調(diào)補肺腎三方對香煙煙霧提取物和脂多糖刺激人中性粒細胞釋放活性氧及彈性蛋白酶的影響

秦燕勤，吳耀松，李建生，陳玉龍，毛筱寧，趙丹瑞

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·論著·

·中醫(yī)·中西醫(yī)結(jié)合研究·

調(diào)補肺腎三方對香煙煙霧提取物和脂多糖刺激人中性粒細胞釋放活性氧及彈性蛋白酶的影響

秦燕勤，吳耀松，李建生，陳玉龍，毛筱寧，趙丹瑞

目的 分析調(diào)補肺腎(補肺益腎、補肺健脾、益氣滋腎)三方對香煙煙霧提取物(CSE)和脂多糖(LSP)刺激人中性粒細胞釋放活性氧(ROS)及彈性蛋白酶(NE)的影響。方法 2014年9月—2015年12月，將常規(guī)飼養(yǎng)1周的普通級日本大耳白兔96只按照隨機數(shù)字表法分為正常組、補肺益腎組、補肺健脾組、益氣滋腎組，24只/組。補肺益腎組、補肺健脾組、益氣滋腎組大耳白兔分別采用補肺益腎方劑、補肺健脾方劑、益氣滋腎方劑灌胃，正常組采用等量的0.9%氯化鈉溶液灌胃，4組均于第7次灌胃后取腹主動脈血，分離血清，-70 ℃保存?zhèn)溆?。取來自河南中醫(yī)藥大學(xué)健康成年男性(年齡20～25歲)志愿者外周血，分離人中性粒細胞。CSE刺激下，將人中性粒細胞分為正常對照組(加20%的正常組兔血清)，CSE模型組(加20%的正常組兔血清+10% CSE)，補肺益腎血清組、補肺健脾血清組、益氣滋腎血清組(分別添加20%相應(yīng)的含藥兔血清+10% CSE)。LPS刺激下，將人中性粒細胞分為正常對照組(加20%的正常組兔血清)，LPS模型組(加20%的正常組兔血清+2 μg/ml LPS)，補肺益腎血清組、補肺健脾血清組、益氣滋腎血清組(分別添加20%相應(yīng)的含藥兔血清+2 μg/ml LPS)。分別采用流式細胞儀和酶聯(lián)免疫吸附實驗(ELISA)法檢測各組ROS活性、NE水平。結(jié)果 與正常對照組比較，CSE模型組、LPS模型組ROS活性增強，NE水平升高(P<0.05)。與CSE模型組比較，補肺益腎血清組ROS活性減弱(P<0.05)，補肺益腎血清組、補肺健脾血清組、益氣滋腎血清組NE水平均降低(P<0.05)。與LPS模型組比較，補肺益腎血清組、益氣滋腎血清組ROS活性減弱(P<0.05)；補肺健脾血清組、益氣滋腎組血清組NE水平降低(P<0.05)。結(jié)論 調(diào)補肺腎三方含藥血清對CSE、LPS刺激下人中性粒細胞釋放ROS和NE水平均有影響；CSE刺激下，補肺益腎方含藥血清降低ROS活性及NE水平的效果最好；LPS刺激下，益氣滋腎方含藥血清降低ROS活性氧及NE水平的效果最好。

中草藥；調(diào)補肺腎三方；人中性粒細胞；活性氧；彈性蛋白酶

秦燕勤，吳耀松，李建生，等.調(diào)補肺腎三方對香煙煙霧提取物和脂多糖刺激人中性粒細胞釋放活性氧及彈性蛋白酶的影響[J].中國全科醫(yī)學(xué)，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.

炎性反應(yīng)在慢性阻塞性肺疾病(chronic obstructive pulmonary disease，COPD)的病理進程中起重要作用[1]。炎性反應(yīng)涉及多種炎性細胞因子，炎癥的發(fā)生、發(fā)展與蛋白酶-抗蛋白酶失衡、氧化-抗氧化損傷等密切相關(guān)，且中性粒細胞在其中起重要作用[2-4]。中醫(yī)治療COPD具有較好的效果，研究發(fā)現(xiàn)調(diào)補肺腎三方在改善COPD穩(wěn)定期臨床癥狀方面療效顯著[5]。為了深入探討調(diào)補肺腎三方的抗炎機制，本研究以香煙提取物(cigarette smoke extract,CSE)和脂多糖(lipopolysaccharide,LPS)作為刺激物，以人中性粒細胞為研究對象，觀察調(diào)補肺腎三方對人中性粒細胞釋放活性氧(reactive oxygen species,ROS)及彈性蛋白酶(neutrophil elastase,NE)的影響。

