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.
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