梁 君， 高 強(qiáng)， 崔梅花， 郁衛(wèi)東， 侯曉琳， 楊 成， 李 想

1.北京大學(xué)航天臨床醫(yī)學(xué)院 航天中心醫(yī)院消化科，北京100049； 2.河南科技大學(xué)第一附屬醫(yī)院消化科； 3.北京大學(xué)人民醫(yī)院臨床分子生物學(xué)研究所； 4.北京大學(xué)人民醫(yī)院動(dòng)物實(shí)驗(yàn)室

論著·腸相關(guān)疾病

TUDCA緩解小鼠腸炎對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激與雙氧化酶2表達(dá)的研究

梁 君1， 高 強(qiáng)2， 崔梅花1， 郁衛(wèi)東3， 侯曉琳1， 楊 成1， 李 想4

1.北京大學(xué)航天臨床醫(yī)學(xué)院 航天中心醫(yī)院消化科，北京100049； 2.河南科技大學(xué)第一附屬醫(yī)院消化科； 3.北京大學(xué)人民醫(yī)院臨床分子生物學(xué)研究所； 4.北京大學(xué)人民醫(yī)院動(dòng)物實(shí)驗(yàn)室

目的 探討特異性內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑?；切苋パ跄懰?Tauroursodeoxycholate, TUDCA)緩解DSS誘導(dǎo)的小鼠腸炎對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress, ERS)蛋白與腸黏膜過(guò)氧化氫產(chǎn)生酶雙氧化酶2(dual oxidase2， Duox2)表達(dá)的研究。方法 7周C57BL/6J雄性小鼠適應(yīng)喂養(yǎng)1周后隨機(jī)分為對(duì)照組、炎癥組、干預(yù)組。炎癥組和干預(yù)組飲用2.5%葡聚糖硫酸鈉(dextran sulphate sodium, DSS)溶液誘導(dǎo)小鼠腸炎，干預(yù)組再以500 mg/kg的TUDCA灌胃。8 d后處死小鼠，收集結(jié)腸作HE和免疫組化染色，Western blotting檢測(cè)Duox2及ERS相關(guān)蛋白Grp78、Atf6、P-Ire1α/Ire1α、Ire1β、P-Perk/Perk的表達(dá)。結(jié)果 TUDCA明顯減輕DSS誘導(dǎo)的小鼠腸炎。Western blotting結(jié)果顯示炎癥組Grp78、P-Perk/Perk蛋白及Duox2表達(dá)均升高，干預(yù)組這三種蛋白表達(dá)恢復(fù)到對(duì)照組水平，其余ERS相關(guān)蛋白表達(dá)無(wú)變化。免疫組化結(jié)果顯示Grp78和Duox2三組表達(dá)水平與Western blotting結(jié)果相一致。結(jié)論 TUDCA緩解小鼠腸炎可能與抑制內(nèi)質(zhì)網(wǎng)Grp78-Perk通路有關(guān)，該通路與Duox2表達(dá)相互影響。