1 材料與方法

1.1 實驗材料

1.1.1 實驗藥物 補肺益腎方由人參9 g、山茱萸12 g、五味子9 g、陳皮9 g等組成；補肺健脾方由黨參15 g、白術(shù)12 g、茯苓12 g、炙甘草6 g等組成；益氣滋腎方由人參9 g、枸杞子12 g、麥冬15 g、炙甘草6 g等組成。藥物均由河南中醫(yī)藥大學(xué)第一臨床醫(yī)學(xué)院中藥制劑與劑型改革基地提供。

1.1.2 主要試劑及儀器 LPS(美國Sigma公司)；RPMI 1640培養(yǎng)基、人外周血中性粒細胞分離液試劑盒(北京索萊寶科技有限公司)；活性氧檢測試劑盒(南京凱基生物公司)；人抗中性粒細胞彈性蛋白酶抗體(ANEA)酶聯(lián)免疫吸附實驗(ELISA)試劑盒(Elabscience公司，E-EL-H0383c)。BD FACSCalibur流式細胞儀(美國Becton Dickinson公司)；全自動酶標儀ELx800(美國BIO-TEK寶特)。

1.1.3 血液及實驗動物 人外周血均來自河南中醫(yī)藥大學(xué)健康成年男性(年齡20～25歲)志愿者。日本大耳白兔96只，普通級，雌雄各半，體質(zhì)量2.0～2.5 kg，由河南康達實驗動物有限公司提供(許可證號：SYXK豫2010-0001)。

1.2 研究時間 2014年9月—2015年12月。

1.3 方法

1.3.1 含藥血清制備 所有大耳白兔飼養(yǎng)于河南中醫(yī)藥大學(xué)動物實驗中心普通級飼養(yǎng)室，每籠1只，室溫為(25±2)℃，相對濕度為(50±10)%，噪聲≤60 db，通風(fēng)良好，滅菌飼料喂養(yǎng)，每天更換墊料，保持環(huán)境安靜。常規(guī)飼養(yǎng)以穩(wěn)定大耳白兔應(yīng)激狀態(tài)，飼養(yǎng)1周后稱重，按照隨機數(shù)字表法將大耳白兔分為正常組、補肺益腎組、補肺健脾組、益氣滋腎組，24只/組。補肺益腎組、補肺健脾組、益氣滋腎組大耳白兔分別采用補肺益腎方劑、補肺健脾方劑、益氣滋腎方劑灌胃，灌胃劑量以60 kg成人生藥量公斤體質(zhì)量的9倍(兔和人系數(shù)為3∶1，再乘血清稀釋倍數(shù)3)，即按W家兔/W人W家兔/W人×每副生藥量×出膏率×9/每毫升含膏量進行計算，正常組大耳白兔采用等量的0.9%氯化鈉溶液灌胃，2次/d，每次間隔12 h，于第7次灌胃后1 h取腹主動脈血。取血前大耳白兔禁食12 h，不禁水，靜置4 h后以3 000 r/min離心15 min(離心半徑15 cm)，分離血清，56 ℃、30 min滅活后過濾除菌，-70 ℃保存?zhèn)溆谩?/p>

1.3.2 CSE制備 將YANG等[6]研究方法進行改良，采用50 ml注射器以0.75 kg/s的水平拉力連續(xù)6次抽吸一支去掉過濾嘴燃燒的香煙，推入盛有10 ml RPMI 1640培養(yǎng)基的負壓裝置，紫外分光光度計測其320 nm處的OD值，取OD值為1.8～2.0的懸液為100% CSE，0.22 μm濾器過濾除菌，避光保存，2 h內(nèi)使用。

1.3.3 人中性粒細胞的分離 取志愿者外周血，將細胞梯度分離液、全血及組織稀釋液、新鮮外周血按3∶2∶5依次加入離心管中，制成梯度界面，以2 500 r/min離心25 min(離心半徑10 cm)，小心分離中間乳白色細胞層，加入3倍于細胞懸液體積的紅細胞裂解液，將紅細胞裂解，加入10 ml清洗液，以1 500 r/min離心10 min(離心半徑10 cm)，重復(fù)操作1次，即得到人中性粒細胞。

1.3.4 分組和藥物干預(yù) 在CSE刺激下，將人中性粒細胞分為正常對照組(加20%的正常組兔血清)、CSE模型組(加20%的正常組兔血清+10% CSE)、補肺益腎血清組、補肺健脾血清組、益氣滋腎血清組(分別添加20%相應(yīng)的含藥兔血清+10% CSE)；LPS刺激下，將人中性粒細胞分為正常對照組(加20%的正常組兔血清)、LPS模型組(加20%的正常組兔血清+2 μg/ml LPS)、補肺益腎血清組、補肺健脾血清組、益氣滋腎血清組(分別添加20%相應(yīng)的含藥兔血清+2 μg/ml LPS)。

