猪舍空气细颗粒物对猪肺泡巨噬细胞精氨酸代谢和糖酵解的影响

Effects of fine particulate matters(PM_(2.5))from pig house on argininemetabolism and glycolysis in porcine alveolar macrophage

[目的]本试验旨在探究猪舍空气细颗粒物(PM_(2.5))对猪肺泡巨噬细胞极化相关的精氨酸代谢和糖酵解以及TLR4-NF-κB信号通路的影响。[方法]选用猪肺泡巨噬细胞系(3D4/21)作为细胞模型,设置不同质量浓度(0、25、50和100μg·mL^(-1))的PM2.5和不同处理时间(4、8、12、16、20和24 h)处理细胞,收集细胞测定精氨酸代谢、糖酵解以及TLR4-NF-κB信号通路相关指标。[结果]PM_(2.5)处理细胞12 h后,细胞上清液中一氧化氮(NO)浓度随PM2.5处理浓度的升高而逐渐上升,当PM_(2.5)浓度大于50μg·mL^(-1)时,NO浓度极显著升高(P<0.01)。以50μg·mL^(-1)PM_(2.5)处理细胞培养不同时间,发现PM_(2.5)处理16 h内细胞上清液中NO浓度逐渐升高;随着细胞处理时间的延长,PM_(2.5)对细胞的影响逐渐减轻。此外,不同浓度PM_(2.5)处理12 h,细胞内精氨酸酶活性降低,其中,50μg·mL^(-1)PM_(2.5)处理组与对照组相比差异极显著(P<0.01)。同时,随着PM_(2.5)浓度的升高,细胞内乳酸生成量/葡萄糖消耗量(L/G)值升高(P<0.05),标志着细胞内葡萄糖向乳酸转化的比率增高,糖酵解相关基因GLUT-1和GAPDH mRNA表达水平显著提高(P<0.05);100μg·mL^(-1)PM_(2.5)处理显著提高了细胞内IL-1β mRNA表达水平(P<0.01);50μg·mL^(-1)PM_(2.5)处理后M2巨噬细胞标志物CD206 mRNA表达水平显著降低(P<0.05),100μg·mL^(-1)PM_(2.5)显著增加细胞TLR4和MyD88 mRNA表达水平(P<0.05)和p-p65蛋白表达水平(P<0.05),激活TLR4-NF-κB信号通路。[结论]猪舍空气PM_(2.5)能够诱导猪肺泡巨噬细胞发生炎症反应,促进细胞内精氨酸和葡萄糖代谢向M1表型特征的代谢转换。

[Objectives]The present study aimed to explore the effects of fine particulate matters(PM_(2.5))from pig house on polarization-related metabolism including arginine metabolism and glycolysis and the TLR4-NF-κB signaling pathway in porcine alveolar macrophages.[Methods]The experiment used porcine alveolar macrophage cell line(3D4/21)as the cell model,and set different concentrations of PM_(2.5)(0,25,50 and 100μg·mL^(-1))and different treatment time(4,8,12,16,20 and 24 h)for the cell treatment,and the cells were collected to measure arginine metabolism,glycolysis and related indicators of the TLR4-NF-κB signaling pathway.[Results]The results showed that after 12 hours of PM_(2.5) treatment,the concentration of nitric oxide(NO)in the cell supernatant gradually increased with the raising concentration of PM_(2.5).When the PM_(2.5) concentration was higher than 50μg·mL^(-1),the NO concentration reached a significant level as compared with the control group(P<0.01).The cells were cultured for different time with 50μg·mL^(-1) PM_(2.5),the results showed that the concentration of NO in cell supernatant gradually increased within 16 hours treatment,with the prolongation of the cell treatment time,the effect of PM_(2.5) on the cells gradually decreased.Further,the treatment of PM_(2.5) reduced the activity of arginase in the cells,and the 50μg·mL^(-1) treatment of PM_(2.5) showed a significant difference(P<0.01).At the same time,with the raising concentration of PM_(2.5),the value of lactate production/glucose consumption(L/G)was increased(P<0.05),indicating that the ratio of intracellular glucose to lactate conversion increased,and the mRNA expression levels of glycolysis-related genes GLUT-1 and GAPDH significantly increased(P<0.05);100μg·mL^(-1) PM_(2.5) significantly increased the mRNA expression level of IL-1β(P<0.01);and after 50μg·mL^(-1) PM_(2.5) treatment,the mRNA expression level of the M2 macrophage marker CD206 significantly decreased(P<0.05).The TLR4-NF-κB signaling pathway was significantly activated after PM_(2.5) treatment(P<0.05).[Conclusions]These results indicated that PM_(2.5) from pig house promoted the occurrence of inflammatory response in porcine alveolar macrophages,and at the same time promoted the metabolic conversion of intracellular arginine and glucose metabolism to M1 phenotypic characteristics.