潰瘍性結(jié)腸炎；內(nèi)質(zhì)網(wǎng)應(yīng)激；?；切苋パ跄懰?；雙氧化酶2

潰瘍性結(jié)腸炎(ulcerative colitis, UC)是西方常見(jiàn)病，近年來(lái)在我國(guó)的發(fā)病率呈明顯上升趨勢(shì)[1]。臨床上UC主要表現(xiàn)為腹痛、腹瀉、黏液膿血便；腸外癥狀如體質(zhì)量減輕、貧血、關(guān)節(jié)疼痛；并發(fā)癥有中毒性巨結(jié)腸和結(jié)腸癌等。內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress, ERS)是指細(xì)胞處于缺氧、氧化應(yīng)激、毒素等環(huán)境時(shí)，未折疊或錯(cuò)誤折疊的蛋白質(zhì)在內(nèi)質(zhì)網(wǎng)腔中聚集，Ca離子儲(chǔ)存釋放平衡失調(diào)，脂質(zhì)代謝合成紊亂的狀態(tài)。ERS有三條信號(hào)通路，起始蛋白分別為肌醇需求酶1(inositol-requiring enzyme-1, Ire1)、蛋白激酶R樣內(nèi)質(zhì)網(wǎng)激酶(protein kinase R-like ER kinase, Perk)、活化轉(zhuǎn)錄因子6(activating transcription factor 6，Atf6)，其中Ire1在體內(nèi)有兩種形式，Ire1α廣泛表達(dá)，Ire1β表達(dá)于呼吸道和胃腸道表面。當(dāng)細(xì)胞處于穩(wěn)態(tài)時(shí)，這3種應(yīng)激感受蛋白都與葡萄糖調(diào)節(jié)蛋白78(glucose regulatory protein 78, Grp78)結(jié)合[2]。內(nèi)質(zhì)網(wǎng)應(yīng)激出現(xiàn)時(shí)，Grp78從這3種感受器上解離而去結(jié)合錯(cuò)誤折疊的蛋白，Grp78表達(dá)升高。使得Ire1、Perk和Atf6通路激活而表達(dá)有所變化。對(duì)于Perk和Ire1α通路蛋白，激活后轉(zhuǎn)變?yōu)榱姿峄问郊碢-Perk和P-Ire1α。已有大量研究[3-4]表明ERS在UC發(fā)生、發(fā)展過(guò)程中起重要作用。而腸上皮細(xì)胞表達(dá)的雙氧化酶2(Duox2)主要功能是產(chǎn)生過(guò)氧化氫(H2O2)，是腸道黏膜H2O2的重要來(lái)源[5]。H2O2作為活性氧(reactive oxygen species, ROS)的一種，可以誘導(dǎo)ERS發(fā)生。研究[6]證實(shí)ROS在結(jié)腸炎模型和患者黏膜中產(chǎn)生增加，且ROS在疾病早期階段被觀察到，并與疾病嚴(yán)重程度及進(jìn)展相關(guān)[6]。本研究采用ERS特異性抑制劑牛磺熊去氧膽酸(TUDCA)[7]干預(yù)腸炎小鼠后，觀察研究ERS各指標(biāo)的變化及腸上皮Duox2的變化，探討ERS與Duox2可能的相互影響。

1 材料與方法

1.1 動(dòng)物模型的建立健康雄性7周C57BL/6J小鼠，體質(zhì)量20～23 g，購(gòu)自北京華阜康公司，飼養(yǎng)于北京大學(xué)人民醫(yī)院SPF級(jí)動(dòng)物實(shí)驗(yàn)室。適應(yīng)性喂養(yǎng)1周后，隨機(jī)分為對(duì)照組、炎癥組、干預(yù)組，每組14只。對(duì)照組自由飲水，炎癥組和干預(yù)組飲用2.5%葡聚糖硫酸鈉(dextran sulphate sodium, DSS)(美國(guó)MP biomedicals公司，分子量36 000～50 000 Da)，共8 d。干預(yù)組自飲用DSS第1天起，用500 mg/kg TUDCA(購(gòu)自美國(guó)EMD chemicals公司)每天灌胃，TUDCA溶于無(wú)菌磷酸鹽緩沖液(phosphate buffered saline, PBS)；對(duì)照組和炎癥組用等量無(wú)菌PBS灌胃。8 d后頸椎脫臼法處死小鼠，測(cè)量結(jié)腸長(zhǎng)度，自遠(yuǎn)端向近端取0.5 cm結(jié)腸置于4%多聚甲醛作HE染色和免疫組化染色，其余部分立即置于液氮中再轉(zhuǎn)入-80 ℃冰箱保存用作蛋白免疫印跡(Western blotting)。

1.2 結(jié)腸炎癥及TUDCA干預(yù)效果評(píng)價(jià)小鼠的觀察內(nèi)容包括疾病活動(dòng)指數(shù)(disease activity index, DAI)[8]、組織學(xué)損傷評(píng)分(histological index, HI)[9]和體質(zhì)量變化。隱血狀況用聯(lián)苯胺法試劑盒(南京建成公司)檢測(cè)。

1.3 Western blotting檢測(cè)相關(guān)蛋白表達(dá)Western blotting檢測(cè)方法為：液氮研磨組織，常規(guī)裂解后進(jìn)行總蛋白提取及變性，每孔上樣50 μg。Western blotting數(shù)據(jù)分析為：目的蛋白灰度值除以內(nèi)參β-actin的灰度值以校正誤差，所得結(jié)果為某樣品目的蛋白相對(duì)含量。而以磷酸化形式激活的蛋白與其總蛋白之比(如P-Perk/Perk、P-Ire1α/Ire1α)為此蛋白的相對(duì)含量。

