一氧化氮抑制缺氧誘導(dǎo)因子α亞基表達(dá)對大鼠缺氧性肺動(dòng)脈高壓的作用*

陳云榮, 戴愛國， 胡瑞成

(湖南省老年醫(yī)院-湖南省老年醫(yī)學(xué)研究所呼吸疾病研究室， 湖南 長沙 410016)

一氧化氮抑制缺氧誘導(dǎo)因子α亞基表達(dá)對大鼠缺氧性肺動(dòng)脈高壓的作用*

陳云榮, 戴愛國△， 胡瑞成

(湖南省老年醫(yī)院-湖南省老年醫(yī)學(xué)研究所呼吸疾病研究室， 湖南 長沙 410016)

目的研究缺氧誘導(dǎo)因子α亞基(HIF-α)在一氧化氮(NO)抑制缺氧性肺動(dòng)脈高壓形成中的作用。方法32只成年雄性SD大鼠隨機(jī)分為4組：常氧對照組(C組)、單純?nèi)毖踅M(H組)、缺氧加左旋精氨酸組(L-Arg組)、缺氧加左旋精氨酸甲酯組(L-NAME組)。3個(gè)缺氧組常壓缺氧(10%)21 d并每天1次腹腔注射相應(yīng)藥物。測定各組大鼠平均肺動(dòng)脈壓(mPAP)、右室肥大指數(shù)(RVHI)、血管形態(tài)學(xué)指標(biāo)；原位雜交和RT-PCR檢測HIF-α、誘導(dǎo)型一氧化氮合酶(iNOS) mRNA的表達(dá)，免疫組化和Western blotting檢測HIF-α、iNOS蛋白質(zhì)的表達(dá)。結(jié)果3個(gè)缺氧組肺組織NO濃度較C組降低，且L-Arg組肺組織NO濃度高于H組，L-NAME組肺組織NO濃度低于H組。3個(gè)缺氧組mPAP、RVHI、血管壁面積與血管面積比值(WA%)、肺動(dòng)脈中膜厚度(PAMT)都較對照組增高(P<0.05)。L-Arg組mPAP和PAMT較H組低(P<0.05)，RVHI和WA%與H組比較差異無顯著。L-NAME組mPAP、RVHI、WA%和PAMT均較H組高(P<0.05)。3個(gè)缺氧組HIF-1α和HIF-3α mRNA表達(dá)較C組增高(P<0.05)，3個(gè)缺氧組間HIF-1α mRNA表達(dá)差異無顯著，而L-NAME組HIF-3α mRNA表達(dá)高于L-Arg組(P<0.05)，其它組間無顯著差異。L-NAME組HIF-2α mRNA高于C組，其它組之間差異無顯著。3個(gè)缺氧組HIF-1α、HIF-2α和HIF-3α蛋白質(zhì)表達(dá)較C組均增高(P<0.05)，且L-Arg組表達(dá)低于H組(P<0.05)，L-NAME組高于H組(P<0.05)。直線相關(guān)分析表明，大鼠肺組織NO濃度與HIF-α蛋白質(zhì)、iNOS mRNA及蛋白質(zhì)表達(dá)水平、mPAP、RVHI、WA%和PAMT呈負(fù)相關(guān)(均P<0.05)。結(jié)論在缺氧性肺動(dòng)脈高壓大鼠模型中NO主要在轉(zhuǎn)錄后下調(diào)HIF-α的表達(dá)，NO下調(diào)HIF-α的表達(dá)可能是其抑制缺氧性肺動(dòng)脈高壓形成的重要機(jī)制。

