廖劍雄, 羅杰, 劉琳, 張謙△, 劉維佳△
IL-6及GATA-6甲基化抑制劑在動(dòng)脈型肺動(dòng)脈高壓大鼠肺組織中的作用及意義*
廖劍雄1, 羅杰2, 劉琳3, 張謙1△, 劉維佳3△
(1貴州省人民醫(yī)院急診內(nèi)科,貴州 貴陽(yáng) 550002,2貴州大學(xué)醫(yī)學(xué)院,貴州 貴陽(yáng) 550003,3貴州省人民醫(yī)院呼吸與危重癥醫(yī)學(xué)科,貴州 貴陽(yáng) 550002)
通過(guò)野百合堿(MCT)誘導(dǎo)的大鼠動(dòng)脈型肺動(dòng)脈高壓(PAH)模型研究給予白細(xì)胞介素6(IL-6)及GATA結(jié)合蛋白6(GATA-6)甲基化抑制劑后大鼠肺組織中IL-6、GATA-6及GATA-6甲基化的變化及意義。將40只成年雄性SD大鼠隨機(jī)分成對(duì)照組(按60 mg/kg單次腹腔內(nèi)注射2∶8的無(wú)水乙醇生理鹽水混合液)、MCT組(1% MCT按60 mg/kg單次腹腔內(nèi)注射)、MCT+二甲基亞砜(DMSO)組(等量1% MCT腹腔內(nèi)注射后每周3次腹部皮下注射1 mg/kg DMSO)、MCT+IL-6抗體(anti-IL-6)組(等量1% MCT腹腔內(nèi)注射后每天腹部皮下注射40 μg/kg anti-IL-6)和MCT+5-氮雜-2'-脫氧胞苷(5-Aza)組(等量1% MCT腹腔內(nèi)注射后每周3次皮下注射1 mg/kg 5-Aza),每組8只。于造模后第21天做右心室壓力監(jiān)測(cè),計(jì)算右心室肥厚指數(shù),肺組織HE染色觀察肺病理改變,免疫熒光及Western blot檢測(cè)肺組織中IL-6和GATA-6的表達(dá),甲基化特異性PCR(MSP)檢測(cè)肺組織中GATA-6甲基化程度。與對(duì)照組比較,MCT組、MCT+anti-IL-6組和MCT+5-Aza組右心室平均壓力顯著升高(<0.05);與MCT組比較,MCT+anti-IL-6組和MCT+5-Aza組右心室平均壓力顯著降低(<0.05)。與對(duì)照組比較,MCT組右心室肥厚指數(shù)顯著增高(<0.05);與MCT組比較,MCT+anti-IL-6組和MCT+5-Aza組右心室肥厚指數(shù)顯著減低(<0.05)。IL-6免疫熒光及Western blot結(jié)果顯示,與對(duì)照組比較,MCT組和MCT+5-Aza組IL-6表達(dá)顯著增多(<0.05);與MCT組比較,MCT+anti-IL-6組和MCT+5-Aza組IL-6表達(dá)顯著減少(<0.05)。GATA-6免疫熒光及Western blot結(jié)果顯示,與對(duì)照組比較,MCT組和MCT+5-Aza組GATA-6表達(dá)顯著減少(<0.05);與MCT組比較,MCT+anti-IL-6組和MCT+5-Aza組GATA-6表達(dá)顯著增多(<0.05)。MSP結(jié)果顯示,對(duì)照組肺組織中GATA-6未發(fā)生明顯甲基化;MCT組及MCT+DMSO組GATA-6呈高度甲基化;MCT+anti-IL-6組及MCT+5-Aza組GATA-6呈低度甲基化。在PAH大鼠肺組織中給予anti-IL-6后IL-6的表達(dá)顯著減少,給予5-Aza后GATA-6的表達(dá)顯著增多,而GATA-6甲基化程度顯著降低。IL-6的高表達(dá)可能是導(dǎo)致GATA-6低表達(dá)的原因,其機(jī)制可能是IL-6誘導(dǎo)了GATA-6的甲基化。
動(dòng)脈型肺動(dòng)脈高壓;白細(xì)胞介素6;GATA結(jié)合蛋白6;5-氮雜-2'-脫氧胞苷
動(dòng)脈型肺動(dòng)脈高壓(pulmonary arterial hypertension, PAH)是一種進(jìn)行性加重而不可治愈的慢性肺部疾病,因其導(dǎo)致患者的生活質(zhì)量差、住院率及死亡率高而成為近年來(lái)研究的熱點(diǎn)[1]。PAH的發(fā)病機(jī)制較為復(fù)雜,現(xiàn)有研究顯示炎癥反應(yīng)及肺動(dòng)脈平滑肌細(xì)胞(pulmonary artery smooth muscle cell, PASMC)異常增殖為主要發(fā)病機(jī)制[2-3]。之前我們?cè)赑AH動(dòng)物模型的研究已經(jīng)證實(shí)白細(xì)胞介素6(interleukin-6, IL-6)及GATA結(jié)合蛋白6(GATA-binding protein-6, GATA-6)在上述發(fā)病機(jī)制中可能起到作用,同時(shí)發(fā)現(xiàn)肺組織中IL-6與GATA-6表達(dá)呈顯著負(fù)相關(guān),而GTAT-6持續(xù)低表達(dá)可能與GATA-6甲基化有關(guān)[4-5]。
