任倩倩,汪麗佩,趙 煒,陸 紅,謝強(qiáng)敏,張水娟(.浙江中醫(yī)藥大學(xué)藥學(xué)院,浙江杭州 005;.浙江中醫(yī)藥大學(xué)基礎(chǔ)醫(yī)學(xué)院,浙江杭州 005;.浙江中醫(yī)藥大學(xué)附屬第一醫(yī)院,浙江杭州0006;.浙江大學(xué)醫(yī)學(xué)院呼吸藥物研究實(shí)驗(yàn)室,浙江杭州 0058)
甘草查爾酮A抑制ERK1/2/NF-κB途徑減輕吸煙誘導(dǎo)的小鼠急性肺損傷
任倩倩1,汪麗佩2,趙煒3,陸紅1,謝強(qiáng)敏4,張水娟1
(1.浙江中醫(yī)藥大學(xué)藥學(xué)院,浙江杭州310053;2.浙江中醫(yī)藥大學(xué)基礎(chǔ)醫(yī)學(xué)院,浙江杭州310053;
3.浙江中醫(yī)藥大學(xué)附屬第一醫(yī)院,浙江杭州310006;4.浙江大學(xué)醫(yī)學(xué)院呼吸藥物研究實(shí)驗(yàn)室,浙江杭州310058)
網(wǎng)絡(luò)出版時(shí)間:2016-4-26 11:06網(wǎng)絡(luò)出版地址:http://www.cnki.net/kcms/detail/34.1086.R.20160426.1106.022.html
目的探討甘草查爾酮A(LA)對(duì)吸煙誘導(dǎo)的急性肺損傷小鼠的保護(hù)作用及相關(guān)機(jī)制。方法整體實(shí)驗(yàn):香煙煙霧暴露構(gòu)建小鼠急性肺損傷模型,取肺泡灌洗液(BALF)進(jìn)行白細(xì)胞計(jì)數(shù),測(cè)定肺內(nèi)角質(zhì)形成細(xì)胞衍生趨化因子(KC)、腫瘤壞死因子α(TNF-α)、白介素-1β(IL-1β)和基質(zhì)金屬蛋白酶9(MMP-9)的mRNA和蛋白表達(dá)水平,用試劑盒測(cè)定肺組織中過(guò)氧化物髓化酶(MPO)、超氧化物歧化酶(SOD)活力以及谷胱甘肽(GSH)水平。肺組織病理切片進(jìn)行HE染色。離體實(shí)驗(yàn):香煙煙霧提取物(CSE)誘導(dǎo)上皮細(xì)胞損傷,測(cè)定細(xì)胞內(nèi)白介素-8(IL-8)、MMP-9 mRNA的表達(dá)。Western blot分析細(xì)胞外信號(hào)調(diào)節(jié)激酶l/2(ERK1/2)、p38分裂原活化蛋白激酶(p38 MAPK)、c-Jun氨基末端激酶(JNK)的磷酸化水平和轉(zhuǎn)錄因子NF-κB p65的活性。結(jié)果整體實(shí)驗(yàn)顯示LA組肺內(nèi)炎癥細(xì)胞數(shù)量及浸潤(rùn)程度均明顯低于模型組。模型組肺內(nèi)KC、TNF-α、IL-1β、MMP-9 mRNA和蛋白表達(dá)較正常對(duì)照組均明顯升高,經(jīng)LA處理后,上述指標(biāo)相比模型組明顯降低。與正常對(duì)照組相比,模型組肺內(nèi)MPO活性明顯上升,而SOD活性和GSH水平明顯下降。LA能逆轉(zhuǎn)這種變化,明顯降低MPO活性,升高SOD活性和GSH水平。離體實(shí)驗(yàn)顯示,LA(2.5、5 μmol·L-1)能明顯降低CSE刺激的細(xì)胞內(nèi)IL-8和MMP-9 mRNA的高表達(dá)。CSE可以誘導(dǎo)細(xì)胞ERK1/2磷酸化和胞核內(nèi)NF-κB p65的表達(dá),經(jīng)LA預(yù)處理后,上述指標(biāo)可明顯抑制。結(jié)論LA可能通過(guò)阻斷ERK1/ 2/NF-κB途徑來(lái)抑制炎癥介質(zhì)的高表達(dá)、調(diào)節(jié)氧化/抗氧化失衡、下調(diào)金屬蛋白酶,從而發(fā)揮對(duì)急性肺損傷小鼠的保護(hù)作用。
甘草;甘草查爾酮A;肺損傷;吸煙;肺上皮細(xì)胞;細(xì)胞因子;信號(hào)通路
