張永紅,李鳳娟,李少軍,謝新明,柯 蕊,李芳偉,朱燕亭,劉 璐,李滿祥*

(1西安交通大學(xué)第二附屬醫(yī)院呼吸內(nèi)科,西安 710004;2西安交通大學(xué)第一附屬醫(yī)院呼吸與危重癥醫(yī)學(xué)科;*通訊作者,E-mail:manxiangli@hotmail.com)

Notch3信號(hào)通路介導(dǎo)MCT誘導(dǎo)的PAH大鼠肺血管ECM的重塑以及分子機(jī)制

張永紅1,李鳳娟1,李少軍2,謝新明2,柯 蕊1,李芳偉2,朱燕亭2,劉 璐2,李滿祥2*

(1西安交通大學(xué)第二附屬醫(yī)院呼吸內(nèi)科,西安 710004;2西安交通大學(xué)第一附屬醫(yī)院呼吸與危重癥醫(yī)學(xué)科;*通訊作者,E-mail:manxiangli@hotmail.com)

目的 探討Notch3信號(hào)通路介導(dǎo)野百合堿(monocrotaline,MCT)誘導(dǎo)的肺動(dòng)脈高壓(pulmonary arterial hypertension,PAH)大鼠肺血管細(xì)胞外基質(zhì)(extracellular matrix,ECM)重塑以及分子機(jī)制。 方法 32只4周齡SD雄性大鼠被隨機(jī)分為3組,對(duì)照組(Con組)10只;MCT誘導(dǎo)的PAH模型組(MCT組)12只,一次性腹腔注射2% MCT 60 mg/kg;3,5-二氟苯乙酰-L-丙氨酰-S-苯基甘氨酸t(yī)-丁酯(N-[N-(3,5-difluorophena-cetyl-L-alanyl)]-S-phenylglycine t-butyl ester,DAPT)處理的PAH模型組(MCT+DAPT組)10只,一次性腹腔注射2% MCT 60 mg/kg后立即給予腹腔注射Notch抑制劑DAPT 20 mg/kg,1次/d,連續(xù)治療28 d。第28天麻醉各組大鼠,測(cè)量右心室收縮壓和右心室肥大指數(shù),Masson染色觀察肺血管ECM的分布,明膠酶譜法檢測(cè)肺組織基質(zhì)金屬蛋白酶-2/9(matrix metalloproteinases-2/9,MMP-2/9)的活性。Western blot方法檢測(cè)肺組織中Notch3、NICD3,以及TIMP-1蛋白質(zhì)水平。 結(jié)果 在MCT誘導(dǎo)的PAH模型組大鼠的肺組織中,Notch3以及其裂解片段NICD3蛋白水平較對(duì)照組明顯升高(P<0.05),伴有肺血管ECM過(guò)量沉積,MMP-2/9活性較對(duì)照組明顯增加(P<0.05),TIMP1蛋白水平亦較對(duì)照組增加(P<0.05);在MCT+DAPT組大鼠的肺組織中,Notch3和NICD3蛋白水平較MCT組明顯下降(P<0.05),MMP-2/9活性亦較MCT組降低(P<0.05),TIMP1蛋白水平亦較MCT組下降(P<0.05),伴有肺血管壁ECM較MCT組明顯減少。 結(jié)論 Notch3信號(hào)通路可能通過(guò)上調(diào)MMP-2/9活性和TIMP1蛋白的表達(dá),介導(dǎo)野百合堿誘導(dǎo)的大鼠肺血管壁ECM的重塑。

肺動(dòng)脈高壓; 細(xì)胞外基質(zhì); Notch3信號(hào)通路

肺動(dòng)脈高壓(pulmonary arterial hypertension,PAH)是以肺血管阻力增加,肺動(dòng)脈壓力升高,最終導(dǎo)致右心功能不全為主要表現(xiàn)的臨床綜合征。肺血管重塑是PAH最主要的發(fā)病機(jī)制,其特征為肺血管細(xì)胞增殖和細(xì)胞外基質(zhì)(extracellular matrix,ECM)重塑。在PAH患者的肺血管壁中,ECM過(guò)量沉積導(dǎo)致肺血管增厚變硬,肺血管阻力增加,以及血管反射波疊加,增加右心室后負(fù)荷[1]。

