同型半胱氨酸调控MEK-ERK-MLCK通路影响结肠炎大鼠肠黏膜通透性的实验研究

丁少桢，丁　浩，梅　俏，刘晓昌，胡　静，胡咏梅，许建明

(安徽医科大学第一附属医院消化内科，安徽 合肥　230022)



同型半胱氨酸调控MEK-ERK-MLCK通路影响结肠炎大鼠肠黏膜通透性的实验研究

丁少桢，丁浩，梅俏，刘晓昌，胡静，胡咏梅，许建明

(安徽医科大学第一附属医院消化内科，安徽 合肥230022)

摘要：目的探讨同型半胱氨酸(homocysteine, Hcy)是否通过调控MEK-ERK-MLCK通路影响结肠炎大鼠肠黏膜通透性的机制。方法SD大鼠分为4组，A组为正常对照组(NS皮下注射+NS灌肠)，B组为正常对照+Hcy注射组(Hcy皮下注射+NS灌肠)，C组为TNBS模型组(NS皮下注射+TNBS灌肠)，D组为TNBS模型+Hcy注射组(Hcy皮下注射+TNBS灌肠)。建立高Hcy血症的实验性结肠炎大鼠模型，实验结束时取大鼠结肠组织病理学检查，并进行结肠匀浆检测MPO活性，采用Western blot方法检测大鼠小肠组织中MEK、ERK、p-ERK、MLCK、p-MLCK的蛋白表达水平，采用RT-qPCR方法检测大鼠小肠组织中MLCK mRNA表达。结果与正常对照组及模型对照组相比，TNBS模型+Hcy皮下注射组大鼠DAI及HI评分增高，结肠匀浆MPO活性增高，小肠黏膜组织MEK、ERK、p-ERK、MLCK、p-MLCK蛋白表达水平增加，MLCK mRNA相对表达量增加。结论Hcy增加实验性结肠炎大鼠肠黏膜通透性，可能与调控MEK-ERK-MLCK信号通路有关。

关键词：同型半胱氨酸；炎症性肠病；肠黏膜通透性；紧密连接；肌球蛋白轻链激酶；细胞外信号调节激酶

肠黏膜通透性增加在炎症性肠病(inflammatory bowel disease，IBD)的病理生理过程中具有重要作用[1]。紧密连接(tight junction，TJ)是构成肠黏膜上皮细胞机械屏障的关键结构[2]，TJ损伤引起肠黏膜屏障破坏，导致肠黏膜通透性增加，参与IBD的病理生理过程。TJ的结构蛋白主要由跨膜蛋白家族(包括Claudin及Occludin)和外周支架蛋白ZO构成。研究发现，肌球蛋白轻链激酶(myosin light chain kinase，MLCK)通过调节Claudin、Occludin及ZO蛋白表达，可以引起肠黏膜通透性增加[3]，MLCK在TJ通透性动态调节过程中发挥重要作用[4]。调控MLCK的信号通路主要包括ERK/MAPK信号转导通路，MEK激活ERK引起ERK磷酸化，ERK1/2通路激活导致Elk-1的激活与磷酸化，激活的Elk-1迁移到细胞核内，与MLCK结合并激活MLCK[5]。

同型半胱氨酸(homocysteine，Hcy)是一种含硫氨基酸，是体内蛋氨酸代谢的重要中间产物，IBD患者血浆和结肠黏膜组织中Hcy含量明显增高[6-7]。研究发现，Hcy通过促进基质金属蛋白酶(MMPs)合成，破坏内皮细胞屏障，增加内皮细胞通透性，这一过程与调控ERK-MLCK通路有关[8]。但Hcy是否通过调控MEK-ERK-MLCK通路影响肠上皮细胞紧密连接结构及功能尚不明确。因此，本研究拟在建立大鼠TNBS/乙醇结肠炎模型的基础上，探讨Hcy是否通过调控MEK-ERK-MLCK信号通路影响肠黏膜通透性的机制。