DNA甲基化是表觀遺傳學(xué)的一種化學(xué)修飾形式,會(huì)導(dǎo)致靶基因沉默而抑制其表達(dá),這個(gè)過(guò)程中DNA甲基轉(zhuǎn)移酶(DNA methyltransferases, DNMTs)作為關(guān)鍵酶起到?jīng)Q定性作用[6]。5-氮雜-2'-脫氧胞苷(5-aza-2'-deoxycytidine, 5-Aza)是一種被FDA批準(zhǔn)DNMTs抑制劑,它通過(guò)催化DNMT1去甲基化依賴蛋白酶體的形成來(lái)降解DNMT1,從而實(shí)現(xiàn)靶基因的去甲基化,臨床常用于治療骨髓增生異常綜合征[7]。為此本研究以野百合堿(monocrotaline, MCT)誘發(fā)大鼠PAH模型,并以5-Aza和IL-6抗體(anti-IL-6)為抑制劑,通過(guò)檢測(cè)大鼠肺組織中IL-6和GATA-6表達(dá)及GATA-6甲基化程度,探討三者在PAH發(fā)展中的作用及關(guān)系。
健康成年清潔級(jí)雄性SD大鼠40只,體重250~300 g,第三軍醫(yī)大學(xué)動(dòng)物實(shí)驗(yàn)室提供并飼養(yǎng),本研究已通過(guò)貴州省人民醫(yī)院倫理委員會(huì)審核(編號(hào):2018-001),所有操作均遵守實(shí)驗(yàn)動(dòng)物倫理規(guī)范。大鼠籠盒飼養(yǎng),保持環(huán)境溫度25 ℃,濕度60%~70%,自由進(jìn)食、飲水。所有動(dòng)物實(shí)驗(yàn)均在貴州醫(yī)科大學(xué)動(dòng)物實(shí)驗(yàn)中心進(jìn)行,許可證號(hào):SYXK(黔)2018-0001。
MCT(Lot#41616)和5-Aza(Lot#29075)均購(gòu)于MCE;IL-6 Ⅰ抗(GR3190303-16)和GATA-6 Ⅰ抗(GR297606-4)均購(gòu)于Abcam;山羊抗兔Ⅱ抗(ZF-0316)購(gòu)于北京中杉金橋生物有限公司;一步法快速WB(HRP)試劑盒(兔,20330;鼠,30324)均購(gòu)于康為世紀(jì)公司;PageRuler? Plus Prestained Protein Ladder(00459134)購(gòu)于Thermo;Methylation-Gold Kit[D5005S(10)]購(gòu)于ZYMO;DNA提取試劑盒(DP304)購(gòu)于天根生化科技(北京)有限公司。
2.1實(shí)驗(yàn)動(dòng)物分組、模型建立及檢測(cè)時(shí)點(diǎn)確認(rèn)大鼠自由飼養(yǎng)1周后于造模前1 d用混合溶劑(無(wú)水乙醇∶生理鹽水=2∶8)將MCT溶解、稀釋?zhuān)涑?% MCT溶液37 ℃過(guò)夜;造模當(dāng)天用二甲基亞砜(dimethyl sulfoxide, DMSO)將5-Aza溶解、稀釋?zhuān)涑? g/L的5-Aza溶液。采用隨機(jī)數(shù)字表法將大鼠分成5組,即對(duì)照組(于造模當(dāng)天按60 mg/kg單次腹腔內(nèi)注射2∶8的無(wú)水乙醇生理鹽水混合液)、MCT組(于造模當(dāng)天1% MCT按60 mg/kg單次腹腔內(nèi)注射)、MCT+DMSO組(于造模當(dāng)天等量1% MCT腹腔內(nèi)注射后每周3次腹部皮下注射1 mg/kg DMSO,持續(xù)3周)、MCT+anti-IL-6組(于造模當(dāng)天等量1% MCT腹腔內(nèi)注射后每天腹部皮下注射40 μg/kg anti-IL-6,持續(xù)3周)和MCT+5-Aza組(于造模當(dāng)天等量1% MCT腹腔內(nèi)注射后每周3次皮下注射1 mg/kg 5-Aza,持續(xù)3周),每組8只。記造模當(dāng)天為第1天,于第21天[8]將大鼠做檢測(cè)。