研究證實(shí),Notch3信號(hào)通路在PAH患者以及PAH動(dòng)物模型的發(fā)生發(fā)展中均起著重要的作用,PAH患者的Notch3受體表達(dá)明顯增高,Notch3信號(hào)通路活性增強(qiáng)[2]。過(guò)度激活Notch3信號(hào)通路介導(dǎo)ECM的重塑[3],肺血管ECM的合成和降解處于動(dòng)態(tài)平衡,MMP-2/9是ECM的主要降解酶,參與肺血管ECM的重塑[4]。腫瘤細(xì)胞的研究證實(shí),Notch3信號(hào)通路通過(guò)調(diào)節(jié)MMP-2/9的活性參與調(diào)節(jié)細(xì)胞的生命活動(dòng)[5,6]。但目前仍不清楚Notch3信號(hào)通路是否通過(guò)調(diào)控MMP-2/9的活性參與肺血管ECM的重塑。本研究探索Notch3信號(hào)通路是否介導(dǎo)MCT誘導(dǎo)的大鼠肺血管ECM以及可能的分子機(jī)制。

1 材料與方法

1.1 主要試劑

野百合堿購(gòu)自美國(guó)Sigma公司。DAPT購(gòu)自美國(guó)Santa Cruz公司。Notch3一抗購(gòu)自英國(guó)Abcam公司,TIMP1一抗購(gòu)自美國(guó)Bioworld公司。GAPDH一抗購(gòu)自美國(guó)Sigma公司。HRP標(biāo)記的二抗購(gòu)自美國(guó)Sigma公司。

1.2 實(shí)驗(yàn)動(dòng)物與分組

32只清潔級(jí)SD雄性大鼠購(gòu)自西安交通大學(xué)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心,飼養(yǎng)于西安交通大學(xué)醫(yī)學(xué)院動(dòng)物中心。分籠分組飼養(yǎng),室內(nèi)溫度為(20±2)℃、濕度為40%-60%,12 h交替晝夜循環(huán),動(dòng)物墊料經(jīng)高壓蒸汽滅菌,自主進(jìn)食飲水,適應(yīng)性飼養(yǎng)1周后SD大鼠體重為170-180 g進(jìn)行實(shí)驗(yàn)。實(shí)驗(yàn)大鼠隨機(jī)分為三組:對(duì)照組(Con組)正常飼養(yǎng)28 d;MCT誘導(dǎo)的PAH模型組(MCT組):給予60 mg/kg MCT一次性腹腔注射后正常飼養(yǎng)28 d;DAPT處理的PAH模型組(MCT+DAPT組):給予60 mg/kg MCT一次性腹腔注射后立即給予DAPT 10 mg/kg腹腔注射,1次/d,持續(xù)治療28 d。

1.3 右心室收縮壓測(cè)定

野百合堿注射后第28天,10%水合氯醛腹腔注射麻醉各組大鼠,行右頸外靜脈插管,經(jīng)頸總靜脈、上腔靜脈、右心房進(jìn)入右心室,測(cè)定并記錄右心室收縮壓(right ventricle systolic pressure,RVSP)。

1.4 右心室肥大指數(shù)測(cè)定

右心室收縮壓檢測(cè)完畢后,立即分離心臟與肺臟,沿室間溝邊緣分離右心室(RV),生理鹽水沖洗,濾紙吸除多余水分,分別稱取右心室及左心室加室間隔(LV+S)的重量,計(jì)算右心室肥大指數(shù)(index of right ventricular hypertrophy,RVH),即RV/(LV+S)。

1.5 肺組織Masson染色

取肺組織甲醛固定,石蠟包埋,切片。將石蠟切片脫蠟、水化后,浸入Weigert鐵蘇木素染液染色5 min,經(jīng)1%鹽酸乙醇分化、Masson藍(lán)化液反藍(lán)后,麗春紅品紅染色染5 min,苯胺藍(lán)溶液染色2 min,弱酸溶液洗1 min,最后經(jīng)無(wú)水乙醇脫水,二甲苯透明,中性樹(shù)膠封片。

