·論著·

肌球蛋白輕鏈激酶在急性壞死性胰腺炎大鼠小腸黏膜的表達(dá)及其作用

石慧榮唐國(guó)都覃蒙斌梁志海

【摘要】目的探討肌球蛋白輕鏈激酶(MLCK)在急性壞死性胰腺炎(ANP)大鼠小腸黏膜的表達(dá)及其作用機(jī)制。方法56只雄性SD大鼠按數(shù)字表法隨機(jī)分為對(duì)照組和ANP組。采用4%?；悄懰徕c胰膽管逆行注射法制備ANP大鼠模型，以僅翻動(dòng)胰腺作為對(duì)照組。造模后6、12、24、48 h分批處死大鼠，取血測(cè)定血清淀粉酶、TNF-α、IL-1β、二胺氧化酶(DAO)水平；取胰腺、小腸組織行病理學(xué)檢查；電鏡下觀察小腸黏膜腸上皮細(xì)胞的超微結(jié)構(gòu)及上皮細(xì)胞間緊密連接(TJ)；免疫組織化學(xué)法測(cè)定小腸黏膜MLCK蛋白表達(dá)。結(jié)果與對(duì)照組比較，ANP組12 h點(diǎn)大鼠血清淀粉酶活性、TNF-α水平、IL-1β水平、DAO活性均顯著升高，分別為(4 978±1 574)U/L比(1 176±124))U/L、(47.88±15.85)μg/L比(17.24±1.99)μg/L、(132.48±68.54)μg/L比(23.51±6.44)μg/L、(95.96±30.84)U/L比(38.06±17.73)U/L；胰腺、小腸病理學(xué)評(píng)分顯著增加[(12.2±1.80)比(4.68±0.35)分，(2.58±0.52)比(0.58±0.26)分]；小腸黏膜組織MLCK蛋白表達(dá)顯著上調(diào)(0.1863±0.0230比0.1636±0.0049)，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.05)。其他各時(shí)間點(diǎn)上述指標(biāo)兩組間差異也均有統(tǒng)計(jì)學(xué)意義(P值均<0.05)。ANP組大鼠腸上皮細(xì)胞的超微結(jié)構(gòu)破壞明顯，上皮細(xì)胞間TJ顯著增寬。結(jié)論ANP大鼠血清TNF-α、IL-1β、DAO水平上調(diào)，小腸黏膜組織MLCK蛋白表達(dá)上調(diào)，由此可能通過破壞腸上皮細(xì)胞間緊密連接的完整性引起腸黏膜屏障功能障礙。

【關(guān)鍵詞】胰腺炎，急性壞死性；緊密連接部；肌球蛋白輕鏈激酶；腸屏障

DOI:10.3760/cma.j.issn.1674-1935.2015.02.009

基金項(xiàng)目：國(guó)家自然科學(xué)基金(81260087)

收稿日期：(2014-09-24)

Expression and role of MLCK in small intestine mucosa in rats with acute necrotizing pancreatitisShiHuirong,TangGuodu,QinMengbin,LiangZhihai.DepartmentofGastroenterology,FirstAffiliatedHospital,GuangxiMedicalUniversity,Nanning530021,China