2.2右心導(dǎo)管法測(cè)右心室壓力于第21天分別取各組大鼠8只,手術(shù)前禁食8 h,10%水合氯醛(0.04 mL/kg)腹腔注射麻醉,將PE10管預(yù)充肝素鹽水采用右心導(dǎo)管法經(jīng)右側(cè)頸外靜脈插管至右心室,用BL-420S生物信號(hào)采集分析系統(tǒng)(成都泰盟軟件有限公司生產(chǎn))檢測(cè)右心室壓力,并計(jì)算右心室平均壓力。
2.4稱重法計(jì)算右心室肥厚指數(shù)各組大鼠測(cè)完右心室壓力后均斷頭處死開(kāi)胸后完整取出心臟,剪去左右心房及心耳,在肺動(dòng)脈出口處剪開(kāi)右心室游離壁組織(RV),其余則為左心室(LV)+室間隔組織(S),洗去血污,吸干水分,然后分別稱重,按公式[右心肥厚指數(shù)=RV/(LV+S)]計(jì)算。
2.5肺組織HE染色病理學(xué)檢測(cè)檢測(cè)右心室壓力后,取肺組織用生理鹽水通過(guò)肺動(dòng)脈沖洗剩余的血液,分離右側(cè)第三葉肺組織放入4%多聚甲醛固定,常規(guī)脫水、石蠟包埋、切片、HE染色、封片。在數(shù)碼三目攝像顯微鏡(BA400Digital,麥克奧迪實(shí)業(yè)集團(tuán)有限公司)下對(duì)切片進(jìn)行圖像采集及分析。
2.6免疫熒光法檢測(cè)肺組織IL-6和GATA-6表達(dá)檢測(cè)右心室壓力后,生理鹽水沖洗干凈肺組織,取右側(cè)第三葉肺組織放入4%多聚甲醛固定、常規(guī)脫水、包埋、切片、脫蠟、抗原修復(fù)后進(jìn)行如下操作:滴加新生牛血清封閉液室溫孵育30 min;分別滴加GATA-6 Ⅰ抗(1∶200)和IL-6 Ⅰ抗(1∶100)4 ℃過(guò)夜;滴加羅丹明標(biāo)記的Ⅱ抗(山羊抗兔原液),37 ℃孵育30 min;滴加DAPI復(fù)染,室溫孵育10 min;抗熒光衰減封片劑封片和鏡檢;采用熒光掃描顯微鏡攝像系統(tǒng)對(duì)切片進(jìn)行圖像采集;應(yīng)用Image-Pro Plus 6.0圖像分析系統(tǒng)將綠色/紅色熒光單色照片轉(zhuǎn)換為黑白圖片然后選取相同的黑色作為判斷陽(yáng)性的統(tǒng)一標(biāo)準(zhǔn),計(jì)算累積光密度和面積,并求出面密度,使用3張圖像的平均面密度再計(jì)算平均數(shù),得出每例樣本的平均面密度。
2.7Western blot檢測(cè)肺組織IL-6和GATA-6蛋白表達(dá)稱取適量肺組織,RIPA裂解液與PMSF體積比按99∶1混勻,充分裂解組織,冰上孵育20 min后,10 000 r/min離心20 min,收集上清。根據(jù)BCA蛋白試劑盒測(cè)蛋白濃度,垂直電泳后,轉(zhuǎn)膜2 h,封閉20 min,IL-6和GATA-6 Ⅰ抗(1∶1 000)4 ℃過(guò)夜,HRP鼠IL-6 Ⅱ抗和HRP兔GATA-6 Ⅱ抗(1∶200)室溫孵育2 h,IL-6的內(nèi)參照為β-actin(1∶200),GATA-6的內(nèi)參照為histone H3(1∶200),ECL發(fā)光液化學(xué)發(fā)光,膠片曝光。采用Gel-Pro Analyzer 4圖像分析軟件測(cè)定各條帶的灰度值并定量分析。
2.8甲基化特異性PCR(methylation-specific PCR, MSP)法檢測(cè)肺組織GATA-6甲基化程度將肺組織處理為細(xì)胞懸液,按組織基因組DNA提取試劑盒說(shuō)明步驟提取基因組DNA,根據(jù)Methylation-Gold Kit試劑合步驟對(duì)基因組DNA進(jìn)行重亞硫酸鹽修飾。設(shè)置反應(yīng)體系為:10× PCR Buffer 2.5 μL,2.5 mmol/L dNTPs 2.5 μL,10 μmol/L引物2.0 μL,基因組DNA 3.0 μL,5 U/μL Taq HS 0.1 μL,ddH2O 14.9 μL。設(shè)置反應(yīng)條件為:95 ℃預(yù)變性5 min;95 ℃變性30 s,55 ℃退火30 s,72 ℃延伸30 s,共45個(gè)循環(huán);最后72 ℃末段延伸5 min。所有引物均交由武漢金開(kāi)瑞生物工程有限公司設(shè)計(jì)合成(表1)。結(jié)果經(jīng)2%瓊脂凝膠電泳顯像,并觀察條帶情況,照相記錄。