慢性阻塞性肺部疾?。╟hronic obstructive pulmonary diseases,COPD)是一種以氣流受限為特征的疾病,它與肺部對(duì)有害氣體或有毒顆粒的異常炎癥反應(yīng)有關(guān)[1]。COPD目前已成為影響人類生活質(zhì)量的全球性健康問(wèn)題,發(fā)病率呈現(xiàn)逐年增加的趨勢(shì),迄今尚無(wú)藥物能遏制COPD病情的進(jìn)行性發(fā)展,因此迫切需要發(fā)展新的COPD治療方法。從中藥和植物藥中尋找新的有效成分,或從已有的有效成分中發(fā)現(xiàn)新的藥理作用是一個(gè)重要方向。盡管COPD發(fā)病機(jī)制尚未完全闡明,但目前普遍認(rèn)為,吸煙是COPD發(fā)生的最主要因素之一[2],吸煙能誘導(dǎo)炎癥并直接導(dǎo)致肺組織的損傷[3]。COPD的病理生理除小氣道的炎癥外,氧化與抗氧化失衡也是一個(gè)很重要的因素[4]。因此,從中藥中尋找抗炎和抗氧化的治療COPD的新藥是一個(gè)值得探索的途徑。甘草是中藥中傳統(tǒng)的鎮(zhèn)咳祛痰藥,入藥歷史悠久,性味甘平,歸脾、胃、心、肺經(jīng),具有益氣補(bǔ)中、潤(rùn)肺止咳、清熱解毒、緩急止痛、調(diào)和諸藥等功效。甘草中主要含有甘草酸、甘草次酸、黃酮、生物堿和氨基酸等活性成分[5]。甘草中提取的黃酮單體甘草查爾酮 A (LA)經(jīng)研究表明具有抗炎、抗氧化和抗腫瘤等作用,能通過(guò)調(diào)節(jié)絲裂原活化蛋白激酶(MAPK)信號(hào)通路,誘導(dǎo)人胃癌細(xì)胞的凋亡[6-8]。目前LA對(duì)肺損傷的研究也有報(bào)道,研究發(fā)現(xiàn)其能抑制細(xì)菌內(nèi)毒素脂多糖(LPS)氣道滴入引起的小鼠急性肺損傷,降低肺內(nèi)腫瘤壞死因子α(TNF-α)、白介素-1β(IL-1β)和白介素-6(IL-6)表達(dá)。進(jìn)一步的研究發(fā)現(xiàn),LA主要是通過(guò)NF-κB和p38/ERK MAPK信號(hào)途徑起作用[9-10],但LA與香煙煙霧所引起的疾病之間的關(guān)系研究,無(wú)論是整體上還是離體上均未見(jiàn)到相關(guān)的報(bào)道。為此,本研究采用香煙煙霧誘導(dǎo)建立小鼠急性肺損傷模型,同時(shí)用香煙煙霧提取物(CSE)誘導(dǎo)肺上皮細(xì)胞損傷,觀察LA對(duì)肺損傷病理改變、炎癥因子、氧化/抗氧化水平、MAPK信號(hào)途徑和胞核內(nèi)NF-κB p65表達(dá)的影響,探索LA對(duì)香煙煙霧誘導(dǎo)的小鼠急性肺損傷的保護(hù)作用及其相關(guān)機(jī)制。
1.1實(shí)驗(yàn)動(dòng)物與藥品C57BL/6清潔級(jí)小鼠,♀,體質(zhì)量(20±2)g,由上海斯萊克實(shí)驗(yàn)動(dòng)物有限責(zé)任公司提供,實(shí)驗(yàn)動(dòng)物質(zhì)量合格證:SCXK(滬)2012-0002,飼養(yǎng)于浙江大學(xué)動(dòng)物實(shí)驗(yàn)中心SPF級(jí)動(dòng)物房。人肺上皮細(xì)胞BEAS-2B細(xì)胞株,購(gòu)自美國(guó)菌種保藏中心(ATCC)細(xì)胞庫(kù)。LA委托沈陽(yáng)藥科大學(xué)中藥學(xué)院趙余慶教授課題組提取和純化,純度>98%。3R4F標(biāo)準(zhǔn)香煙購(gòu)自美國(guó)肯塔基大學(xué)煙草研究所(批號(hào)200712)。RT-PCR相關(guān)試劑購(gòu)自日本TaKaRa公司。ELISA試劑盒購(gòu)于eBioscience公司。過(guò)氧化物髓化酶(MPO)、超氧化物歧化酶(SOD)以及谷胱甘肽(GSH)測(cè)定試劑盒購(gòu)自南京建成生物技術(shù)有限公司。ERK1/2、p-ERK1/2、p-p38、p38、JNK、p-JNK、NF-κB p65抗體購(gòu)于Cell Signaling公司。
1.2模型制備與分組給藥整體實(shí)驗(yàn)中,將60只♀C57BL/6清潔級(jí)小鼠隨機(jī)分為正常對(duì)照組、模型組、LA低、中、高劑量組(10、20、40 mg·kg-1)、地塞米松對(duì)照組(Dex,1 mg·kg-1)。依據(jù)我們以前報(bào)道的方法,建立香煙煙霧誘導(dǎo)的小鼠急性肺損傷模型[11]。香煙煙霧連續(xù)暴露4 d,正常對(duì)照組使用空氣進(jìn)行暴露。各組小鼠預(yù)給藥2 d,每天給藥1次,而后在連續(xù)4 d的煙霧暴露前1 h灌胃給予藥物。末次香煙煙霧暴露結(jié)束后18h處理動(dòng)物。
