Abstract: Objective　To analyze the effects of three methods for regulating and invigorating the lung and kidney（lung invigorating and kidney tonifying,lung invigorating and spleen strengthening,and Qi supplementing and kidney nourishing） on the neutrophil-released reactive oxygen species（ROS） and neutrophil elastase（NE） stimulated by cigarette smoke extract（CSE） and Lipopolysaccharide（LSP）.Methods　From September 2014 to December 2015,96 conventional Japanese big ear rabbits were fed routinely for 1 week,then they were equally divided into 4 groups（normal group,lung invigorating and kidney tonifying group,lung invigorating and spleen strengthening group,and Qi supplementing and kidney nourishing group） according to random number table method,there were 24 rabbits in each group.Rabbits in lung invigorating and kidney tonifying group,lung invigorating and spleen strengthening group,and Qi supplementing and kidney nourishing group were intragastric administrated with lung invigorating and kidney tonifying,lung invigorating and spleen strengthening,and Qi supplementing and kidney nourishing formulas,respectively,rabbits in normal group were intragastric administrated with the same amount of normal saline.Blood samples are obtained from abdominal aorta after the 7th gavage,and the serum was segregated,then was saved in -70 ℃ for detection.Fresh blood was drawn from healthy male adult volunteers of Henan University of Chinese Medicine,and neutrophils were segregated.Under the stimulating of CSE,neutrophils were divided into normal group（20% normal group rabbit serum was added）,CSE model group（20% normal group rabbit serum and 10% CSE were added）,lung invigorating and kidney tonifying serum group,lung invigorating and spleen strengthening serum group,and Qi supplementing and kidney nourishing serum group（20% rabbit serum containing relevant formula and 10% CSE were added）.Under the stimulating of LPS,neutrophils were divided into normal group（20% normal rabbit serum was added）,LPS model group（20% normal rabbit serum and 2 μg/ml LPS were added）,lung invigorating and kidney tonifying serum group,lung invigorating and spleen strengthening serum group,and Qi supplementing and kidney nourishing serum group（20% rabbit serum containing relevant formula and 2 μg/ml LPS were added）.The activity of ROS and level of NE were detected by flow cytometry instrument and enzyme-linked immunosorbent assay（ELISA）.Results　The activity of ROS and level of NE in CSE and LPS group were significantly higher than those in normal group，respectively （P<0.05）.The activity of ROS in lung invigorating and kidney tonifying serum group,lung invigorating serum group was significantly lower than that in CSE model group,respectively （P<0.05）.While the level of NE significantly decreased in lung invigorating and kidney tonifying serum group,lung invigorating and spleen strengthening serum group,and Qi supplementing and kidney nourishing group （P<0.05）.The activity of ROS in lung invigorating and kidney tonifying serum group，Qi supplementing and kidney nourishing serum group was significantly lower than that in LPS model group （P<0.05）.While the level of NE significantly decreased in lung invigorating and spleen strengthening serum group and Qi supplementing and kidney nourishing serum group （P<0.05）.Conclusion　The serum with three methods of regulating and invigorating the lung and kidney can influence neutrophil-released ROS and NE stimulated by CSE and LPS.Under the stimulating of CSE,the serum containing lung invigorating and kidney tonifying drug is better than others in reducing the activity of ROS and level of NE.Under the stimulating of LPS,the serum containing Qi supplementing and kidney nourishing drug is the best in reducing the activity of ROS and level of NE.

Key words: Drugs,Chinese herbal, Three methods for regulating and invigorating the lung and kidney, Neutrophil, Reactive oxygen species, Neutrophil elastase

摘要： 目的　分析调补肺肾（补肺益肾、补肺健脾、益气滋肾）三方对香烟烟雾提取物（CSE）和脂多糖（LSP）刺激人中性粒细胞释放活性氧（ROS）及弹性蛋白酶（NE）的影响。方法　2014年9月—2015年12月，将常规饲养1周的普通级日本大耳白兔96只按照随机数字表法分为正常组、补肺益肾组、补肺健脾组、益气滋肾组，24只/组。补肺益肾组、补肺健脾组、益气滋肾组大耳白兔分别采用补肺益肾方剂、补肺健脾方剂、益气滋肾方剂灌胃，正常组采用等量的0.9%氯化钠溶液灌胃，4组均于第7次灌胃后取腹主动脉血，分离血清，-70 ℃保存备用。取来自河南中医药大学健康成年男性（年龄20~25岁）志愿者外周血，分离人中性粒细胞。CSE刺激下，将人中性粒细胞分为正常对照组（加20%的正常组兔血清），CSE模型组（加20%的正常组兔血清+10% CSE），补肺益肾血清组、补肺健脾血清组、益气滋肾血清组（分别添加20%相应的含药兔血清+10% CSE）。LPS刺激下，将人中性粒细胞分为正常对照组（加20%的正常组兔血清），LPS模型组（加20%的正常组兔血清+2 μg/ml LPS），补肺益肾血清组、补肺健脾血清组、益气滋肾血清组（分别添加20%相应的含药兔血清+2 μg/ml LPS）。分别采用流式细胞仪和酶联免疫吸附实验（ELISA）法检测各组ROS活性、NE水平。结果　与正常对照组比较，CSE模型组、LPS模型组ROS活性增强，NE水平升高（P<0.05）。与CSE模型组比较，补肺益肾血清组ROS活性减弱（P<0.05），补肺益肾血清组、补肺健脾血清组、益气滋肾血清组NE水平均降低（P<0.05）。与LPS模型组比较，补肺益肾血清组、益气滋肾血清组ROS活性减弱（P<0.05）；补肺健脾血清组、益气滋肾组血清组NE水平降低（P<0.05）。结论　调补肺肾三方含药血清对CSE、LPS刺激下人中性粒细胞释放ROS和NE水平均有影响；CSE刺激下，补肺益肾方含药血清降低ROS活性及NE水平的效果最好；LPS刺激下，益气滋肾方含药血清降低ROS活性氧及NE水平的效果最好。

关键词: 中草药, 调补肺肾三方, 人中性粒细胞, 活性氧, 弹性蛋白酶