白介素6在痤疮囊肿皮损中的表达及痤疮丙酸杆菌体外对THP-1细胞白介素6水平的影响

Expression of interleukin-6 in cystic lesions of patients with acne vulgaris and in vitro effect of Propionibacterium acnes on the production of interleukin- 6 by human THP- 1 monocytes

目的 检测寻常痤疮患者囊肿皮损中白介素6（IL-6）的表达，并观察痤疮丙酸杆菌体外对人急性单核细胞白血病细胞系THP-1信号分子p38MAPK及白介素6水平的影响。方法 实时荧光定量PCR检测6例痤疮患者囊肿皮损和6例健康人皮肤组织中IL-6 mRNA表达水平。用2 × 106、2 × 107、2 × 108 CFU/ml灭活痤疮丙酸杆菌菌悬液或脂多糖（100 μg/L）刺激THP-1细胞，同时设培养基对照组和p38MAPK抑制剂SB203580组（先用20 μmol/L SB203580处理，再用2 × 108 CFU/ml痤疮丙酸杆菌刺激），作用不同时间（1 ~ 6 h）后，实时荧光定量PCR检测IL-6 mRNA表达水平，酶联免疫吸附试验检测上清液中IL-6含量。2 × 108 CFU/ml灭活痤疮丙酸杆菌菌悬液刺激THP-1细胞15、30、60 min或脂多糖（100 μg/L）刺激30 min后，Western印迹法检测p38MAPK和磷酸化p38MAPK表达水平。结果 痤疮囊肿皮损和健康对照皮肤IL-6 mRNA水平分别为3.680 ± 0.790、1.155 ± 0.250，两组比较差异有统计学意义（t = 3.047，P 〈 0.05）。双因素方差分析显示，不同浓度痤疮丙酸杆菌组、脂多糖组、培养基对照组IL-6 mRNA表达水平差异有统计学意义（F = 532.3，P 〈 0.001，ν = 4），痤疮丙酸杆菌刺激1、3、6 h之间差异也有统计学意义（F = 526.6，P 〈 0.001，ν = 2）。2 × 108 CFU/ml痤疮丙酸杆菌组、脂多糖组、培养基对照组上清液中IL-6浓度分别为（1 618.22 ± 32.23）、（3 212.06 ± 353.00）、（147.10 ± 0.53） ng/L，3组间差异有统计学意义（ν = 2，F = 102.35，P 〈 0.01）。2 × 108 CFU/ml痤疮丙酸杆菌作用于THP-1细胞15、30、60 min后磷酸化p38MAPK蛋白水平升高。SB203580组THP-1细胞IL-6 mRNA水平与未抑制组比较明显降低（t = 15.91，P = 0.004）。结论 寻常痤疮患者囊肿中IL-6 mRNA水平显著升高。痤疮丙酸杆菌体外可激活人THP-1细胞信号分子p38MAPK，促进其分泌IL-6。

Objective To determine the of interleukin-6（IL-6） in cystic lesions of patients with acne vulgaris, and to evaluate the in vitro effect of Propionibacterium acnes （P. acnes） on the production of IL-6 and activation of p38 mitogen-activated protein kinase （p38MAPK） in the human acute monocytic leukemia cell line THP-1. Methods Real-time fluorescence-based quantitative PCR was performed to determine the mRNA of IL-6 in cystic lesions of 6 patients with acne vulgaris, as well as in skin tissues of 6 healthy persons. Some cultured THP-1 cells were divided into 5 groups to be treated with 2 × 106 CFU/ml, 2 × 107 CFU/ml and 2 × 108 CFU/ml heat-killed P. acnes suspensions （P. acnes groups）, 100 μg/L lipopolysaccharide （LPS group） and RPMI 1640 medium （control group） respectively. After 1-, 3- and 6-hour treatment, real-time fluorescence-based quantitative PCR was conducted to determine the mRNA of IL-6 in the above groups. Enzyme-linked immunosorbent assay （ELISA） was performed to detect the level of IL-6 in the culture supernatant of cells in the 2 × 108-CFU/ml P. acnes group, LPS group and control group at 24 hours after the treatment. Western blot analysis was conducted to determine the protein of p38MAPK and phosphorylated p38MAPK in the 2 × 108-CFU/ml P. acnes group after 15-, 30- and 60-minute treatment, as well as in the LPS group after 30-minute treatment and in the control group. Some other THP-1 cells were divided into 3 groups： 2 × 108-CFU/ml P. acnes group treated with 2 × 108 CFU/ml P. acnes suspensions, SB203580 （an inhibitor of p38MAPK） group treated with 20 μmol/L SB203580 for 30 minutes followed by the treatment with 2 × 108 CFU/ml P. acnes suspensions, and control group treated with RPMI 1640 medium alone. After 6-hour treatment, the mRNA of IL-6 in the above 3 groups was measured by real-time fluorescence-based quantitative PCR. Results The mRNA of IL-6 was significantly higher in the cystic lesions of acne vulgaris than in the normal skin tissues （3.680 ± 0.790 vs. 1.155 ± 0.250, t = 3.047, P 〈 0.05）. Two-way analysis of variance showed that there were significant difference in the mRNA of IL-6 among the 2 × 106-CFU/ml, 2 × 107-CFU/ml and 2 × 108-CFU/ml P. acnes groups, LPS group and control group （F = 532.3, P 〈 0.001, v = 4）, and the mRNA of IL-6 significantly differed among different time points （F = 526.6, P 〈 0.001, v = 2）. There were also significant differences in the IL-6 level in the culture supernatant of cells among the 2 × 108-CFU/ml P. acnes group （[1 618.22 ± 32.23] ng/L）, LPS group （[3 212.06 ± 353.00] ng/L） and control group （[147.10 ± 0.53] ng/L; v = 2, F = 102.35, P 〈 0.01）. After 15-, 30- and 60-minute treatment with 2 × 108 CFU/ml P. acnes suspensions, the protein of phosphorylated p38MAPK obviously increased. The mRNA of IL-6 in THP-1 cells was significantly lower in the SB203580 group than in the 2 × 108-CFU/ml P. acnes group （t = 15.91, P = 0.004）. Conclusions The mRNA of IL-6 evidently increases in the cystic lesions of patients with acne vulgaris. P. acnes can activate the signaling molecule p38MAPK in THP-1 cells, and promote the production of IL-6 by THP-1 cells.