缺氧誘導(dǎo)因子； 一氧化氮； 高血壓，肺性； 缺氧

缺氧誘導(dǎo)因子(HIF)是由α、β亞基組成的異二聚體，其β亞基為結(jié)構(gòu)亞基，對氧穩(wěn)定；α亞基(HIF-α)是功能亞基，對氧敏感。HIF-α家族包括3個(gè)成員HIF-1α，HIF-2α、HIF-3α，在缺氧性肺動(dòng)脈高壓形成過程中有重要作用，我們最近研究表明，缺氧時(shí)肺小血管HIF-α蛋白質(zhì)表達(dá)增高，在缺氧大鼠和慢性阻塞性肺疾病患者的HPH發(fā)病中起著重要作用[1-3]。一氧化氮(nitric oxide,NO) 可通過調(diào)節(jié)血管張力和血管平滑肌細(xì)胞的增殖而影響HPH的發(fā)生、發(fā)展。最近研究表明：缺氧時(shí)NO可降低HIF-α蛋白質(zhì)的穩(wěn)定性，阻止缺氧誘導(dǎo)的HIF-α的蓄積。HIF-α可調(diào)節(jié)誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthase,iNOS)的表達(dá)從而影響NO的合成。 NO和HIF-α在缺氧性肺動(dòng)脈高壓形成過程中的相互調(diào)控值得研究。本實(shí)驗(yàn)通過觀察大鼠HPH模型中左旋精氨酸(L-arginine,L-Arg)和左旋硝基精氨酸甲酯(Nω-nitro-L-arginine methyl ester,L-NAME)對肺組織、肺小血管HIF-α及表達(dá)的影響，探討NO是否通過調(diào)節(jié)HIF-α表達(dá)抑制缺氧性肺動(dòng)脈高壓形成及NO和HIF-α在缺氧性肺動(dòng)脈高壓形成過程中的相互調(diào)控，為HPH的防治提供新的理論依據(jù)。

材 料 和 方 法

1動(dòng)物模型復(fù)制及分組

32只清潔級(jí)成年雄性健康SD大鼠 (湖南中醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供)，體重(270±30)g，隨機(jī)數(shù)字表法分為4組，每組8只：常氧對照組，3個(gè)缺氧組。常氧對照組(C組)每天腹腔注射生理鹽水1 mL。3個(gè)缺氧組分別為：單純?nèi)毖踅M(H組)，每天缺氧前腹腔注射生理鹽水1 mL;缺氧加L-Arg組(L-Arg組)，每天缺氧前腹腔注射L-Arg(Sigma)500 mg/kg;缺氧加L-NAME組(L-NAME組),每天缺氧前腹腔注射L-NAME(Sigma)5 mg/kg。3個(gè)缺氧組按本研究室傳統(tǒng)方法[2]每天(10.0±0.5)%氧濃度常壓缺氧8 h。

2平均肺動(dòng)脈壓(meanpulmonaryarterialpressure,mPAP)測定及右室肥厚指數(shù)(rightventriclehypertrophyindex,RVHI)的測定

按照我室的傳統(tǒng)方法[2]大鼠經(jīng)1%戊巴比妥鈉(40 mg/kg) 腹腔麻醉后，經(jīng)右頸外靜脈插入微導(dǎo)管,通過壓力傳感器接上Medlab生物信號(hào)采集系統(tǒng)(南京美易科技有限公司) 測大鼠mPAP。經(jīng)以上檢測后將大鼠處死,取出心臟置4 %多聚甲醛固定48 h，分別稱取右室(RV) 和左室+室間隔(LV+S) 的濕重,按RVHI=RV/ (LV+S) 計(jì)算RVHI。

3肺小血管形態(tài)學(xué)分析

大鼠左側(cè)肺組織以4%多聚甲醛固定,石蠟包埋,切片,厚度4 μm，蘇木精-伊紅(HE) 染色，每只大鼠選3張肺組織切片,每張切片以單盲法選取斷面積較圓的直徑100 μm左右的肺細(xì)小動(dòng)脈5支,用病理圖像分析軟件(PIPS22020型,重慶天海醫(yī)療設(shè)備有限公司)測定肺動(dòng)脈管壁面積/管總面積(ratio of vascular wall area to total vascular area,WA%)和肺動(dòng)脈中膜厚度(pulmonary artery media thickness,PAMT)，作為肺小血管重塑指標(biāo)。