1.6 明膠酶譜法檢測(cè)MMP-2、MMP-9活性

將肺組織提取的總蛋白用BCA蛋白定量試劑盒定量。取等量蛋白樣品上樣到含有0.1%明膠的10% SDS-PAGE凝膠,電泳。電泳結(jié)束后,將分離膠切下,置于含2.5% Triton X-100的洗脫液中,室溫下于搖床上振蕩洗脫2次,每次40 min。隨后將凝膠置于孵育液中,37 ℃震蕩孵育42 h。孵育結(jié)束后,將含有0.1%明膠的凝膠置于考馬斯亮藍(lán)染液中染色至少3 h,將凝膠移入脫色液中脫色,在藍(lán)色背景色顯示出清晰透亮帶為止,置于雙蒸水中終止脫色?？梢?jiàn)72 kD(MMP-2)和92 kD(MMP-9)處為透亮帶,將所得結(jié)果于凝膠成像系統(tǒng)照相。

1.7 Western bolt檢測(cè)Notch3、NICD3、TIMP-1水平

將肺組織提取的總蛋白用BCA蛋白定量試劑盒定量。取等量蛋白樣品上樣到10% SDS-PAGE凝膠,電泳。將凝膠上的蛋白轉(zhuǎn)移至NC膜,封閉后分別加Notch3、NICD3、TIMP-1及GAPDH一抗溶液,4 ℃孵育過(guò)夜。TBST洗膜,滴加二抗溶液,室溫孵育2 h,TBST洗膜后加入發(fā)光液,曝光,顯影,定影,晾干,用Quantity One 4.6.2(美國(guó)Bio-Rad公司)凝膠成像分析儀采集底片圖像并進(jìn)行定量分析。

1.8 統(tǒng)計(jì)學(xué)分析

實(shí)驗(yàn)數(shù)據(jù)應(yīng)用SPSS13.0軟件統(tǒng)計(jì)分析,正態(tài)分布的計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差,非正態(tài)分布資料經(jīng)正態(tài)轉(zhuǎn)換后再進(jìn)行統(tǒng)計(jì)學(xué)分析,多組間比較應(yīng)用單因素方差分析Tukey post hoc檢驗(yàn),以P<0.05認(rèn)為組間差異具有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 野百合堿誘導(dǎo)的PAH大鼠肺組織Notch3和NICD3蛋白的水平明顯升高

本研究應(yīng)用Western blot法檢測(cè)了三組實(shí)驗(yàn)大鼠肺組織Notch3、NICD3和內(nèi)參GAPDH蛋白的水平,在MCT誘導(dǎo)的PAH大鼠肺組織中,Notch3蛋白水平明顯增加,是對(duì)照組的1.83倍(P<0.05),DAPT可明顯降低PAH模型組大鼠肺組織中的Notch3水平,為對(duì)照組的0.98倍(P<0.05,見(jiàn)圖1)。Notch3細(xì)胞內(nèi)片段NICD3的水平變化趨勢(shì)與Notch3相似(見(jiàn)圖1C),在MCT誘導(dǎo)的PAH大鼠肺組織中,NICD3蛋白水平明顯增加,是對(duì)照組的1.82倍(P<0.05),DAPT可顯著降低PAH模型組NICD3的水平,為對(duì)照組的0.83倍(P<0.05),提示在MCT誘導(dǎo)的PAH大鼠肺組織中,Notch3信號(hào)通路活性增強(qiáng)。

A.Western blot結(jié)果 B.應(yīng)用GAPDH內(nèi)參校正后,三組大鼠肺組織Notch3蛋白質(zhì)相對(duì)水平 C.應(yīng)用GAPDH內(nèi)參校正后,三組大鼠肺組織NICD3蛋白質(zhì)相對(duì)水平與對(duì)照組比較,*P<0.05;與MCT組(野百合堿誘導(dǎo)的PAH模型組)比較,#P<0.05圖1 大鼠肺組織Notch3和NICD3蛋白水平的變化(n=3)Figure 1 The protein level of Notch3 and NICD3 in rat lung (n=3)

