熊友誼，時(shí)維靜，俞 浩，張孝林

(安徽科技學(xué)院 食品藥品學(xué)院， 安徽 鳳陽 233100)

研究論文

紫花前胡苷抑制哮喘小鼠氣道炎性反應(yīng)和NF-κB信號(hào)傳導(dǎo)通路

熊友誼*，時(shí)維靜，俞 浩，張孝林

(安徽科技學(xué)院 食品藥品學(xué)院， 安徽 鳳陽 233100)

目的觀察紫花前胡苷抗過敏性哮喘鼠氣道炎性反應(yīng)作用。方法BALB/c小鼠被隨機(jī)分為對(duì)照、模型、紫花前胡苷和地塞米松4組。除對(duì)照組外，所有小鼠腹腔注射和霧化吸入卵清蛋白以致敏和誘導(dǎo)氣道炎性反應(yīng)。于每次霧化前1h，給紫花前胡苷組灌胃10 mg/kg紫花前胡苷；給地塞米松組腹腔注射1 mg/kg地塞米松。動(dòng)物肺功能儀分析氣道高反應(yīng)性；細(xì)胞計(jì)數(shù)器和Diff-Quick染色計(jì)數(shù)支氣管灌洗液中白細(xì)胞總數(shù)及分類；酶聯(lián)免疫吸附法檢測血清或BALF中炎性介質(zhì)的水平；蘇木精-伊紅染色法觀察氣道病理改變；電泳遷移率和免疫印跡法檢測肺組織NF-κВ信號(hào)通路中蛋白的活力和表達(dá)。結(jié)果與對(duì)照組比較，模型組呈現(xiàn)明顯的氣道炎性反應(yīng)，氣道反應(yīng)性顯著增高 (Plt;0.05)；血清或BALF中IgE、IL-4、IL-5和IL-13的水平顯著增加 (Plt;0.01)；細(xì)胞系P65、p-P65的水平顯著增加(Plt;0.05)；細(xì)胞質(zhì)P65和IκBα顯著減少，NF-κB DNA-結(jié)合力顯著增加 (Plt;0.05)。與模型組比較，紫花前胡苷能夠顯著抑制氣道炎性反應(yīng)和氣道高反應(yīng) (Plt;0.05)；降低血清或BALF中IgE、IL-4、IL-5和IL-13的水平(Plt;0.01)；抑制細(xì)胞系P65、p-P65水平(Plt;0.05)；增加細(xì)胞質(zhì)P65、IκBα蛋白和減弱NF-κB DNA-結(jié)合力(Plt;0.05)。結(jié)論紫花前胡苷具有抗過敏性哮喘鼠氣道炎性反應(yīng)。

紫花前胡苷；紫花前胡；哮喘；核因子κB

哮喘是一種以嗜酸性粒細(xì)胞為主的多種炎性細(xì)胞浸潤的慢性氣道炎性反應(yīng)。主要病理生理改變?yōu)闅獾栏叻磻?yīng)、黏液分泌增多和間歇性氣道阻塞[1]。哮喘是臨床常見病多發(fā)病，尚無有效的根治方法[2]。紫花前胡苷是一種具有代表性的線性四氫呋喃型香豆素苷類化合物，以傳統(tǒng)抗哮喘中藥紫花前胡中含量高。研究表明紫花前胡苷具有抗炎、抗過敏、抗氧化等多種生物活性[3～5]?？紤]到哮喘多屬過敏性氣道炎性反應(yīng)，以及紫花前胡抗哮喘的傳統(tǒng)功效。本研究以雌性BALB/c小鼠模仿人類的哮喘模型，研究紫花前胡苷的抗哮喘作用，為其新藥開發(fā)提供理論依據(jù)。

