胡 蓉 劉學(xué)淵 姚晴晴 李笑天

(復(fù)旦大學(xué)附屬婦產(chǎn)科醫(yī)院產(chǎn)科 上海 200011)

子癇前期宮內(nèi)環(huán)境導(dǎo)致子代大鼠海馬HPA軸相關(guān)基因表達(dá)改變

胡 蓉 劉學(xué)淵 姚晴晴 李笑天△

(復(fù)旦大學(xué)附屬婦產(chǎn)科醫(yī)院產(chǎn)科 上海 200011)

目的 研究子癇前期宮內(nèi)環(huán)境對(duì)子代SD大鼠海馬下丘腦-垂體-腎上腺(hypothalamic-pituitary-adrenal,HPA)軸相關(guān)基因表達(dá)的影響。方法 通過(guò)給妊娠第14天SD大鼠連續(xù)皮下注射亞硝基左旋精氨酸甲酯(L-NAME)至分娩的方法建立子癇前期大鼠動(dòng)物模型,對(duì)照組予生理鹽水1 mL皮下注射。用酶聯(lián)免疫檢測(cè)(ELISA)方法檢測(cè)新生子代大鼠血皮質(zhì)酮(corticosterone,CORT)濃度。用Real-time PCR方法和Western bolt分別檢測(cè)8周齡子代大鼠海馬組織糖皮質(zhì)激素受體(glucocorticoid receptor,GR)、促腎上腺皮質(zhì)激素釋放激素(corticotropin releasing hormone,CRH)、CRH受體1 (CRH receptor 1,CRHR1)和IL-6的mRNA水平及其蛋白質(zhì)水平。結(jié)果 與正常子代新生鼠相比,子癇前期子代新生鼠血CORT濃度升高(P<0.05)。與正常子代鼠相比,子癇前期青少年子代鼠海馬GR、CRH 和CRHR1 mRNA和蛋白質(zhì)水平均增加(P<0.05),炎性因子IL-6 mRNA和蛋白質(zhì)水平均增加(P<0.05)。結(jié)論 子癇前期宮內(nèi)環(huán)境影響子代大鼠海馬HPA軸相關(guān)基因的表達(dá),在子代學(xué)習(xí)記憶能力受損中起作用。

子癇前期; 子代大鼠; 糖皮質(zhì)激素受體; CRH; CRHR1; IL-6

子癇前期是妊娠期最常見(jiàn)的以高血壓、蛋白尿?yàn)橹饕卣鞯牟l(fā)癥,國(guó)外報(bào)道發(fā)病率達(dá)到7%～10%,我國(guó)大約為9.4%。子癇前期對(duì)胎兒最重要的影響是慢性宮內(nèi)缺氧的應(yīng)激環(huán)境,其對(duì)子代各臟器結(jié)構(gòu)和功能的程序化影響是多方面的[1-2],其中學(xué)習(xí)記憶能力和生活質(zhì)量密切相關(guān)。

臨床流行病學(xué)研究發(fā)現(xiàn),與正常人群子代相比,子癇前期的子代幼年期、青少年期和成年后學(xué)習(xí)記憶能力下降、IQ平均值較低[2-5]。我們運(yùn)用子癇前期的大鼠動(dòng)物模型研究,發(fā)現(xiàn)子代鼠青少年期和成年期智力受損、學(xué)習(xí)記憶和空間定位能力降低[6],伴海馬神經(jīng)干細(xì)胞增殖受到抑制[7]。也有研究發(fā)現(xiàn)子癇前期的子代鼠表現(xiàn)為大腦缺氧改變、神經(jīng)元遷移和凋亡的改變。但子癇前期宮內(nèi)環(huán)境導(dǎo)致子代學(xué)習(xí)記憶能力受損的分子機(jī)制尚不清楚。

