藏好晶,康玉明,王桂琴(山西醫(yī)科大學(xué)微生物學(xué)與免疫學(xué)教研室,太原 03000;西安交通大學(xué)醫(yī)學(xué)部生理學(xué)系;通訊作者,E-mail:guiqinwang3@63.com)

阻斷中樞NF-κB合成對(duì)高血壓大鼠下丘腦IL-1β表達(dá)水平的影響

藏好晶1,康玉明2,王桂琴1*
(1山西醫(yī)科大學(xué)微生物學(xué)與免疫學(xué)教研室,太原 030001;2西安交通大學(xué)醫(yī)學(xué)部生理學(xué)系;*通訊作者,E-mail:guiqinwang321@163.com)

目的 觀察核轉(zhuǎn)錄因子κB(NF-κB)拮抗劑阻斷中樞NF-κB合成對(duì)高血壓大鼠下丘腦室旁核(PVN)白介素1β(IL-1β)表達(dá)水平的影響,探討中樞NF-κB在高血壓發(fā)病中的作用。 方法 雄性SD大鼠24只被隨機(jī)分為4組:高血壓干預(yù)組(AngⅡ+PDTC)、高血壓對(duì)照組(AngⅡ+aCSF)、假手術(shù)干預(yù)組(NS+PDTC),假手術(shù)對(duì)照組(NS+aCSF),每組6只。記錄3 d基礎(chǔ)血壓后,高血壓組麻醉狀態(tài)下肩胛區(qū)皮下給予Ang Ⅱ200 ng/(kg·min),假手術(shù)組給予生理鹽水(NS)。干預(yù)組通過(guò)下丘腦室旁核插管給予NF-κB抑制劑吡咯烷二硫基甲酸鹽(PDTC),對(duì)照組給予人工腦脊液(aCSF)。4周后,測(cè)量血流動(dòng)力學(xué)指標(biāo),免疫熒光法檢測(cè)下丘腦室旁核IL-1β水平。 結(jié)果 與假手術(shù)對(duì)照組比較,高血壓對(duì)照組單位鏡下的下丘腦室旁核激活的IL-1β陽(yáng)性細(xì)胞數(shù)明顯增加,平均動(dòng)脈壓(MAP)及左室舒張末壓(LVEDP)升高,左心室壓力最大上升、下降速率(±dp/dtmax)絕對(duì)值低于正常組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。高血壓干預(yù)組與高血壓對(duì)照組比較,單位鏡下IL-1β陽(yáng)性細(xì)胞數(shù)減少,MAP、LVEDP降低,±dp/dtmax升高,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。 結(jié)論 高血壓大鼠下丘腦室旁核IL-1β表達(dá)水平升高,中樞NF-κB通過(guò)上調(diào)下丘腦IL-1β水平參與高血壓的發(fā)病。

高血壓； 下丘腦室旁核； 血管緊張素Ⅱ； 核轉(zhuǎn)錄因子κB； 白介素1β

高血壓發(fā)病機(jī)制復(fù)雜,是遺傳因素與環(huán)境因素相互作用的結(jié)果。越來(lái)越多的研究顯示,高血壓可能是一種慢性炎癥狀態(tài)。臨床研究證實(shí),高血壓時(shí)血清炎性細(xì)胞因子水平升高,并且對(duì)高血壓疾病的發(fā)展及預(yù)后有著重要影響[1]。

近年來(lái),人們逐漸認(rèn)識(shí)到中樞調(diào)控在心血管疾病發(fā)生發(fā)展中的作用,研究表明高血壓時(shí)中樞也有免疫細(xì)胞浸潤(rùn)及明顯的炎癥反應(yīng)。核轉(zhuǎn)錄因子κB(NF-κB)是調(diào)節(jié)基因轉(zhuǎn)錄的關(guān)鍵因子之一,在外周及中樞神經(jīng)系統(tǒng)中參與許多免疫和炎癥反應(yīng)的調(diào)節(jié),并且在心血管疾病的發(fā)生、發(fā)展中起著重要作用[2]。研究已證實(shí)NF-κB激活后可調(diào)控的靶基因產(chǎn)物多為炎癥反應(yīng)介質(zhì),其中包括炎性細(xì)胞因子。本研究將視角轉(zhuǎn)向中樞,下丘腦室旁核給予高血壓大鼠NF-κB阻滯劑PDTC處理,觀察高血壓大鼠下丘腦白介素1β表達(dá)水平及血流動(dòng)力學(xué)的變化,驗(yàn)證NF-κB是否通過(guò)調(diào)節(jié)下丘腦IL-1β水平引起血壓升高。