Correspondingauthor：TangGuodu,Email：tguodu02@126.com

Abstract【】ObjectiveTo explore the expression and function of myosin light streptokinase (MLCK) in small intestine mucosa of acute necrotizing pancreatitis (ANP) rats. MethodsFifty-six male SD rats were randomly assigned to control group and ANP group. A rat model of ANP was reproduced by retrograde injection of 4% sodium taurocholate into the biliopancreatic duct, while the control group underwent a sham operation. The rats were sacrificed at 6th, 12th, 24th, 48th hour after ANP induction. Serum amylase、TNF α, IL 1β, diamine oxidase (DAO) were measured. The pathological scores in the pancreas and small intestine were observed. The ultrastructure and tight junction (TJ) changes in the small intestine mucosa were observed with an electron microscope. The localization and expression of MLCK in small intestine mucosa was determined by immunohistochemistry method. ResultsCompared to the control group, the serum amylase, TNF-α, IL-1β, DAO level, in the ANP group were all significantly increased; [(4 978±1 574)U/L vs (1 176±124))U/L, (47.88±15.85)μg/L vs (17.24±1.99)μg/L, (132.48±68.54)μg/L vs (23.51±6.44)μg/L, (95.96±30.84)μg/Lvs(38.06±17.73)U/L at 12 h], and the pathology scores of pancreas and small intestine were both significantly elevated [12 h: (12.2±1.80) vs (4.68±0.35), (2.58±0.52) vs (0.58±0.26)] (P<0.05); the MLCK protein expression in small intestine mucosa was significantly increased in ANP group (12 h: 0.1863±0.0230vs0.1636±0.0049), and the difference was statistically significant (P<0.05). The small intestine ultrastructure was seriously damaged and TJ was widened significantly in ANP Group. ConclusionsThe increased serum TNF alpha and IL-1β concentration and DAO activity and up-regulated MLCK protein expression in small intestine mucosa may damage the integrity of tight junction of intestinal epithelial cell and cause intestine mucosa barrier dysfunction.

作者單位：530021南寧，廣西醫(yī)科大學(xué)第一附屬醫(yī)院消化科

通信作者：唐國(guó)都， Email：tguodu02@126.com

【Key words】Pancreatitis, acute necrotizing;Tight junctions;Myosin-light-chain kinase;Intestine barrier

重癥急性胰腺炎(SAP)是臨床常見急危重癥之一，病程發(fā)展迅速，病情兇險(xiǎn)，病死率高。SAP早期腸道缺血-再灌注可導(dǎo)致腸屏障功能損害，腸壁通透性增加，細(xì)菌或內(nèi)毒素易位，造成腸源性感染，使胰腺組織繼發(fā)感染，進(jìn)而啟動(dòng)全身炎癥反應(yīng)綜合征(SIRS)，并引起多器官功能障礙綜合征(MODS)[1-2]，加重病情。因此，積極恢復(fù)和維持腸黏膜屏障功能在SAP治療中非常必要。上皮細(xì)胞間緊密連接(tight junction, TJ)是維持腸黏膜屏障的重要結(jié)構(gòu)基礎(chǔ)，是決定腸壁通透性大小的主要因素。由肌球蛋白輕鏈激酶(myosin light chain, MLCK)誘導(dǎo)的肌球蛋白輕鏈(myosin light chain, MLC)磷酸化導(dǎo)致的TJ開放和細(xì)胞骨架的收縮是腸上皮屏障破壞的必需因素[3]。本研究檢測(cè)急性壞死性胰腺炎(ANP)大鼠小腸MLCK蛋白表達(dá)，觀察腸上皮細(xì)胞間TJ結(jié)構(gòu)改變，探討MLCK對(duì)小腸上皮細(xì)胞間TJ的調(diào)節(jié)作用及其對(duì)腸屏障功能的影響。

材料和方法

一、實(shí)驗(yàn)動(dòng)物及分組

雄性SD大鼠56只，體質(zhì)量250～300 g，清潔級(jí)，購自廣西醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心。大鼠適應(yīng)性飼養(yǎng)1周，術(shù)前禁食12 h，自由飲水。按數(shù)字表法隨機(jī)分為對(duì)照組、ANP組。采用4%牛磺膽酸鈉(日本TCI公司)1 ml/kg體質(zhì)量逆行胰膽管注射的方法制備ANP模型。對(duì)照組大鼠于開腹后用鑷子輕翻動(dòng)十二指腸及胰腺后關(guān)腹。術(shù)后皮下注射生理鹽水10 ml/kg體質(zhì)量。術(shù)后6、12、24、48 h分批處死大鼠，每個(gè)時(shí)點(diǎn)7只。右心房采血，離心取上清，置-80℃冰箱備用。取胰腺及回盲部以上5 cm左右的小腸組織，部分置甲醛液固定，部分置3%戊二醛液固定。