下丘腦-垂體-腎上腺軸(hypothalamic-pituitary-adrenal,HPA軸)對(duì)海馬依賴的學(xué)習(xí)記憶能力有調(diào)控作用,同時(shí)受孕期宮內(nèi)應(yīng)激環(huán)境的程序化影響[8],宮內(nèi)應(yīng)激的作用時(shí)間、種類和強(qiáng)度的不同,子代可能表現(xiàn)為應(yīng)激損傷或者對(duì)應(yīng)激的適應(yīng)性改變,并引起腦內(nèi)HPA軸相關(guān)基因表達(dá)的改變。例如胚胎發(fā)育期的應(yīng)激會(huì)引起海馬糖皮質(zhì)激素受體(glucocorticoid receptor,GR)的過(guò)度激活和海馬損傷,對(duì)HPA軸的負(fù)反饋抑制減弱,導(dǎo)致HPA軸過(guò)度興奮,下丘腦分泌更多的促腎上腺皮質(zhì)激素釋放激素(corticotropin releasing hormone,CRH),激活垂體產(chǎn)生促腎上腺皮質(zhì)激素(adrenocortico tropic hormone,ACTH),ACTH作用于腎上腺皮質(zhì)促進(jìn)皮質(zhì)酮(corticoserone,CORT)的生成和分泌,并進(jìn)一步損傷海馬造成惡性循環(huán)[9]。CRH過(guò)表達(dá)的轉(zhuǎn)基因小鼠存在學(xué)習(xí)記憶缺陷,給予促腎上腺皮質(zhì)激素釋放激素受體(CRHR)拮抗劑能糾正應(yīng)激引起的記憶障礙以及海馬樹(shù)突丟失[10]。炎性因子白介素-6 (IL-6)在GR受體通路以及大腦缺血缺氧改變的研究中發(fā)揮作用,孕期高脂飲食導(dǎo)致子代大腦GR信號(hào)通路發(fā)生改變,IL-6在其中發(fā)揮作用[11]。

因此,我們研究了子癇前期宮內(nèi)環(huán)境對(duì)青少年子代大鼠海馬HPA軸相關(guān)基因GR、CRH和CRHR1表達(dá)的影響,和對(duì)子代海馬炎性因子IL-6表達(dá)的影響,以探討子癇前期宮內(nèi)環(huán)境對(duì)子代學(xué)習(xí)記憶能力影響的分子機(jī)制,為出生后干預(yù)尋找可能的方向。

材 料 和 方 法

動(dòng)物實(shí)驗(yàn) 清潔級(jí)雌性SD大鼠8只(斯萊克實(shí)驗(yàn)動(dòng)物有限公司),體質(zhì)量230～260 g,于室溫18～20 ℃自由飲食。于發(fā)情期與雄性SD大鼠8只合籠飼養(yǎng),陰栓脫落日定為妊娠第1天,分籠飼養(yǎng)。母鼠隨機(jī)分為2組。在妊娠第14天開(kāi)始,實(shí)驗(yàn)組每天給予亞硝基左旋精氨酸甲酯(美國(guó)Sigma公司) 125 mg/kg,對(duì)照組給予生理鹽水1 mL皮下注射,每天1次直至分娩。自然分娩后,部分仔鼠以斷頭法取血,血樣以2 000×g/min離心10 min,血清-80 ℃保存。部分仔鼠和母鼠一起飼養(yǎng),每籠1只母鼠所帶仔鼠數(shù)目相同。各組仔鼠由母鼠哺乳至21天斷奶后,分籠飼養(yǎng)至8周齡(56天)以頸椎脫位法處死,取海馬組織置于-80 ℃冰箱保存?zhèn)溆谩?/p>

酶聯(lián)免疫檢測(cè)(ELISA法) 取血清按酶聯(lián)免疫吸附試驗(yàn)檢測(cè)試劑盒說(shuō)明書(shū)檢測(cè)血清CORT。