1 材料與方法

1.1 主要試劑

血管緊張素Ⅱ(Sigma公司),吡咯烷二硫基甲酸鹽/PDTC(Sigma公司),兔抗大鼠IL-1β一抗(博奧森生物技術(shù)有限公司),熒光標(biāo)記羊抗兔IgG二抗(博奧森生物技術(shù)有限公司)。

1.2 動(dòng)物模型制備

清潔級(jí)雄性SD大鼠24只,本校實(shí)驗(yàn)動(dòng)物中心提供,體重250-275 g,7周齡,許可證編號(hào):scxk(晉)2015-0001。隨機(jī)分為4組:高血壓干預(yù)組(AngⅡ+PDTC)、高血壓對(duì)照組(AngⅡ+aCSF)、假手術(shù)干預(yù)組(NS+PDTC),假手術(shù)對(duì)照組(NS+aCSF)。給予10%水合氯醛(0.3 ml/100 g)腹腔麻醉,肩胛區(qū)皮下植入微型滲透泵,高血壓模型大鼠通過(guò)微型滲透泵給予Ang Ⅱ[200 ng/(kg·min)]誘導(dǎo)高血壓,假手術(shù)大鼠給予生理鹽水。

1.3 下丘腦室旁核給藥

動(dòng)物模型制備結(jié)束后,將大鼠固定于腦立體定位儀上(ZH-藍(lán)星B型,淮北正華),剪開(kāi)頭部皮膚暴露顱骨。在前囟向后I.8 mm、正中線旁開(kāi)0.2 mm處插管,深度由硬腦膜起7.9 mm。插管由牙托粉固定,并于頸背部埋置微型滲透泵,通過(guò)硅膠管將插管與埋置于頸部皮下的微型滲透泵連接。通過(guò)滲透泵干預(yù)組給予PDTC(5 μg/h)4周,對(duì)照組給予人工腦脊液(0.25 μl/h)。所有過(guò)程無(wú)菌操作,大鼠清醒后分籠飼養(yǎng)。

1.4 血流動(dòng)力學(xué)檢測(cè)

大鼠術(shù)后4周,檢測(cè)血流動(dòng)力學(xué)。25%烏拉坦(0.4 ml/100 g)腹腔注射,麻醉后固定于手術(shù)臺(tái)上,頸前正中切口,分離出右側(cè)頸總動(dòng)脈并結(jié)扎遠(yuǎn)心端,動(dòng)脈夾阻斷近心端血流,用眼科剪在阻斷的頸總動(dòng)脈部分剪一V字形口,將充滿肝素生理鹽水的導(dǎo)管插入右側(cè)頸總動(dòng)脈。導(dǎo)管的另一端由三通管連接壓力換能器,壓力信號(hào)輸入BL-420S生物信號(hào)記錄分析系統(tǒng)記錄頸動(dòng)脈壓力曲線,打開(kāi)動(dòng)脈夾,出現(xiàn)動(dòng)脈波形后,固定好插管。待波形穩(wěn)定后,記錄平均動(dòng)脈壓(MAP)15 min。將插管插入心室,生物機(jī)能實(shí)驗(yàn)系統(tǒng)記錄左室內(nèi)壓力曲線。測(cè)量左心室舒張末壓(LVEDP)、左心室內(nèi)壓最大上升和下降速率(±dp/dtmax)。

1.5 免疫熒光染色法檢測(cè)下丘腦室旁核IL-1β表達(dá)水平

1.6 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 各組大鼠血流動(dòng)力學(xué)參數(shù)比較

術(shù)后4周進(jìn)行大鼠血流動(dòng)力學(xué)檢測(cè),與假手術(shù)對(duì)照組比較,高血壓對(duì)照組LVEDP升高,MAP升高,±dp/dtmax絕對(duì)值降低,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。下丘腦室旁核給予PDTC治療4周,與高血壓對(duì)照組相比,高血壓干預(yù)組LVEDP、MAP降低,±dp/dtmax絕對(duì)值升高,差異有統(tǒng)計(jì)學(xué)意義(P<0.05,見(jiàn)表1)。