1.4 觀察指標 分別觀察CSE、LPS刺激物下，正常對照組、CSE模型組、LPS模型組、補肺益腎血清組、補肺健脾血清組、益氣滋腎血清組ROS活性、NE水平。

1.4.1 ROS活性檢測

1.4.1.1 2′，7′-二氯熒光素二乙酸酯(DCFH-DA)探針裝載 上述各組細胞37 ℃、5% CO2培養(yǎng)箱孵育1.5 h后，用RPMI 1640培養(yǎng)基按照1∶1 000稀釋DCFH-DA，使終濃度為10 μmol/L。將細胞懸浮于稀釋好的DCFH-DA中，于37 ℃培養(yǎng)箱孵育20 min，每隔3～5 min顛倒混勻一下，使探針與細胞充分接觸，洗滌細胞3次，充分除去未進入細胞內(nèi)的DCFH-DA，最后用500 μl PBS重懸細胞。

1.4.1.2 流式細胞儀檢測ROS活性 采用空白對照管在FSC和SSC二維點陣圖中選取人中性粒細胞區(qū)域，以FL1(RHO)直方圖檢測RHO陽性的人中性粒細胞為產(chǎn)生ROS的人中性粒細胞，以FL1(DCF)直方圖檢測DCF陽性的人中性粒細胞為產(chǎn)生ROS的人中性粒細胞。每個樣品收集1×104個細胞,采用CellQuest軟件分析MFI。實驗獨立重復(fù)3次。

1.4.2 NE水平檢測 上述各組細胞37 ℃、5% CO2培養(yǎng)箱孵育24 h后，去除上清液，加500 μl細胞裂解液充分裂解細胞，1 000 r/min離心5 min(離心半徑10 cm)，收集細胞裂解液。嚴格按照ELISA說明書進行操作。實驗獨立重復(fù)3次。

2 結(jié)果

2.1 正常對照組、CSE模型組、LPS模型組ROS活性及NE水平比較 正常對照組ROS活性及NE水平分別為(1.00±0.00)、(27.71±0.80)，CSE模型組、LPS模型組ROS活性及NE水平分別見表1、2。3組ROS活性及NE水平比較，差異有統(tǒng)計學(xué)意義(F值分別為224.848、121.086，P<0.05)；CSE模型組、LPS模型組ROS活性強于正常對照組，NE水平高于正常對照組，差異有統(tǒng)計學(xué)意義(P<0.05)。

2.2CSE刺激下，CSE模型組、補肺益腎血清組、補肺健脾血清組、益氣滋腎血清組ROS活性及NE水平比較 4組ROS活性及NE水平比較，差異有統(tǒng)計學(xué)意義(P<0.05)；補肺益腎血清組ROS活性弱于CSE模型組，差異有統(tǒng)計學(xué)意義(P<0.05)；補肺益腎血清組、補肺健脾血清組、益氣滋腎血清組NE水平低于CSE模型組，差異有統(tǒng)計學(xué)意義(P<0.05，見表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

組別ROS活性NECSE模型組224±01811337±000補肺益腎血清組161±027a2696±055a補肺健脾血清組233±0292716±335a益氣滋腎血清組198±0633706±709aF值4219338561P值0017<0001

注：CSE=香煙煙霧提取物，ROS=活性氧，NE=彈性蛋白酶；與CSE模型組比較，aP<0.05

2.3 LPS刺激下，LPS模型組、補肺益腎血清組、補肺健脾血清組、益氣滋腎血清組ROS活性及NE水平比較 4組ROS活性及NE水平比較，差異有統(tǒng)計學(xué)意義(P<0.05)；補肺益腎血清組、益氣滋腎血清組ROS活性弱于LPS模型組，差異有統(tǒng)計學(xué)意義(P<0.05)；補肺健脾血清組、益氣滋腎血清組NE水平低于LPS模型組，差異有統(tǒng)計學(xué)意義(P<0.05，見表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

組別ROS活性NELPS模型組118±0488539±1188補肺益腎血清組085±025a6493±3431補肺健脾血清組100±0312823±247a益氣滋腎血清組079±013a2875±255aF值40977173P值00200012

注：LPS=脂多糖；與LPS模型組比較，aP<0.05

3 討論

COPD屬于中醫(yī)“咳證”“喘證”“肺脹”等范疇。素體正虛，肺臟感邪，遷延失治，痰瘀稽留，復(fù)傷正氣，肺、脾、腎虛損，正虛衛(wèi)外不固，外邪易反復(fù)侵襲，從而誘發(fā)COPD，其病理變化為本虛標實，病理機制為“正虛積損”。故治當從“虛”入手，扶正兼驅(qū)邪，大量研究表明，調(diào)補肺腎三方治療COPD穩(wěn)定期的臨床效果明顯[7-8]。