Real-time PCR檢測(cè) 細(xì)胞總RNA用Trizol常規(guī)抽提。逆轉(zhuǎn)錄用PrimeScriptTMRT試劑盒(日本TAKARA公司),操作步驟嚴(yán)格按照說(shuō)明書(shū)。real-time PCR 在Applied Biosystems FTC3000HT 熒光定量PCR儀(加拿大Funglyn Biotech公司)上完成。PCR樣品總反應(yīng)體積為25 μL,反應(yīng)體系包括cDNA模板1 μL、引物各0.8 μL、SYBRgreen mix (2×) 10 μL,ROX (50×) 0.4 μL,ddH2O (滅菌蒸餾水) 7 μL。經(jīng)95 ℃ 30 s預(yù)變性后,95 ℃ 5 s、60 ℃ 30～34 s循環(huán)40個(gè),每個(gè)循環(huán)結(jié)束前LightCycle系統(tǒng)自動(dòng)檢測(cè)熒光產(chǎn)物的量,所有循環(huán)結(jié)束后,繪制融解曲線判斷擴(kuò)增產(chǎn)物的特異性。為排除基因組DNA的污染每個(gè)樣品均設(shè)置相應(yīng)未經(jīng)逆轉(zhuǎn)錄的模板作為陰性對(duì)照。每組實(shí)驗(yàn)重復(fù)3次,每次各樣品均設(shè)復(fù)孔。利用2-ΔΔCt法計(jì)算相對(duì)表達(dá)量。

表1 PCR引物序列

Western blot檢測(cè) 以RIPA裂解組織,4 ℃下10 000×g離心5 min后取上清液。BCA法蛋白質(zhì)定量后經(jīng)SDS-PAGE分離,轉(zhuǎn)膜后,用含5%脫脂奶粉的TBST溶液封閉1 h,加人對(duì)應(yīng)一抗于4 ℃孵育過(guò)夜,以TBST洗滌3次后,加入二抗于室溫孵育1 h,再以TBST洗滌3次后,采用ECL試劑曝光顯色。應(yīng)用一抗如下:GR (ab3579,英國(guó)Abcam公司)、CRH (ab8901,美國(guó)Abcam公司)、CRHR1 (sc-12383,美國(guó)Santa Cruz公司),IL-6 (ab9324,英國(guó)Abcam公司),內(nèi)參選用GAPDH (ab-9485,美國(guó)Abcam公司)。

結(jié) 果

子癇前期宮內(nèi)環(huán)境導(dǎo)致子代新生大鼠HPA軸血清CORT濃度的變化 一氧化氮(nitricoxide,NO)合成減少與子癇前期發(fā)病相關(guān)。Yallampalli等首次用NO合成抑制劑L-NAME建立子癇前期的大鼠動(dòng)物模型,本研究同樣運(yùn)用孕鼠皮下注射L-NAME的方法建立模型。ELlSA檢測(cè)結(jié)果顯示,與正常子代P0期新生鼠相比,子癇前期子代新生大鼠血清CORT濃度明顯升高(P < 0.05,圖1)。有研究發(fā)現(xiàn)子癇前期的新生兒血漿皮質(zhì)醇濃度較高[12],而L-NAME的使用可能會(huì)導(dǎo)致血清皮質(zhì)酮含量降低[13],因此子代新生大鼠血清CORT濃度升高,提示了子癇前期宮內(nèi)環(huán)境引起子代高CORT的內(nèi)環(huán)境。

Compared with normal neonatal rats,the serum CORT concentration of neonatal rats with preeclampsia was increased.