在離體實(shí)驗(yàn)中,依據(jù)我們以前報(bào)道的方法制作CSE,用CSE刺激人肺上皮細(xì)胞(BEAS-2B),誘導(dǎo)上皮細(xì)胞損傷[12]。采用不同濃度的 LA(1、2.5、5 μmol·L-1)預(yù)處理1 h,而后加入2.5%CSE誘導(dǎo)上皮細(xì)胞損傷,48 h后提取細(xì)胞RNA進(jìn)行細(xì)胞因子測(cè)定;2.5%CSE誘導(dǎo)30min后提取細(xì)胞蛋白進(jìn)行信號(hào)通路研究。
1.3小鼠肺泡灌洗液(BALF)制備和炎癥細(xì)胞計(jì)數(shù)末次香煙煙霧暴露結(jié)束后18 h,腹腔注射6 g· kg-1烏拉坦處死各組小鼠。分離小鼠氣道,插管,結(jié)扎左肺,進(jìn)行肺泡灌洗,取50 μL肺泡灌洗液,加同體積白細(xì)胞計(jì)數(shù)液,100倍光鏡下進(jìn)行白細(xì)胞計(jì)數(shù);其余肺泡灌洗液4℃ 2 000 r·min-1離心10min,沉淀做細(xì)胞涂片,室溫干燥后瑞氏-吉姆薩染色,400倍光鏡下依據(jù)形態(tài)學(xué)標(biāo)準(zhǔn)進(jìn)行細(xì)胞分類計(jì)數(shù)。
1.4肺組織病理學(xué)觀察按照我們以前報(bào)道的方法[11-12],將肺組織福爾馬林固定,石蠟包埋,切片,HE染色,200倍鏡下檢查肺組織炎癥細(xì)胞浸潤(rùn)程度。
1.5ELISA取小鼠肺泡灌洗液,根據(jù)ELISA試劑盒說(shuō)明書,分別檢測(cè)灌洗液中角質(zhì)形成細(xì)胞衍生趨化因子(KC)、TNF-α、IL-1β和基質(zhì)金屬蛋白酶9 (MMP-9)蛋白含量。
1.6MPO、SOD和GSH水平測(cè)定取肺組織,制成5%肺勻漿,按試劑盒說(shuō)明書分別測(cè)定上清液中MPO、SOD活力以及GSH的水平。
1.7熒光實(shí)時(shí)定量PCR測(cè)定KC、TNF-α、IL-1β、MMP-9和白介素-8(IL-8)mRNA表達(dá)水平用TRIzol試劑提取小鼠肺組織及細(xì)胞樣本中總RNA,參照逆轉(zhuǎn)錄試劑盒說(shuō)明書合成cDNA,引物序列由上海生工生物技術(shù)有限公司合成,詳見(jiàn)Tab 1。用cDNA模板對(duì)相關(guān)基因分別進(jìn)行PCR擴(kuò)增,擴(kuò)增條件:95℃ 預(yù)變性2 min,95℃變性10 s,退火58℃10 s,72℃延伸30 s,共40個(gè)循環(huán)。以β-actin為內(nèi)參照,采用ΔΔCt值法測(cè)定基因表達(dá)水平。
Tab 1 Primer sequences used in the present study
1.8Western blot將處于對(duì)數(shù)生長(zhǎng)期的BEAS-2B細(xì)胞接種至6孔板中,相應(yīng)處理后,棄培養(yǎng)液,提取細(xì)胞蛋白。制備SDS-PAGE凝膠,上樣、電泳、轉(zhuǎn)膜,室溫下用5%脫脂奶粉封閉2 h,取出硝酸纖維素膜,放入兔抗ERK1/2、p-ERK1/2、p-p38、p38、JNK、p-JNK、NF-κB p65一抗液中,4℃過(guò)夜。次日洗膜后加入熒光二抗,室溫避光搖動(dòng)2 h,用Odyssey熒光掃描儀,拍攝條帶,采用Quantity one軟件計(jì)算灰度值。
1.9統(tǒng)計(jì)學(xué)處理采用SPSS(20.0版)統(tǒng)計(jì)軟件包進(jìn)行數(shù)據(jù)分析,實(shí)驗(yàn)數(shù)據(jù)以±s表示,采用單因素方差分析(one-way ANOVA)比較多組均數(shù)以及SNK檢驗(yàn)進(jìn)行兩兩之間的比較。