4NO濃度測定

5肺小血管壁HIF-α和iNOS的原位雜交

寡核苷酸探針,地高辛標(biāo)記的多相寡核苷酸探針(武漢博士德生物工程有限公司提供) 探針序列。HIF-1α:(1)5’-TTATG AGCTT GCTCA TCAGT TGCCA CTTCC-3’; (2)5’-CTCAG TTTGA ACTAA CTGGA CACAG TGTGT-3’; (3)5’-GGCCG CTCAA TTTAT GAATA TTATC ATGCT-3’。HIF-2α:(1) 5’-CGAAC ACATA AACTC CTGTC TTCAG TGTGC-3’;(2)5’-ATCCG AGAGA ACCTG ACACT CAAAA CTGGC-3’; (3)5’-GGGCA AGTGA GAGTC TACAA CAACT GCCCC-3’.HIF-3α:(1)5’-CGCAT GCACC GCCTC TGCGC TGCAG GGGAG-3’;(2)5’-ACATG GCTTA CCTGT CGGAA AATGT CAGCA-3’;(3)5’-ATATG AGGGC CTACA AGCCC CCTGC ACAGA-3’。iNOS:(1)5’-GTGGC GTAAA GTATG TGTCT GCAGA TATGC TGGAA-3’;(2)5’-GAAGC CATGA CCTTC CGCAT TAGCA GAGAA GCAAA-3’; (3)5’-TCTTC GGGCTTCAGG TTATT GATCC AAGTG CTGCA-3’。參照說明書及Hu等[4]雜交步驟，二氨基聯(lián)苯胺(DAB) 顯色棕黃色為陽性結(jié)果(主要在胞漿)。結(jié)果觀察: 每只大鼠選2張結(jié)構(gòu)完整的切片,每張切片以單盲法選3支直徑100 μm左右的肺小動(dòng)脈，圖像分析軟件(PIPS22020型,重慶天海醫(yī)療設(shè)備有限公司)檢測肺細(xì)小動(dòng)脈管壁平均吸光度(A) 值作為管壁HIF-1α、HIF-2α、HIF-3α和iNOS表達(dá)相對含量。

6RT-PCR測肺組織中HIF-α和iNOSmRNA的轉(zhuǎn)錄

取右肺組織0.1 g，按說明書用Trizol試劑(上海生工)提取總RNA, 逆轉(zhuǎn)錄后進(jìn)行PCR，擴(kuò)增引物由上海生物公司合成。PCR 反應(yīng)條件： 94 ℃預(yù)變性5 min，94 ℃變性30 s，適宜溫度退火30 s，72 ℃延伸60 s，反應(yīng)適當(dāng)循環(huán)，最后72 ℃延伸10 min。RT-PCR產(chǎn)物經(jīng)1.5 % 的瓊脂糖凝膠(含0.5 mg/L 溴化乙啶) 電泳，凝膠圖像分析系統(tǒng)(上海Tanon Gis-2010)對擴(kuò)增產(chǎn)物條帶吸光度半定量，計(jì)算待測條帶的吸光度與內(nèi)參照β-actin的吸光度比值。引物序列、退火溫度、產(chǎn)物長度、循環(huán)數(shù)見表1。

表1 各目的基因的引物序列、退火溫度、產(chǎn)物長度、循環(huán)數(shù)

7肺小血管壁HIF-α和iNOS免疫組織化學(xué)

Ⅰ抗:HIF-1α，HIF-2α、HIF-3α、iNOS均為兔抗大鼠多克隆抗體(Santa Cruz)，1∶200稀釋。Ⅱ抗SABC測檢試劑盒(購自武漢博士德公司)。操作步驟參照李啟芳等[2]的方法, DAB顯色陽性結(jié)果(主要在胞漿) 顯棕黃色。結(jié)果觀察:肺小動(dòng)脈血管選擇以及管壁HIF-α和iNOS的蛋白表達(dá)相對含量檢測同原位雜交。

8Westernblotting檢測肺組織中HIF-α和iNOS蛋白質(zhì)表達(dá)水平

取大鼠右側(cè)肺組織0.1 g，剪碎加入1 mL改良RIPA裂解液[50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl、0.5%脫氧膽酸鈉、1 mmol/L EDTA、1% NP-40、0.1 mg/L PMSF和2 mg/L亮抑素]，冰浴條件下組織勻漿; 4 ℃ 10 000×g離心，棄除沉淀，上清液即為肺組織總蛋白。Bradford法測定上清液總蛋白濃度，樣品分裝、貯存于-80 ℃中備用。每孔加入等量蛋白質(zhì)樣品，7.5%十二烷基硫酸鈉-丙烯酰胺凝膠200伏恒壓電泳后，轉(zhuǎn)印至硝酸纖維膜。5%脫脂奶粉室溫封閉2 h后,與1∶1 000稀釋的Ⅰ抗(Santa Cruz) 4 ℃溫育過夜。封閉液漂洗后加入1∶2 000稀釋的Ⅱ抗(Santa Cruz),室溫下?lián)u床雜交1 h。漂洗后增強(qiáng)化學(xué)發(fā)光法發(fā)光，X線膠片顯影，圖像分析系統(tǒng)(上海Tanon Gis-2010)對條帶吸光度半定量，計(jì)算待測條帶的吸光度與內(nèi)參照β-actin的吸光度比值。