COPD主要病理變化為炎性反應(yīng)，細菌、香煙煙霧等有害顆粒等致炎物質(zhì)進入肺內(nèi)，導(dǎo)致中性粒細胞、巨噬細胞等炎性細胞在肺內(nèi)活化，從而產(chǎn)生炎性因子，持續(xù)的炎性反應(yīng)會造成持續(xù)的氧化應(yīng)激、受損的細胞修復(fù)和細胞死亡及細胞外基質(zhì)的破壞和細胞凋亡等[9-10]。中性粒細胞是炎性反應(yīng)中起關(guān)鍵作用的效應(yīng)細胞，其分泌的NE水平與COPD的嚴重程度直接相關(guān)。研究表明，COPD肺部中性粒細胞浸潤性增加，活化的中性粒細胞數(shù)目和中性粒細胞分泌的NE水平影響COPD患者肺氣腫的嚴重程度[11]。NE水解及前炎癥特征在COPD組織病理和炎癥募集中伴有重要角色，同時被香煙煙霧等致炎物質(zhì)激活的中性粒細胞可產(chǎn)生ROS，ROS活性增強會進一步損傷組織，從而加快機體炎性反應(yīng)進程。此外，中性粒細胞壽命延長、活性增強，NE、ROS等物質(zhì)的釋放增加均易引起肺損傷和腺體增生，且NE及其抑制劑還會導(dǎo)致嚴重的肺血管重構(gòu)[12]。近年來，大量研究表明，調(diào)補肺腎三方在阻抑炎性反應(yīng)方面起重要作用[13-15]，其作用與降低炎性細胞的活化程度、抑制炎性遞質(zhì)的釋放、促進炎性分泌物的吸收及調(diào)控炎癥通路有關(guān)，但大多是從動物實驗和上皮細胞和巨噬細胞實驗著手，本研究以人中性粒細胞為研究對象，觀察在CSE、LPS刺激時調(diào)補肺腎三方對ROS和NE的影響。

本研究結(jié)果顯示，在10% CSE和2 μg/ml LPS刺激作用下，人中性粒細胞ROS活性顯著增強，NE水平顯著升高，提示香煙煙霧和細菌內(nèi)毒素可直接或間接引起中性粒細胞炎性反應(yīng)，造成肺組織損傷。CSE刺激下，補肺益腎血清組ROS活性較CSE模型組明顯減弱；補肺益腎血清組、補肺健脾血清組、益氣滋腎血清組NE水平均顯著低于CSE模型組。LPS刺激下，補肺益腎血清組、益氣滋腎血清組ROS活性明顯弱于LPS模型組；補肺健脾血清組、益氣滋腎血清組NE水平顯著低于LPS模型組；表明益氣滋腎血清組效果更好。以上結(jié)果表明在抑制ROS的釋放方面，調(diào)補肺腎三方均可不同程度地降低ROS活性和NE水平，這與既往報道的調(diào)補肺腎三方均可通過抑制炎性反應(yīng)治療COPD一致[16-17]。但可能由于調(diào)補肺腎三方的作用靶點不同，造成調(diào)補肺腎三方含藥血清對兩種刺激物的效應(yīng)不一。

綜上所述，CSE、LPS刺激下人中性粒細胞均會釋放ROS及NE，調(diào)補肺腎三方均可在一定程度上影響人中性粒細胞ROS及NE的釋放。CSE刺激下，補肺益腎方降低ROS活性及NE水平的效果最好；LPS刺激下，益氣滋腎方降低ROS活性及NE水平的效果最好。但本研究未對調(diào)補肺腎三方的具體作用機制和作用靶點進一步研究，有待繼續(xù)補充。

作者貢獻：秦燕勤進行實驗設(shè)計與實施、資料收集整理、撰寫論文并對文章負責(zé)；秦燕勤、吳耀松、毛筱寧、趙丹瑞進行實驗實施、評估、資料收集；李建生、陳玉龍進行質(zhì)量控制與審校。

本文無利益沖突。

本研究不足之處：

本研究未涉及相關(guān)基因與信號轉(zhuǎn)導(dǎo)通路檢測，擬于后續(xù)研究中進行該方面的研究。

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(本文編輯：毛亞敏)

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

國家自然科學(xué)基金資助項目(81130062)

450046河南省鄭州市，河南中醫(yī)藥大學(xué)呼吸疾病診療與新藥研發(fā)河南省協(xié)同創(chuàng)新中心(秦燕勤，李建生,毛筱寧，趙丹瑞)；河南中醫(yī)藥大學(xué)分子生物學(xué)實驗中心(吳耀松，陳玉龍)

李建生，450046河南省鄭州市，河南中醫(yī)藥大學(xué)呼吸疾病診療與新藥研發(fā)河南省協(xié)同創(chuàng)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)