两性霉素B对人THP-1细胞产生肿瘤坏死因子α、白细胞介素8及p38MAPK活化的影响

Effect of amphotericin B on the production of tumor necrosis factor- a and interleukin- 8 and activation of signaling molecule p38MAPK in human THP- 1 cells

目的 探讨两性霉素B对人急性单核细胞白血病细胞系THP-1细胞系分泌肿瘤坏死因子α（TNF-α）和白细胞介素8（IL-8）以及p38丝裂原活化蛋白激酶（MAPK）活化的影响。方法 将THP-1细胞分为空白对照组、两性霉素B组（分别加入2、4、8 mg/L两性霉素B处理）；阳性对照组（用100 μg/L β葡聚糖或者100 mg/L脂多糖处理）。实时荧光定量PCR分析不同刺激物和THP-1细胞作用一定时间后TNF-α、IL-8 mRNA表达水平。酶联免疫吸附试验检测8 mg/L两性霉素B刺激THP-1细胞24 h后上清液中TNF-α分泌量。Western印迹检测8 mg/L两性霉素B作用THP-1细胞后p38MAPK和磷酸化p38MAPK的水平。结果 2、4、8 mg/L两性霉素B刺激6 h，TNF-α mRNA水平分别为7.55 ± 1.17、19.47 ± 2.91、57.22 ± 0.65，与空白对照组比较，差异均有统计学意义（P 〈 0.01、0.001、0.001）。IL-8 mRNA水平分别为2.98 ± 0.04、5.22 ± 1.35、11.82 ± 1.66，与空白对照组比较，差异均有统计学意义（P 〈 0.01、0.001、0.001）。8 mg/L两性霉素B刺激THP-1细胞1、3、6 h后TNF-α mRNA水平分别为8.61 ± 0.30、10.75 ± 0.08、56.98 ± 2.43，与空白对照组比较，差异均有统计学意义（P 〈 0.05、0.01、0.001）。IL-8 mRNA水平分别为2.63 ± 0.28、5.35 ± 0.98、11.73 ± 1.18，与空白对照组比较，差异均有统计学意义（P 〈 0.05、0.01、0.001）。8 mg/L两性霉素B刺激THP-1细胞24 h上清液中TNF-α蛋白水平为（4 039.06 ± 223.87） ng/L，与空白对照组比较差异有统计学意义（P 〈 0.001）。结论 两性霉素B体外可促进人THP-1细胞p38MAPK蛋白磷酸化及TNF-α和IL-8分泌，具有免疫调节作用。

Objective To evaluate the effect of amphotericin B on the production of tumor necrosis factor-α （TNF-α） and interleukin-8 （IL-8） and activation of p38 mitogen-activated protein kinases （p38MAPK） in a human acute monocytic leukemia cell line （THP-1）. Methods Cultured THP-1 cells were divided into several groups： blank control group receiving no treatment, amphotericin B groups treated with 2, 4 and 8 mg/L amphotericin B separately, positive control group treated with 100 μg/L β-glucosan or 100 mg/L lipopolysaccharide. Real-time fluorescence-based quantitative PCR was performed to determine the mRNA of TNF-α and IL-8 after the THP-1 cells were treated with different stimuli for some durations. Enzyme-linked immunosorbent assay （ELISA） was conducted to detect the level of TNF-α in the culture supernatant of THP-1 cells after 24-hour treatment with 8 mg/L amphotericin B, and Western blot analysis to measure the levels of p38MAPK and phosphorylated p38MAPK after 30-minute treatment with 8 mg/L amphotericin B. Results After 6-hour treatment with 2, 4 and 8 mg/L amphotericin B separately, the mRNA levels of TNF-α in THP-1 cells （7.55 ± 1.17, 19.47 ± 2.91, 57.22 ± 0.65） and IL-8 （2.98 ± 0.04, 5.22 ± 1.35, 11.82 ± 1.66） were all significantly higher than those in the blank control group （TNF-α： 1.00 ± 0.07, P 〈 0.01, 0.001, 0.001 respectively; IL-8： 1.01 ± 0.23, P 〈 0.01, 0.001, 0.001 respectively）. After the treatment with 8 mg/L amphotericin B for 1, 3, 6 hours, the mRNA levels of TNF-α （8.61 ± 0.30, 10.75 ± 0.08, 56.98 ± 2.43） and IL-8 （2.63 ± 0.28, 5.35 ± 0.98, 11.73 ± 1.18） in THP-1 cells were all significantly higher than those in the blank control group （TNF-α： 1.18 ± 0.17, P 〈 0.05, 0.01, 0.001; IL-8： 1.23 ± 0.11, P 〈 0.05, 0.01, 0.001）. After 24-hour treatment with 8 mg/L amphotericin B, the level of TNF-α in the culture supernatant of THP-1 cells was significantly higher than that in the blank control group （4 039.06 ± 223.87 ng/L vs. 96.31 ± 0.26 ng/L, P 〈 0.001）. Conclusion Amphotericin B can promote the p38MAPK phosphorylation and increase the levels of TNF-α and IL-8 in human THP-1 cells in vitro, suggesting its immunomodulatory effects.