1 材料與方法

1.1 動(dòng)物與試劑

SPF級(jí)雌性BALB/c小鼠，6～8周齡，體質(zhì)量22～25 g[北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司，動(dòng)物合格證號(hào):SCXK(京)20060009]。Ⅰ級(jí)卵白蛋白(OVA) 試劑盒、氯化乙酰膽堿、紫花前胡苷和地塞米松(Sigma公司)；IL-4、IL-5和IL-13 ELISA試劑盒(Bender Medsystems公司)；抗P65、抗p-P65 (Ser536)、羊抗兔IgG(碧云天生物技術(shù)公司)；抗IκBα(Cell Signaling公司)；EMSA試劑盒(Pierce公司)。

1.2 實(shí)驗(yàn)分組與模型

用隨機(jī)表將小鼠分成對(duì)照、模型、紫花前胡苷和地塞米松4組，每組8只。于實(shí)驗(yàn)第0、7和14 d，模型、紫花前胡苷和地塞米松組腹腔注射10 μg OVA混懸液(含50 μg氫氧化鋁凝膠)，自第21 d霧化吸入1% OVA，1次/d，20 min/次，連續(xù)5 d；對(duì)照組腹腔注射50 μg氫氧化鋁凝膠和霧化吸入0.9%氯化鈉注射液。于霧化前20 min，紫花前胡苷組灌胃給予10 mg/kg紫花前胡苷(溶劑為0.5% 羧甲基纖維素鈉)，地塞米松組腹腔注射1 mg/kg地塞米松(Dex)，對(duì)照組和模型灌胃給予等容的0.5% 羧甲基纖維素鈉。

1.3 肺功能檢測

小鼠末次霧化24 h后，用60 mg/kg戊巴比妥鈉麻醉，行氣管插管術(shù)，后被置于動(dòng)物呼吸機(jī)檢測肺功能。每隔5 min，小鼠尾靜脈順次注射10、30、90和270 μg氯化乙酰膽堿誘導(dǎo)氣道高反應(yīng)，紀(jì)錄每次注射后1 min的最大氣道阻力。具體操作方法按AniRes2005肺功能儀(北京貝蘭博科技有限公司)說明書進(jìn)行。

1.4 白細(xì)胞計(jì)數(shù)和細(xì)胞因子檢測

小鼠肺功能檢測完畢先后被摘球取血和支氣管灌洗。灌洗液(BALF)離心上清和血清分別用于IL-4、IL-5、IL-13 和IgE 酶聯(lián)免疫吸附法測定。BALF離心沉淀用細(xì)胞計(jì)數(shù)器和Diff-Quick染色法計(jì)數(shù)。

1.5 肺組織病理改變

小鼠左下肺組織經(jīng)4%中性多聚甲醛溶液固定送至南京醫(yī)科大學(xué)作病理分析。

1.6 Western blot檢測p-P65、P65和IκВα蛋白

參照Heijink的實(shí)驗(yàn)方法[6]，結(jié)果以目的蛋白和內(nèi)參電泳條帶的吸光度比值來表示。

1.7 NF-κB DNA結(jié)合力

按試劑盒的操作說明逐步進(jìn)行，結(jié)果以目的蛋白與突變探針NF-κB電泳條帶的吸光度百分比來表示。

2 結(jié)果

2.1 氣道高反應(yīng)實(shí)驗(yàn)

與對(duì)照組比較，模型組的氣道阻力顯著增加(Plt;0.05)。與模型組比較，紫花前胡苷組氣道阻力顯著降低(Plt;0.05) (表1)。

2.2 肺組織病理改變

對(duì)照氣道上皮平整，黏膜下無炎性細(xì)胞浸潤，肺泡腔內(nèi)無滲出。模型組氣道氣道有大量炎性滲出，氣道和血管周圍有大量炎性細(xì)胞浸潤。紫花前胡苷組炎性滲出和炎性細(xì)胞浸潤減少 (圖1)。

2.3 BALF中白細(xì)胞總數(shù)及分類計(jì)數(shù)

模型組BALF中白細(xì)胞總數(shù)、嗜酸性粒細(xì)胞和淋巴細(xì)胞計(jì)數(shù)較對(duì)照組顯著增加(Plt;0.01)；紫花前胡苷組較模型組顯著回降(Plt;0.05或Plt;0.01) (表2)。