9統(tǒng)計(jì)學(xué)處理

結(jié) 果

1NO抑制缺氧性肺血管重塑和肺動(dòng)脈壓升高

3個(gè)缺氧組肺組織勻漿NO濃度均降低，L-Arg組肺組織勻漿NO濃度高于H組，L-NAME組低于H組,見表2；3個(gè)缺氧組mPAP、RVHI都增高，并發(fā)生血管重塑。L-Arg組mPAP、RVHI較H組低(P<0.05)，血管重塑不如H組明顯，L-NAME組mPAP、RVHI較H組高(P<0.05)，血管重塑比H組更明顯，見表2。

表2各組大鼠肺組織NO濃度及mPAP、RVHI(%)、WA%

GroupNO(mol/gprotein)mPAP(mmHg)RVHI(%)WA(%)PAMT(μm)Control12.49±2.4816.9±2.523.8±3.834.8±5.58.4±1.3L-Arg7.95±1.25*24.8±3.2*26.8±2.5*55.6±5.6*16.5±2.2*Hypoxia5.47±0.91*△30.4±4.5*△28.9±1.9*61.2±4.9*19.8±3.1*△L-NAME2.08±0.36*△#35.1±3.9*△#32.0±1.6*△#69.8±7.0*△#23.3±2.9*△#

*P<0.05vscontrol;△P<0.05vsL-Arg;#P<0.05vshypoxia.

2NO抑制缺氧誘導(dǎo)的缺氧誘導(dǎo)因子升高

2.1不同組HIF-α mRNA表達(dá)變化 原位雜交示肺小動(dòng)脈HIF-1α mRNA在對照組呈陽性表達(dá)，缺氧組HIF-1α mRNA表達(dá)增高，呈較強(qiáng)陽性或強(qiáng)陽性表達(dá)，但H組、L-Arg組、L-NAME組3個(gè)缺氧組間HIF-1α mRNA表達(dá)差異無顯著,見圖2；RT-PCR示肺組織HIF-1α mRNA表達(dá)變化規(guī)律與肺小動(dòng)脈相同，見表3、圖1。原位雜交示各組大鼠肺小動(dòng)脈HIF-2α mRNA呈陽性表達(dá)，L-NAME組HIF-2α mRNA表達(dá)略高于C組，見表3、圖3。其它組間表達(dá)差異無顯著；RT-PCR示4組大鼠肺組織HIF-2α mRNA均有明顯表達(dá)，但各組間表達(dá)無明顯差異，見圖1。原位雜交示HIF-3α mRNA在對照組呈弱陽性表達(dá)，3個(gè)缺氧組HIF-3α mRNA均呈陽性或較強(qiáng)陽性表達(dá)，見表3、圖4，且L-NAME組HIF-3α mRNA表達(dá)高于L-Arg組(P<0.05)，L-Arg組、L-NAME組與H組間比較差異均無顯著，RT-PCR示對照組HIF-3α mRNA表達(dá)較弱，3個(gè)缺氧組HIF-3α mRNA表達(dá)均較C組增高，見圖1。

Figure 1. Reverse transcription-polymerase chain reaction analysis of hypoxia-inducible factor (HIF)-α subunits (HIF-1α, HIF-2α and HIF-3α)and iNOS mRNA C:control; H, hypoxia for 21 d； L-Arg: hypoxia for 21 d with administration of L-Arg; L-NAME: hypoxia for 21d with administration of L-NAME. The amplification of β-actin was used as a control.

圖1RT-PCR檢測各組大鼠肺組織HIF-1α、HIF-2α、HIF-3α和iNOSmRNA的表達(dá)

Figure 2.Insituhybridization analysis of hypoxia-inducible factor (HIF)-1α mRNA expression in rat pulmonary arteries after exposure to hypoxia for 0 d (A) (control), 21 d(B), or hypoxia for 21 d with administration of L-Arg(C) and L-NAME(D)(DAB,×400). HIF-1α mRNA was positively stained in control (A), and increased a little after exposure to hypoxia(B) (P<0.05), but no significant differences were observed among the three hypoxia groups(B, C and D).

