调补肺肾三方对香烟烟雾提取物和脂多糖刺激人中性粒细胞释放活性氧及弹性蛋白酶的影响

秦燕勤，吴耀松，李建生，陈玉龙，毛筱宁，赵丹瑞



·论著·

·中医·中西医结合研究·

调补肺肾三方对香烟烟雾提取物和脂多糖刺激人中性粒细胞释放活性氧及弹性蛋白酶的影响

秦燕勤，吴耀松，李建生，陈玉龙，毛筱宁，赵丹瑞

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。

中草药；调补肺肾三方；人中性粒细胞；活性氧；弹性蛋白酶

秦燕勤，吴耀松，李建生，等.调补肺肾三方对香烟烟雾提取物和脂多糖刺激人中性粒细胞释放活性氧及弹性蛋白酶的影响[J].中国全科医学，2016，19(36)：4515-4519.[www.chinagp.net]

QIN Y Q,WU Y S,LI J S,et al.Effect of three prescription for regulating and invigorating the lung and kidney on neutrophil-released reactive oxygen species and neutrophil elastase stimulated by cigarette smoke extract and lipopolysaccharide[J].Chinese General Practice，2016，19(36)：4515-4519.

炎性反应在慢性阻塞性肺疾病(chronic obstructive pulmonary disease，COPD)的病理进程中起重要作用[1]。炎性反应涉及多种炎性细胞因子，炎症的发生、发展与蛋白酶-抗蛋白酶失衡、氧化-抗氧化损伤等密切相关，且中性粒细胞在其中起重要作用[2-4]。中医治疗COPD具有较好的效果，研究发现调补肺肾三方在改善COPD稳定期临床症状方面疗效显著[5]。为了深入探讨调补肺肾三方的抗炎机制，本研究以香烟提取物(cigarette smoke extract,CSE)和脂多糖(lipopolysaccharide,LPS)作为刺激物，以人中性粒细胞为研究对象，观察调补肺肾三方对人中性粒细胞释放活性氧(reactive oxygen species,ROS)及弹性蛋白酶(neutrophil elastase,NE)的影响。

1 材料与方法

1.1 实验材料

1.1.1 实验药物 补肺益肾方由人参9 g、山茱萸12 g、五味子9 g、陈皮9 g等组成；补肺健脾方由党参15 g、白术12 g、茯苓12 g、炙甘草6 g等组成；益气滋肾方由人参9 g、枸杞子12 g、麦冬15 g、炙甘草6 g等组成。药物均由河南中医药大学第一临床医学院中药制剂与剂型改革基地提供。

1.1.2 主要试剂及仪器 LPS(美国Sigma公司)；RPMI 1640培养基、人外周血中性粒细胞分离液试剂盒(北京索莱宝科技有限公司)；活性氧检测试剂盒(南京凯基生物公司)；人抗中性粒细胞弹性蛋白酶抗体(ANEA)酶联免疫吸附实验(ELISA)试剂盒(Elabscience公司，E-EL-H0383c)。BD FACSCalibur流式细胞仪(美国Becton Dickinson公司)；全自动酶标仪ELx800(美国BIO-TEK宝特)。

1.1.3 血液及实验动物 人外周血均来自河南中医药大学健康成年男性(年龄20～25岁)志愿者。日本大耳白兔96只，普通级，雌雄各半，体质量2.0～2.5 kg，由河南康达实验动物有限公司提供(许可证号：SYXK豫2010-0001)。

1.2 研究时间 2014年9月—2015年12月。

1.3 方法

1.3.1 含药血清制备 所有大耳白兔饲养于河南中医药大学动物实验中心普通级饲养室，每笼1只，室温为(25±2)℃，相对湿度为(50±10)%，噪声≤60 db，通风良好，灭菌饲料喂养，每天更换垫料，保持环境安静。常规饲养以稳定大耳白兔应激状态，饲养1周后称重，按照随机数字表法将大耳白兔分为正常组、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，灌胃剂量以60 kg成人生药量公斤体质量的9倍(兔和人系数为3∶1，再乘血清稀释倍数3)，即按W家兔/W人W家兔/W人×每副生药量×出膏率×9/每毫升含膏量进行计算，正常组大耳白兔采用等量的0.9%氯化钠溶液灌胃，2次/d，每次间隔12 h，于第7次灌胃后1 h取腹主动脉血。取血前大耳白兔禁食12 h，不禁水，静置4 h后以3 000 r/min离心15 min(离心半径15 cm)，分离血清，56 ℃、30 min灭活后过滤除菌，-70 ℃保存备用。

1.3.2 CSE制备 将YANG等[6]研究方法进行改良，采用50 ml注射器以0.75 kg/s的水平拉力连续6次抽吸一支去掉过滤嘴燃烧的香烟，推入盛有10 ml RPMI 1640培养基的负压装置，紫外分光光度计测其320 nm处的OD值，取OD值为1.8～2.0的悬液为100% CSE，0.22 μm滤器过滤除菌，避光保存，2 h内使用。