二、血淀粉酶、TNF-α、IL-1β、二胺氧化酶檢測(cè)

血清淀粉酶活性用全自動(dòng)生物化學(xué)檢測(cè)儀測(cè)定。TNF-α、IL-1β水平采用ELISA方法測(cè)定，試劑盒購自欣博盛生物公司。二胺氧化酶(diamine oxidase, DAO)活性采用ELISA方法測(cè)定，試劑盒購自南京建成生物工程研究所，按說明書操作。

三、胰腺、小腸組織病理學(xué)檢查

取固定的胰腺、小腸組織，常規(guī)石蠟包埋、切片，HE染色。參考Schmidt等[4]標(biāo)準(zhǔn)從水腫、炎性細(xì)胞浸潤(rùn)、出血及壞死4個(gè)方面對(duì)胰腺組織進(jìn)行病理評(píng)分；參考Chiu等[5]標(biāo)準(zhǔn)從黏膜、絨毛損傷及炎癥、出血等方面對(duì)小腸組織進(jìn)行病理評(píng)分。

四、小腸組織超微結(jié)構(gòu)觀察

取固定的小腸組織，制備超薄切片，于透射電鏡下觀察腸上皮細(xì)胞的超微結(jié)構(gòu)及細(xì)胞間TJ。

五、小腸黏膜組織MLCK表達(dá)檢測(cè)

采用常規(guī)免疫組化法檢測(cè)小腸組織MLCK蛋白的表達(dá)。兔抗大鼠MLCK多克隆抗體購自Sigma公司，工作濃度1∶200；即用型SP-9000免疫組化檢測(cè)試劑盒及DAB試劑盒均購自北京中杉金橋公司。用PBS代替一抗作為陰性對(duì)照。腸上皮細(xì)胞胞質(zhì)內(nèi)出現(xiàn)棕黃色顆粒為陽性表達(dá)。每張切片在高倍鏡下隨機(jī)取5個(gè)視野，應(yīng)用美國(guó)Imagepro plus 6.0專業(yè)圖像分析軟件獲取每個(gè)視野的光密度值，取均值。

六、統(tǒng)計(jì)學(xué)處理

結(jié)　　果

一、血淀粉酶、TNF-α、IL-1β、DAO水平的變化

與對(duì)照組比較，ANP組各時(shí)間點(diǎn)大鼠的血清淀粉酶、TNF-α、IL-1β、DAO水平均于造模后明顯上升，顯著高于相應(yīng)時(shí)間點(diǎn)的對(duì)照組(P值均<0.05)。淀粉酶于術(shù)后6 h達(dá)峰值，TNF-α于12 h達(dá)峰值，IL-1β、DAO于24 h達(dá)峰值，并持續(xù)處于高水平(表1)。

指 標(biāo)時(shí)間點(diǎn)(h)對(duì)照組(28只)ANP組(28只)t值P值淀粉酶(U/L)61841±1816015±1216-8.3390.001121176±1244978±1574-5.9000.002241360±2784225±647-9.2260.001481148±912125±805-2.9830.015TNF-α(μg/L)6 11.26±2.7629.43±5.83-5.6310.00112 17.24±1.99 47.88±15.85-3.8360.03024 9.19±3.98 47.69±21.11-3.5850.01248 12.45±3.5224.53±6.59-3.2070.018IL-1β(μg/L)6 26.91±16.01 91.25±44.01-2.7480.03312 23.51±6.44132.48±68.54-3.1660.04924 18.02±3.11204.06±76.71-5.1000.00248 37.67±21.26 85.78±44.27-2.8390.049DAO(U/L)6 36.96±9.36 79.62±15.03-5.5970.00112 38.06±17.73 95.96±30.84-3.3140.01324 57.76±8.02 153.30±30.67-6.7910.00148 33.35±11.12 45.66±12.52-2.6670.037