2.2 抑制Notch3信號(hào)通路對(duì)野百合堿誘導(dǎo)大鼠的右心室收縮壓的影響

在無(wú)肺動(dòng)脈瓣狹窄或右心室流出道梗阻的情況下,肺動(dòng)脈壓相當(dāng)于右心室收縮壓,因此本研究用右心室收縮壓評(píng)估大鼠肺動(dòng)脈壓的變化。SD雄性大鼠腹腔注射MCT后,第28天右心室收縮壓為(39.0±2.6)mmHg,明顯高于對(duì)照組(21.7±1.5)mmHg(P<0.05,見(jiàn)圖2)。MCT誘導(dǎo)大鼠的肺動(dòng)脈壓力符合PAH的生理學(xué)特征,提示PAH模型建立成功。給予DAPT處理后PAH模型大鼠的右心室收縮壓下降為(26.6±1.3)mmHg,與野百合堿誘導(dǎo)的PAH模型組比較,差異有統(tǒng)計(jì)學(xué)意義(P<0.05),提示抑制Notch3信號(hào)通路可降低MCT誘導(dǎo)大鼠的右心室收縮壓。

2.3 抑制Notch3信號(hào)通路對(duì)野百合堿誘導(dǎo)大鼠的右心室肥大指數(shù)的影響

右心肥大指數(shù)是右心室重量與左心室加室間隔重量的比值,是衡量右心室肥大的重要指標(biāo),大鼠腹腔注射MCT后,第28天SD大鼠右心室肥大指數(shù)為(53.4±5.3)%,明顯高于對(duì)照組(22.2±2.2)%(P

<0.05,見(jiàn)圖3),經(jīng)DAPT治療后,PAH模型大鼠右心肥大指數(shù)下降為(33.5±2.6)%,與野百合堿誘導(dǎo)的PAH模型組比較,差異有統(tǒng)計(jì)學(xué)意義(P<0.05),提示抑制Notch3信號(hào)通路可以抑制MCT誘導(dǎo)大鼠的右心室肥大。

RVSP:right ventricle systolic pressure,右心室收縮壓;與對(duì)照組比較,*P<0.05;與MCT組(野百合堿誘導(dǎo)的PAH模型組)比較,#P<0.05圖2 Notch3信號(hào)通路對(duì)MCT誘導(dǎo)大鼠右心室收縮壓的影響 (n=9-10)Figure 2 Effect of Notch3 signaling pathway on right ventricle systolic pressure in MCT-induced PAH rats (n=9-10)

RV/LV+S:右心室重量與左心室加室間隔重量的比值與對(duì)照組比較,*P<0.05;與MCT組(野百合堿誘導(dǎo)的PAH模型組)比較,#P<0.05圖3 Notch3信號(hào)通路對(duì)MCT誘導(dǎo)大鼠右心室肥大指數(shù)的影響 (n=9-10)Figure 3 Effect of Notch3 signaling pathway on right ventricular hypertrophy index in MCT-induced PAH rats (n=9-10)

2.4 抑制Notch3信號(hào)通路對(duì)野百合堿誘導(dǎo)的PAH大鼠肺血管ECM重塑的影響

Notch3信號(hào)通路參與PAH的發(fā)生,肺血管重塑是PAH的重要發(fā)病機(jī)制,肺血管重塑主要表現(xiàn)為肺血管細(xì)胞增加以及肺血管ECM的過(guò)量沉積,過(guò)量沉積的ECM導(dǎo)致肺血管壁增厚僵硬,管腔狹窄,血管阻力增加。本研究為了探索Notch3信號(hào)通路是否介導(dǎo)MCT誘導(dǎo)的PAH大鼠肺血管ECM重塑,大鼠肺組織切片Masson染色觀察其膠原分布情況。對(duì)照組大鼠肺血管壁、細(xì)支氣管壁及肺泡間隔可見(jiàn)少量的膠原沉積(見(jiàn)圖4A);MCT誘導(dǎo)的PAH大鼠肺血管內(nèi)彈膜厚薄不均斷裂,血管壁有較多的藍(lán)染膠原沉積(見(jiàn)圖4B);DAPT處理的PAH模型組大鼠的肺血管壁藍(lán)染膠原較PAH模型組明顯減少(見(jiàn)圖4C)。