表1 甲基化特異性PCR的引物序列
M: methylated; U: unmethylated.
應(yīng)用SPSS 20.0軟件包進(jìn)行統(tǒng)計(jì)分析。計(jì)量資料采用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。組間比較采用單因素方差分析,多重比較均采用LSD法。以<0.05為差異有統(tǒng)計(jì)學(xué)意義。
與對(duì)照組比較,MCT、MCT+anti-IL-6和MCT+5-Aza組右心室平均壓力顯著升高(<0.05);MCT+DMSO組與MCT組比較無(wú)顯著差異(>0.05);與MCT組比較,MCT+anti-IL-6和MCT+5-Aza組右心室平均壓力顯著降低(<0.05),見(jiàn)圖1。
Figure 1.Comparison of mean right ventricular pressure in the mice of each group. A: control group; B: MCT group; C: MCT+DMSO group; D: MCT+anti-IL-6 group; E: MCT+5-Aza group. Mean±SD. n=8. #P<0.05 vs A; *P<0.05 vs B.
與對(duì)照組比較,MCT組右心室肥厚指數(shù)顯著增高(<0.05),MCT+anti-IL-6組和MCT+5-Aza組未見(jiàn)顯著差異(>0.05);MCT+DMSO組與MCT組比較無(wú)顯著差異(>0.05);與MCT組比較,MCT+anti-IL-6組和MCT+5-Aza組右心室肥厚指數(shù)顯著減低(<0.05),見(jiàn)圖2。
Figure 2.Comparison of right ventricular hypertrophy index in the mice of each group. A: control group; B: MCT group; C: MCT+DMSO group; D: MCT+anti-IL-6 group; E: MCT+5-Aza group. Mean±SD. n=8. #P<0.05 vs A; *P<0.05 vs B.
對(duì)照組各級(jí)動(dòng)脈壁結(jié)構(gòu)清晰,厚度正常,血管周?chē)鸁o(wú)明顯炎癥細(xì)胞浸潤(rùn),肺泡內(nèi)無(wú)明顯巨噬細(xì)胞浸潤(rùn);MCT組血管周?chē)装Y細(xì)胞呈環(huán)狀浸潤(rùn),形成血管袖套(黑色箭頭),平滑肌增生肥厚,管壁肌型化(紅色箭頭),組織小范圍可見(jiàn)肺泡性水腫,大量肺泡內(nèi)充滿嗜酸性蛋白樣物質(zhì)(綠色箭頭),并伴較多炎性細(xì)胞滲出(黃色箭頭);MCT+DMSO組血管周?chē)装Y細(xì)胞呈環(huán)狀浸潤(rùn),血管壁結(jié)構(gòu)疏松,層次不清(紅色箭頭),組織可見(jiàn)肺泡性水腫,大量肺泡內(nèi)充滿嗜酸性蛋白樣物質(zhì)(綠色箭頭),并伴較多炎癥細(xì)胞滲出(黃色箭頭);MCT+anti-IL-6組動(dòng)脈管壁結(jié)構(gòu)清晰,血管周?chē)鸁o(wú)炎癥細(xì)胞浸潤(rùn),少量動(dòng)脈壁明顯增厚,平滑肌增生肥厚,管壁肌型化(黑色箭頭),管壁周?chē)g隙增寬,伴蛋白樣液滲出(紅色箭頭),少量肺泡內(nèi)可見(jiàn)泡沫狀巨噬細(xì)胞(黃色箭頭);MCT+5-Aza組大量血管周?chē)装Y細(xì)胞呈環(huán)狀浸潤(rùn),形成血管袖套(黑色箭頭),局部肺泡內(nèi)較多泡沫狀巨噬細(xì)胞聚集(黃色箭頭),見(jiàn)圖3。
Figure 3.staining of Lung tissue in the mice of each group (HE staining, ×400). A: control group; B: MCT group; C: MCT+DMSO group; D: MCT+anti-IL-6 group; E: MCT+5-Aza group.