2.1LA對(duì)肺部炎癥細(xì)胞的影響對(duì)吸煙小鼠肺泡灌洗液中炎癥細(xì)胞進(jìn)行總數(shù)和分類計(jì)數(shù),如Fig 1所示,與正常對(duì)照組小鼠相比,模型組小鼠BALF中白細(xì)胞總數(shù)、中性粒細(xì)胞、淋巴細(xì)胞和巨噬細(xì)胞數(shù)均有明顯增加(P<0.01)。LA能明顯抑制吸煙誘導(dǎo)小鼠BALF中白細(xì)胞總數(shù)、中性粒細(xì)胞的增加,呈明顯的量效關(guān)系。LA 40 mg·kg-1組對(duì)淋巴細(xì)胞和巨噬細(xì)胞的增加亦有明顯抑制作用。小鼠肺組織HE染色后發(fā)現(xiàn),香煙煙霧暴露的小鼠肺組織中性粒細(xì)胞和巨噬細(xì)胞浸潤(rùn)增加,而LA能明顯抑制吸煙小鼠中性粒細(xì)胞和巨噬細(xì)胞在肺部的浸潤(rùn),見(jiàn)Fig 2。
2.2LA對(duì)肺內(nèi)細(xì)胞因子的影響為了探討LA是否通過(guò)抑制炎癥反應(yīng)而保護(hù)小鼠急性肺損傷,應(yīng)用熒光實(shí)時(shí)定量PCR檢測(cè)各組小鼠肺組織細(xì)胞因子mRNA表達(dá)及ELISA法檢測(cè)BALF中蛋白水平。結(jié)果顯示,與正常對(duì)照組相比,模型組小鼠肺組織中KC、TNF-α、IL-1β和 MMP-9 mRNA的表達(dá)(Fig 3)及BALF中蛋白含量(Fig 4)均明顯升高。與模型組相比,LA 20、40 mg·kg-1組上述指標(biāo)均明顯降低。
2.3LA的抗氧化效應(yīng)為了探討LA是否通過(guò)調(diào)節(jié)氧化應(yīng)激反應(yīng)而保護(hù)吸煙誘導(dǎo)的急性肺損傷小鼠,用試劑盒測(cè)定各組小鼠肺組織中MPO、SOD和GSH的水平。結(jié)果顯示,與正常對(duì)照組相比,模型組小鼠肺組織中MPO水平明顯上升(P<0.01),SOD和GSH水平明顯下降(P<0.05~0.01)。LA能逆轉(zhuǎn)這種變化,明顯降低MPO水平,升高SOD和GSH水平,見(jiàn)Fig 5。 Fig 1LA inhibits inflammatory cell accumulation in BALF of cigarette smoke-exposed mice(±s,n=10)
A:Total leukocyte cell counts;B:Differential cell counts.##P<0.01 vs control group;*P<0.05,**P<0.01 vs model group.Licochalcone A(LA);Dexamethasone(Dex)
Fig 2 LA inhibits inflammatory cells’infiltration in lungs of cigarette smoke-exposed mice(HE,×200)
Fig 3 LA inhibits expression of cytokines mRNA in lung tissues of cigarette smoke-exposed mice(±s,n=10)
2.4LA抑制CSE刺激BEAS-2B細(xì)胞表達(dá)IL-8 和MMP-9 mRNA利用熒光實(shí)時(shí)定量PCR檢測(cè)各組細(xì)胞中IL-8和MMP-9 mRNA的表達(dá)。結(jié)果顯示,與空白對(duì)照組相比,2.5%CSE刺激細(xì)胞48 h后,IL-8和MMP-9 mRNA的表達(dá)明顯上升(P<0.01),而2.5、5 μmol·L-1的LA能明顯抑制兩者的上升(P<0.05~0.01)。不加CSE刺激,只單獨(dú)給予5 μmol·L-1LA的細(xì)胞,IL-8和MMP-9 mRNA的表達(dá)與空白對(duì)照組細(xì)胞(不加CSE,不加藥物)相當(dāng),無(wú)明顯變化,見(jiàn)Fig 6。
2.5LA對(duì)CSE刺激BEAS-2B細(xì)胞信號(hào)通路活化的影響通過(guò)Western blot方法檢測(cè)BEAS-2B細(xì)胞p38、JNK和ERK1/2磷酸化表達(dá),探索LA治療小鼠急性肺損傷的作用機(jī)制。如Fig 7所示,CSE可以誘導(dǎo)p38、JNK和ERK1/2的磷酸化,而LA能劑量依賴性抑制由CSE誘導(dǎo)的肺上皮細(xì)胞ERK1/2磷酸化(Fig 7C),但對(duì)p38(Fig 7A)和JNK(Fig 7B)磷酸化無(wú)明顯抑制作用。采用核質(zhì)分離的方法檢測(cè)細(xì)胞核內(nèi)NF-κB亞基p65的表達(dá),以進(jìn)一步探索LA治療小鼠急性肺損傷的作用機(jī)制。如Fig 7D所示,2.5%CSE能明顯誘導(dǎo)p65的表達(dá),LA可明顯抑制其表達(dá)。