圖1 子癇前期宮內(nèi)環(huán)境對(duì)子代新生大鼠血清CORT濃度的影響

Fig 1 Effect of preeclampsia on serum CORT concentration in neonatal offspring rats

子癇前期宮內(nèi)環(huán)境導(dǎo)致8周齡子代大鼠海馬GR、CRH和CRHR1表達(dá)的改變 分別取實(shí)驗(yàn)組和對(duì)照組8周齡的子代鼠(相當(dāng)于人類青春期的年齡)海馬組織,Real-time PCR檢測(cè)發(fā)現(xiàn),子癇前期8周齡子代大鼠海馬GR mRNA的水平增加(P<0.05),CRH和CRHR1 mRNA的水平均增加(P<0.05,圖2)。 Western blot檢測(cè)發(fā)現(xiàn),子癇前期8周齡子代大鼠海馬GR、CRH、CRHR1蛋白質(zhì)水平均增加(P<0.05,圖3)。

Compared with normal adolescent offspring,the expression of GR,CRH and CRHR1 mRNA was increased in the hippocampus of adolescent rats with preeclampsia.

圖2 子癇前期宮內(nèi)環(huán)境導(dǎo)致8周齡子代大鼠海馬GR、CRH和CRHR1 mRNA水平的改變

Fig 2 Effect of preeclampsia on expressions of GR,CRH and CRHR1 mRNA in the hippocampus of 8-week offspring rats

Compared with normal adolescent offspring,the expression of GR,CRH and CRHR1 protein was increased in the hippocampus of adolescent rats with preeclampsia.

圖3 子癇前期宮內(nèi)環(huán)境導(dǎo)致8周齡子代大鼠海馬GR、CRH和CRHR1蛋白水平的改變

Fig 3 Effect of preeclampsia on expressions of GR,CRH and CRHR1 protein in the hippocampus of 8-week offspring rats

子癇前期宮內(nèi)環(huán)境導(dǎo)致8周齡子代大鼠海馬炎性因子IL-6 表達(dá)的變化 本研究也檢測(cè)了GR相關(guān)的IL-6的表達(dá),Real-time PCR和Western blot檢測(cè),與對(duì)照組8周齡子代大鼠相比,子癇前期子代大鼠海馬中促炎性因子IL-6 mRNA和蛋白質(zhì)水平均增加(P< 0.05,圖4～5)。

Compared with normal adolescent offspring,the expression of IL-6 mRNA was increased in the hippocampus of adolescent rats with preeclampsia.

圖4 子癇前期宮內(nèi)環(huán)境導(dǎo)致8周齡子代大鼠海馬炎性因子IL-6 mRNA水平的變化

Fig 4 Effect of preeclampsia on expressions of IL-6 mRNA in the hippocampus of 8-week offspring rats

圖5 子癇前期宮內(nèi)環(huán)境導(dǎo)致8周齡子代大鼠海馬炎性因子IL-6蛋白水平的變化

討 論

本研究發(fā)現(xiàn),首先子癇前期子代新生大鼠血清皮質(zhì)酮濃度升高,其次8周齡子代大鼠海馬GR、CRH和CRHR1 mRNA水平均增加,8周齡子代大鼠海馬促炎性因子IL-6 mRNA水平也增加。提示子癇前期宮內(nèi)環(huán)境導(dǎo)致子代大鼠出生時(shí)血清高皮質(zhì)酮,以及8周齡海馬HPA軸相關(guān)基因表達(dá)的改變,可能在子代學(xué)習(xí)記憶能力受損中發(fā)揮作用。

子癇前期子代可能處于高糖皮質(zhì)激素的宮內(nèi)環(huán)境水平,依據(jù):(1)子癇前期的新生兒血漿皮質(zhì)醇濃度較高[12];(2)胎盤局部11β-HSD2酶可以降解糖皮質(zhì)激素,作為胎盤屏障,避免胎兒處于高糖皮質(zhì)激素的環(huán)境,子癇前期由于胎盤11β-HSD2酶表達(dá)的減少,導(dǎo)致局部的屏障作用受損,胎兒暴露于高糖皮質(zhì)激素環(huán)境中[14];(3)子癇前期孕婦的早產(chǎn)兒,與其他同胎齡兒相比,肺透明膜病變發(fā)病率較低。本研究運(yùn)用子癇前期動(dòng)物模型,發(fā)現(xiàn)子癇前期子代新生大鼠出生后血清皮質(zhì)酮濃度升高,提示了子癇前期子代存在宮內(nèi)高糖皮質(zhì)激素的環(huán)境,并可能對(duì)HPA軸產(chǎn)生影響。有研究發(fā)現(xiàn)子癇前期子代到了青少年期,血漿皮質(zhì)酮水平也較高,提示子癇前期子代HPA軸活性的過(guò)度激活[15]。