Western blot和免疫熒光結(jié)果均顯示,與對(duì)照組比較,MCT組和MCT+5-Aza組IL-6的表達(dá)顯著增高(<0.05),MCT+anti-IL-6組無(wú)顯著差異(>0.05);MCT+DMSO組與MCT組比較無(wú)顯著差異(>0.05);與MCT組比較,MCT+anti-IL-6組和MCT+5-Aza組IL-6的表達(dá)顯著減低(<0.05),見(jiàn)圖4、5。
Figure 4.Western blot detection of IL-6 protein in the mice of each group. A: control group; B: MCT group; C: MCT+DMSO group; D: MCT+anti-IL-6 group; E: MCT+5-Aza group. Mean±SD. n=8. #P<0.05 vs A; *P<0.05 vs B.
Figure 5.Immunofluorescence of IL-6 protein in the mice of each group. A: control group; B: MCT group; C: MCT+DMSO group; D: MCT+anti-IL-6 group; E: MCT+5-Aza group.
Western blot和免疫熒光結(jié)果均顯示,與對(duì)照組比較,MCT組和MCT+5-Aza組GATA-6的表達(dá)顯著減低(<0.05),MCT+anti-IL-6組無(wú)顯著差異(>0.05);MCT+DMSO組與MCT組比較無(wú)顯著差異(>0.05);與MCT組比較,MCT+anti-IL-6組和MCT+5-Aza組GATA-6的表達(dá)顯著增高(<0.05),見(jiàn)圖6、7。
Figure 6.Western blot detection of GATA-6 protein in the mice of each group. A: control group; B: MCT group; C: MCT+DMSO group; D: MCT+anti-IL-6 group; E: MCT+5-Aza group. Mean±SD. n=8. #P<0.05 vs A; *P<0.05 vs B.
Figure 7.Immunofluorescence comparison GATA-6 of protein in the mice of each group. A: control group; B: MCT group; C: MCT+DMSO group; D: MCT+anti-IL-6 group; E: MCT+5-Aza group. Mean±SD. n=8. #P<0.05 vs A; *P<0.05 vs B.
對(duì)照組大鼠肺組織中GATA-6未發(fā)生明顯甲基化;MCT組及MCT+DMSO組大鼠肺組織中GATA-6呈高度甲基化;MCT+anti-IL-6組及MCT+5-Aza組肺組織中GATA-6呈低甲基化,與MCT組比較甲基化程度明顯減弱,見(jiàn)圖8。
Figure 8.GATA-6 MSP electrophoretogram in the mice of each group. A: control group; B: MCT group; C: MCT+DMSO group; D: MCT+anti-IL-6 group; E: MCT+5-Aza group; M: methylated; U: unmethylated.