表1血流動(dòng)力學(xué)結(jié)果比較

Table1Changesofhemodynamicparametersinfourgroups

GroupnMAP(mmHg)LVEDP(mmHg)+dp/dtmax(mmHg/s)-dp/dtmax(mmHg/s) 高血壓干預(yù)組6127 09±6 31?#10 13±1 74?#4627 57±390 21?#-4115 50±275 99?# 高血壓對(duì)照組6152 18±6 39?15 76±1 40?3800 05±350 46?-3356 26±320 41? 假手術(shù)干預(yù)組692 65±7 585 58±0 895987 95±478 98-5209 31±355 65 假手術(shù)對(duì)照組696 21±7 326 19±0 875975 54±514 59-5237 04±442 96

與相應(yīng)假手術(shù)組相比,*P<0.05；與高血壓對(duì)照組相比,#P<0.05

2.2 各組大鼠下丘腦室旁核IL-1β表達(dá)水平比較

與假手術(shù)對(duì)照組比較,高血壓對(duì)照組單位鏡下的下丘腦室旁核IL-1β陽(yáng)性細(xì)胞數(shù)明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。下丘腦室旁核給予PDTC治療4周,與高血壓對(duì)照組比較,高血壓干預(yù)組下丘腦室旁核IL-1β陽(yáng)性細(xì)胞數(shù)顯著降低,差異有統(tǒng)計(jì)學(xué)意義(P<0.05,見(jiàn)圖1)。

與相應(yīng)假手術(shù)組(NS+PDTC或NS+aCSF)相比,*P<0.05;與高血壓對(duì)照組(AngⅡ+aCSF)相比,#P<0.05

圖1 免疫熒光法檢測(cè)下丘腦室旁核中IL-1β表達(dá)(紅色熒光) (n=6)
Figure 1 IL-1β expression in paraventricular nucleus of hypothalamus (red) by immunofluorescene (n=6)

3 討論

近來(lái)研究表明,中樞調(diào)控在高血壓的發(fā)生發(fā)展中起到重要作用。下丘腦室旁核作為神經(jīng)內(nèi)分泌和自主神經(jīng)這兩大系統(tǒng)的整合部位,在調(diào)節(jié)機(jī)體呼吸、血壓和心血管活動(dòng)等方面起了非常重要的作用[3]。有研究發(fā)現(xiàn),損毀PVN能使高血壓大鼠的平均動(dòng)脈壓降低,同時(shí)能抑制或中止大鼠血壓繼續(xù)升高。

越來(lái)越多的研究證實(shí),免疫調(diào)節(jié)機(jī)制在心血管疾病中起著重要作用[4]。細(xì)胞因子是由免疫細(xì)胞和某些非免疫細(xì)胞合成、分泌的一類低分子量的糖蛋白或多肽。根據(jù)細(xì)胞因子在炎性反應(yīng)性疾病中的作用不同,可將其分為炎性細(xì)胞因子和抗炎細(xì)胞因子,IL-1β則屬于炎性細(xì)胞因子。IL-1β主要由活化的單核-巨噬細(xì)胞產(chǎn)生,在傳遞信息、激活與調(diào)節(jié)免疫細(xì)胞,介導(dǎo)T、B細(xì)胞活化、增殖與分化以及炎癥反應(yīng)中發(fā)揮重要作用[5]。有研究表明,高血壓是慢性炎癥過(guò)程,而IL-1β是重要的炎癥反應(yīng)標(biāo)志物[6]。高血壓的病變程度與IL-1β具有相關(guān)性[7]。生理情況下外周及中樞炎性細(xì)胞因子維持在一個(gè)較低水平,而在各種心血管疾病中,炎性細(xì)胞因子如IL-1β明顯增加,并對(duì)疾病的預(yù)后有重要的影響。

