陸韋,王蕾,譙明,王玉,江吉富,吳中明

(1.遵義醫(yī)學(xué)院附屬醫(yī)院兒科,遵義 563099;2.遵義醫(yī)學(xué)院微免教研室,遵義 563003)

地塞米松對(duì)哮喘小鼠支氣管肺泡灌洗液中IL-25和IFN-γ的影響*

陸韋1,王蕾1,譙明1,王玉2,江吉富2,吳中明2

(1.遵義醫(yī)學(xué)院附屬醫(yī)院兒科,遵義 563099;2.遵義醫(yī)學(xué)院微免教研室,遵義 563003)

目的 通過檢測支氣管肺泡灌洗液(BALF)中白細(xì)胞介素-25(IL-25)和γ-干擾素(IFN-γ)的水平,探討地塞米松對(duì)小鼠支氣管哮喘的治療作用機(jī)制。方法清潔級(jí)Balb/c小鼠隨機(jī)分為正常對(duì)照組、哮喘組和地塞米松組。以雞卵清蛋白(OVA)致敏激發(fā)法建立哮喘小鼠模型。地塞米松組在每次激發(fā)前1 h予地塞米松腹腔注射。每次激發(fā)時(shí)觀察小鼠的表現(xiàn)。于末次激發(fā)24 h處死小鼠,取右肺作蘇木精-伊紅(HE)染色病理切片,顯微鏡下觀察炎癥情況;收集左肺BALF,鏡下計(jì)數(shù)白細(xì)胞總數(shù)、嗜酸性粒細(xì)胞(EOS)絕對(duì)數(shù)目,計(jì)算EOS百分比;用酶聯(lián)免疫吸附法(ELISA)測定BALF中IL-25和IFN-γ的水平,并做相關(guān)性分析。結(jié)果哮喘組小鼠BALF中白細(xì)胞總數(shù)、EOS數(shù)目和百分比分別與正常對(duì)照組、地塞米松組比較均明顯增加(P＜0.05),而以上3個(gè)指標(biāo)在正常對(duì)照組和地塞米松組間差異無統(tǒng)計(jì)學(xué)意義。哮喘組小鼠BALF中IL-25水平高于正常對(duì)照組和地塞米松組(P＜0.05),而地塞米松組的含量也高于正常對(duì)照組;IFN-γ水平低于正常對(duì)照組和地塞米松組(P＜0.05),而后兩組差異無統(tǒng)計(jì)學(xué)意義。各組小鼠BALF中IL-25和IFN-γ水平都呈負(fù)相關(guān)。結(jié)論地塞米松治療哮喘病的部分機(jī)制是減輕肺部炎癥和促進(jìn)IFN-γ的產(chǎn)生,同時(shí)可能抑制IL-25的表達(dá)。

地塞米松;哮喘;白細(xì)胞介素-25;γ-干擾素

哮喘的發(fā)病機(jī)制尚未完全清楚,一般認(rèn)為與免疫、神經(jīng)、精神、內(nèi)分泌和遺傳學(xué)背景等因素密切相關(guān)。在免疫發(fā)病的機(jī)制中最重要的環(huán)節(jié)是Th1/Th2細(xì)胞的失衡[1-2]。γ-干擾素(interferon-γ,IFN-γ)是重要的Th1型細(xì)胞因子,而關(guān)于Th2型細(xì)胞因子的研究大多集中于白細(xì)胞介素(interleukin,IL)-4、IL-5、IL-10和IL-13等,但一些非經(jīng)典的細(xì)胞因子也同樣發(fā)揮一定的作用,IL-25就是其中的一種。有研究表明,IL-25具有促進(jìn)Th2型免疫反應(yīng)的作用[3-7]。目前還沒有根治哮喘病的方法,糖皮質(zhì)激素仍是首選藥物,但尚不清楚其對(duì)IL-25具有何種作用。因此,筆者在本實(shí)驗(yàn)中擬建立哮喘小鼠模型,設(shè)立地塞米松組,檢測地塞米松對(duì)哮喘小鼠支氣管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中IL-25和IFN-γ水平的影響,進(jìn)一步探討地塞米松對(duì)哮喘的治療機(jī)制。