IL-6作為PAH發(fā)病過(guò)程中最重要的因子之一已被國(guó)內(nèi)外大多學(xué)者認(rèn)可,一方面IL-6通過(guò)激活信號(hào)轉(zhuǎn)導(dǎo)及轉(zhuǎn)錄激活因子3(signal transducer and activator of transcription 3, STAT3)在Y705位點(diǎn)的磷酸化,導(dǎo)致細(xì)胞內(nèi)DNA結(jié)合、二聚體形成、核易位來(lái)增強(qiáng)細(xì)胞收縮能力,延長(zhǎng)細(xì)胞存活,從而促進(jìn)PASMC增殖[9];另一方面IL-6可以干預(yù)骨形態(tài)發(fā)生蛋白(BMP)信號(hào)通路中BMP9的活性使得內(nèi)皮細(xì)胞表型異常轉(zhuǎn)化,從而導(dǎo)致內(nèi)皮-間質(zhì)轉(zhuǎn)化(EndMT)過(guò)程中EndMT信號(hào)延長(zhǎng),其結(jié)果是細(xì)胞內(nèi)促炎癥反應(yīng)信號(hào)持續(xù)加強(qiáng)(如TGF-β1、p-Smad2/3等)、細(xì)胞缺氧加重及凋亡加速,最終導(dǎo)致PASMC異常增殖[10-11]。一份來(lái)自美國(guó)國(guó)立衛(wèi)生研究院肺動(dòng)脈高壓生物庫(kù)(PAHB)和科羅拉多州兒童醫(yī)院(CHC)共236位PAH患者的血清隊(duì)列研究顯示IL-6與患者的死亡率、肺移植的成功率、姑息性手術(shù)時(shí)機(jī)顯著相關(guān),此后該研究還通過(guò)對(duì)患者平均肺動(dòng)脈壓力、肺動(dòng)脈楔壓、右心室輸出功率、左心輸出量等血流動(dòng)力學(xué)指標(biāo)進(jìn)行分析推測(cè)IL-6作為PAH的早期標(biāo)記物能更早的預(yù)警PAH導(dǎo)致的心力衰竭,其敏感性甚至高于N末端B型利鈉肽前體(NT-proBNP)[12-13]。同時(shí)Hernández-Sánchez等[14]的臨床研究顯示給予IL-6受體抑制劑后能有效降低肺動(dòng)脈壓力并改善PAH患者的生活質(zhì)量,提示IL-6也是目前治療PAH潛在研究靶點(diǎn)。我們的研究結(jié)果顯示在PAH模型建立成功后MCT組肺組織HE染色有大量炎癥細(xì)胞及肺水腫表現(xiàn),且肺組織中IL-6的Western blot及免疫熒光結(jié)果顯示與對(duì)照組比較顯著增高,再次證實(shí)IL-6在PAH形成中發(fā)揮著重要作用,同時(shí)我們給予MCT+anti-IL-6組大鼠IL-6抑制劑后發(fā)現(xiàn)MCT+anti-IL-6組大鼠右心室平均壓力、右心室肥厚指數(shù)、肺組織中IL-6的Western blot及免疫熒光表達(dá)較MCT組均顯著降低,HE染色也顯示給予IL-6抑制劑后肺組織炎癥反應(yīng)及肺水腫均明顯好轉(zhuǎn),其結(jié)果與上述文獻(xiàn)報(bào)道一致,也提示IL-6在PAH的發(fā)展中所涉及的機(jī)制可能是成為治療PAH潛在靶點(diǎn)。
研究證實(shí)GATA-6作為鋅指結(jié)構(gòu)轉(zhuǎn)錄因子其高表達(dá)可以維持正常血管平滑肌細(xì)胞的穩(wěn)定,但低表達(dá)則促進(jìn)了VSMC的損傷,機(jī)制可能為損傷導(dǎo)致了GATA-6 的沉默,而GATA-6的沉默可通過(guò)內(nèi)皮細(xì)胞-GATA-6-PDGF(血小板衍生的生長(zhǎng)因子)-B通路誘導(dǎo)VSMC表型轉(zhuǎn)換的失調(diào)來(lái)實(shí)現(xiàn)VSMC的損傷,所以推測(cè)GATA-6可能是發(fā)生VSMC異常增殖的主要靶點(diǎn)[15]。最新報(bào)道顯示BMPR2突變可以引起PASMC增殖異常[16],但抑制人類(lèi)直系同源物(TWIST1)則可逆轉(zhuǎn)這一現(xiàn)象,主要原因?yàn)門(mén)WIST1的抑制可以促進(jìn)GATA-6合成所依賴的結(jié)合蛋白釋放,而GATA-6的大量合成則直接導(dǎo)致了BMPR2的表達(dá)受限(GATA-6被證實(shí)為BMPR2啟動(dòng)子區(qū)下游靶點(diǎn))從而抑制了PASMC的異常增殖[17]。從Lentjes等[18]的綜述中我們發(fā)現(xiàn)GATA-6作為胚體內(nèi)胚層的代表基因可以促進(jìn)心血管上皮細(xì)胞的分化及發(fā)育,并且能防止該類(lèi)細(xì)胞的過(guò)分化,此后一項(xiàng)胚胎干細(xì)胞的研究顯示GATA-6的沉默主要是GATA-6啟動(dòng)子區(qū)發(fā)生了甲基化的結(jié)果,而5-Aza作為去甲基化劑可以逆轉(zhuǎn)GATA-6的沉默,恢復(fù)GATA-6的功能[19]。我們的研究在PAH模型建立成功后MCT組大鼠肺組織中Western blot及免疫熒光結(jié)果顯示GATA-6表達(dá)較對(duì)照組均顯著降低,而GATA-6的MSP結(jié)果提示對(duì)照組大鼠并未出現(xiàn)明顯甲基化,而MCT組大鼠出現(xiàn)了高度的甲基化,提示PAH發(fā)生過(guò)程中GATA-6的低表達(dá)可能是因?yàn)镚ATA-6甲基化所致,在此基礎(chǔ)上我們給予5-Aza處理MCT+5-Aza組大鼠發(fā)現(xiàn)該組大鼠右心室平均壓力及右心室肥厚指數(shù)較MCT組顯著降低,再次對(duì)MCT+5-Aza組肺組織進(jìn)行Western Blot及免疫熒光檢測(cè)發(fā)現(xiàn)GATA-6的表達(dá)較MCT組顯著增高,MCT+5-Aza組GATA-6的MSP結(jié)果也顯示甲基化程度較MCT組顯著減弱,而且MCT+5-Aza組HE染色結(jié)果顯示肺組織中炎癥反應(yīng)及肺水腫情況與MCT組比較明顯改善,提示大鼠PAH有大幅度好轉(zhuǎn),與上述文獻(xiàn)報(bào)道結(jié)果相似,再次證實(shí)GATA-6在PAH的發(fā)展中可能起到不可忽略的作用。
