孕期尼古丁暴露所致子2代大鼠神經(jīng)內(nèi)分泌代謝編程改變的可遺傳效應(yīng)

郭自景, 徐 丹,2, 羅瀚文,3, 鄧梓辛, 鐘衛(wèi)華, 汪 暉,2

(武漢大學 1. 基礎(chǔ)醫(yī)學院, 2. 發(fā)育源性疾病湖北省重點實驗室, 3. 中南醫(yī)院骨科, 湖北 武漢 430071)

孕期尼古丁暴露所致子2代大鼠神經(jīng)內(nèi)分泌代謝編程改變的可遺傳效應(yīng)

郭自景1, 徐 丹1,2, 羅瀚文1,3, 鄧梓辛1, 鐘衛(wèi)華1, 汪 暉1,2

(武漢大學 1. 基礎(chǔ)醫(yī)學院, 2. 發(fā)育源性疾病湖北省重點實驗室, 3. 中南醫(yī)院骨科, 湖北 武漢 430071)

目的 探討孕期尼古丁暴露所致宮內(nèi)發(fā)育遲緩(IUGR)子代大鼠神經(jīng)內(nèi)分泌代謝編程改變的跨代遺傳效應(yīng)。方法 Wistar大鼠孕11 d起每天sc給予尼古丁2 mg·kg-1至分娩。子1代(F1)正常組和尼古丁組IUGR交叉配對而得子2代(F2)：CC組(親氏為正常F1)、CN組(父為正常F1，母為IUGR F1)、NC組(父為IUGR F1，母為正常F1)和NN組(親氏為IUGR F1)。成年F2給予2周冰水游泳刺激，收集刺激前、后血樣，采用放免試劑盒檢測血清促腎上腺皮質(zhì)激素(ACTH)水平，ELISA試劑盒檢測皮質(zhì)酮(CORT)水平，生化試劑盒檢測葡萄糖、甘油三酯(TG)和總膽固醇(TCH)水平。結(jié)果 慢性刺激前，NN組雄性子代血清CORT較CC組顯著降低，為CC組的73.9%(P<0.05)，CN和NC組雄性子代血清TG分別升高到CC組的1.43和1.52倍(P<0.05)，同時CN, NC和NN組雌性子代血清TG分別升高到CC組的1.71, 1.80和1.81倍(P<0.05)；慢性刺激后，CC組雄性子代血清CORT增加率為-1.67%，而NN組雄性子代血清CORT增加率為36.0%，NC組雄性及CN組雌性子代血糖顯著升高，分別升高至各自CC對照組的1.61和1.62倍(P<0.01)，同時各尼古丁組雌、雄中子代血清TG增加率均較CC組顯著降低(P<0.05)，具體表現(xiàn)為CN, NC和NN組雄性子代血清TG增加率分別降低至CC組的46.4%, 16.7%和7.7%，而相應(yīng)雌性子代血清TG增加率分別降低至CC組的20.6%, 4.0%和8.4%。與CC組相比，慢性刺激前，NN組雌、雄性子代血清TCH分別下降40.5%和21.9%(P<0.01)；慢性刺激后，雌性子代TCH增長率升高49.7%(P<0.05)。結(jié)論 孕期尼古丁暴露致大鼠神經(jīng)內(nèi)分泌代謝編程改變具有跨代遺傳效應(yīng)，且具有一定的性別和親源性差異。

