA, and HSP 72 expression was detected by Western blotting. Results ①HSP 72 expression was detected in all RAW264.7 macrophages 4 h after LPS stimulation. Compared with 0 and 0.5 mmol/L Gln, there was significant increase in HSP 72 expression under culture with 8 mmol/L of Gln （P 〈 0.01 ）, while there was no significant difference among different concentrations of Gln at 24 h （P 〉 0.05）. The production of TNF-α was increased by Gln in a concentration- and time-dependent manner. ②Heat stress significantly promoted Gln-induced HSP 72 expression （P 〈 0.01） and suppressed Gln-induced TNF-α production, but the production of TNF-α still increased with the concentrations of Gln. ③ DON pretreatment significantly suppressed TNF-① production （P 〈 0.01） with no effect on HSP 72 expression. ④After short-term culture with Gln, HSP 72 expression of those cultured with 0.5 and 8 mmol/L Gln was more significant than that cultured with 0 mmol/L Gln （P 〈 0. 01 ）. TNF-α production increased with Gln concentrations, while there was no significant difference among those cultured with different concentrations of Gln （P 〉 0.05）. Conclusion Gln can increase HSP 72 expression of RAW264. 7 macrophages, while it can not suppress TNF-α production. Besides induction of HSP 72 expression, there may be additional pathways by which Gln modulates inflammatory reaction in macrophages during sepsis.