Objective: To explore the effects of endotoxin/lipopolysaccharide (LPS) on early apoptosis of human neutrophil through PIM3. Methods: Venous blood samples were collected from a healthy adult volunteer to isolate neutrophils. The neutrophils were divided into control group, LPS group, and LPS+ PIM447 group according to the random number table. No treatment was given to the cells in control group. The cells in LPS group underwent LPS stimulation (1 μL, 1 μg/mL). The cells in LPS+ PIM447 group underwent PIM447 (1 μL, final amount-of-substance concentration of 5 μmol/L) intervention 30 min before having the same LPS stimulation as that in LPS group. After conventional culture for 1 h, the early cell apoptosis rate was determined with flow cytometer; the generation level of reactive oxygen species (ROS) was assessed with dihydrogenrhodamine 123 fluorescent probe staining method; and the level of PIM3 was detected by Western blotting. After conventional culture for 2 h, the cell chemotaxis distance was measured by agarose chemotaxis cell model. The sample numbers of each group in the 4 experiments were all 5. Data were processed with one-way analysis of variance and Student-Newman-Keuls test. Results: (1) The early apoptosis rate of cells in LPS group [(0.891±0.012)%] was close to that in control group [(1.351±0.183)%, P>0.05)]. The early apoptosis rate of cells in LPS+ PIM447 group [(82.057±0.121)%] was higher than that in LPS group (P<0.01). (2) The cell chemotaxis distance of cells in LPS group [(984±5) μm] was significantly shorter than that in control group [(2 241±7) μm, P<0.01]. The cell chemotaxis distance of cells in LPS+ PIM447 group [(1 785±11) μm]was significantly longer than that in LPS group (P<0.05). (3) The generation level of ROS in cells of LPS group was significantly higher than that in control group (P<0.05). The generation level of ROS in cells of LPS+ PIM447 group was significantly lower than that in LPS group (P<0.05). (4) The expression level of PIM3 in cells of LPS group (1.297±0.015) was significantly higher than that in control group (0.789±0.021, P<0.05). The expression level of PIM3 in cells of LPS+ PIM447 group (0.731±0.011) was significantly lower than that in LPS group (P<0.05). Conclusions: LPS stimulation can reduce the early apoptosis of human neutrophils. Pre-intervention with PIM447 can significantly increase the early apoptosis of neutrophils after LPS stimulation, recover the chemotaxis, and inhibit the production of ROS. The mechanism may be related to LPS promoting the expression of PIM3.
目的: 探讨内毒素/脂多糖(LPS)通过PIM3对人中性粒细胞早期凋亡的影响。 方法: 采集1名健康成年志愿者静脉血,分离出中性粒细胞,采用随机数字表法分为对照组、LPS组、LPS+PIM447组。对照组不行任何处理,LPS组采用1 μL 1 μg/mL的LPS刺激,LPS+PIM447组在采用上述LPS刺激的前30 min,用1 μL终物质的量浓度为5 μmol/L的PIM447预干预30 min。常规培养1 h后,采用流式细胞仪检测细胞早期凋亡率,二氢罗丹明123荧光探针法检测细胞的活性氧生成水平,蛋白质印迹法检测细胞内PIM3的表达水平。常规培养2 h后,琼脂糖细胞趋化模型检测细胞趋化距离。以上4个实验每组样本数均为5。对数据行单因素方差分析、SNK检验。 结果: (1)LPS组细胞早期凋亡率[(0.891±0.012)%]与对照组[(1.351±0.183)%]相近(P>0.05),LPS+PIM447组细胞早期凋亡率[(82.057±0.121)%]明显高于LPS组(P<0.01)。(2)LPS组细胞趋化距离[(984±5)μm]明显小于对照组[(2 241±7)μm,P<0.01],LPS+PIM447组细胞趋化距离[(1 785±11)μm]明显大于LPS组(P<0.05)。(3)LPS组细胞活性氧生成水平较对照组明显增加(P<0.05),LPS+PIM447组细胞活性氧生成水平较LPS组明显降低(P<0.05)。(4)LPS组细胞PIM3表达水平(1.297±0.015)较对照组(0.789±0.021)明显增加(P<0.05),LPS+PIM447组细胞PIM3表达水平(0.731±0.011)较LPS组明显降低(P<0.05)。 结论: LPS刺激可以降低人中性粒细胞的早期凋亡,使用PIM447预干预可以明显增加LPS刺激后中性粒细胞的早期凋亡,恢复趋化功能,且很好地抑制活性氧的产生,其机制可能与LPS促进PIM3的表达有关。.
Keywords: Apoptosis; Lipopolysaccharides; Neutrophils; PIM3; Sepsis.