宮內(nèi)應(yīng)激或其他不良環(huán)境會(huì)影響子代HPA軸的功能。孕晚期反復(fù)遭受社會(huì)壓力應(yīng)激的子代,青少年期出現(xiàn)胰島素抵抗、成年后出現(xiàn)應(yīng)激時(shí)HPA軸過(guò)度激活、海馬GR和CRH mRNA水平均增加[16-17]。孕期高糖低蛋白飲食導(dǎo)致子代的HPA功能異常和成年后高血壓等代謝綜合征發(fā)病增加[18]。宮內(nèi)缺氧應(yīng)激可導(dǎo)致子代鼠HPA軸敏感性的改變和crhr1 DNA CpG島甲基化的改變[19]。但也有海馬GR過(guò)表達(dá)導(dǎo)致負(fù)反饋增強(qiáng)和HPA軸功能減弱的研究結(jié)果[20],例如出生后獲得充分母愛(ài)的仔鼠,遇到應(yīng)激時(shí)GR表達(dá)增加,CRH、ACTH和糖皮質(zhì)激素分泌均減少,表現(xiàn)為HPA軸的負(fù)反饋增加,減少應(yīng)激時(shí)HPA軸的反應(yīng)性[21-22]。本研究發(fā)現(xiàn)子癇前期的子代大鼠8周齡時(shí)GR、CRH和CRHR1 表達(dá)增加,提示8周齡時(shí)子代表現(xiàn)為對(duì)HPA軸的負(fù)反饋抑制減弱和HPA軸的過(guò)度激活,進(jìn)一步可通過(guò)CRHR的拮抗劑或激動(dòng)劑來(lái)檢測(cè)應(yīng)激狀態(tài)下HPA軸的功能。

HPA軸與海馬依賴的學(xué)習(xí)記憶能力有關(guān)。有研究發(fā)現(xiàn)胚胎發(fā)育期應(yīng)激會(huì)導(dǎo)致CRH和CRHR過(guò)表達(dá)[23],給予CRHR拮抗劑能糾正應(yīng)激引起的記憶障礙以及海馬樹(shù)突丟失[10]。小劑量CRH能增強(qiáng)大鼠的空間學(xué)習(xí)能力,CRH直接注入大鼠腦可導(dǎo)致神經(jīng)元長(zhǎng)時(shí)程突觸效應(yīng)的持續(xù)增強(qiáng),該效應(yīng)能被CRHR拮抗劑所阻斷,提示CRH通過(guò)其受體參與調(diào)節(jié)海馬神經(jīng)元的長(zhǎng)時(shí)程突觸效應(yīng),進(jìn)而影響海馬依賴的學(xué)習(xí)記憶功能。CRH通過(guò)CRHRl抑制神經(jīng)元突起的發(fā)育[24],并涉及AC/PKC以及MAPK信號(hào)轉(zhuǎn)導(dǎo)通路,有NMDA、AMPA和GABA受體參與[25-26]。因此我們推測(cè)子癇前期宮內(nèi)環(huán)境導(dǎo)致子代CRH/CRHR1表達(dá)的增加,提示了子癇前期子代HPA軸的過(guò)度激活,與出生后學(xué)習(xí)記憶能力受損有關(guān)。