1.3.3 人中性粒细胞的分离 取志愿者外周血，将细胞梯度分离液、全血及组织稀释液、新鲜外周血按3∶2∶5依次加入离心管中，制成梯度界面，以2 500 r/min离心25 min(离心半径10 cm)，小心分离中间乳白色细胞层，加入3倍于细胞悬液体积的红细胞裂解液，将红细胞裂解，加入10 ml清洗液，以1 500 r/min离心10 min(离心半径10 cm)，重复操作1次，即得到人中性粒细胞。

1.3.4 分组和药物干预 在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)。

1.4 观察指标 分别观察CSE、LPS刺激物下，正常对照组、CSE模型组、LPS模型组、补肺益肾血清组、补肺健脾血清组、益气滋肾血清组ROS活性、NE水平。

1.4.1 ROS活性检测

1.4.1.1 2′，7′-二氯荧光素二乙酸酯(DCFH-DA)探针装载 上述各组细胞37 ℃、5% CO2培养箱孵育1.5 h后，用RPMI 1640培养基按照1∶1 000稀释DCFH-DA，使终浓度为10 μmol/L。将细胞悬浮于稀释好的DCFH-DA中，于37 ℃培养箱孵育20 min，每隔3～5 min颠倒混匀一下，使探针与细胞充分接触，洗涤细胞3次，充分除去未进入细胞内的DCFH-DA，最后用500 μl PBS重悬细胞。

1.4.1.2 流式细胞仪检测ROS活性 采用空白对照管在FSC和SSC二维点阵图中选取人中性粒细胞区域，以FL1(RHO)直方图检测RHO阳性的人中性粒细胞为产生ROS的人中性粒细胞，以FL1(DCF)直方图检测DCF阳性的人中性粒细胞为产生ROS的人中性粒细胞。每个样品收集1×104个细胞,采用CellQuest软件分析MFI。实验独立重复3次。

1.4.2 NE水平检测 上述各组细胞37 ℃、5% CO2培养箱孵育24 h后，去除上清液，加500 μl细胞裂解液充分裂解细胞，1 000 r/min离心5 min(离心半径10 cm)，收集细胞裂解液。严格按照ELISA说明书进行操作。实验独立重复3次。

2 结果

2.1 正常对照组、CSE模型组、LPS模型组ROS活性及NE水平比较 正常对照组ROS活性及NE水平分别为(1.00±0.00)、(27.71±0.80)，CSE模型组、LPS模型组ROS活性及NE水平分别见表1、2。3组ROS活性及NE水平比较，差异有统计学意义(F值分别为224.848、121.086，P<0.05)；CSE模型组、LPS模型组ROS活性强于正常对照组，NE水平高于正常对照组，差异有统计学意义(P<0.05)。

2.2CSE刺激下，CSE模型组、补肺益肾血清组、补肺健脾血清组、益气滋肾血清组ROS活性及NE水平比较 4组ROS活性及NE水平比较，差异有统计学意义(P<0.05)；补肺益肾血清组ROS活性弱于CSE模型组，差异有统计学意义(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水平低于CSE模型组，差异有统计学意义(P<0.05，见表1)。

Table 1 Comparison of activity of ROS and level of NE among CSE model group,lung invigorating and kidney tonifying serum group,lung invigorating and spleen strengthening serum group,and Qi supplementing and kidney nourishing serum group under the stimulating of CSE

组别ROS活性NECSE模型组224±01811337±000补肺益肾血清组161±027a2696±055a补肺健脾血清组233±0292716±335a益气滋肾血清组198±0633706±709aF值4219338561P值0017<0001

注：CSE=香烟烟雾提取物，ROS=活性氧，NE=弹性蛋白酶；与CSE模型组比较，aP<0.05

2.3 LPS刺激下，LPS模型组、补肺益肾血清组、补肺健脾血清组、益气滋肾血清组ROS活性及NE水平比较 4组ROS活性及NE水平比较，差异有统计学意义(P<0.05)；补肺益肾血清组、益气滋肾血清组ROS活性弱于LPS模型组，差异有统计学意义(P<0.05)；补肺健脾血清组、益气滋肾血清组NE水平低于LPS模型组，差异有统计学意义(P<0.05，见表2)。

Table 2 Comparison of activity of ROS and level of NE among LPS model group,lung invigorating and kidney tonifying serum group,lung invigorating and spleen strengthening serum group,and Qi supplementing and kidney nourishing serum group under the stimulating of LPS

组别ROS活性NELPS模型组118±0488539±1188补肺益肾血清组085±025a6493±3431补肺健脾血清组100±0312823±247a益气滋肾血清组079±013a2875±255aF值40977173P值00200012