1 材料與方法

1.1 動(dòng)物 6～8周齡清潔級(jí)Balb/c雄性小鼠36只,體質(zhì)量(18±2)g,購于中國科學(xué)院上海斯萊克實(shí)驗(yàn)動(dòng)物中心,合格證號(hào):SCXK(滬)2007-0005。

1.2 試劑 雞卵清清蛋白(ovalbumin,OVA)Ⅴ級(jí),氫氧化鋁凝膠(Sigma公司,批號(hào):MKBC0623),布地奈德混懸液(澳大利亞Astra Zeneca公司,批號(hào):305144),地塞米松注射液(四川科倫藥業(yè)有限公司,批號(hào):130601),0. 9%氯化鈉溶液(四川科倫藥業(yè)有限公司,批號(hào): H11021313),小鼠IL-25、IFN-γ ELISA試劑盒(深圳欣博盛生物科技有限公司,批號(hào):060728,101221)。

1.3 儀器 超聲霧化器(德國百瑞,型號(hào): PARIDOY),普通顯微鏡(日本NIKON&YS100),低溫離心機(jī)(中國飛鴿,型號(hào):TGL-16G),電熱恒溫培養(yǎng)箱(上海恒科技有限公司,型號(hào):DHP-9162)。

1.4 建立哮喘模型及藥物干預(yù) 用隨機(jī)數(shù)字表法將36只Balb/c小鼠分為3組,即正常對(duì)照組、哮喘組和地塞米松組各12只。哮喘組在第0天、第10天腹腔注射致敏液0.1 mL(含OVA25 μg及氫氧化鋁凝膠2 mg)。在第14天開始用1%OVA溶液行超聲霧化,每次30 min,連續(xù)7 d,此即激發(fā)[8]。地塞米松組除按哮喘組操作外,在每次激發(fā)前1 h予地塞米松2 mg·kg-1腹腔注射[9]。正常對(duì)照組用0.9%氯化鈉溶液致敏和霧化,方法同哮喘組。

1.5 標(biāo)本制備 小鼠于末次激發(fā)后24 h腹腔注射10%水合氯醛(每只0.075 mL)后固定,暴露腹腔后腹主動(dòng)脈,放血處死,打開胸腔,立即結(jié)扎右肺,并做以下兩步操作。第一步,取右肺,按常規(guī)制作蘇木精-伊紅(hematoxylin-eosinstaining,HE)染色病理切片。第二步,充分暴露小鼠頸部氣管,用22G留置針行氣管插管,輕柔注入4℃、0.9%氯化鈉溶液0.5 mL,反復(fù)回抽3次后收集該液體,此即為BALF,以上操作重復(fù)3次(回收率＞80%)。收集的BALF放入-20℃保存。

1.6 觀察和檢測項(xiàng)目 ①小鼠的激發(fā)表現(xiàn):在每次激發(fā)時(shí)觀察小鼠呼吸、神智、鼻腔分泌物、行動(dòng)等臨床表現(xiàn)。②觀察肺組織炎癥改變:用普通顯微鏡觀察各組小鼠肺組織病理切片的炎癥情況。③記錄細(xì)胞數(shù):吸取BALF滴在計(jì)數(shù)板上,低倍鏡下記錄并計(jì)算白細(xì)胞計(jì)數(shù)(white blood cell,WBC)。單盲法高倍鏡下數(shù)至少200個(gè)細(xì)胞作嗜酸性粒細(xì)胞(eosinophils,EOS)絕對(duì)計(jì)數(shù)和計(jì)算EOS在白細(xì)胞中的百分比。④酶聯(lián)免疫吸附測定法(enzyme-linked imruno-sorbent assay, ELISA):將BALF 4℃離心10 min,取上清液,按說明書測定IL-25和IFN-γ的水平。