二、胰腺、小腸組織病理改變

對(duì)照組大鼠胰腺小葉完整，未見出血、壞死，偶有輕度水腫及少量炎性細(xì)胞浸潤(rùn)；ANP組大鼠胰腺間質(zhì)增寬，腺小葉結(jié)構(gòu)破壞，炎性細(xì)胞浸潤(rùn)，局灶或大片出血壞死。對(duì)照組大鼠小腸黏膜結(jié)構(gòu)完整，未見固有層水腫及絨毛脫落，偶見絨毛頂端上皮下間隙增寬；ANP組大鼠小腸黏膜結(jié)構(gòu)破壞，上皮下間隙增寬，固有層水腫、裸露，絨毛脫落，固有層出血及炎癥細(xì)胞大量聚集。與對(duì)照組比較，ANP組各時(shí)間點(diǎn)大鼠的胰腺、小腸組織病理評(píng)分均顯著高于同時(shí)間點(diǎn)的對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.05)。 胰腺組織損傷于術(shù)后12 h最嚴(yán)重，小腸組織損傷于24 h最嚴(yán)重 (表2)。

組織來源時(shí)間點(diǎn)(h)對(duì)照組(28只)ANP組(28只)t值P值胰腺63.90±1.148.80±1.08-7.6350.001124.68±0.3512.20±1.80-10.6490.001244.43±0.5310.83±0.85-17.0510.001484.00±0.5210.60±1.14-12.7670.001小腸60.50±0.222.17±0.21-13.4840.001120.58±0.262.58±0.52-8.4850.001240.88±0.133.21±0.64-8.7140.001480.56±0.192.49±0.24-15.7950.001

三、小腸組織超微結(jié)構(gòu)及上皮細(xì)胞間TJ改變

對(duì)照組大鼠小腸黏膜超微結(jié)構(gòu)未見明顯改變，腸上皮微絨毛發(fā)達(dá), 排列整齊；細(xì)胞內(nèi)線粒體、內(nèi)質(zhì)網(wǎng)等結(jié)構(gòu)未見異常；細(xì)胞間TJ可見，寬度無明顯變化。ANP組小腸黏膜微絨毛破壞、減少、變短、缺失；細(xì)胞內(nèi)線粒體腫脹、空泡變性，內(nèi)質(zhì)網(wǎng)擴(kuò)張、脫粒, 細(xì)胞器明顯減少；腸上皮細(xì)胞間TJ增寬(圖1)。與對(duì)照組比較，ANP組各時(shí)點(diǎn)大鼠小腸上皮細(xì)胞間TJ均顯著增寬，以24 h時(shí)的TJ增寬最明顯。

圖1　對(duì)照組(上)及ANP組(下)大鼠6(1A)、12(1B)、24(1C)、48 h(1D)時(shí)小腸黏膜上皮細(xì)胞的超微結(jié)構(gòu)及細(xì)胞間TJ的改變(×50000)

四、小腸黏膜組織MLCK表達(dá)

對(duì)照組大鼠小腸組織不表達(dá)或弱陽性表達(dá)MLCK蛋白。ANP組大鼠小腸上皮細(xì)胞胞質(zhì)內(nèi)出現(xiàn)棕黃色顆粒，呈陽性表達(dá)(圖2)。對(duì)照組大鼠6、12、24、48 h點(diǎn)小腸黏膜組織MLCK的相對(duì)表達(dá)量分別為0.1568±0.0028、0.1636±0.0049、0.1929±0.0024、0.1865±0.0074；ANP組大鼠分別為0.1680±0.0089、0.1863±0.0230、0.1995±0.0105、0.1947±0.0054。與對(duì)照組比較，ANP組大鼠小腸黏膜MLCK的表達(dá)量于造模后即上升，24 h達(dá)峰值，并持續(xù)處于高水平，較對(duì)照組同時(shí)間點(diǎn)顯著增加，差異均有統(tǒng)計(jì)學(xué)意義(F值分別為-5.272、-3.340、-2.270、-2.445，P值均<0.05)。