注：LPS=脂多糖；与LPS模型组比较，aP<0.05

3 讨论

COPD属于中医“咳证”“喘证”“肺胀”等范畴。素体正虚，肺脏感邪，迁延失治，痰瘀稽留，复伤正气，肺、脾、肾虚损，正虚卫外不固，外邪易反复侵袭，从而诱发COPD，其病理变化为本虚标实，病理机制为“正虚积损”。故治当从“虚”入手，扶正兼驱邪，大量研究表明，调补肺肾三方治疗COPD稳定期的临床效果明显[7-8]。

COPD主要病理变化为炎性反应，细菌、香烟烟雾等有害颗粒等致炎物质进入肺内，导致中性粒细胞、巨噬细胞等炎性细胞在肺内活化，从而产生炎性因子，持续的炎性反应会造成持续的氧化应激、受损的细胞修复和细胞死亡及细胞外基质的破坏和细胞凋亡等[9-10]。中性粒细胞是炎性反应中起关键作用的效应细胞，其分泌的NE水平与COPD的严重程度直接相关。研究表明，COPD肺部中性粒细胞浸润性增加，活化的中性粒细胞数目和中性粒细胞分泌的NE水平影响COPD患者肺气肿的严重程度[11]。NE水解及前炎症特征在COPD组织病理和炎症募集中伴有重要角色，同时被香烟烟雾等致炎物质激活的中性粒细胞可产生ROS，ROS活性增强会进一步损伤组织，从而加快机体炎性反应进程。此外，中性粒细胞寿命延长、活性增强，NE、ROS等物质的释放增加均易引起肺损伤和腺体增生，且NE及其抑制剂还会导致严重的肺血管重构[12]。近年来，大量研究表明，调补肺肾三方在阻抑炎性反应方面起重要作用[13-15]，其作用与降低炎性细胞的活化程度、抑制炎性递质的释放、促进炎性分泌物的吸收及调控炎症通路有关，但大多是从动物实验和上皮细胞和巨噬细胞实验着手，本研究以人中性粒细胞为研究对象，观察在CSE、LPS刺激时调补肺肾三方对ROS和NE的影响。

本研究结果显示，在10% CSE和2 μg/ml LPS刺激作用下，人中性粒细胞ROS活性显著增强，NE水平显著升高，提示香烟烟雾和细菌内毒素可直接或间接引起中性粒细胞炎性反应，造成肺组织损伤。CSE刺激下，补肺益肾血清组ROS活性较CSE模型组明显减弱；补肺益肾血清组、补肺健脾血清组、益气滋肾血清组NE水平均显著低于CSE模型组。LPS刺激下，补肺益肾血清组、益气滋肾血清组ROS活性明显弱于LPS模型组；补肺健脾血清组、益气滋肾血清组NE水平显著低于LPS模型组；表明益气滋肾血清组效果更好。以上结果表明在抑制ROS的释放方面，调补肺肾三方均可不同程度地降低ROS活性和NE水平，这与既往报道的调补肺肾三方均可通过抑制炎性反应治疗COPD一致[16-17]。但可能由于调补肺肾三方的作用靶点不同，造成调补肺肾三方含药血清对两种刺激物的效应不一。

综上所述，CSE、LPS刺激下人中性粒细胞均会释放ROS及NE，调补肺肾三方均可在一定程度上影响人中性粒细胞ROS及NE的释放。CSE刺激下，补肺益肾方降低ROS活性及NE水平的效果最好；LPS刺激下，益气滋肾方降低ROS活性及NE水平的效果最好。但本研究未对调补肺肾三方的具体作用机制和作用靶点进一步研究，有待继续补充。

作者贡献：秦燕勤进行实验设计与实施、资料收集整理、撰写论文并对文章负责；秦燕勤、吴耀松、毛筱宁、赵丹瑞进行实验实施、评估、资料收集；李建生、陈玉龙进行质量控制与审校。

本文无利益冲突。

本研究不足之处：

本研究未涉及相关基因与信号转导通路检测，拟于后续研究中进行该方面的研究。

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(本文编辑：毛亚敏)

Effect of Three Prescription for Regulating and Invigorating the Lung and Kidney on Neutrophil-released Reactive Oxygen Species and Neutrophil Elastase Stimulated by Cigarette Smoke Extract and Lipopolysaccharide

QIN Yan-qin,WU Yao-song,LI Jian-sheng,CHEN Yu-long，MAO Xiao-ning，ZHAO Dan-rui.

Henan University of Chinese Medicine，Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province，Zhengzhou 450046,China

LI Jian-sheng,Henan University of Chinese Medicine，Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province，Zhengzhou 450046,China； E-mail：li_js8@163.com

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.

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

国家自然科学基金资助项目(81130062)

450046河南省郑州市，河南中医药大学呼吸疾病诊疗与新药研发河南省协同创新中心(秦燕勤，李建生,毛筱宁，赵丹瑞)；河南中医药大学分子生物学实验中心(吴耀松，陈玉龙)

李建生，450046河南省郑州市，河南中医药大学呼吸疾病诊疗与新药研发河南省协同创新中心；

E-mail：li_js8@163.com

R 563.9

A

10.3969/j.issn.1007-9572.2016.36.022

2016-06-13；

2016-11-01)