A study of apoptosis of murine platelet induced by lipopolysaccharide derived fromEscherichia coli in vitro / 中华危重病急救医学

ABSTRACT

ObjectiveTo observe whether lipopolysaccharide (LPS) derived fromEscherichia coli (E.coli) can induce apoptosis of murine platelets in vitro.Methods Washed platelet suspension was prepared and adjusted to the final concentration of 3×108/mL. According to the difference in stimulants, samples were divided into control group (non-calcium Tyrode buffer), thrombin-treated group (1 U/mL final concentration and non-calcium TB) and LPS in different concentrations treated groups (1, 10 and 100μg/mL final concentration respectively and non-calcium TB). To each specimental group corresponding stimulus was added and incubated 30 minutes at room temperature. Chemiluminescence was adopted to determine the concentration of adenosine triphosphate (ATP) and the activity of cysteinyl aspartate specific proteinase-3 (caspase-3). The percentage of Annexin V positive platelets was determined by flow cytometry to reflect the level of phosphatidylserine (PS) exposure. Mean channel fluorescence (MCF) of platelets was determined by flow cytometry for reflecting the level of mitochondrial inner transmembrane potential (ΔΨm) depolarization.Results Compared with control group, the ATP concentration in thrombin-treated group was decreased obviously [relative light unit (RLU) (5.46±0.14)×105 vs. (6.25±0.26)×105,P< 0.05], Annexin V positive ratio [(50.43±2.45)% vs. (1.58±0.25)%,P< 0.05] and caspase-3 activity [RLU (26.92±1.60)×103 vs. (1.30±0.10) ×103,P< 0.05] were increased obviously, and platelets MCF was lowered significantly [(8.32±0.58)×104 vs. (13.05±1.10)×104,P< 0.05], suggesting an increase inΔΨm depolarization. After being treated with different concentrations of LPS, ATP concentration, Annexin V positive ratio and caspase-3 activity were increased obviously, platelet MCF was decreased obviously, suggestingΔΨm depolarization was increased in a concentration-dependent manner. Compared with control group, 1μg/mL LPS could increase Annexin V positive ratio [(10.45±1.08)% vs. (1.58±0.25)%,P< 0.05], elevate caspase-3 activity [RLU (14.06±0.61)×103 vs. (1.30±0.10)×103,P< 0.05], and decrease MCF significantly [(9.48±0.50)×104 vs. (13.05±1.10)×104,P< 0.05]. The ATP concentration, Annexin V positive ratio and caspase-3 activity reached maximum levels after the treatment with 100μg/mL LPS, and they were higher obviously than those of the control group [ATP (RLU) (7.00±0.03)×105 vs. (6.25±0.26)×105, Annexin V positive ratio (55.35±2.42)% vs. (1.58±0.25)%, casepase-3 (RLU) (32.00±3.75)×103 vs. (1.30± 0.10)×103, allP< 0.05], and platelets MCF reached trough levels, and they were obviously lower than those of the control group [(4.69±0.55)×104 vs. (13.05±1.10)×104,P< 0.05].ConclusionE.coli LPS can induce an increase in ATP, PS exposure,ΔΨm depolarization and activity increase of caspase-3 on mouse platelet in vitro, which indicate that LPS can induce apoptosis of platelets in a concentration-dependent manner.