2 結(jié)果

2.1 小鼠的激發(fā)表現(xiàn) 哮喘組小鼠經(jīng)OVA激發(fā)后,出現(xiàn)煩躁不安、鼻腔分泌物增多、撓鼻和呼吸急促等癥狀,嚴(yán)重者呼吸減慢或節(jié)律不規(guī)則,反應(yīng)遲鈍,四肢癱軟。地塞米松組的表現(xiàn)較哮喘組明顯減輕,其中5只小鼠與正常對(duì)照組無區(qū)別。

2.2 小鼠肺部炎癥改變 哮喘組小鼠支氣管、細(xì)支氣管和肺泡腔縮小,肺泡間隔增寬,管壁及肺間質(zhì)有炎性細(xì)胞浸潤,以嗜酸性粒細(xì)胞為主,支氣管、細(xì)支氣管及肺泡腔內(nèi)可見滲出物,支氣管和細(xì)支氣管黏膜上皮部分壞死脫落,伴杯狀細(xì)胞增生。地塞米松組炎癥反應(yīng)明顯減輕。見圖1。

2.3 記錄細(xì)胞數(shù) 結(jié)果表明:BALF中白細(xì)胞總數(shù)、EOS數(shù)目和百分比在哮喘組比正常對(duì)照組明顯增加,在正常對(duì)照組和地塞米松組進(jìn)行兩兩比較均差異無統(tǒng)計(jì)學(xué)意義。見表1。

A.正常對(duì)照組;B.哮喘組;C.地塞米松組圖1 3組小鼠肺組織的病理改變(HE,×400)A.normal control group;B.asthma group;C.dexamethasone groupFig.1 Pathological changes of lungs in three groups of mice(HE,×400)

表1 3組BALF中白細(xì)胞總數(shù)、EOS絕對(duì)計(jì)數(shù)和百分比Tab.1 Changes of total white cell count,absolute number of EOS and EOS%in BALF of three groups ±s

表1 3組BALF中白細(xì)胞總數(shù)、EOS絕對(duì)計(jì)數(shù)和百分比Tab.1 Changes of total white cell count,absolute number of EOS and EOS%in BALF of three groups ±s

與正常對(duì)照組比較,*1P＜0.01;與哮喘組比較,*2P＜0.01Compared with normal control group,*1P＜0.01;compared with asthma group,*2P＜0.01

組別小鼠/只WBC/ (×104個(gè)·mL-1) EOS (×104個(gè)·mL-1)%正常對(duì)照組124.98±2.120.55±0.301.85±1.06哮喘組1296.14±46.60*118.34±3.16*14.52±2.83*1地塞米松組127.28±4.30*23.30±2.14*21.81±1.23*2F 19.25631.3219.685 P 0.0000.0000.000

2.4 IL-25的水平測定 結(jié)果表明:哮喘組小鼠BALF中IL-25的水平較正常對(duì)照組增高,地塞米松組能使IL-25降低,但不能降至正常水平。見圖2。

與正常對(duì)照組比較,*1P＜0.05;與哮喘組比較,*2P＜0.05圖2 3組小鼠BALF中IL-25的濃度Compared with normal control group,*1P＜0.05;compared with asthma group,*2P＜0.05Fig.2 IL-25 concentration in BALF in three groups of mice

2.5 ＼IFN-γ水平的測定 結(jié)果表明:哮喘組小鼠BALF中IFN-γ的水平較正常對(duì)照組減低,地塞米松能使其增加至正常水平。見圖3。

2.6 相關(guān)性分析 正常對(duì)照組、哮喘組和地塞米松組小鼠BALF中IL-25和IFN-γ的濃度均呈負(fù)相關(guān)關(guān)系,r分別為-0.948,-0.963,-0.580(P＜0.01,n=12)。

與哮喘組比較,*1P＜0.05圖3 3組小鼠BALF中IFN-γ的濃度Compared with asthma group,*1P＜0.05Fig.3 IFN-γ concentration in BALF in three groups of mice