糖皮質(zhì)激素有促進(jìn)炎癥或者抗炎癥的作用,因此本研究也檢測(cè)了海馬中與GR信號(hào)轉(zhuǎn)導(dǎo)通路和宮內(nèi)應(yīng)激環(huán)境改變有關(guān)的炎癥基因IL-6 的水平[27]。發(fā)現(xiàn)子癇前期的8周齡子代大鼠的IL-6 水平增加,提示GR下游的炎癥促進(jìn)因子可能在子癇前期子代學(xué)習(xí)記憶能力抑制的過(guò)程中發(fā)揮作用。在神經(jīng)退行性疾病研究中發(fā)現(xiàn),慢性糖皮質(zhì)激素高暴露導(dǎo)致海馬IL-6、NF-κB和IL-18等水平增加,提示糖皮質(zhì)激素導(dǎo)致的海馬炎癥反應(yīng)與神經(jīng)退行性改變有關(guān)[28]。研究發(fā)現(xiàn),抑郁癥人群中糖皮質(zhì)激素暴露導(dǎo)致血IL-6水平升高與海馬體積縮小有關(guān),提示了IL-6在海馬神經(jīng)細(xì)胞分化生成異常和神經(jīng)可塑性的異常中起作用[29]。

目前胎源性成人疾病已成為研究熱點(diǎn),子癇前期是妊娠期最常見(jiàn)的并發(fā)癥之一,隨著圍產(chǎn)醫(yī)學(xué)的發(fā)展,重度子癇前期圍產(chǎn)兒出生率的增加,子癇前期宮內(nèi)環(huán)境對(duì)子代的影響深遠(yuǎn),包括代謝性疾病發(fā)病率的增加等,其中學(xué)習(xí)記憶能力的影響引起我們的關(guān)注。本研究結(jié)果初步提示,子癇前期大鼠宮內(nèi)高糖皮質(zhì)激素的環(huán)境影響了子代的HPA軸的功能,可能在子代學(xué)習(xí)記憶能力受損中起作用,為出生后干預(yù)提供了可能的環(huán)節(jié)。但本研究未對(duì)子代HPA軸應(yīng)激情況下的功能進(jìn)行研究,是后續(xù)要開(kāi)展的內(nèi)容。

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Preeclampsia alters the expression of HPA axis related genes in the hippocampus of offspring rats

HU Rong, LIU Xue-yuan, YAO Qing-qing, LI Xiao-tian△

(DepartmenofObstetrics,Obstetrics&GynecologyHospital,FudanUniversity,Shanghai200011,China)

Objective To explore the effect of preeclampsia on the expression of hypothalamic-pituitary-adrenal (HPA) axis related genes in the hippocampus of adolescent offspring rats. MethodsThe pregnant Sprague-Dawley rats were randomly divided into two groups to be given 125 mg·kg-1·d-1L-NAME or 1 mL pure saline by daily injection from day 14 to delivery.Serum corticosterone (CORT) levels in neonatal offspring rats were detected by ELISA.The mRNA and protein levels of glucocorticoid receptor (GR),corticotropin releasing hormone (CRH),CRH receptor 1 (CRHR1) and IL-6 in the hippocampus of 8-week offspring rats were determined by real-time PCR and Western blot,respectively. Results Compared with normal neonatal rats,the serum CORT concentration of neonatal rats with preeclampsia was increased (P<0.05).Compared with normal adolescent offspring,the levels of GR,CRH and CRHR1 mRNA and protein were increased in the hippocampus of adolescent rats with preeclampsia (P<0.05),accompanied by increased IL-6 mRNA and protein (P<

0.05),as a downstream inflammatory gene of the GR. Conclusions We found that adolescent SD rats exposed to preeclampsia showed alteration in the expression of HPA related genes in the hippocampus,which played a role in the impairment of learning and memory in children.

preeclampsia; offspring rats; glucocorticoid receptor; CRH; CRHR1; IL-6

國(guó)家自然科學(xué)基金(81571460,81270712)

R714.24+5

A

10.3969/j.issn.1672-8467.2017.04.007

2016-12-06;編輯:沈玲)

△Corresponding author E-mail:xiaotianli555@163.com

*This work was supported by the National Natural Science Foundation of China (81571460,81270712).