A.對(duì)照組大鼠肺血管壁可見(jiàn)少量的膠原沉積 B.MCT誘導(dǎo)的PAH(MCT組)大鼠肺血管壁有大量廣泛分布的膠原沉積 C.DAPT處理的PAH模型(MCT+DAPT組)大鼠肺血管膠原沉積較MCT組明顯減少圖4 Notch3信號(hào)通路對(duì)MCT誘導(dǎo)的PAH大鼠肺血管膠原沉積的影響 (Masson染色,×400)Figure 4 Effect of Notch3 signaling pathway on pulmonary arterial vascular ECM remodeling in MCT-induced PAH rats (Masson,×400)

2.5 Notch3信號(hào)通路對(duì)野百合堿誘導(dǎo)的PAH大鼠肺組織MMP-2/9活性的影響

本研究為了探討Notch3信號(hào)通路是否調(diào)控MCT誘導(dǎo)的PAH大鼠肺組織MMP-2/9活性,應(yīng)用明膠酶譜法檢測(cè)各組實(shí)驗(yàn)大鼠肺組織MMP-2/9活性,結(jié)果顯示,在MCT誘導(dǎo)的PAH大鼠肺組織中,MMP-9活性明顯增加(P<0.05,見(jiàn)圖5),是對(duì)照組的1.86倍,DAPT可明顯降低PAH模型組大鼠的MMP-9活性(P<0.05),其活性降為對(duì)照組的1.21倍。在MCT誘導(dǎo)的PAH大鼠肺組織中,MMP-2活性明顯增加,是對(duì)照組的3.38倍(P<0.05,見(jiàn)圖5C),DAPT抑制Notch3信號(hào)通路可明顯降低PAH模型組大鼠的MMP-2活性(P<0.05),其活性降為對(duì)照組的1.76倍。

2.6 Notch3信號(hào)通路對(duì)野百合堿誘導(dǎo)的PAH大鼠肺組織TIMP1蛋白表達(dá)的影響

TIMP1是重要的膠原酶抑制劑,調(diào)控ECM的重塑。本研究應(yīng)用Western blot法檢測(cè)三組大鼠肺組織TIMP1和內(nèi)參GAPDH蛋白水平結(jié)果顯示,在MCT誘導(dǎo)的PAH大鼠肺組織中,TIMP1蛋白水平明顯增加,是對(duì)照組的1.74倍(P<0.05,見(jiàn)圖6),DAPT抑制Notch3信號(hào)通路可明顯降低PAH模型組大鼠肺組織中的TIMP1蛋白水平(P<0.05),降為對(duì)照組的1.22倍。

3 討論

本研究探討了Notch3信號(hào)通路在MCT誘導(dǎo)的PAH大鼠肺血管ECM重塑中的作用及其分子機(jī)制。在MCT誘導(dǎo)的PAH大鼠肺組織中,Notch3信號(hào)活性增強(qiáng),伴有肺血管ECM過(guò)量沉積,通過(guò)DAPT抑制Notch3信號(hào)通路,可顯著減輕MCT誘導(dǎo)的PAH大鼠肺血管ECM的過(guò)量沉積,提示Notch3信號(hào)通路介導(dǎo)MCT誘導(dǎo)的PAH大鼠肺血管ECM的重塑。在MCT誘導(dǎo)的PAH大鼠肺組織中,MMP-2/9膠原酶活性增強(qiáng),TIMP1表達(dá)上調(diào),導(dǎo)致MMP/TIMP比例失衡,誘發(fā)肺血管ECM的重塑,抑制Notch3信號(hào)通路可重建MMP/TIMP失衡,減輕MCT大鼠肺血管ECM的過(guò)量沉積,提示Notch3信號(hào)通路可能通過(guò)上調(diào)MMP-2/9活性和TIMP1蛋白的表達(dá),促進(jìn)肺血管壁ECM的重塑。

A.三組大鼠肺組織MMP2和MMP-9明膠酶譜圖 B.應(yīng)用明膠酶譜法檢測(cè)大鼠肺組織MMP-2相對(duì)明膠酶活性 C.應(yīng)用明膠酶譜法檢測(cè)大鼠肺組織MMP-2相對(duì)明膠酶活性與對(duì)照組比較,*P<0.05;與MCT組(野百合堿誘導(dǎo)的PAH模型組)比較,#P<0.05圖5 Notch3信號(hào)通路對(duì)MCT誘導(dǎo)的PAH大鼠肺組織MMP-2和MMP-9明膠酶活性的影響(n=3) Figure 5 Effect of Notch3 signaling pathway on activity of MMP-2/9 from lung tissue lysates inMCT-induced PAH rats (n=3)