圖2肺小血管壁HIF-1αmRNA表達(dá)

Figure 3.Insituhybridization analysis of hypoxia-inducible factor (HIF)-2α mRNA expression in rat pulmonary arteries after exposure to hypoxia for 0 d (A) (control), 21 d(B), or hypoxia for 21 d with administration of L-Arg(C) and L-NAME(D) (DAB, ×400). HIF-2α mRNA was positively stained in control(A),and increased a little after exposure to hypoxia with administration of L-NAME(D) (P<0.05), but no significant differences were observed among A,B and C).

圖3肺小血管壁HIF-2αmRNA表達(dá)

Figure 4.Insituhybridization analysis of hypoxia-inducible factor (HIF)-3α mRNA expression in rat pulmonary arteries after exposure to hypoxia for 0 d (A) (control), 21 d(B), or hypoxia for 21 d with administration of L-Arg(C) and L-NAME(D)(DAB, ×400). HIF-3α mRNA was poor positively stained in control (A), and increased significantly after exposure to hypoxia.

圖4肺小血管壁HIF-3αmRNA表達(dá)

2.2HIF-α protein表達(dá)變化 Western blotting示HIF-1α、HIF-2α蛋白在C組大鼠肺組織中有表達(dá)，HIF-3α在C組大鼠肺組織中呈較弱表達(dá)，缺氧組大鼠HIF-1α、HIF-2α、HIF-3α表達(dá)均明顯增高，且它們在L-Arg組大鼠肺組織表達(dá)水平低于C組，而L-NAME組大鼠，肺組織表達(dá)水平高于C組,見圖5。免疫組化示HIF-1α蛋白在對照組肺小血管表達(dá)不明顯，在肺小血管中L-Arg組呈弱陽性表達(dá)，H組、L-NAME組呈陽性表達(dá)，且L-NAME組表達(dá)更明顯，見表3、圖6。HIF-2α蛋白在對照組肺小血管呈弱陽性表達(dá)，L-Arg組呈陽性表達(dá)，H組、L-NAME組呈強(qiáng)陽性表達(dá)，L-NAME組表達(dá)更明顯，見表3、圖7。HIF-3α蛋白在對照組肺小血管未見明顯表達(dá)(支氣管上皮呈弱陽性表達(dá))，L-Arg組呈陽性表達(dá)，H組、L-NAME組呈較強(qiáng)陽性表達(dá)，且L-NAME組表達(dá)更明顯，見表3、圖8。

3NO抑制缺氧誘導(dǎo)的iNOS表達(dá)升高

原位雜交示及免疫組化示對照組大鼠肺小血管iNOS mRNA和蛋白質(zhì)呈陽性表達(dá),見表3、圖9、10， H組iNOS mRNA和蛋白質(zhì)呈強(qiáng)陽性表達(dá)。H組iNOS與L-NAME組、L-Arg組比較差別無顯著，但L-NAME組iNOS與L-Arg組比較差異顯著。RT-PCR結(jié)果與原位雜交結(jié)果一致，見圖5。

Figure 5. Western blotting analysis of hypoxia-inducible factor (HIF)-α subunits (HIF-1α, HIF-2α and HIF-3α) and iNOS protein levels in rat lung tissues during normoxia (control) and hypoxia for 21 d with administration of L-Arg and L-NAME. C: control; H: hypoxia for 21 d; L-Arg: hypoxia for 21d with administration of L-Arg; L-NAME: hypoxia for 21 d with administration of L-NAME. The amplification of β-actin was used as a control.

圖5各組大鼠肺組織HIF-1α、HIF-2α、HIF-3α和iNOS蛋白質(zhì)的表達(dá)

Figure 6. Immunohistochemistry analysis of hypoxia-inducible factor (HIF)-1α protein expression in rat pulmonary arteries after exposure to hypoxia for 0 d (A) (control), 21 d(B), or hypoxia for 21 d with administration of L-Arg(C) and L-NAME(D)(DAB,×400). HIF-1α protein was induced in all three hypoxia groups, and it was significantly lower in L-Arg group, but significantly higher in L-NAME group than that in H group.