尼古丁; 宮內(nèi)發(fā)育遲緩; 跨代遺傳; 糖代謝; 脂代謝

流行病學調(diào)查顯示，20%～50%的婦女在妊娠期吸煙，而50%不吸煙的孕婦也存在被動吸煙[1-2]。尼古丁是煙草中脂溶性較高的生物堿，可被快速吸收并能透過胎盤在胎兒體內(nèi)蓄積，被認為是煙草中危害胎兒健康的主要毒性成分[3]。本課題組前期動物實驗表明，孕期尼古丁暴露可致胎兒宮內(nèi)發(fā)育遲緩(intrauterine growth retardation, IUGR)和胎鼠母源性糖皮質(zhì)激素(glucocorticoid, GC)過暴露，后者可介導胎下丘腦-垂體-腎上腺軸(hypothalamic-pituitary-adrenal axis, HPA)相關(guān)的宮內(nèi)神經(jīng)內(nèi)分泌代謝編程改變及代謝綜合征(metabolic syndrome, MS)易感[4-5]，表現(xiàn)為宮內(nèi)胎HPA軸功能發(fā)育抑制、糖脂代謝功能及血表型改變，出生后HPA軸低基礎(chǔ)活性和高應(yīng)激敏感性改變，同時伴隨糖脂代謝血表型的GC依賴性改變[6-7]。孕期不良環(huán)境對子代的跨代遺傳效應(yīng)已經(jīng)被證實，但是其潛在的機制仍是近年來研究的熱點[8]。研究證實，孕期不良環(huán)境可對子代HPA軸功能產(chǎn)生不利影響，并具有跨代遺傳效應(yīng)[9-13]，但是孕期尼古丁暴露所致子代HPA軸相關(guān)神經(jīng)內(nèi)分泌代謝編程改變是否具有遺傳效應(yīng)還未見報道。本研究擬在前期研究基礎(chǔ)上，通過觀察孕期尼古丁暴露的子2代(F2)大鼠在慢性刺激前、后的HPA軸活性及糖脂代謝改變，探討尼古丁所致子代生殖發(fā)育毒性及神經(jīng)內(nèi)分泌代謝編程的跨代遺傳效應(yīng)。

1 材料與方法

1.1 藥品和試劑尼古丁購自美國Sigma-Aldrich公司；大鼠促腎上腺皮質(zhì)激素(adrenocorticotropic hormone, ACTH)放免試劑盒購自北京北方生物技術(shù)研究所；血皮質(zhì)酮(corticosterone, CORT)ELISA試劑盒購自美國Assaypro公司；葡糖氧化酶分析試劑盒購自上海Mind生物工程有限公司；甘油三酯(triglycerides, TG)和總膽固醇(total cholesterol, TCH)分析試劑盒購自上海生工生物工程股份有限公司；異氟烷購自美國巴克斯特醫(yī)療保健有限公司；其余化學試劑均為國產(chǎn)分析純。

1.2 動物和分組實驗大鼠由湖北省預(yù)防科學院實驗動物中心(許可證號：SCXK 2010-2011)提供。相關(guān)動物實驗均按照中國動物福利委員會的使用原則執(zhí)行。親代(P)：健康成年Wistar雌性大鼠(180～220 g)和雄性大鼠(260～300 g)，適應(yīng)性喂養(yǎng)1周后，每晚18:00進行合籠交配(雌∶雄=2∶1)，次晨進行雌鼠陰道涂片，在顯微鏡下檢見精子時記為受孕0 d(gestational day 0 , GD0)。確定受孕后，將孕鼠隨機分為2組。尼古丁組于GD11開始sc給予尼古丁2 mg·kg-1，直至分娩。對照組給予等量生理鹽水。給藥體積為10 mL·kg-1。子1代(F1)：待P的孕鼠自然分娩后，按仔鼠出生日期的不同分批，分別編號并篩選，其篩選原則為：① 在產(chǎn)仔數(shù)≥10只孕鼠中挑選；② 對照組保留非IUGR仔鼠，尼古丁組保留IUGR仔鼠；③ 盡量使挑選仔鼠的雌雄比率為1∶1。最終獲得的對照組和尼古丁組F1仔鼠各12只，記錄出生后每周(postnatal week, PW)體質(zhì)量變化。F2∶F1的仔鼠出生4個月(postnatal month 4, PM4)時按雌∶雄為2∶1比率進行合籠交配，正常生產(chǎn)得到4組F2，分別為CC組(上一代均為正常F1)、CN組(父為正常F1，母為IUGR F1)、NC組(父為IUGR F1，母為正常F1)和NN組(上一代均為IUGR F1)，詳見表1。

Tab.1 F1 mating principle and F2 grouping

F1(number)♂♀F2Normal(6)Normal(6)Normal♂×Normal♀(CC)Normal(6)IUGR(6)Normal♂×IUGR♀(CN)IUGR(6)Normal(6)IUGR♂×Normal♀(NC)IUGR(6)IUGR(6)IUGR♂×IUGR♀(NN)

IUGR: intrauterine growth retardation.