A.三組大鼠肺組織TIMP1和GADPH蛋白的Western blot結(jié)果 B.應(yīng)用GAPDH內(nèi)參校正后,三組大鼠肺組織TIMP1蛋白相對(duì)水平與對(duì)照組比較,*P<0.05;與MCT組(野百合堿誘導(dǎo)的PAH模型組)比較,#P<0.05圖6 Notch3信號(hào)通路對(duì)MCT誘導(dǎo)的PAH大鼠肺組織TIMP1蛋白水平的影響 (n=3)Figure 6 Effect of Notch3 signaling pathway on the level of TIMP1 from lung tissue lysates in MCT-induced PAH rats (n=3)

肺血管重塑是PAH最主要的發(fā)病機(jī)制,其特征為肺血管細(xì)胞增殖和ECM重塑。既往的研究表明Notch3信號(hào)通路參與ECM的重塑。在高血壓腎纖維化患者的腎臟組織中,Notch3表達(dá)明顯升高[7],此外γ分泌酶抑制劑抑制Notch信號(hào)通路可以減輕糖尿病腎病的腎臟纖維化[8],在硬皮病患者中,Notch3信號(hào)通路參與調(diào)節(jié)ECM的合成和分泌,過(guò)度激活的Notch3信號(hào)通路誘導(dǎo)成纖維細(xì)胞轉(zhuǎn)化為肌成纖維細(xì)胞,并分泌大量的Ⅰ和Ⅲ型膠原蛋白等ECM,促進(jìn)硬皮病的進(jìn)展[9]。腺病毒介導(dǎo)的Notch3-shRNA下調(diào)Notch3信號(hào)可以抑制上皮-間質(zhì)轉(zhuǎn)化,抑制四氯化碳誘導(dǎo)的肝纖維化[3]。本研究結(jié)果提示在MCT誘導(dǎo)的PAH大鼠肺組織中,Notch3信號(hào)通路活性增強(qiáng),伴有肺血管ECM的過(guò)量沉積;抑制Notch3信號(hào)通路可以減輕百合堿誘導(dǎo)的PAH大鼠肺血管ECM的過(guò)量沉積。提示Notch3信號(hào)通路介導(dǎo)MCT誘導(dǎo)的PAH大鼠肺血管ECM的重塑。

生理狀態(tài)下,ECM的合成與降解處于動(dòng)態(tài)平衡,其主要的降解酶為MMPs,它是一組鋅依賴性多功能蛋白酶。TIMP是MMPs內(nèi)源性抑制劑,是調(diào)控MMPs活性的一個(gè)關(guān)鍵因素,防止ECM的過(guò)度降解。MMPs/TIMP動(dòng)態(tài)調(diào)節(jié)ECM的平衡,參與廣泛生理過(guò)程,包括胚胎發(fā)育,血管生成/再生,組織修復(fù)/重塑[10]。研究證實(shí)MMPs/TIMP失衡導(dǎo)致肺血管ECM過(guò)量沉積參與PAH的發(fā)生和發(fā)展[11]。MCT誘導(dǎo)的PAH大鼠MMP-2和MMP-9表達(dá)上調(diào),活性增加,抑制MMP-2/9可減輕MCT誘導(dǎo)的PAH大鼠肺血管ECM的重塑[12,13],提示MMPs在PAH肺血管ECM的重塑過(guò)程起著不可或缺的作用。本研究結(jié)果證實(shí),MCT誘導(dǎo)的PAH大鼠肺血管ECM過(guò)量沉積,伴Notch3信號(hào)通路活性增高,MMP-2/9酶活性增加,TIMP1的表達(dá)上調(diào),DAPT抑制Notch3信號(hào)通路可減輕MCT誘導(dǎo)的上述變化,減輕MCT誘導(dǎo)的PAH大鼠肺血管ECM過(guò)量沉積。在MCT誘導(dǎo)的PAH大鼠肺組織中,Notch3信號(hào)通路的活性增強(qiáng),伴有MMP2/9活性增高,抑制Notch3信號(hào)通路可以降低MMP2/9活性,提示Notch3信號(hào)通路介導(dǎo)MCT誘導(dǎo)的PAH大鼠肺組織MMP2/9活性增加,其機(jī)制可能為一方面Notch3信號(hào)通路依賴CBF1/RBPJ途徑正性調(diào)節(jié)NF-κB的表達(dá)[14-16];另一方面Notch3信號(hào)通路通過(guò)Ras正性調(diào)節(jié)ERK1/2磷酸化水平[17],激活的ERK1/2可以進(jìn)一步磷酸化核轉(zhuǎn)錄因子NF-κB,二者均促進(jìn)MMP2/9的表達(dá)[18-20]。MMP-2/9是調(diào)節(jié)ECM降解的重要金屬蛋白酶,參與PAH動(dòng)物模型肺血管ECM的重塑。綜上所述,Notch3信號(hào)通路可能通過(guò)多種信號(hào)通路正性調(diào)節(jié)MMP-2/9的活性介導(dǎo)MCT誘導(dǎo)的PAH大鼠肺血管ECM的重塑。