最后我們?cè)赑AH模型建立成功后給予IL-6抑制劑處理MCT+anti-IL-6組大鼠發(fā)現(xiàn),與MCT組比較MCT+anti-IL-6組大鼠右心室平均壓力及右心室肥厚指數(shù)顯著降低(Western Blot及免疫熒光結(jié)果顯示肺組織中IL-6的表達(dá)顯著減低,而GATA-6的表達(dá)顯著增加,HE染色結(jié)果也顯示MCT+anti-IL-6組大鼠肺組織炎癥反應(yīng)與肺水腫較MCT組大幅度好轉(zhuǎn),同時(shí)GATA-6的MSP結(jié)果顯示MCT+anti-IL-6組大鼠GATA-6呈低甲基化,再將MCT+anti-IL-6組大鼠右心室肥厚指數(shù)、肺組織中IL-6及GATA-6的表達(dá)與對(duì)照組比較甚至發(fā)現(xiàn)無(wú)統(tǒng)計(jì)學(xué)意義,而這些結(jié)果與MCT組大鼠肺組織檢測(cè)結(jié)果恰恰相反。查閱相關(guān)文獻(xiàn)我們發(fā)現(xiàn)關(guān)于IL-6誘導(dǎo)靶基因甲基化導(dǎo)致靶基因沉默的研究屢見(jiàn)不鮮,例如:(1)在腸預(yù)激綜合征(IBS)的研究中發(fā)現(xiàn)IL-6增加了組蛋白H3賴氨酸9(H3K9)的甲基化,使得腸上皮細(xì)胞緊密連接蛋白(TJ)表達(dá)下調(diào),導(dǎo)致細(xì)胞通透性增加而產(chǎn)生內(nèi)臟痛覺(jué)過(guò)敏[20]:(2)膿毒癥的相關(guān)研究顯示對(duì)單核細(xì)胞中與膿毒癥發(fā)病相關(guān)的全基因組進(jìn)行甲基化分析發(fā)現(xiàn)隨著IL-6水平的增加出現(xiàn)了2492個(gè)CpG位點(diǎn)高甲基化及909個(gè)CpG位點(diǎn)低甲基化[21];(3)在骨骼肌疾病的研究中發(fā)現(xiàn)IL-6通過(guò)增加DNMT的表達(dá)間接性的導(dǎo)致微小RNA142-3p(miR142-3p)啟動(dòng)子的高度甲基化,從而促進(jìn)膠質(zhì)母細(xì)胞瘤的生長(zhǎng)[22]。所以結(jié)合本次研究結(jié)果,我們有理由懷疑PAH的形成中GATA-6的高度甲基化可能是高表達(dá)的IL-6誘導(dǎo)的,而該機(jī)制可能在PAH的發(fā)展過(guò)程中起到作用。
綜上所述,在PAH大鼠肺組織中給予IL-6抗體后IL-6的表達(dá)顯著減低,給予5-Aza后GATA-6的表達(dá)顯著增高,而GATA-6甲基化程度顯著減低;同時(shí)IL-6的高表達(dá)可能是導(dǎo)致GATA-6低表達(dá)的原因,其機(jī)制可能為IL-6誘導(dǎo)了GATA-6的甲基化,而該機(jī)制是否在PAH的發(fā)展中起到作用有望本研究的體內(nèi)實(shí)驗(yàn)進(jìn)一步明確。
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(責(zé)任編輯:盧萍,羅森)
Role and significance of IL-6 and GATA-6 methylation inhibitors in lung tissue of rats with pulmonary arterial hypertension
LIAO Jian-xiong1, LUO Jie2, LIU Lin3, ZHANG Qian1△, LIU Wei-jia3△
(1,,550002,;2,,550003,;3,,550002,)
To study the changes and significance of interleukin-6 (IL-6), GATA-binding protein-6 (GATA-6) and GATA-6 methylation in monocrotaline (MCT)-induced rat pulmonary arterial hypertension (PAH) model.Forty adult male SD rats were randomly divided into control group, MCT group, MCT+dimethyl sulfoxide (DMSO) group, MCT+IL-6 antibody (anti-IL-6) group and MCT+5-aza-2'-deoxycytidine (5-Aza) group, with 8 rats in each group. The rats in control group
a single intraperitoneal injection of 2∶8 anhydrous ethanol and physiological saline mixture at 60 mg/kg. The rats in MCT group received a single intraperitoneal injection of 1% MCT at 60 mg/kg. The rats in MCT+DMSO group were intraperitoneally injected with the same amount of 1% MCT and subcutaneously injected DMSO at 1 mg/kg into the abdomen 3 times a week. The rats in MCT+anti-IL-6 group were