圖6肺小血管壁HIF-1α蛋白質(zhì)表達(dá)

Figure 7. Immunohistochemistry analysis of hypoxia-inducible factor (HIF)-2α protein expression in rat pulmonary arteries after exposure to hypoxia for 0 d (A) (control), 21 d(B), or hypoxia for 21 d with administration of L-Arg(C) and L-NAME(D) (DAB,×400). HIF-2α protein were induced in all three hypoxia groups, and it was significantly lower in L-Arg group, but significantly higher in L-NAME group than that in H group.

圖7肺小血管壁HIF-2α蛋白質(zhì)表達(dá)

Figure 8. Immunohistochemistry analysis of hypoxia-inducible factor (HIF)-3α protein expression in rat pulmonary arteries after exposure to hypoxia for 0 d (A) (control), 21d(B), or hypoxia for 21d with administration of L-Arg(C) and L-NAME(D) (DAB,×400). HIF-3α protein were induced in all three hypoxia groups, and it was significantly lower in L-Arg group, but significantly higher in L-NAME group than that in H group.

圖8肺小血管壁HIF-3α蛋白質(zhì)表達(dá)

表3NO對大鼠肺小動(dòng)脈HIF-1α、HIF-2α、HIF-3α和iNOSmRNA和蛋白表達(dá)的影響

GroupHIF-1αmRNAHIF-2αmRNAHIF-3αmRNAHIF-1αproteinHIF-2αproteinHIF-3αproteiniNOSmRNAiNOSproteinControl0.143±0.0180.130±0.0180.090±0.0050.082±0.0110.096±0.0070.050±0.0070.125±0.0170.130±0.020 L-Arg0.173±0.018*0.153±0.0160.220±0.020*0.112±0.012*0.184±0.019*0.141±0.017*0.232±0.029*0.223±0.030*Hypoxia0.172±0.020*0.152±0.0180.239±0.028*0.145±0.017*△0.239±0.028*△0.182±0.029*△0.248±0.041*0.244±0.025*L-NAME0.185±0.018*0.159±0.016*0.257±0.029*△0.205±0.021*△#0.294±0.028*△#0.233±0.025*△#0.283±0.032*△#0.272±0.032*△#

*P<0.05vscontrol;△P<0.05vsL-Arg;#P<0.05vshypoxia.

Figure 9.Insituhybridization analysis of iNOS mRNA expression in rat pulmonary arteries after exposure to hypoxia for 0 d (A)(control), 21 d(B), or hypoxia for 21 d with administration of L-Arg(C) and L-NAME(D) (DAB,×400). iNOS mRNA was increased in all three hypoxia groups, and was significantly higher in L-NAME group than that in H group, but no significant difference was observed between L-Arg group and H group.

圖9肺小血管壁iNOSmRNA表達(dá)

Figure 10. Immunohistochemistry analysis of iNOS mRNA expression in rat pulmonary arteries after exposure to hypoxia for 0 d (A)(control), 21 d(B), or hypoxia for 21 d with administration of L-Arg(C) and L-NAME(D) (DAB,×400). iNOS protein was increased in all three hypoxia groups, and was significantly higher in L-NAME group than than in H group, but no significant difference was observed between L-Arg group and H group.

圖10肺小血管壁iNOS蛋白質(zhì)表達(dá)

4NO濃度與HIF-α蛋白質(zhì)、iNOS表達(dá)及非小血管重塑指標(biāo)的相關(guān)性

直線相關(guān)分析表明，大鼠肺組織NO濃度與HIF-1α(r=-0.85)、HIF-2α(r=-0.89)、HIF-3α(r=-0.88)蛋白質(zhì)、iNOS mRNA(r=-0.82)、iNOS蛋白質(zhì)(r=-0.84)表達(dá)水平及PAMT(r=-0.87)、RVHI(r=-0.74)、mPAP(r=-0.83)、WA%(r=-0.87)呈負(fù)相關(guān)(均P<0.01)。