2.4 血清或BALF中炎性介質(zhì)的水平

模型組IgE、IL-4、IL-5和IL-13較對(duì)照組顯著增加 (Plt;0.01)；紫花前胡苷組較模型組明顯降低 (Plt;0.05或Plt;0.01) (表3)。

2.5 肺組織p-P65、P65、Iκβα的表達(dá)水平

模型組細(xì)胞系P65、p-P65較對(duì)照組顯著增加(Plt;0.05)，紫花前胡苷組較模型組顯著回降(Plt;0.05)；模型組細(xì)胞質(zhì)P65、Iκβα較對(duì)照組顯著減少(Plt;0.05), 紫花前胡苷組P65、Iκβα較模型組顯著回升(Plt;0.05) (圖2)。

表1 紫花前胡苷對(duì)氯乙酰膽堿誘導(dǎo)的哮喘鼠氣道高反應(yīng)的作用

#Plt;0.05,##Plt;0.01 compared with control group;*Plt;0.05,**Plt;0.05 compared with model group.

圖1 紫花前胡苷對(duì)OVA 誘導(dǎo)的哮喘鼠肺組織炎性反應(yīng)的作用Fig 1 Effects of nodakenin on OVA-induced airway inflammation in asthmatic mice(×400)

grouptotalleukocyteseosinophilslymphocytescontrol10±3.000.3±0.2model72.7±18.2#41.3±10.9#17.6±3.0#nodakenin29.3±7.3*12.8±3.2*5.4±1.3*Dex14.3±3.0*7.9±1.2*2.6±0.2*

#Plt;0.01 compared with control group;*Plt;0.01 compared with model group.

表3 紫花前胡苷對(duì)OVA誘導(dǎo)的哮喘鼠血清或BALF中炎性介質(zhì)的作用

#Plt;0.01 compared with control group;*Plt;0.05,**Plt;0.01 compared with model group.

#Plt;0.05 compared with control group; *Plt;0.05 compared with model group 圖2 紫花前胡苷對(duì)OVA誘導(dǎo)的哮喘鼠肺組織NF-κВ信號(hào)傳導(dǎo)蛋白的作用Fig 2 Effects of nodakenin on proteins of NF-κВ signaling pathway in asthmatic mice induced by OVA(±s,n=8)

#Plt;0.05 compared with control group; *Plt;0.05; **Plt;0.01 compared with model group圖3 紫花前胡苷對(duì)OVA誘導(dǎo)的哮喘鼠肺組織NF-κВ-DNA結(jié)合力的作用Fig 3 Effects of nodakenin on the NF-κВ-DNA binding activity in asthmatic mice induced by OVA(±s,n=8)

2.6 肺組織NF-κВ-DNA結(jié)合力

模型組NF-κВ-DNA結(jié)合力較對(duì)照組顯著增加(Plt;0.05)，紫花前胡苷組NF-κВ-DNA結(jié)合力較模型組明顯降低(Plt;0.05或Plt;0.01) (圖3)。

3 討論

目前中國哮喘病患者約2 500萬，隨著氣候和自然環(huán)境的改變，發(fā)病率呈現(xiàn)上升趨勢[7]。糖皮質(zhì)激素是抑制哮喘氣道炎性反應(yīng)最有效的藥物，但長期使用會(huì)導(dǎo)致機(jī)體對(duì)藥物產(chǎn)生耐受性，藥效降低，并產(chǎn)生一些嚴(yán)重的不良反應(yīng)[8]。開發(fā)低毒、療效好的抗哮喘天然藥物具有重大意義。本次研究表明紫花前胡苷能顯著抑制了哮喘鼠氣道炎性反應(yīng)，減少氣道分泌物和氣道高反應(yīng)，具有明顯的抗哮喘作用。