討　　論

腸黏膜上皮屏障是體內(nèi)重要的生物防御屏障，在抵御腸腔內(nèi)細(xì)菌或內(nèi)毒素易位中起重要作用。它主要包括完整的腸上皮細(xì)胞(intestinalepithelialcell，IEC)和上皮細(xì)胞間連接，包含TJ、黏附連接和縫隙連接等，其中TJ為最主要連接方式。TJ位于上皮細(xì)胞膜外側(cè)頂部，是多蛋白結(jié)構(gòu)復(fù)合體，其主要功能是只允許離子及小分子可溶性物質(zhì)通過，不允許毒性大分子及微生物通過。此特殊生理功能在SAP病程中能防止細(xì)菌、內(nèi)毒素、炎癥介質(zhì)的侵入。本研究建立了ANP大鼠模型，電鏡下觀察到ANP大鼠小腸黏膜上皮細(xì)胞的超微結(jié)構(gòu)發(fā)生明顯改變，腸黏膜微絨毛被破壞，腸上皮細(xì)胞間TJ增寬，提示ANP大鼠腸黏膜屏障功能損傷存在小腸上皮細(xì)胞間TJ的改變。

圖2　對(duì)照組(2A)及ANP組大鼠6(2B)、12(2c)、24(2D)、48 h(2E)時(shí)小腸黏膜組織MLCK的表達(dá)(免疫組化　×400)

MLCK是絲氨酸/蘇氨酸蛋白激酶家族成員，是位于胞膜的鈣調(diào)素依賴酶，能調(diào)節(jié)MLC磷酸化。MLC發(fā)生磷酸化后可活化肌球蛋白重鏈頭部ATP，產(chǎn)生的能量使細(xì)胞骨架肌動(dòng)蛋白微絲滑動(dòng)，細(xì)胞收縮，通透性改變。因此，MLC磷酸化在細(xì)胞間TJ相關(guān)蛋白的調(diào)控及腸上皮屏障功能紊亂中起著重要作用[6-7]。MLCK誘導(dǎo)的MLC磷酸化水平升高被認(rèn)為是腸上皮屏障通透性增加的重要分子基礎(chǔ)[8]，對(duì)維持TJ的完整性起著重要作用[9]。Shen等[10]建立胞膜MLCK持續(xù)高表達(dá)的體外細(xì)胞模型，觀察到腸黏膜屏障功能受損。Clayburgh等[11]通過敲除MLCK基因和利用特異性MLCK抑制劑的方法均能減輕腸上皮細(xì)胞TJ的損傷。Moriez等[12]研究表明，MLCK特異性抑制劑能抑制內(nèi)毒素血癥狀態(tài)下的腸黏膜菌群易位、減輕腸黏膜通透性的增加，并減輕細(xì)胞間TJ的損傷。

TNF-α和IL-1β可增加MLC磷酸酶轉(zhuǎn)錄和激活MLCK活性，上調(diào)MLCK蛋白表達(dá)，引起MLC磷酸化增加及TJ相關(guān)蛋白重新分布，改變TJ的結(jié)構(gòu)及完整性[13-14]，導(dǎo)致腸上皮細(xì)胞TJ的收縮和開放,增加腸道通透性[15-16]。DAO是位于腸黏膜上絨毛細(xì)胞中高度活性的細(xì)胞內(nèi)酶，主要存在于回腸黏膜絨毛，其活性與黏膜細(xì)胞的核酸和蛋白質(zhì)合成密切相關(guān)，能反映腸黏膜完整性和損傷程度[17]。當(dāng)小腸上皮完整性受到破壞，小腸黏膜上皮內(nèi)的DAO則會(huì)釋放入血，因此，血液中DAO水平可以間接反映腸黏膜上皮細(xì)胞的完整性及腸屏障功能[18]。本研究結(jié)果表明，ANP大鼠中血清TNF-α、IL-1β、DAO水平均升高，與小腸病理損傷及超微結(jié)構(gòu)動(dòng)態(tài)改變一致，進(jìn)一步證實(shí)ANP大鼠存在腸屏障功能障礙。

參考文獻(xiàn)

[1]Liu H, Li W, Wang X, et al. Early gut mucosal dysfunction in patients with acute pancreatitis[J]. Pancreas, 2008,36(2):192-196.