1.4 免疫組化染色常規(guī)組織切片脫蠟后進(jìn)行，一抗與Western blotting所用來(lái)源為同一抗體。陰性對(duì)照玻片只滴加PBS。評(píng)分標(biāo)準(zhǔn)[10]：每張切片隨機(jī)選擇10個(gè)視野，每個(gè)視野觀察100個(gè)細(xì)胞。染色強(qiáng)度：細(xì)胞質(zhì)無(wú)染色0分，淡黃色1分，棕色2分，棕褐色3分；染色范圍：<5%計(jì)0分，5%～25%計(jì)1分，26%～50%計(jì)2分，51%～75%計(jì)3分，76%～100%計(jì)4分；兩項(xiàng)標(biāo)準(zhǔn)乘積為每張切片的評(píng)分結(jié)果。

2 結(jié)果

2.1 小鼠結(jié)腸炎癥情況對(duì)照組小鼠正?；顒?dòng)，毛發(fā)光澤，體質(zhì)量上升。炎癥組于第4天出現(xiàn)活動(dòng)減少，稀便，肉眼血便，毛色無(wú)光澤，第5天始平均體質(zhì)量明顯下降。干預(yù)組于第5天平均體質(zhì)量開(kāi)始明顯下降，出現(xiàn)隱血便，但始終未出現(xiàn)稀便，且活動(dòng)正常，毛色光澤。第5～8天炎癥組平均體質(zhì)量下降幅度大于干預(yù)組(見(jiàn)圖1)。對(duì)照組結(jié)腸黏膜完整、腺體排列整齊、結(jié)構(gòu)正常。炎癥組結(jié)腸黏膜炎癥嚴(yán)重，炎癥細(xì)胞浸潤(rùn)，黏膜不完整，腺體正常結(jié)構(gòu)消失，排列紊亂。干預(yù)組可見(jiàn)炎癥明顯減輕，雖有腺體部分消失，但上皮完整，炎癥細(xì)胞浸潤(rùn)不明顯(見(jiàn)圖2)。炎癥組小鼠結(jié)腸長(zhǎng)度明顯縮短，TUDCA治療后結(jié)腸長(zhǎng)度有所恢復(fù)(見(jiàn)表1)。

2.2 Western blotting結(jié)果DSS誘導(dǎo)的腸炎小鼠結(jié)腸組織Duox2、Grp78、P-Perk/Perk蛋白表達(dá)量高，經(jīng)TUDCA干預(yù)后表達(dá)明顯下降，對(duì)照組表達(dá)量低(P<0.05)。而P-Ire1α/Ire1α、Atf6、Ire1β蛋白均在三組間表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05，見(jiàn)圖3)。

圖1 三組小鼠體質(zhì)量-時(shí)間變化比較

Fig 1 Comparison of the body weight-time of mice among three groups

注：與對(duì)照組比較，aP<0.05；與干預(yù)組比較，bP<0.05。

圖2 8 d時(shí)三組小鼠結(jié)腸HE染色(400×)

注：與對(duì)照組比較，aP<0.05；與干預(yù)組比較，bP<0.05。

2.3 免疫組化染色結(jié)果Grp78和Duox2蛋白進(jìn)行免疫組化染色，炎癥組表達(dá)較對(duì)照組明顯升高，經(jīng)TUDCA干預(yù)后Grp78和Duox2蛋白的表達(dá)下降(見(jiàn)表2)。Grp78蛋白對(duì)照組表達(dá)于結(jié)腸上皮細(xì)胞質(zhì)，炎癥組Grp78在炎癥區(qū)域明顯染色，干預(yù)組染色強(qiáng)度和范圍明顯低于炎癥組。Duox2蛋白在對(duì)照組主要表達(dá)于腸上皮刷狀緣和細(xì)胞質(zhì)，在炎癥組的炎癥區(qū)域內(nèi)大范圍染色，在干預(yù)組表達(dá)比炎癥組明顯降低，于結(jié)腸上皮刷狀緣及部分炎性細(xì)胞的胞質(zhì)染色(見(jiàn)圖4)。