Th2優(yōu)勢反應(yīng)是哮喘發(fā)病機(jī)制中的關(guān)鍵因素，Th2細(xì)胞通過分泌IL-4、IL-5、IL-13等炎性因子在誘導(dǎo)EOS早期分化、活化及向炎性反應(yīng)區(qū)域的募集，高反應(yīng)性和黏液高分泌的產(chǎn)生過程中發(fā)揮重要作用[9-10]。紫花前胡苷具有顯著下調(diào)BALF中IL-4、IL-5、IL-13 等炎性因子的作用。說明紫花前胡苷抑制哮喘氣道炎性反應(yīng)和高反應(yīng)與抑制Th2 優(yōu)勢有關(guān)，對(duì)免疫起調(diào)節(jié)作用。

另外，NF-κB作為哮喘發(fā)生發(fā)展過程中促炎信號(hào)通路中的轉(zhuǎn)錄因子，參與了多種炎性介質(zhì)的表達(dá)，包括對(duì)Th2細(xì)胞因子[11]。P50/P65二聚體是NF-κB信號(hào)通路中重要的信號(hào)物質(zhì)，在細(xì)胞的靜息狀態(tài)下，P50/P65被IκB蛋白家族結(jié)合滯留于細(xì)胞質(zhì)中而失活。當(dāng)細(xì)胞在受到過敏原等各種刺激下，IκBα發(fā)生磷酸化進(jìn)而被降解，P50/P65被激活進(jìn)入核內(nèi)促進(jìn)致炎性反應(yīng)物質(zhì)基因的表達(dá)。另外，NF-κB活性受其核內(nèi)磷酸化化水影響[12]。本研究結(jié)果顯示，紫花前胡苷能顯著抑制P65的核轉(zhuǎn)位和抑制核P65的磷酸化，以及抑制NF-κB P65的DNA結(jié)合力，提示是其抗哮喘鼠氣道炎性反應(yīng)和高反應(yīng)的重要機(jī)制?？傊?，通過本文研究表明紫花前胡苷具有抗哮喘作用，具有很好的研究和開發(fā)價(jià)值。

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Inhibitory effects of nodakenin on the airwayinflammation and NF-κB signaling pathway in a murine asthmatic model

XIONG You-yi*, SHI Wei-jing, YU Hao, ZHANG Xiao-lin

(College of Food and Drug, Anhui Science and Technology University, Fengyang 233100, China)

ObjectiveTo observe the effects of nodakenin on airway inflammation in a mouse model of allergic asthma.MethodsBALB/c mice were assigned randomly to one of the following four experimental groups: control, model, nodakenin and dexamethasone. All test mice were sensitized and challenged by OVA to induce airway inflammation. One hour before OVA challenge, nodakenin group was intragastrically administered with nodakenin at a dose of 10 mg/kg, and dexamethasone group was intraperitoneally injected with dexamethasone a dose of 1 mg/kg. Airway responsiveness was measured by a lung function analysis systems. The number of total leukocytes in BALF was counted using a hemocytometer, and differential cells were counted using Diff-Quick-stained smears. Histopathology of lung tissue was analyzed by Hematoxylin-eosin staining. Levels of inflammatory mediators in serum or BALFs were measured by ELISA. The activity and expression of proteins in NF-κB signaling pathway was respectively evaluated by EMSA and western blot analysis.ResultsCompared with control group, the model group

exhibited obvious airway inflammation, airway reactivity was significantly increased, the level of total cells and differential cells was significantly increased, levels of IL-4, IL-5, IL-13, IgE were significantly increased, levels of nuclear P65 and p-P65 protein was significantly enhanced, level of cytoplasmic P65 and IκBα protein was significantly decreased, and the NF-κB DNA binding activity was significantly increased. Compared with model group, nodakenin significantly suppressed airway inflammation, airway hyperreactivity, reduced levels of IL-4, IL-5 and IL-13 in BALF, and IgE in serum, decreased levels of nuclear P65 and p-P65 protein, increased cytoplasmic P65 and IκBα protein, and increased the NF-κB DNA binding activity.ConclusionsNodakenin efficiently inhibits antigen-induced airway inflammation in asthmatic mouse.

nodakenin；peucedanum decursivum maxim；asthma；NF-κB

2013-11-21

2014-01-16

*通信作者(correspondingauthor)： xyytc1@163.com

1001-6325(2014)05-0690-05

R 541.4

A