益肺活血方对香烟提取物诱导巨噬细胞炎症反应的影响

目的探讨益肺活血方对香烟提取物诱导巨噬细胞炎症反应的影响。方法用12-豆蔻酸-13-乙酸佛波醇将U937细胞诱导为巨噬细胞,用1.2、2.4、4.8、9.6 g/L的益肺活血方及5、10、20、50、100 mL/L的香烟提取物处理巨噬细胞,于12、24、48 h用细胞增殖和毒性检测(CCK-8)法测定细胞活性。巨噬细胞分5组:正常细胞组、香烟提取物组、香烟提取物+益肺活血方低、中、高浓度组,酶联免疫吸附测定(ELISA)检测培养上清白细胞介素-8(IL-8)和肿瘤坏死因子-α(TNF-α)含量,RT-PCR法检测胞内IL-8和TNF-αmRNA水平。机制实验分4组:正常细胞组、香烟提取物组、香烟提取物+益肺活血方(2.4 g/L)组及香烟提取物+NF-κB抑制剂组,蛋白印迹法(Western blot)检测胞内核转录因子-κB(NF-κB)p50、p65及p-IκB表达;免疫荧光法观察胞核内NF-κB p65及胞浆内p-IκB表达。结果益肺活血方(9.6 g/L)作用细胞24、48 h后及香烟提取物(100 mL/L)于各时间点均有细胞毒性(P0.05)。与正常细胞组相比,香烟提取物组IL-8及TNF-α表达上调,胞内NF-κB p50、p65及p-IκB表达亦升高(P0.05),NF-κB核转位增加(P0.05);益肺活血方可抑制诱导后的IL-8和TNF-α的表达以及NF-κB活性(P0.05)。结论益肺活血方可减少香烟提取物诱导的巨噬细胞表达IL-8、TNF-α,抑制NF-κB活化可能是其机制之一。

Effects of Yifei Huoxue Fang on the inflammatory response in macrophages induced by cigarette smoke extract

Objective To explore the effects of Yifei Huoxue Fang (YFHXF, lung-invigorating blood-activating formula) on the inflammatory response in macrophages induced by cigarette smoke extract.Methods U937 monocytic cells were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA).These PMA-induced macrophages were then treated with YFHXF at various concentrations (1.2,2.4,4.8,9.6 g/L) or exposed to cigarette smoke extract (CSE)(5,10,20,50,100 mL/L).At 12, 24, and 48 h, viability of the cells was assessed by using the cell counting kit-8 (CCK-8).Macrophages were divided into 5 groups: normal group, CSE group, CSE+ YFHXF low-dose (1.2 g/L) group, CSE+ YFHXF medium-dose (2.4 g/L) group, and CSE+ YFHXF high-dose (4.8 g/L) group.Levels of IL-8 and TNF-α in cell culture supernatant were detected by using enzyme-linked immunosorbent assay (ELISA), and gene expression of IL-8 and TNF-α were also measured with real-time polymerase chain reaction (RT-PCR).For the study of mechanism, macrophages were divided into 4 groups: normal group, CSE group, CSE+ YFHXF (2.4 g/L) group, and CSE+NF-κB inhibifant(parthenolide) group.The expression of nuclear factor-κB (NF-κB) p50/p65 and p-IκB was examined by using Western blot method.Meanwhile, the expression of nucleus p65 and plasma p-IκB in macrophages was also measured by using the immunofluorescent assay.Results Cytotoxicity was present in macrophages at all timing points induced by CSE (100 ml/L) and at 24, and 48 hours after treated with YFHXF (9.6 g/L).Compared with normal cell group, IL-8 and TNF-α of CSE group, and NF-κB p50/p65 and p-IκB expression were all upregulated (P<0.05).NF-κB nuclear translocation was increased (P<0.05).Yifei Huoxue Fang could inhibit the expression of IL-8 and TNF-α induced by CSE, and viability of NF-κB (P<0.05).Conclusion Yifei Huoxue Fang could inhibit the expression of IL-8 and TNF-α induced by CSE, possibly by inhibiting NF-κB activation.