3 討論

IL-25主要來源于Th2細(xì)胞,也可由肥大細(xì)胞、巨噬細(xì)胞、嗜酸性粒細(xì)胞和嗜堿性粒細(xì)胞等產(chǎn)生。屬于IL-17家族,受體是IL-17RB。它的靶細(xì)胞主要有兩種,一種是記憶性Th2細(xì)胞,另一種是一類非T非B細(xì)胞的附屬細(xì)胞群,如髓源性肥大細(xì)胞、肺泡巨噬細(xì)胞等[6-7,10-11]。既往研究顯示,IL-25可以誘導(dǎo)哮喘病的炎癥前和炎癥反應(yīng),引起肺部嗜酸性粒細(xì)胞和Th2細(xì)胞增多,血清IgE、IgA和IgG1增加,IL-4、IL-5和IL-13增加,氣道高反應(yīng)性,肺上皮細(xì)胞增生肥大和黏液高分泌,以及氣道重建[4-5,12-13]。本實(shí)驗(yàn)哮喘組小鼠BALF中IL-25水平增高,驗(yàn)證了IL-25參與到哮喘病炎癥發(fā)病的過程之中。IL-25增高的可能機(jī)制是:致哮喘病的各種始動(dòng)因素促使BALF中白細(xì)胞數(shù)目增加,分泌IL-25的細(xì)胞(如Th2細(xì)胞、嗜酸性粒細(xì)胞和嗜堿性粒細(xì)胞等)也相應(yīng)增多,同時(shí),IL-25基因的表達(dá)增強(qiáng)。

本實(shí)驗(yàn)中地塞米松可抑制IL-25的表達(dá),說明地塞米松可通過抑制IL-25生成,使哮喘病緩解?？赡艿臋C(jī)制是:①地塞米松抑制IL-25基因轉(zhuǎn)錄和表達(dá),使IL-25 mRNA產(chǎn)生減少;②抑制IL-25的合成和釋放;③降低IL-25的穩(wěn)定性;④下調(diào)IL-25的靶細(xì)胞和信號(hào)傳導(dǎo)通路的功能。但是,地塞米松組小鼠BALF的IL-25并沒有完全恢復(fù)至正常水平。其原因可能是:①IL-25的清除尚需要一段時(shí)間;②某些促炎因素使IL-25的穩(wěn)定性增加,降解減少;③不能完全抑制IL-25基因的轉(zhuǎn)錄和表達(dá)。哮喘組小鼠BALF中IFN-γ水平低于正常對(duì)照組,這與許卓謙等[10]的結(jié)果相同。表明IFN-γ受到抑制,也驗(yàn)證了哮喘病時(shí)Th1型細(xì)胞因子的產(chǎn)生是受到抑制的。IFN-γ被抑制的機(jī)制可能是:Th2細(xì)胞分泌的IL-4和IL-10不但使IFN-γ蛋白分泌減少,而且還抑制IFN-γ基因的表達(dá)并降低其穩(wěn)定性[14]。地塞米松可增加IFN-γ的產(chǎn)生,使其濃度恢復(fù)到正常水平,支持了糖皮質(zhì)激素可以通過上調(diào)Th1型細(xì)胞因子的表達(dá)治療哮喘病的理論。糖皮質(zhì)激素促進(jìn)IFN-γ產(chǎn)生的機(jī)制可能是:①促進(jìn)IFN-γ基因的轉(zhuǎn)錄;②多途徑地促進(jìn)IFN-γ的表達(dá)和合成;③抑制Th2型細(xì)胞因子的產(chǎn)生,從而減輕該類細(xì)胞因子對(duì)IFN-γ的抑制作用;④增加IFN-γ的穩(wěn)定性,減少其降解;⑤上調(diào)IFN-γ的靶細(xì)胞和信號(hào)傳導(dǎo)通路的功能[15]。INF-γ在哮喘組和地塞米松組存在動(dòng)態(tài)變化,提示其參與了哮喘病的發(fā)病?？赡軝C(jī)制是:INF-γ抑制B細(xì)胞產(chǎn)生IgE;抑制IL-4 mRNA的轉(zhuǎn)錄水平;抑制氣道炎癥細(xì)胞的滲出和Th2型細(xì)胞因子的分泌[16-17]。本實(shí)驗(yàn)中各個(gè)組IFN-γ和IL-25均呈負(fù)相關(guān)關(guān)系,說明二者有相互抑制的關(guān)系,它們的作用方向相反,共同參與哮喘病的發(fā)展。