COPD以遍及氣道、肺實(shí)質(zhì)和肺血管的慢性炎癥為特征。吸煙是誘發(fā)COPD的主要因素之一,香煙煙霧中含有數(shù)以萬(wàn)計(jì)的有毒有害物質(zhì),這些有毒化合物一旦進(jìn)入肺部,就難以代謝排出,從而誘導(dǎo)炎癥并直接導(dǎo)致肺組織的損傷[13]。本研究重點(diǎn)觀察LA對(duì)吸煙誘導(dǎo)的小鼠急性肺損傷的作用及作用機(jī)制。
Fig 4 LA inhibits expression of cytokines protein in BALF of cigarette smoke-exposed mice(±s,n=10)
COPD的氣道炎癥不同于哮喘,其炎癥細(xì)胞主要為中性粒細(xì)胞、CD8+T淋巴細(xì)胞和巨噬細(xì)胞,肺損傷后炎癥細(xì)胞的浸潤(rùn)、活化導(dǎo)致促炎細(xì)胞因子的異常表達(dá),與COPD的病理生理過(guò)程密切相關(guān)。KC是人IL-8的同源物,通過(guò)其受體CXCR2產(chǎn)生作用,是肺中性粒細(xì)胞趨化的重要介質(zhì)。有研究顯示,通過(guò)小分子抑制劑抑制CXCR2受體,能明顯抑制吸煙誘導(dǎo)動(dòng)物模型中肺中性粒細(xì)胞炎癥[14]。前炎細(xì)胞因子,如TNF-α和IL-1β,能增強(qiáng)炎癥反應(yīng)。吸煙可介導(dǎo)COPD患者全身性炎癥反應(yīng),與非吸煙者相比,吸煙的COPD患者血清中TNF-α表達(dá)明顯升高[15]。最近,IL-1β因其參與炎癥反應(yīng)的長(zhǎng)期持續(xù)過(guò)程而廣受關(guān)注[16]。IL-1β能明顯激活COPD患者體內(nèi)的巨噬細(xì)胞,釋放炎癥細(xì)胞因子、趨化因子和MMP-9[17]。本研究中,香煙暴露4 d后,小鼠肺部炎癥細(xì)胞(中性粒細(xì)胞和巨噬細(xì)胞)大量聚集,肺泡灌洗液和肺組織中炎癥因子KC、TNF-α及IL-1β的蛋白和mRNA水平明顯上升。LA能有效抑制這些炎癥細(xì)胞的聚集和炎癥因子的釋放,提示其對(duì)肺損傷的保護(hù)作用可能與抑制炎癥反應(yīng)有關(guān)。
體內(nèi)氧化/抗氧化平衡失調(diào)是COPD慢性損傷的重要原因。人們發(fā)現(xiàn),無(wú)論在急性發(fā)作期還是在緩解期均存在兩者作用失調(diào)。煙草中含有大量的氧化劑,刺激炎癥細(xì)胞生成內(nèi)源性氧化物,減少抗氧化物生成,促進(jìn)氧化應(yīng)激反應(yīng)。SOD和GSH是氣道內(nèi)的抗氧化物質(zhì),研究表明香煙煙霧可以降低內(nèi)源性抗氧化物的活性。如在人支氣管肺泡上皮細(xì)胞實(shí)驗(yàn)中,發(fā)現(xiàn)香煙提取物能降低GSH的含量[18],而另一項(xiàng)臨床研究發(fā)現(xiàn),吸煙者血漿中 SOD的活性下降[19]。研究顯示,LA能抑制由叔丁基過(guò)氧化氫誘導(dǎo)的氧化應(yīng)激反應(yīng),降低氧基自由基離子(ROS)水平,減少GSH的消耗[20]。我們的研究發(fā)現(xiàn),LA不僅能夠抑制炎癥因子的釋放,而且能夠有效降低MPO活性,同時(shí)升高SOD活性和GSH水平。我們的結(jié)果表明,在香煙誘導(dǎo)的小鼠模型中,LA對(duì)肺損傷的保護(hù)作用可能與調(diào)節(jié)氧化和抗氧化的平衡有關(guān)。