intraperitoneally injected with the same amount of 1% MCT and then intraperitoneally injected with anti-IL-6 at 40 μg/kg every day. The rats in MCT+5-Aza group were intraperitoneally injected with the same amount of 1% MCT and then injected subcutaneously with 5-Aza at 1 mg/kg 3 times a week. On the 21st day after modeling, the rats in each group were monitored for right ventricular pressure and right ventricular hypertrophy index. Lung tissue HE staining was performed to observe lung pathological changes. The IL-6 and GATA-6 expression in lung tissues was detected by immunofluorescence and Western blot. The GATA-6 methylation in lung tissues was detected by methylation-specific PCR (MSP).Compared with control group, the average right ventricular pressure in MCT, MCT+anti-IL-6 and MCT+5-Aza groups was significantly increased (<0.05). Compared with MCT group, the average pressure of right ventricle in MCT+anti-IL-6 and MCT+5-Aza groups was significantly decreased (<0.05). Compared with control group, the right ventricular hypertrophy index in MCT group was significantly increased (<0.05). Compared with MCT group, the right ventricular hypertrophy index in MCT+anti-IL-6 and MCT+5-Aza groups was significantly decreased (<0.05). Immunofluorescence and Western blot results showed that the expression of IL-6 in MCT and MCT+5-Aza groups was significantly increased compared with control group (<0.05), while that in MCT+anti-IL-6 and MCT+5-Aza groups was significantly reduced compared with MCT group (<0.05). The expression of GATA-6 in MCT and MCT+5-Aza groups was significantly reduced compared with control group (<0.05), while that in MCT+anti-IL-6 and MCT+5-Aza groups was significantly increased compared with MCT group (<0.05). The MSP results showed that GATA-6 in lung tissues was highly methylated in MCT and MCT+DMSO groups, hypomethylated in MCT+anti-IL-6 and MCT+5-Aza groups, and unmethylated in control group.In the lung tissue of rats with PAH, the IL-6 expression is significantly reduced after anti-IL-6 treatment, the GATA-6 expression is significantly increased after 5-Aza treatment, and the degree of GATA-6 methylation is significantly reduced. The high expression of IL-6 may be a result of low GATA-6 expression, which is caused by GATA-6 methylation.
Pulmonary arterial hypertension; Interleukin-6; GATA-binding protein-6; 5-Aza-2'-deoxycytidine
R543.1; R363.2
A
10.3969/j.issn.1000-4718.2022.03.017
1000-4718(2022)03-0517-09
2021-08-26
2021-12-24
[基金項(xiàng)目]貴州省科技計(jì)劃項(xiàng)目(黔科合基礎(chǔ)[2018]1408號(hào))
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