F1自然分娩后，每窩隨機選取1～2只仔鼠，按出生日期不同分批編號，記錄其出生后每周體質(zhì)量變化。F2大鼠于PM4～PM4.5期間每天給予3～5 min冰水游泳慢性刺激。慢性刺激前，分2次對F2 PM4大鼠進行鼠尾取血，每次300 μL，共600 μL；慢性刺激結(jié)束后，分2次在游泳完畢后1 h內(nèi)進行鼠尾取血，每次300 μL，共600 μL。待血液室溫凝固后，4℃下16 099×g離心10 min，吸取上層血清，-80℃凍存?zhèn)溆谩?/p>

1.3 檢測指標和方法取-80℃凍存?zhèn)溆玫腇2大鼠慢性刺激前、后血清，采用放免同位素試劑盒測定大鼠血清ACTH濃度[14]，其批間和批內(nèi)變異系數(shù)分別為10%和15%。采用ELISA試劑盒檢測大鼠CORT濃度[15]，其批間和批內(nèi)變異系數(shù)分別為5.0%和7.2%。血清葡萄糖、TG和TCH濃度檢測按照生化分析試劑盒的說明書進行檢測。計算血清ACTH、CORT、葡萄糖和TG濃度增長率。濃度增長率(%)=(慢性刺激后濃度- 慢性刺激前濃度)/ 慢性刺激前濃度×100%。

2 結(jié)果

2.1 孕期尼古丁暴露對F1仔鼠體質(zhì)量的影響如圖1所示，與正常對照組相比，尼古丁組F1出生后(PW3～PW14)體質(zhì)量降低或有降低趨勢，雌性F1在PW3～PW8顯著降低(圖1A,P<0.05)，而雄性F1在PW3～PW5顯著降低(圖1B,P<0.05)。

2.2 孕期尼古丁暴露對F2仔鼠體質(zhì)量的影響如圖2所示，對于雌性F2，與CC組相比，在PW1～PW16內(nèi)，尼古丁各組體質(zhì)量均低于CC組(P<0.01)，其中CN組在PW1～PW8顯著降低(P<0.05)，NC組和NN組在PW8顯著降低(P<0.05, P<0.01)，提示孕期尼古丁暴露可致雌性F2大鼠體質(zhì)量降低且CN組存在追趕性生長。與CC組相比，雄性F2 NC和NN組體質(zhì)量顯著升高(P<0.05, P<0.01)。

2.3 孕期尼古丁暴露對子代HPA軸神經(jīng)內(nèi)分泌代謝編程的影響

2.3.1 血清ACTH和CORT濃度變化如圖3所示，對于雌性F2，與CC組相比，慢性

刺激前，血清ACTH濃度在尼古丁各組均無明顯變化，血清CORT濃度在NN組顯著升高(P<0.01)；慢性刺激后，血清ACTH和CORT濃度在尼古丁各組均無顯著性改變。對于雄性F2，與CC組相比，慢性刺激前，NN組血清ACTH濃度顯著升高(P<0.01)，血清CORT濃度顯著降低(P<0.05)；慢性刺激后，血清ACTH濃度及其增加率在NN組顯著降低(P<0.01)或有降低趨勢，但血清CORT濃度變化率顯示一定的升高趨勢。

2.3.2 血糖濃度變化如圖4所示，對于雌性F2，與CC組相比，慢性刺激前，尼古丁各組血糖濃度均無顯著性改變；慢性刺激后，CN組血糖濃度顯著升高(P<0.05)。對于雄性F2，與CC組相比，慢性刺激前，尼古丁各組血糖濃度均無顯著性改變；慢性刺激后，NC組血糖濃度及增加率顯著升高(P<0.05,P<0.01)。

2.3.3 血脂濃度變化2.3.

3.1 血清TG濃度變化如圖5所示，對于雌性F2，與CC組相比，慢性刺激前，尼古丁各組血清TG濃度顯著升高(P<0.05, P<0.01)，刺激后NC組和NN組TG濃度及尼古丁各組TG增長率顯著降低(P<0.05)。對于雄性F2，與CC組相比，CN組和NC組刺激前TG濃度顯著升高(P<0.05)，刺激后NN組TG濃度顯著降低(P<0.01)，NC組和NN組TG增加率降低(P<0.05, P<0.01)。2.3.