討 論

早期研究發(fā)現(xiàn)NO和L-Arg可通過抑制內(nèi)皮素1(ET-1)、血管內(nèi)皮生長因子(VEGF)等的表達(dá)[5]，抑制HPH和HPSR的發(fā)生發(fā)展。HIF對常氧和缺氧狀態(tài)下介導(dǎo)生理和病理過程起非常關(guān)鍵的作用，Manalo等[6]認(rèn)為肺動(dòng)脈內(nèi)皮細(xì)胞可能有5%的基因受HIF-1調(diào)控。我們研究發(fā)現(xiàn)HPH發(fā)展的不同階段HIF-α亞基基因表達(dá)在肺血管壁的動(dòng)態(tài)變化有明顯差異[7]，缺氧時(shí)HIF-1α可調(diào)控其靶基因iNOS、血紅素氧合酶1等表達(dá)，參與缺氧大鼠和慢性阻塞性肺疾病(COPD)患者缺氧性肺血管重塑(HPSR)的形成。我們研究還發(fā)現(xiàn)有絲分裂原激活蛋白激酶(MAPKs)、磷脂酰肌醇3激酶(PI3K)通路調(diào)控HIFs，參與在缺氧大鼠和COPD患者HPSR的形成[8]。低濃度NO可減少缺氧[9,10]及去鐵胺[11]誘導(dǎo)的HIF-1α及其靶基因在培養(yǎng)細(xì)胞內(nèi)的表達(dá)增高。本實(shí)驗(yàn)中L-Arg組大鼠HIF-α表達(dá)均降低，L-NAME組大鼠HIF-α表達(dá)均增高，且HIF-1α、HIF-2α、HIF-3α蛋白質(zhì)水平與肺組織NO濃度均呈負(fù)相關(guān)，提示缺氧性肺動(dòng)脈高壓大鼠模型中NO可能通過調(diào)節(jié)HIF-α的表達(dá)，進(jìn)而調(diào)節(jié)其靶基因的表達(dá)。

我們先前研究表明，缺氧時(shí)肺組織HIF-1α在肺組織蓄積，可誘導(dǎo)iNOS表達(dá)增高，使NO合成增加[4]。本實(shí)驗(yàn)中iNOS表達(dá)變化的趨勢與HIF-α一致，3個(gè)缺氧組(L-Arg組、單純?nèi)毖踅M、L-NAME組)iNOS水平均增高，且L-Arg組表達(dá)低于H組，L-NAME組高于H組。iNOS的表達(dá)與HIF-α呈正相關(guān)，而與NO呈負(fù)相關(guān)，提示NO可能通過抑制HIF-α的表達(dá)，降低iNOS的表達(dá)水平，使NO合成減少。

缺氧對HIF-α的表達(dá)調(diào)控主要通過影響HIF-α蛋白質(zhì)的穩(wěn)定性，我們研究發(fā)現(xiàn)大鼠缺氧性肺動(dòng)脈高壓發(fā)生發(fā)展過程中HIF-1α、HIF-2α mRNA僅輕度升高，而蛋白質(zhì)表達(dá)水平明顯升高，說明其表達(dá)主要發(fā)生在蛋白質(zhì)水平。低濃度NO可減少缺氧及去鐵胺誘導(dǎo)的HIF-1α在培養(yǎng)細(xì)胞內(nèi)的蓄積，而對HIF-1 α mRNA水平無影響，說明NO主要通過對HIF-α的蛋白質(zhì)穩(wěn)定性調(diào)節(jié)HIF-α的蛋白質(zhì)水平[8-10]。本實(shí)驗(yàn)中3個(gè)缺氧組(H組、L-Arg組、和L-NAME組)肺組織和肺小血管HIF-α蛋白質(zhì)水平均增高，且L-Arg組表達(dá)低于H組，L-NAME組高于H組，而3個(gè)缺氧組間mRNA表達(dá)水平差異無顯著，提示NO對HIF-α表達(dá)的調(diào)控發(fā)生在蛋白質(zhì)水平。

