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目的探讨香烟烟雾浓缩物(CSC)对人类支气管上皮细胞系(16-HBE)表达4-羟基壬烯醛(4-HNE)的影响以及银杏内酯 B 的干预作用;并观察4-HNE 对中性粒细胞的趋化作用。方法采用免疫细胞化学和 Western blot 方法。(1)不同浓度(1、10μg/ml CSC)CSC 刺激16-HBE 细胞4h后4-HNE 加成蛋白的表达水平,设正常对照组(无血清培养基代替 CSC);(2)10μg/ml CSC 刺激不同时间(1、4、8、12、24、30h)后,16-HBE 中4-HNE 加成蛋白的水平,设正常对照组(无血清培养基代替 CSC);(3)用100μmol/L 银杏内酯 B 孵育16-HBE 后,CSC 刺激1、4、8、12h,检测4-HNE 加成蛋白表达的变化,设正常对照组(无银杏内酯 B 孵育);(4)以趋化实验检测0.1、1、10μmol/L 4-HNE 对兔中性粒细胞的趋化作用。结果(1)1、10μg/ml CSC 刺激4h 后,16-HBE 细胞表达4-HNE 加成蛋白水平(免疫化学为2.12±0.38、2.69±0.42,Western blot 为100.2±6.3、72.3±6.1)均较正常对照组显著增加(免疫化学为1.25±0.37,Western blot 为122.4±4.2,P 均<0.01)。(2)10μg/ml CSC 刺激1、4、8、12、24、30h后,16-HBE 细胞表达4-HNE 加成蛋白(免疫化学为2.67±0.46、2.69±0.42、2.71±0.48、2.72±0.56、2.93±0.11、2.92±0.20,Western blot 为73.2±8.3,72.3±6.4,72.6±9.2,71.5±8.1,54.4±3.6,56.7±4.4)较正常对照组(免疫化学为1.25±0.35,Western blot 为122.4±4.1)显著增加(P 均<0.01),刺激24h 和30h,细胞内4-HNE 加成蛋白较其他时间点增加。(3)50、100μmol/L 银杏内酯 B 孵育后再以 CSC 刺激时,16-HBE 细胞内4-HNE 加成蛋白表达水平(Westernblot 为84.6±4.4、101.2±4.4)明显低于未以内酯 B 孵育组,但高于正常对照组(Western blot 为72.5±6.4,P 均<0.01)。(4)0.1、1、10μmol/L 4-HNE 对兔中性粒细胞趋化数目(92±12、104±16、131±12)较正常对照组(72±12)均显著增加,10 μmol/L 4-HNE 趋化数目显著高于0.1和1μmol/L4-HNE,差异均有统计学意义(P 均<0.01)。结论香烟所引起肺中性粒细胞趋化的结果可能与其促使4-HNE 产生增加有关,银杏内酯 B 可以抑制 CSC 对4-HNE 的诱生。
Objective To investigate the effect of cigarette smoke concentrate (CSC) on the expression of 4-hydroxynonenal (4-HNE) in human bronchial epithelial cell line (16-HBE) and the intervention of ginkgolide B; The chemotaxis of granulocytes. Methods Immunocytochemistry and Western blot methods were used. (1) 4-HNE-adjuvant protein expression levels in 16-HBE cells stimulated with CSC at different concentrations (1, 10 μg/ml CSC) for 4 h, set as normal control group (serum-free medium instead of CSC); (2) 10 μg/ml CSC different times after stimulation (1,4,8,12,24,30h), 16-HBE addition of 4-HNE protein levels, normal control group (instead of serum free media CSC); (. 3) with 100μmol after / L of ginkgolide B was incubated 16-HBE, CSC stimulation 1,4,8,12h, protein expression was detected by the addition of 4-HNE, normal control group (no incubation ginkgolide B); (4) Chemotaxis assays were used to detect the chemotaxis of rabbit neutrophils by 0.1,1,10 μmol/L 4-HNE. Results (1) 1 and 10 μg/ml CSCs stimulated for 4 h, 16-HBE cells expressed 4-HNE adipin level (immunochemistry was 2.12±0.38, 2.69±0.42, Western blot was 100.2±6.3, 72.3±6.1). Compared with the normal control group, there was a significant increase (immunochemistry 1.25±0.37, Western blot 122.4±4.2, P<0.01). (2) 10μg / ml CSC after stimulation 1,4,8,12,24,30h, 16-HBE cells express 4-HNE protein adduct (Immunochemistry of 2.67 ± 0.46,2.69 ± 0.42,2.71 ± 0.48,2.72 ± 0.56,2.93 ± 0.11,2.92 ± 0.20, Western blot was 73.2 ± 8.3,72.3 ± 6.4,72.6 ± 9.2,71.5 ± 8.1,54.4 ± 3.6,56.7 ± 4.4) compared with normal control group (immunochemistry was 1.25 ± 0.35, Western Significantly increased (12.4%±4.1.4%) of blots (P<0.01). At 24h and 30h, intracellular 4-HNE addition protein increased compared with other time points. (3) When the 50 and 100 μmol/L ginkgolide B was incubated with CSC, the 4-HNE-adjuvant protein expression level in Western blot (Western blot is 84.6±4.4, 101.2±4.4) was significantly lower than that of lactone-free lactone. The B group was incubated but higher than the normal control group (Western blot, 72.5±6.4, P<0.01). (4) 0.1, 1, 10μmol/L 4-HNE significantly increased the number of neutrophil chemotaxis (92±12, 104±16, 131±12) in rabbits compared with the normal control group (72±12), 10 μmol The number of chemotaxis of /L 4-HNE was significantly higher than that of 0.1 and 1 μmol/L4-HNE, and the difference was statistically significant (P <0.01). Conclusion Cigarette-induced neutrophil chemotaxis may be related to the increase of 4-HNE production. Ginkgolide B can inhibit the induction of 4-HNE by CSC.