注：與對(duì)照組比較，aP<0.05；與干預(yù)組比較，bP<0.05。

圖4 8 d時(shí)三組結(jié)腸免疫組化染色(400×) A：Grp78蛋白；B：Duox2蛋白

3 討論

在UC發(fā)生、發(fā)展過(guò)程中，氧化應(yīng)激被認(rèn)為是導(dǎo)致腸道損傷的關(guān)鍵因素[11]。氧化應(yīng)激是ROS和活性氮類(reactive nitrogen species, RNS)物質(zhì)產(chǎn)生過(guò)多，超出機(jī)體氧化物的清除能力，導(dǎo)致細(xì)胞組織損傷的病理、生理過(guò)程。Duox2屬于NADPH氧化酶家族[12]，Duox2是內(nèi)質(zhì)網(wǎng)定居蛋白，它的轉(zhuǎn)錄后儲(chǔ)存修飾與內(nèi)質(zhì)網(wǎng)功能密切相關(guān)。它可以直接釋放H2O2，是腸道黏膜H2O2的重要來(lái)源。H2O2是ROS的重要成員，臨床研究[13]表明UC患者腸道黏膜Duox2蛋白表達(dá)上調(diào)且ERS在UC發(fā)病中起重要作用。而ROS是造成ERS發(fā)生的重要誘因，可以導(dǎo)致ERS信號(hào)通路激活[14]。

本研究采用DSS誘導(dǎo)小鼠UC模型，以ERS特異性抑制劑TUDCA干預(yù)后，通過(guò)DAI評(píng)分、HE染色鏡下觀察結(jié)腸黏膜病理改變，均表明TUDCA在小鼠結(jié)腸炎模型中，ERS干預(yù)有減輕炎癥的效果[7]。TUDCA為ERS特異性抑制劑，臨床主要以TUDCA治療原發(fā)性膽汁性肝硬化或膽囊膽固醇結(jié)石等[15]。本實(shí)驗(yàn)采用Cao等[7]的方法，結(jié)果為Western blotting炎癥組的結(jié)腸Duox2和Grp78、P-Perk/Perk表達(dá)量明顯升高，而干預(yù)后Duox2、Grp78、P-Perk/Perk表達(dá)恢復(fù)到對(duì)照組水平，而ERS另兩條通路蛋白Atf6、P-Ire1α/Ire1α、Ire1β三組表達(dá)量并無(wú)變化，原因可能是小鼠結(jié)腸炎急性期時(shí)，Grp78-Perk通路激活，而Atf6、Ire1α和Ire1β通路并未激活，或炎癥組小鼠暫時(shí)的結(jié)腸上皮損傷導(dǎo)致了Atf6、Ire1α和Ire1β蛋白表達(dá)被抑制到了正常水平，這可能由于DSS處理后觸發(fā)了細(xì)胞的保護(hù)反應(yīng)[16]。本研究Western blotting結(jié)果顯示同一組內(nèi)，Perk和Duox2變化趨勢(shì)一致，可能是由于腸道炎癥發(fā)生時(shí)，Duox2表達(dá)增加，產(chǎn)生了過(guò)量的H2O2[17]，參與并促進(jìn)了ERS的發(fā)生，使Grp78-Perk通路激活。反之，內(nèi)質(zhì)網(wǎng)通路激活后，影響Duox2的表達(dá)，促進(jìn)了氧化應(yīng)激的發(fā)展[18]。而免疫組化染色與Western blotting結(jié)果一致。顯示Duox2、Grp78蛋白在對(duì)照組表達(dá)低，在炎癥組炎癥區(qū)域內(nèi)染色強(qiáng)度和范圍都明顯增加，可能是參與炎癥的其他細(xì)胞的上述蛋白表達(dá)量增加所致。干預(yù)組Duox2和Grp78表達(dá)顯著低于炎癥組。

非成熟形式的Duox2表達(dá)于內(nèi)質(zhì)網(wǎng)，而經(jīng)加工成熟后的Duox2被運(yùn)送到細(xì)胞膜表面[19],故免疫組化定位可見(jiàn)Duox2表達(dá)于胞漿和腸上皮細(xì)胞刷狀緣。Duox2及其成熟因子Duox2能直接產(chǎn)生H2O2[18]。這對(duì)于殺菌和正常腸道防御是必要的，過(guò)多的H2O2產(chǎn)生則會(huì)對(duì)機(jī)體造成氧化應(yīng)激損傷。有研究[20]發(fā)現(xiàn)，DSS處理小鼠后能直接誘導(dǎo)Duox2表達(dá)升高，表明Duox2可能在組織產(chǎn)生實(shí)質(zhì)病理?yè)p傷前驅(qū)動(dòng)炎癥反應(yīng)的發(fā)生。而破壞細(xì)胞內(nèi)氧化還原平衡狀態(tài)，會(huì)使細(xì)胞對(duì)于ERS的有害作用更敏感，且抑制ROS的產(chǎn)生，可以減輕ERS后造成的細(xì)胞凋亡。核因子E2相關(guān)因子2(nuclear factor E2-related-factor2，Nrf2)是細(xì)胞應(yīng)對(duì)氧化應(yīng)激狀態(tài)的主要調(diào)節(jié)因子[21]。Nrf2還是Perk的下游底物[22]。當(dāng)Perk通路激活時(shí)，可使其特定位點(diǎn)磷酸化，導(dǎo)致Nrf2在細(xì)胞核中的累積，增加Nrf2靶基因的轉(zhuǎn)錄。而Perk-Nrf2的適度激活，可以促進(jìn)氧化還原平衡及ERS后的細(xì)胞存活[23]。Perk-eIF2α-Atf4-Chop作為ERS發(fā)生的重要信號(hào)通路，Perk和其下游eIF2α蛋白重要的特性就是對(duì)氧化還原平衡狀態(tài)的調(diào)節(jié)和使細(xì)胞適應(yīng)因胞內(nèi)ROS形成造成的氧化應(yīng)激狀態(tài)。這些特性主要由增加的Atf4合成和轉(zhuǎn)錄活性介導(dǎo)，導(dǎo)致抗氧化物基因的表達(dá)[24]。