我們研究發(fā)現(xiàn)HIF脯氨酸羥化酶(HIF prolyl hydroxylase,PHD)表達(dá)和活性的改變是導(dǎo)致HIF-α差異性表達(dá)的重要原因，NO降低HIF-α蛋白質(zhì)穩(wěn)定性的作用可能與cGMP-蛋白激酶途徑無關(guān)，而與PHD活性有關(guān)[12]。Hagen等[9]發(fā)現(xiàn)這是由于線粒體細(xì)胞色素C氧化酶對O2的Km值比PHD低，NO抑制細(xì)胞色素氧化酶，氧分子重新分布，細(xì)胞內(nèi)氧濃度增加，PHD活性增強(qiáng)，HIF-1α的降解增加，其它線粒體呼吸抑制劑也有相同作用。Callapina等[10]發(fā)現(xiàn)NO可使細(xì)胞內(nèi)活性氧簇(ROS)形成增加，增強(qiáng)PHD活性，清除ROS后NO的作用減弱。Kozhukhar等[13]認(rèn)為NO損傷線粒體及線粒體呼吸鏈，線粒體內(nèi)2-酮戊二酸、亞鐵離子釋放入胞質(zhì)增加PHD的活性。本實(shí)驗(yàn)中NO增加HIF-α降解的機(jī)制是否通過增強(qiáng)PHD活性尚需進(jìn)一步研究。尚有研究發(fā)現(xiàn)常氧時(shí)NO可以增加HIF-1α的蛋白質(zhì)穩(wěn)定性[14，15]，但本實(shí)驗(yàn)中NO增加了HIF-α的降解，導(dǎo)致HIF-α蛋白質(zhì)水平下降及其靶基因表達(dá)的下調(diào)。

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Inhibitionofhypoxia-induciblefactorαexpressionbynitricoxideinratswithhypoxicpulmonaryhypertension

CHEN Yun-rong, DAI Ai-guo, HU Rui-cheng

(DepartmentofRespiratoryMedicine,HunanInstituteofGerontology,HunanProvinceGeriatricHospital,Changsha410001,China.E-mail:daiaiguo2003@163.com)

AIM: To investigate the effect of nitric oxide (NO) on the expression of hypoxia-inducible factor α(HIF-α) in rats with hypoxic pulmonary hypertension.METHODSL-arginine was used as the NO donor. Male SD rats (n=32) were randomly divided into 4 groups: 1 group of normoxia (C group) and 3 groups of hypoxia. The rats in hypoxia groups were exposed to 10% oxygen merely (H group), and with administration of L-arginine (L-Arg group) or nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME group). Mean pulmonary arterial pressure (mPAP), pulmonary artery morphometry and right ventricle hypertrophy index (RVHI) were measured. RT-PCR andinsituhybridization were used to detect the mRNA expression. Immunohistochemistry and Western blotting were adopted to determine the expression of the proteins.RESULTSThe NO concentrations of the lung tissues in 3 hypoxic groups were lower than that in C group. In H group, the NO concentration was lower than that in L-Arg group, bwt higher than that in L-NAME group. mPAP, RVHI, ratio of vascular wall area to total vascular area(WA%) and pulmonary artery media thickness(PAMT) were higher in hypoxic groups than those in C group. mPAP and PAMT in L-Arg group were significantly lower than those in H group (P<0.05), but those in L-NAME group were signficantly higher (P<0.05) WA% and RVHI in L-NAME group were significantly higher than those in H group(P<0.05), but no significant difference was observed between H group and L-Arg group. Compared with C group, the mRNA expression of HIF-1α and HIF-3α increased in hypoxia groups (P<0.05).For HIF-1α mRNA, no significant difference was observed among the 3 hypoxia groups, while the alteration of HIF-3α mRNA was more obvious in L-NAME group than that in H group (P<0.05). HIF-2α mRNA was higher in L-NAME group than that in H group (P<0.05), but no significant difference was observed among other groups. The proteins of HIF-α were induced in all 3 hypoxia groups (P<0.05), and they were significantly lower in L-Arg group (P<0.05)but significantly higher in L-NAME group (P<0.05) than that in H groups. Linear correlation analysis showed the negative correlations between NO concentration and the HIF-α protein, iNOS mRNA and protein, mPAP, RVHI, WA%, PAMT.CONCLUSIONNitric oxide may down-regulate HIF-α expression via posttranscriptional modification. The NO-mediated increase in HIF-α expression may be potentially involved in the inhibition of developing hypoxic pulmonary hypertension.

Hypoxia-inducible factor; Nitric oxide; Hypertension, pulmonary; Hypoxia

R363

A

10.3969/j.issn.1000-4718.2011.01.003

1000-4718(2011)01-0014-08

2010-06-20

2010-10-23

國家自然科學(xué)基金資助項(xiàng)目 (No.30570815);湖南省自然科學(xué)基金資助項(xiàng)目(No.07JJ3035);湖南省衛(wèi)生廳課題資助項(xiàng)目(No.B2006-178)

△通訊作者 Tel:0731-84762793； E-mail: daiaiguo2003@163.com