正常人體的氣道、肺泡內(nèi)蛋白酶和抗蛋白酶處于平衡狀態(tài),這種平衡的失調(diào),會(huì)導(dǎo)致肺實(shí)質(zhì)的結(jié)構(gòu)破壞和纖維化。目前認(rèn)為肺泡損傷和小氣道纖維化是COPD氣流受限的重要原因[21]?;|(zhì)金屬蛋白酶(matrix metalloproteinases,MMPs)是一組鋅離子依賴性蛋白水解酶家族,能降解細(xì)胞外基質(zhì)蛋白。MMP-9是該家族最重要的成員之一,在纖維化修復(fù)過(guò)程中,它是參與細(xì)胞外基質(zhì)成分降解的關(guān)鍵酶[22]。研究顯示,在香煙煙霧誘導(dǎo)的小鼠肺部炎癥模型中,MMP-9表達(dá)明顯上升[23],另有研究發(fā)現(xiàn)COPD患者的MMP-9表達(dá)較正常人高[24]。本實(shí)驗(yàn)結(jié)果顯示,香煙暴露后,小鼠肺泡灌洗液內(nèi)MMP-9蛋白表達(dá)增加,而LA能夠明顯抑制這種蛋白的表達(dá)。MMP-9 mRNA的表達(dá)變化和其蛋白一致,在整體和離體實(shí)驗(yàn)中,LA均能明顯抑制MMP-9 mRNA的高表達(dá)。我們推測(cè)LA對(duì)香煙誘導(dǎo)的急性肺損傷的保護(hù)作用可能與下調(diào)蛋白酶有關(guān)。
Fig 5 LA inhibits oxidative stress in lung tissues of cigarette smoke-exposed mice(±s,n=10)
?Fig 6 LA suppresses CSE-induced mRNA expression of cytokines in BEAS-2B cells(±s,n=6)
肺上皮屏障是肺抵抗外界環(huán)境的第一道防御系統(tǒng),在這個(gè)屏障里,肺上皮細(xì)胞起很重要的防御作用。為進(jìn)一步探索LA對(duì)急性肺損傷的保護(hù)作用和作用機(jī)制,我們用人肺上皮細(xì)胞株BEAS-2B進(jìn)行了相關(guān)研究。目前的研究[25]和我們的實(shí)驗(yàn)均顯示,在香煙煙霧刺激的人肺上皮細(xì)胞中,IL-8表達(dá)量的升高幅度比其他炎癥因子高。炎癥細(xì)胞的遷移和激活受到細(xì)胞因子和趨化因子的調(diào)節(jié),IL-8又稱CXCL8,是中性粒細(xì)胞趨化因子,在COPD發(fā)展過(guò)程中發(fā)揮重要作用[26]。Schneider等[27]的研究顯示,COPD患者氣道上皮細(xì)胞中 IL-8表達(dá)明顯升高。IL-8通過(guò)其受體CXCR2發(fā)揮作用,用CXCR2拮抗劑可明顯減輕中性粒細(xì)胞炎癥和肺泡損傷。本研究中,2.5%CSE刺激細(xì)胞48 h后,IL-8 mRNA的表達(dá)明顯上升,而LA能明顯抑制其高表達(dá),進(jìn)一步證明了LA對(duì)肺損傷中炎癥反應(yīng)的抑制作用。
Fig 7 Effects of LA on CSE-induced protein expression of MAP kinases and NF-κB in BEAS-2B cells(±s,n=6)
MAPK家族包括ERK(extracellular signal-regulated kinase)、p38和JNK(c-Jun NH2-terminal kinase),這些通路調(diào)節(jié)細(xì)胞的多種生理病理過(guò)程,如細(xì)胞增殖、分化、遷移、存活和死亡等。轉(zhuǎn)錄因子NF-κB是內(nèi)皮激活過(guò)程中重要的調(diào)節(jié)因子,與許多炎癥因子的表達(dá)相關(guān),包括細(xì)胞因子和黏附分子NO、TNF-α、MCP-1和CAMs[28-29]。在細(xì)胞核內(nèi),NF-κB以p65/ p50二聚體形式存在,NF-κB的激活過(guò)程首先是與IκBα解離,然后p65亞基入核,介導(dǎo)炎癥因子的轉(zhuǎn)錄活性[30]。研究報(bào)道,CSE誘導(dǎo)的炎癥反應(yīng)與MAPK信號(hào)通路的激活密切相關(guān),阻斷MAPK通路的激活能保護(hù)上皮細(xì)胞,減輕炎癥反應(yīng)、抗凋亡和抗氧化應(yīng)激反應(yīng)[31-32]。Chu等[9]的研究證明LA能通過(guò)p38/ERK MAPK信號(hào)通路緩解LPS誘導(dǎo)的小鼠急性肺損傷模型的炎癥反應(yīng),F(xiàn)urusawa等[10]還發(fā)現(xiàn)其能明顯抑制LPS誘導(dǎo)的NF-κB轉(zhuǎn)錄活性。我們猜測(cè)LA調(diào)節(jié)CSE誘導(dǎo)的炎癥反應(yīng)可能與MAPK途徑中的多條信號(hào)通路及NF-κB的核轉(zhuǎn)錄有關(guān)。為了驗(yàn)證這一推測(cè),我們采用Western blot方法檢測(cè)BEAS-2B細(xì)胞中p38、JNK和ERK1/2的磷酸化表達(dá)以及NF-κB的活性。實(shí)驗(yàn)結(jié)果表明,LA能明顯抑制CSE誘導(dǎo)的ERK1/2的磷酸化,但對(duì)p38和JNK的磷酸化無(wú)明顯作用,同時(shí)LA也能有效阻斷細(xì)胞核內(nèi)NF-κB p65亞基的激活,提示LA發(fā)揮的抗炎作用可能是通過(guò)阻斷NF-κB p65的激活以及抑制CSE誘導(dǎo)的ERK1/2 MAPK的激活。
