萝卜硫素对慢性阻塞性肺疾病患者巨噬细胞Toll样受体4/髓样分化因子88信号通路的影响

Effects of sulforaphane on Toll-like receptor 4/myeloid differentiation factor 88 pathway of monocyte-derived macrophages from patients with chronic obstructive pulmonary disease

目的 探讨萝卜硫素对慢性阻塞性肺疾病（简称慢阻肺）患者Toll样受体4（TLR4）、髓样分化因子88（MyD88）和下游炎性因子的影响.方法 选择2012年1月至2013年3月兰州大学第一医院慢阻肺稳定期患者32例为慢阻肺组,同期健康体检者30名为非慢阻肺组,分离外周血单核细胞,诱导形成单核细胞源性巨噬细胞（MDM）.将慢阻肺组MDM分为空白对照组、脂多糖组、萝卜硫素组和萝卜硫素联合脂多糖组（简称联合组）,非慢阻肺组不用任何药物干预,每组3×106个细胞.实时荧光定量PCR和免疫印迹法检测MDM中TLR4、MyD88 mRNA和蛋白表达,酶联免疫吸附试验检测细胞培养上清液中肿瘤坏死因子-α（TNF-α）和IL-6含量.多组间比较采用单因素方差分析和LSD-t检验.结果 空白对照组TLR4、MyD88 mRNA和蛋白表达（3.7±0.5、1.9±0.4和0.45±0.18、1.11 ±0.65）及细胞培养上清液中TNF-α和IL-6浓度[（31±4）和（43 ±5） μg/L]显著高于非慢阻肺组[（1.00、1.00和0.26 ±0.14、0.58 ±0.40、（19±2）和（29±4）μg/L],差异均有统计学意义（t值为2.19～12.11,P＜0.05或P＜0.01）;脂多糖组上述指标[5.5±1.1、3.4±1.6和0.65±0.20、1.66±0.64、（47±4）和（54 ±5）μg/L]显著高于空白对照组,差异均有统计学意义（t值为2.39～11.9,P＜0.05或P＜0.01）;萝卜硫素组上述指标[2.2±0.4、1.0±0.6和0.25±0.09、0.62±0.34、（20±3）和（27±4） μg/L]显著低于空白对照组,差异均有统计学意义（t值为2.13 ～8.46,P＜0.05或P＜0.01）;联合组上述指标[3.2±0.5、1.5±0.8和0.33 ±0.11、0.77 ±0.25、（31±3）和（33±4） μg/L]显著低于脂多糖组,差异均有统计学意义（t值为3.87 ～ 12.24,均P＜0.01）.结论 慢阻肺患者的巨噬细胞TLR4/MyD88信号通路活化,下游炎性因子分泌增加.萝卜硫素可抑制TLR4/MyD88通路,减少下游炎性因子的释放,可能对慢阻肺患者有抗炎作用.

Objective To explore the effects of sulforaphane on Toll-like receptor 4 （TLR4）/ myeloid differentiation factor 88 （MyD88） pathway and its downstream inflammatory cytokines in patients with chronic obstructive pulmonary disease （COPD）.Methods From Jan.2012 to Mar.2013,thirty-two stable COPD patients and thirty healthy donors （non-COPD group） from the First Hospital of Lanzhou University were recruited.The peripheral blood monocytes were isolated and induced to macrophages （monocyte-derived macrophages,MDMs）.The MDMs of COPD patients were divided into a blank control group,a LPS group,a sulforaphane group,a sulforaphane and LPS group （combined group）,while the MDMs from the non-COPD group received no drug intervention.The number of cells in each group was 3 ×106.The mRNA and protein expression of TLR4 and MyD88 were measured with real-time PCR and Western blot.The TNF-α and IL-6 levels in the culture supernatant were measured with ELISA.Oneway ANOVA and LSD-t test were used for statistical analysis.Results The levels of mRNA and protein of TLR4 and MyD88 and the contents of TNF-α and IL-6 in the culture supernatant were higher in the blank control group [3.7 ±0.5,1.9±0.4,0.45 ±0.18,1.11 ±0.65,（31 ±4） and （43 ±5） μg/L] than those in the nonCOPDgroup [1.00,1.00,0.26±0.14,0.58±0.40,（19±2） and （29±4） μg/L] （t=2.19-12.11,P ＜0.05 or P ＜0.01）.After LPS treatment （LPS group）,the above parameters [5.5 ± 1.1,3.4 ± 1.6,0.65 ± 0.20,1.66 ± 0.64,（47 ± 4） and （54 ± 5） μg/L] were increased as compared to those in the blank control group （t =2.39-11.9,P ＜ 0.05 or P ＜ 0.01）,but after sulforaphane treatment （Sulforaphane group）,these parameters [2.2 ± 0.4,1.0 ± 0.6,0.25 ± 0.09,0.62 ± 0.34,（20 ± 3） and （27 ±4） μg/L] were decreased as compared to those in the blank control group （t =2.13-8.46,P ＜ 0.05 or P ＜ 0.01）.Similarly,these parameters in the combined group [3.2 ± 0.5,1.5 ± 0.8,0.33 ± 0.11,0.77 ±0.25,（31 ±3） and （33 ±4） μg/L] were also remarkably decreased as compared to those in the LPS group （t =3.87-12.24,all P＜0.01）.Conclusions The TLR4/MyD88 pathway was activated and its downstream inflammatory cytokines were increased in macrophages from COPD patients.Sulforaphane could inhibit the TLR4/MyD88 pathway and reduce the releasing of downstream inflammatory cytokines,suggesting that sulforaphane may have an anti-inflammatory effect in COPD.