本實(shí)驗(yàn)結(jié)果表明，TUDCA作為ERS特異性抑制劑，其減輕小鼠腸炎效果明顯。TUDCA緩解腸炎后ERS通路之一Grp78/Perk表達(dá)降低說(shuō)明TUDCA的治療效果與抑制Grp78/Perk通路有關(guān)，且Duox2表達(dá)與Grp78/Perk一致，說(shuō)明Perk信號(hào)通路可能對(duì)Duox2的表達(dá)有影響，具體作用機(jī)制還需進(jìn)一步探索證明。TUDCA對(duì)小鼠腸炎的干預(yù)效果及對(duì)Duox2和Grp78-Perk通路表達(dá)的影響為今后臨床UC的發(fā)病機(jī)制和治療的研究提供新思路。

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(責(zé)任編輯：馬 軍)

Effect of mouse colitis alleviated by Tauroursodeoxycholate on expressions of endoplasmic reticulum stress and dual oxidase2

LIANG Jun1, GAO Qiang2, CUI Meihua1, YU Weidong3, HOU Xiaolin1, YANG Cheng1, LI Xiang4

1.Department of Gastroenterology and Hepatology, Peking University Aerospace School of Clinical Medicine, Aerospace Center Hospital, Beijing 100049; 2.Department of Gastroenterology and Hepatology, the First Affiliated Hospital of Henan University of Science and Technology; 3.Institute of Clinical Molecular Biology, Peking University People’s Hospital; 4.Animal Center, Peking University People’s Hospital, China

Objective To investigate the effect of Tauroursodeoxycholate (TUDCA), a specific inhibitor of endoplasmic reticulum stress (ERS), which alleviate mouse colitis induced by dextran sulfate sodium (DSS) on the expressions of chaperone proteins of ERS and the dual oxidase2 (Duox2). Methods Seven-week-old C57BL/6J male mice were divided randomly into control group, DSS group, TUDCA treatment group. Mice in the treatment group and inflammation group were used 2.5% DSS to induce colitis. Mice in the treatment group were

500 mg/kg TUDCA by gavage. On 8th day, all mice were sacrificed, the colon tissues were collected, HE staining was used to evaluate pathology of colon, Western blotting and immunohistochemistry (IHC) were used to examine the expressions of proteins including Duox2 and Grp78, Atf6, P-Ire1α/Ire1α, Ire1β, P-Perk/Perk. Results Mice colitis induced by DSS was alleviated by TUDCA. The expressions of Grp78, Duox2, P-Perk/Perk in DSS group were increased, and the expressions of these proteins were down to the level of control in treatment mice. Other proteins were not affected by inflammation or TUDCA. The IHC results were consistent with the results of Grp78 and Duox2 by Western blotting. Conclusion Mouse colitis alleviated by TUDCA could be associated with the inhibition of Grp78-Perk signal pathway, they may be interaction between Duox2 expression and Grp78-Perk signal pathway.

Ulcerative colitis; Endoplasmic reticulum stress; Tauroursodeoxycholate; Dual oxidase2

10.3969/j.issn.1006-5709.2017.01.011

國(guó)家自然科學(xué)基金(81370487)；航天中心醫(yī)院科研基金(YN201310)

梁君，碩士，研究方向：炎癥性腸病。E-mail：daerduo.hi@163.com

崔梅花，博士，主任醫(yī)師，研究方向：消化疾病的診治。E-mail：cuimeih@sina.com

R574.62

A

1006-5709(2017)01-0040-05

2016-02-26