3.2 血清TCH濃度變化如圖6所示，與CC組相比，慢性刺激前，雌、雄F2子代NN組大鼠血清TCH濃度均顯著降低(P<0.01)，刺激后各組血清TCH濃度均無顯著性改 變，NN組雌鼠血清TCH的增加率顯著升高(P<0.05)。

3 討論

本課題組前期研究發(fā)現(xiàn)，孕11 d大鼠sc給予尼古丁后，胎鼠過暴露于母源性GC，從而改變胎鼠生長發(fā)育指標(如體質(zhì)量、身長、尾長以及胎盤重量)和糖脂代謝，且IUGR發(fā)生率增加[16-17]。尼古丁暴露的F1成年子代表現(xiàn)為HPA軸低基礎(chǔ)活性和高應(yīng)激敏感性，同時，慢性刺激前血清TG和TCH濃度升高，慢性刺激后血糖增加率升高，血清TG和TCH增加率均降低[6]。本研究發(fā)現(xiàn)，F(xiàn)1尼古丁組大鼠體質(zhì)量降低，同時，F(xiàn)2尼古丁各組雌性大鼠體質(zhì)量降低，提示孕期尼古丁暴露所致雌性子代的生殖發(fā)育毒性具有可遺傳性。進一步研究發(fā)現(xiàn)，慢性刺激前，F(xiàn)2 NN組雄性大鼠血清CORT顯著降低；慢性刺激后，血清CORT變化率有升高趨勢。提示F2 NN組雄性大鼠存在HPA軸低基礎(chǔ)活性和高應(yīng)激敏感性。對糖脂水平的檢測顯示，慢性刺激前，F(xiàn)2尼古丁各組雌性大鼠，CN組及NC組雄性大鼠血清TG均升高；慢性刺激后，F(xiàn)2 CN組雌性大鼠血糖升高，且NC組雄性大鼠血糖及血糖增加率均升高，尼古丁各組大鼠血清TG增加率顯著降低或有降低趨勢。提示孕期尼古丁暴露所致子代神經(jīng)內(nèi)分泌代謝編程改變具有可遺傳性。已有報道，孕期不良環(huán)境所致子代糖脂代謝改變具有性別差異[18-19]；孕期地塞米松暴露所致的跨代遺傳效應(yīng)具有性別差異，且可增加心血管疾病的風險[20]。本研究也發(fā)現(xiàn)，孕期尼古丁暴露所致神經(jīng)內(nèi)分泌代謝編程改變具有性別差異和親源性差異：F2尼古丁各組雌性子代體質(zhì)量降低，而NC組和NN組雄性子代體質(zhì)量升高，慢性刺激前F2 NN組雌性大鼠血清CORT升高而雄性大鼠血清CORT顯著降低；慢性刺激后，父系尼古丁暴露使雄性子代血糖升高，而母系尼古丁暴露使雌性子代血糖升高。提示孕期尼古丁暴露所致子代體質(zhì)量、血清CORT改變具有性別差異，且血糖改變具有親源性差異。Drake等[13]研究發(fā)現(xiàn)，孕期GC暴露可導致F1大鼠葡萄糖耐受，并增加肝組織中的磷酸烯醇丙酮酸羧激酶，且這些改變可以持續(xù)到F2。大量研究均表明，循環(huán)中GC可以調(diào)控糖脂代謝相關(guān)的多個通路[21-25]。本課題組前期研究發(fā)現(xiàn)，孕期尼古丁暴露所致胎鼠高水平GC可通過下調(diào)胰島素樣生長因子(insulin-like growth factor, IGF)1/胰島素信號通路，并上調(diào)脂聯(lián)素/瘦素信號通路改變糖脂代謝功能。因此，本研究認為，HPA軸功能改變(GC水平變化)可能是糖脂代謝血表型改變(尤其是TG)的重要原因。Ding等[26]研究發(fā)現(xiàn)，妊娠糖尿病所致子代葡萄糖耐受具有跨代遺傳效應(yīng)，可能與生殖細胞中IGF2及其交互印記基因H19的表觀遺傳修飾改變有關(guān)；孕期煙草攝入可改變DNA甲基化水平[27]；孕期尼古丁暴露可導致子代印記基因IGF2差異甲基化區(qū)甲基化水平升高，且雄性子代甲基化差異更明顯[28]。因此初步推測，孕期尼古丁暴露所致大鼠神經(jīng)內(nèi)分泌代謝編程的親源性差異和性別差異可能是由印記基因的表觀遺傳學改變所致。