雖然糖皮質(zhì)激素是目前治療哮喘病的首選藥物,但單純依靠激素卻很難治愈該病,因此,除了聯(lián)合應(yīng)用外源性的IFN-γ外[18],IL-25可能提供了一個(gè)治療的途徑。本實(shí)驗(yàn)中哮喘組小鼠使用地塞米松后IL-25的水平不能降至正常水平,說明激素可能不能完全抑制IL-25的產(chǎn)生,該細(xì)胞因子在哮喘病緩解期仍高于正常水平并繼續(xù)發(fā)揮Th2型免疫反應(yīng)作用,成為哮喘病只能緩解而不能根治的原因之一,因此推測IL-25可能參與到糖皮質(zhì)激素耐藥的機(jī)制中。多項(xiàng)研究表明,可溶性IL-25R和抗IL-25 mAb均可減輕小鼠肺組織的炎癥反應(yīng)。并且,IL-25 mAb在人體內(nèi)也能阻止IL-25和IL-25R的結(jié)合[6,13,19]。因此,可考慮將IL-25作為一條輔助治療哮喘病的途徑。

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DOI 10.3870/yydb.2014.08.005

Influence of Dexamethasone on IL-25 and IFN-γ in Bronchoalveolar Lavage Fluid of Asthmatic Mice

LU Wei1,WANG Lei1,QIAO Ming1,WANG Yu2,JIANG Ji-fu2,WU Zhong-ming2
(1.Department of Pediatrics, Affaliated Hospital,Zunyi 563099,China;2.Department of Immunology,Zunyi Medical University,Zunyi 563003,China)

Objective To investigate the mechanism of therapeutic action of dexamethasone on asthmatic mice by detecting the levels of IL-25 and IFN-γ in bronchoalveolar lavage fluid(BALF).MethodsBalb/c mice with SPF grade were randomly divided into normal control group,asthma group and dexamethasone group.Asthma group and dexamethasone group were sensitized and challenged with ovalbumin(OVA).Dexamethasone group was intraperitoneally injected with dexamethasone one hour before challenging.The mice were executed 24 hours after the last challenge,and the HE stained pathological sections of the right lung were made.Pathological sections of lung were observed.BALF in the left lung was also collected.The total white blood cell count and absolute eosinophile(EOS)count were observed,and the percentage of EOS was calculated.The levels of IL-25 and IFN-γ were measured with ELISA,and correlation analyses were made.ResultsThe counts of total white blood cell and EOS, and the percentage of EOS were significantly higher in the asthma group than in the normal control group and dexamethasone group (P＜0.05).No differences were found between the normal control group and dexamethasone group.The IL-25 level was higher in the asthma group than in the normal control group and dexamethasone group(P＜0.05),and its level in the dexamethasone group was also higher than that in the normal control group.The IFN-γ level was lower in the asthma group than in the normal control group and dexamethasone group(P＜0.05),while there was no significant difference between the normal control group and dexamethasone group.IL-25 was negatively correlated with IFN-γ in each group.ConclusionPart of the mechanisms of dexamethasone acting on asthma are related to its inhibition on the pulmonary inflammation and promotion on the expression of IFN-γ,and possible inhibition of IL-25 expression.

Dexamethasone;Asthma;Interleukin-25;γ-Interferon

R977.1;R965

A

1004-0781(2014)08-0997-04

2013-07-10

2014-01-15

*貴州省科技廳基金資助項(xiàng)目(黔科合J字LKZ[2010]14號(hào))

陸韋(1977-),女,貴州遵義人,副教授,碩士,研究方向:兒童哮喘。電話:(0)13193042247,E-mail:luweizmc @163.com。