NF-κB為調(diào)節(jié)基因轉(zhuǎn)錄的關(guān)鍵因子,參與許多與炎癥反應(yīng)有關(guān)的早期免疫應(yīng)答的基因調(diào)控。靜息狀態(tài)的NF-κB存在于胞質(zhì)中,NF-κB被激活后,從細(xì)胞漿移到細(xì)胞核[8],發(fā)揮轉(zhuǎn)錄調(diào)節(jié)功能。細(xì)胞內(nèi)NF-κB對(duì)環(huán)境變化敏感,可以被多種因素激活。在AngⅡ誘導(dǎo)的高血壓大鼠中,下丘腦室旁核中NF-κB水平升高[9],而激活的NF-κB又能增加炎性細(xì)胞因子的合成。

本實(shí)驗(yàn)結(jié)果顯示,高血壓組下丘腦室旁核中IL-1β水平升高明顯,MAP升高。中樞給予PDTC處理后,高血壓大鼠室旁核IL-1β水平下降,同時(shí)MAP下降。該結(jié)果提示,高血壓時(shí)NF-κB激活,而激活的NF-κB增加了IL-1β的合成,從而引起血壓升高。有研究表明將IL-1β注入穹窿下器可顯著激活外周交感神經(jīng)活動(dòng)[10]。據(jù)此推測(cè),中樞IL-1β可能刺激交感神經(jīng)興奮,引起交感神經(jīng)興奮性血壓升高。本實(shí)驗(yàn)結(jié)果提示,下丘腦室旁核NF-κB與IL-1β參與了高血壓的病理過(guò)程,阻斷中樞NF-κB與IL-1β的表達(dá)水平有助于降低高血壓大鼠的血壓值,對(duì)高血壓發(fā)病機(jī)制的研究具有一定的意義。

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Effectsofblockingcentralnuclearfactor-κBonthehypothalamicinterleukin-1betaexpressioninhypertensiverats

ZANG Haojing1,KANG Yuming2,WANG Guiqin1*
(1DepartmentofMicrobiologyandImmunology,ShanxiMedicalUniversity,Taiyuan030001,China;2DepartmentofPhysiology,Xi’anJiaotongUniversitySchoolofMedicine;*Correspondingauthor,E-mail:guiqinwang321@163.com)

ObjectiveTo investigate the effects of NF-κB blockade on the level of hypothalamic interleukin-1beta(IL-1β)expression in hypertensive rats, to explore its possible role of central NF-κB in the development of hypertension.MethodsAdult male Sprage-Dawley rats were randomly divided into 4 groups(6 rats each group): hypertension treatment group(AngⅡ+PDTC), hypertension control group(AngⅡ+aCSF), sham treatment group(NS+PDTC), sham control group(NS+aCSF). After recording baseline blood pressure for three days, the rats were implanted subcutaneously with an angiotensin Ⅱ 200 ng/(kg·min)-filled osmotic minipump to induce the hypertension. Rats in sham group were administered with normal saline(NS). PVN cannulae were implanted for the injection of PDTC(5 μg/h) or artificial cerebrospinal fluid(aCSF, 0.25 μl/h). At 4 week after treatment, the hemodynamic index was measured, and the level of IL-1β in PVN was detected by immunofluorescence.ResultsImmunofluorescence results showed that, compared with sham group, the number of IL-1β positive cells in PVN increased significantly in hypertension group(P<0.05). The mean arterial pressure(MAP) and left ventricular end-diastolic pressure(LVEDP) were significantly higher in hypertension group than in sham group, while the left ventricular maximal rate of pressure increase and decrease(±dp/dtmax) were lower(P<0.05). After treatment with PDTC for four weeks, compared with sham group, IL-1β positive cells in PVN were significantly reduced in hypertension group, MAP and LVEDP were decreased, while ±dp/dtmaxwere increased(P<0.05).ConclusionThe level of hypothalamic interleukin-1beta(IL-1β) expression increases in hypertensive rats, and the central NF-κB may be involved in hypertension by modulating IL-1β expression in PVN.

hypertension; paraventricular hypothalamic nucleus; angiotensin Ⅱ; NF-κB; IL-1β

山西省科技攻關(guān)基金資助項(xiàng)目(20120313024-3)

藏好晶,女,1985-11生,碩士,助理實(shí)驗(yàn)師,E-mail:haojing406@163.com

2017-07-22

R544.1

A

1007-6611(2017)12-1221-04

10.13753/j.issn.1007-6611.2017.12.004