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(本文編輯: 喬 虹)

Prenatal nicotine exposure induces transgenerationalneuroendocrine metabolic programming alterationin second-generation rats

GUO Zi-jing1, XU Dan1,2, LUO Han-wen1,3, DENG Zi-xin1, ZHONG Wei-hua1, WANG Hui1,2

(1.BasicMedicalSchool, 2.HubeiProvincialKeyLaboratoryofDevelopmentallyOriginatedDisease,3.DepartmentofOrthopaedics,ZhongnanHospital,WuhanUniversity,Wuhan430071,China)

OBJECTIVE To investigate the transgenerational effect of neuroendocrine metabolic programmed alteration in adult intrauterine growth retardation (IUGR) offspring rats with prenatal nicotine exposure. METHODS Pregnant Wistar rats were administered daily with nicotine (2 mg·kg-1) by sc from gestational day 11 until delivery. F1 offspring was fed with a standard diet before four groups in F2 were set up according to the cross-mating between F1 normal adult rats and nicotine-induced IUGR adult rats. CC group was mated by F1 normal adult rats, CN group by F1 normal adult male rats and IUGR adult female rats, NC group by F1 IUGR adult male rats and normal adult female rats, while NN group was mated by F1 IUGR adult rats. F2 adult rats were subjected to a fortnight ice water swimming stimulus. Blood samples were collected before and after stress and then detected for the levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), glucose, triglycerides(TG) and total cholesterol (TCH). RESULTS Before stress, the level of serum CORT in F2 male rats of NN group was decreased to 73.9% of that of the CC group (P<0.05)，while the level of serum TG in F2 male rats of CN and NC groups was increased to 1.43 and 1.52 times that of the CC group, respectively (P<0.05). Meanwhile, the level of serum TG in F2 female rats of CN, NC and NN groups was increased to 1.71, 1.80 and 1.81 times that of the CC group, respectively (P<0.05). After stress, the serum CORT gain rate in F2 male rats of CC group was -1.67%, but was 36.0% in NN group. The serum glucose level in male NC group and in female CN group was increased to 1.61 and 1.62 times that of the corresponded CC groups, respectively (P<0.01). Furthermore, the serum TG gain rate in F2 rats of each nicotine group was decreased markedly in comparison with their corresponding controls (P<0.05),ie, the serum TG gain rates in F2 male rats of CN, NC and NN groups were decreased to 46.4%, 16.7% and 7.7% of the CC group, while the serum TG gain rates in F2 female rats of these groups were decreased to 20.6%, 4.0% and 8.4% of the CC group, respectively. Compared with CC group, TCH level of females and males in NN group was decreased by 40.5% and 21.9%(P<0.01) before stress, respectively, and the TCH gain rate of females in NN group was increased by 49.7%(P<0.05) after stress. CONCLUSIONThe reproductive and developmental toxicities and the neuroendocrine metabolic programming alterations induced by prenatal nicotine exposure are transgenerated to F2 offspring and these effects exhibit gender and parental differences.Key words: nicotine; intrauterine growth retardation; transgenerational inheritance; glucose metabolism; lipid metabolism

XU Dan, Tel: 15972228956, E-mail: xuyidan70188@whu.edu.cn

國家自然科學基金(81220108026)；國家自然科學基金(81430089)；國家自然科學基金(81371483)

郭自景，女，碩士研究生，主要從事發(fā)育源性疾病研究; 徐 丹，女，副教授，博士，主要從事發(fā)育源性疾病研究。

徐 丹, E-mail: xuyidan70188@whu.edu.cn, Tel: 15972228956

Foundation item: The project supported by National Natural Science Foundation of China(81220108026); National Natural Science Foundation of China (81430089); and National Natural Science Foundation of China (81371483)

2015-01-04 接受日期: 2015-03-12)

R394.6

A

1000-3002(2015)02-0277-07

10.3867/j.issn.1000-3002.2015.02.015

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