綜上所述,LA對(duì)香煙煙霧誘導(dǎo)的急性肺損傷具有保護(hù)作用,這種保護(hù)作用可能與抑制炎癥介質(zhì)的高表達(dá)、調(diào)節(jié)氧化/抗氧化失衡、下調(diào)金屬蛋白酶有關(guān),其調(diào)控機(jī)制可能與阻斷ERK1/2/NF-κB途徑有關(guān)。
(致謝:本實(shí)驗(yàn)在浙江大學(xué)醫(yī)學(xué)院呼吸藥物研究實(shí)驗(yàn)室完成。感謝謝強(qiáng)敏教授和實(shí)驗(yàn)室同學(xué)在本課題研究中給予的指導(dǎo)和幫助。感謝沈陽(yáng)藥科大學(xué)中藥學(xué)院趙余慶教授課題組提取和純化甘草查爾酮A。)
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Licochalcone A protects against cigarette smoke-mediated acute lung injury in mice by suppressing ERK1/2/NF-κB pathways
REN Qian-qian1,WANG Li-pei2,ZHAO Wei3,LU Hong1,XIE Qiang-min4,ZHANG Shui-juan1
(1.College of Pharmaceutical Science,2.College of Basic Medical Science,Zhejiang Chinese Medical University,Hangzhou310053,China;
3.the First Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou310006,China;4.Zhejiang Respiratory Drugs Research Laboratory,Zhejiang University School of Medicine,Hangzhou310058,China)
AimTo explore the protective roles of licochalcone A(LA)on mice with cigarette smoke-mediated acute lung injury and the related mechanisms. MethodsIn vivo:Mice were exposed to cigarette smoke(CS)to establish acute lung injury model.The bronchoalveolar lavage fluid(BALF)was conducted for cell counting.The mRNA and protein expression of keratinocyte-derived chemokine(KC),tumor necrosis factor alpha(TNF-α),interleukin 1β(IL-1β)and matrix metalloproteinases(MMP)-9 in lungs were determined.The myeloperoxidase(MPO),superoxide dismutase(SOD)activities and glutathione(GSH)levels in lungs were quantified.The paraffin sections of lungs were prepared and stained with HE.In vitro:Human lung epithelial cells(BEAS-2B)were