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Notch3signalingpathwaymediatedpulmonaryvascularECMremodelinginMCT-inducedpulmonaryarterialhypertensionratsanditsmolecularmechanism

ZHANG Yonghong1,LI Fengjuan1,LI Shaojun2,XIE Xinming2,KE Rui1,LI Fangwei2,ZHU Yanting2,LIU Lu2,LI Manxiang2*

(1DepartmentofRespiratoryMedicine,SecondAffiliatedHospitalofXi’anJiaotongUniversity,Xi’an710004,China:2DepartmentofRespiratoryandCriticalCareMedicine,FirstAffiliatedHospitalofXi’anJiaotongUniversity;*Correspondingauthor,E-mail:manxiangli@hotmail.com)

ObjectiveTo investigate the molecular mechanisms of Notch3 signaling pathway-mediated pulmonary vascular extracellular matrix(ECM) remodeling in monocrotaline(MCT)-induced pulmonary arterial hypertension(PAH) rats.MethodsTotally 32 male 4-week-old SD rats were randomly divided into 3 groups: control group(n=10), MCT-induced PAH model group(MCT,n=12), PAH model+DAPT group(MCT+DAPT,n=10). The PAH model was induced by intraperitoneal injection with MCT(60 mg/kg body weight) at day 1. DAPT(10 mg/kg) was administered by intraperitoneal injection once a day after administrated with MCT at day 1 and continuously treated for 28 d. Rats in each group were anesthetized at day 28 to measure the right ventricle systolic pressure(RVSP) and index of right ventricular hypertrophy(RVH), ECM in pulmonary vessel was localized by Masson staining, and the activity of MMP-2/9 from the lung tissue lysates was assessed by gelatin zymography. The contents of Notch3, NICD3, tissue inhibitor of matrix metalloproteinase-1(TIMP-1)protein were detected by Western blot.ResultsThe levels of Notch3 and its cleavage domain NICD3 protein in lung tissue in MCT group were significantly higher than that in control group. Compared with control group, the deposition of pulmonary vascular ECM increased in MCT group, the activity of MMP-2/9 was enhanced in lung tissue, and TIMP1 protein level from lung tissue lysates was increased. The levels of Notch3 and its cleavage domain NICD3 protein in lung tissue in MCT+DAPT group were significantly lower than those in MCT group(P<0.05), and the activity of MMP-2/9 and the level of TIMP1 protein were also lower(P<0.05), accompanied with decreased deposition ECM of pulmonary vascular wall.ConclusionNotch3 signal may promote the pulmonary vascular ECM remodeling through increasing the activity of MMP-2/9 and the expression of TIMP1.

pulmonary arterial hypertension; extracellular matrix; Notch3 signaling pathway

國(guó)家自然科學(xué)基金面上項(xiàng)目(81170051)

張永紅,男,1981-03生,博士,助理研究員,E-mail:zyh420989344@126.com

2017-06-19

R544.1

A

1007-6611(2017)11-1118-06

10.13753/j.issn.1007-6611.2017.11.007