调补肺肾三法对香烟烟雾提取物和脂多糖刺激A549细胞分泌细胞因子的影响

Effects of nourishing lung and kidney three methods on cytokines of A549 cells induced by CSE and LPS

目的:从调补肺肾三法对香烟烟雾提取物(CSE)和脂多糖(LPS)刺激A549分泌细胞因子变化的影响,揭示调补肺肾三法治疗COPD分子机制。方法:CSE和LPS及两者联合(LC)刺激肺泡上皮细胞A549,每种刺激物下分为正常组(20%正常大鼠血清)、刺激物组(20%正常大鼠血清和5%CSE或75pg/mL LPS,或5%CSE和75pg/mL LPS)、补肺益肾组(BYFS)、补肺健脾组(BFJP)和益气滋肾组(YQZS)(20%相应方药大鼠含药血清和5%CSE或75pg/mL LPS,或5%CSE和75pg/mL LPS),ELISA法检测细胞因子,应用网络工具分析细胞因子的转录因子。结果:与正常组比较,CSE、LPS和LC组A549分泌IL-3和TGF-β减少(P<0.01);CSE组分泌MMP-9增加,分泌IL-8减少(P<0.05);LPS组分泌TIMP-1增加(P<0.05),LC组分泌TNF-α和MMP-9减少(P<0.01,P<0.05)。与CSE组比较,BFYS组分泌IL-1β和PPAP2B增加(P<0.01,P<0.05);BFJP组分泌TNF-α减少(P<0.05),IL-1β和PPAP2B增加(P<0.01,P<0.05);YQZS组分泌L1β增加(P<0.01)。与LPS组比较,BFYS组分泌IL-1β、MMP-9(P<0.01)和PPAP2B增加(P<0.05),分泌TIMP-1、PPAP2B和TNF-α减少(P<0.05);BFJP组分泌TIMP-1和TNF-α减少(P<0.01),分泌IL-1β和SOD增加(P<0.05);YQZS组分泌TIMP-1和TNF-α减少(P<0.05,P<0.01),IL-3、MMP-9和IL-1β分泌增加(P<0.05)。与LC组比较,BFYS组分泌MMP-9、IL-3、SOD、IL-1β、TNF-α、CD36分泌增加(P<0.05);BFJP组分泌MMP9、TGF-β、SOD、IL-1β分泌增加(P<0.05);YQZS组分泌MMP-9、IL-3、SOD、TNF-α、TGF-β增加(P<0.05),3个用药组分泌的TIMP-1皆减少(P<0.01)。结论:CSE和LPS对A549分泌细胞因子影响不同。3个调补肺肾方药对CSE和LPS刺激下分泌细胞因子和转录因子均有一定调控作用,补肺益肾中药在CSE刺激下纠正细胞因子分泌作用较好,益气滋肾中药对LPS与CSE联合状态下调节效果较好。

Objective： To observe effects on cytokines secreted by A549 cells which were stimulated separately with cigarette smoke extract（CSE） and LPS,and reveal molecular mechanism of nourishing lung and kidney（NLK） three methods treatment on COPD.Methods： Monocyte macrophage A549 were stimulated separately with CSE,LPS,and LPS plus CSE（LC）,in which samples were divided into normal group（normal rat serum）,stimulus group（20% normal rat serum with 5%CSE or 75 pg/mL LPS,or 5%CSE and 75pg/mL LPS）,Invigorating Lung and Kidney Prescription group（BYFS）,Nourishing Lung and Spleen Prescription group（BFJP） and Supplementing Qi and Nourishing Kidney group（YQZS）（20% corresponding prescription of NLK three methods serum rat serum with 5%CSE or 75pg/mL LPS,or 5%CSE and 75pg/mL LPS）.The cytokines were detected by ELISA and the transcription factors of cytokines were analyzed with Network tool.Results： Compared with the normal group,the secretion of IL-3 and TGF-β decreased in CSE,LPS and LC groups（P〈0.01）,MMP-9 increased and IL-8 decreased in CSE group（P〈0.05）,TIMP-1 increased in LPS groups（P〈0.05）,while TNF-α and MMP-9 increased in LC group（P〈0.01,P〈0.05）.Compared with the CSE group,IL1B and PPAP2B increased in BFYS and BFJP groups（P〈0.01,P〈0.05）,while TNF-α decreased in BFJP group（P〈0.05） and only increased in YQZS（P〈0.01）.Compared with the LPS group,IL1B,MMP-9（P〈0.01）and PPAP2B（P〈0.05） increased while TIMP-1,PPAP2B and TNF-α decreased in BFYS group（P〈0.05）.IL1B and SOD increased（P〈0.05） and TIMP-1 and TNF-α decreased in BFJP group（P〈0.05,P〈0.01）.IL-3,MMP-9 and IL1B increased（P〈0.05）,at same time,TIMP-1and TNF-α decreased in YQZS group（P〈0.05,P〈0.01）.Compared with the LC group,MMP-9 and SOD increased（P〈0.05） while TIMP-1 decreased（P〈0.01） in BFYS,BFJP and YQZS groups.In addition,IL-3,IL1B,TNF-α,CD36 in BFYS group,MMP-9,TGF-β,SOD,IL1B in BFJP group,and IL-3,SOD,TNF-α,TGF-β in YQZS group all increased（P〈0.05）.Conclusion： There were different effects and interaction on cytokines secretion between CSE and LPS.The three corresponding prescriptions of nourishing lung and kidney methods can regulate cells secretion of cytokines induced by CSE and LPS in some degree,in which effect of BYFS is stronger in CSE group and YQZS is stronger in LPS and LC groups.