exposed to cigarette smoke extract(CSE),which induced cell injury.The releases of interleukin 8(IL-8)and MMP-9 were assessed.The phosphorylation of mitogen-activated protein kinases(MAPKs,including ERK1/2,p38 and JNK)and nuclear factor-κB(NF-κB)p65 protein were analyzed by Western blot.ResultsIn vivo:The accumulation of inflammatory cells was lower in LA groups than that in model group.In comparison with normal control group,the mRNA and protein lev-els of KC,TNF-α,IL-1β and MMP-9 were significantly increased in model group.Following treatment with LA,the above indicators were significantly decreased as compared to model group.In the CS-exposed mice,the MPO activity in lungs was significantly increased,meanwhile the SOD activity and GSH level were significantly decreased compared with the air-exposed animals.CS-induced activity of MPO was significantly inhibited,which were accompanied by increases in SOD and GSH levels by LA.In vitro:CSE-induced mRNA levels of IL-8 and MMP-9 were significantly inhibited by LA at 2.5 and 5 μmol·L-1.The CSE-induced phosphorylation of ERK1/2 and nucleus NF-κB p65 protein expression were prevented by pretreatment with LA.ConclusionsLA has protective effects on CS-exposed acute lung injury in mice by preventing CS-induced pulmonary inflammation,oxidative stress and protease rise.The exploration of the mechanisms suggests that LA exerts protective effects via suppressing ERK1/2/NF-κB pathways.
licorice;licochalcone A;lung injury;cigarette smoke;lung epithelial cell;cytokines;signal pathway
2016-01-07,修回日期:2016-02-02
浙江省教育廳科研項(xiàng)目(No Y201328230);浙江中醫(yī)藥大學(xué)校級(jí)科研基金項(xiàng)目(No 2010ZY07)
任倩倩(1989-),女,碩士生,研究方向:抗炎免疫藥理學(xué),E-mail:61324680@qq.com;張水娟(1980-),女,博士,實(shí)驗(yàn)師,研究方向:抗炎免疫藥理學(xué),通訊作者,E-mail:zsjdxw@163.com
10.3969/j.issn.1001-1978.2016.05.011
A
1001-1978(2016)05-0643-09
R-332;R284.1;R163.1;R322.35;R563.902.5