[2]Mole DJ, Taylor MA, McFerran NV, et al. The isolated perfused liver response to a ‘second hit’ of portal endotoxin during severe acute pancreatitis[J]. Pancreatology, 2005,5(4-5):475-485.

[3]Boivin MA, Ye D, Kennedy JC, et al. Mechanism of glucocorticoid regulation of the intestinal tight junction barrier[J]. Am J Physiol Gastrointest Liver Physiol,2007,29(2):G590-G598.

[4]Schmidt J, Rattner DW, Lewandrowski K, et al. A better model of acute pancreatitis for evaluating therapy[J]. Ann Surg,1992,215(1):44-56.

[5]Chiu CJ, McArdle AH, Brown R, et al. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal[J]. Arch Surg, 1970,101(4):478-483.

[6]Matsumura F, Hartshorne DJ. Myosin phosphatase target subunit: Many roles in cell function[J]. Biochem Biophys Res Commun,2008,369(1):149-156.

[7]Goeckeler ZM, Bridgman PC, Wysolmerski RB. Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity[J]. Am J Physiol Cell Physiol,2008,295(4):C994-C1006.

[8]Turner JR. Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application[J]. Am J Pathol, 2006,169(6):1901-1909.

[9]Shen L, Turner JR. Role of epithelial cells in initiation and propagation of intestinal inflammation. Eliminating the static: tight junction dynamics exposed[J]. Am J physiol Gastrointest Liver Physiol, 2006,290(4):G577-G582.

[10]Shen L, Black ED, Witkowski ED, et al. Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure[J]. J Cell Sci,2006,119(Pt 10):2095-2106.

[11]Clayburgh DR, Barrett TA, Tang Y, et al. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo[J]. J Clin Invest, 2005,115(10):2702-2715.

[12]Moriez R, Salvador-Cartier C, Theodorou V, et al. Myosin light chain kinase is involved in lipopolysaccharide-induced disruption of colonic epithelial barrier and bacterial translocation in rats[J]. Am J Pathol, 2005,167(4):1071-1079.

[13]Ye D, Ma TY. Cellular and molecular mechanisms that mediate basal and tumour necrosis factor-alpha-induced regulation of myosin light chain kinase gene activity[J]. J Cell Mol Med, 2008,12(4):1331-1346.

[14]Gilbert S, Zhang R, Denson L, et al. Enterocyte STAT5 promotes mucosal wound healing via suppression of myosin light chain kinase-mediated loss of barrier function and inflammation[J]. EMBO Mol Med,2012,4(2):109-124.

[15]Chen ML, Ge Z, Fox JG, et al. Disruption of tight junctions and induction of proinflammatory cytokine responses in colonic epithelial cells by Campylobacter jejuni[J]. Infect Immun, 2006,74(12):6581-6589.

[16]Turner JR. Intestinal mucosal barrier function in health and disease[J]. Nat Rev Immunol, 2009,9(11):799-809.

[17]張興文, 王湘英, 盧義展, 等. 清胰湯對(duì)急性出血壞死性胰腺炎大鼠腸屏障功能障礙的保護(hù)作用[J]. 中國(guó)現(xiàn)代醫(yī)學(xué)雜志,2011,21(11):1339-1342.

[18]Goto T, Matsubara T, Yoshizawa Y, et al. Diamine oxidase as blood biomarker in rats and humans to GI tract toxicity of fluorouracil anti-cancer drugs[J]. Gan To Kagaku Ryoho, 2011,38(5):765-769.

(本文編輯：呂芳萍)