1材料与方法

1.1实验动物和试剂SD大鼠，SPF级，♂，体质量(200±20) g，由安徽省动物中心提供，实验动物许可证号：34000200000480，在室温、光照周期12 h：12 h条件下适应性饲养1周使用。TNBS(031 M5021)、DL-同型半胱氨酸(121M39044)均购自Sigma公司。

1.2实验方法

1.2.1实验分组实验大鼠分为4组，每组8只。A组(正常对照组)：生理盐水灌肠+生理盐水皮下注射；B组(正常对照+Hcy注射组)：生理盐水灌肠+Hcy皮下注射；C组(TNBS模型组)：TNBS/乙醇溶液灌肠+生理盐水皮下注射；D组(TNBS模型+Hcy注射组)：TNBS/乙醇溶液灌肠+ Hcy皮下注射。

1.2.2模型制备和给药方法参照文献方法[9]，10%水合氯醛腹腔注射麻醉，用橡胶管经肛门插入大鼠结肠内8 cm左右，注入以等体积乙醇溶解的TNBS(100 mg·kg-1)溶液。对照组灌肠等体积生理盐水。DL-Hcy溶于NS，滴定pH至7.4，参照文献剂量[10]自TNBS模型制备后d1起皮下注射Hcy(0.03 umol·g-1)，每天2次，间隔8 h，连续30 d。对照组皮下注射等体积生理盐水。

1.2.3标本采集10%水合氯醛腹腔注射麻醉，分别切取远端结肠及末段小肠约8 cm，沿纵轴剪开肠管，冰生理盐水冲洗干净后取部分结肠及小肠进行检测。

1.2.4结肠炎症评价实验过程中每日观察大鼠体重变化和大便性状，每日观察或用粪便隐血试纸检测大便带血情况，按文献方法[11]行疾病活动指数(disease activity index，DAI)评分。实验结束后于全麻下处死大鼠，取远端结肠甲醛固定，石蜡包埋后行HE染色，按文献方法[12]行组织学评分(histological index, HI)。

1.3检测方法

1.3.1结肠匀浆MPO活性检测取结肠组织制备10%结肠组织匀浆，采用四甲基联苯胺法[13]检测结肠MPO活性。

1.3.2大鼠结肠匀浆MLCK及Hcy水平检测ELISA法检测大鼠结肠匀浆MLCK，Hcy水平。

1.3.3Western blot法检测大鼠小肠黏膜组织MLCK、p-MLCK、MEK、ERK、p-ERK蛋白表达取小肠黏膜组织，加入RIPA细胞裂解液进行裂解，离心后收集上清液。加入等量的2X SDS-PAGE蛋白上样缓冲液，沸水加热10 min，上样，电泳，用电转印法将电泳条带转移到聚氟乙烯(PVDF)膜上，加入Western封闭液(5%脱脂奶粉)，封闭PVDF膜非特异抗原，依次加一抗、二抗孵育，ECL试剂盒曝光和显影。

1.3.4RT-qPCR法检测大鼠小肠黏膜组织MLCK mRNA表达提取小肠黏膜组织mRNA、去除基因组DNA、合成cDNA后，按试剂盒说明书进行加样：95℃ 5 min，95℃ 10 s，60℃ 30 s退火，72℃延伸，40个循环。分析方法为：relative quantification study，分析指标为：2^-ΔΔCt。MLCK引物序列：Forward primer: 5′-CCTGAGGAGAACAAGGAGCA-3′;Reverse primer, 5′- TTCTGGCTCTCTTCCCACAG-3′，产物长度113 bp。

2结果

2.1Hcy对结肠炎大鼠结肠炎症损伤的影响结肠炎模型组大鼠出现稀便或肉眼血便等症状以及体重下降。结肠组织病理提示结肠黏膜及黏膜下层炎细胞浸润，局部水肿、糜烂，并伴有溃疡形成。与正常对照组及TNBS模型对照组比较，TNBS模型+Hcy皮下注射组大鼠体重明显减轻，DAI及HI评分升高，结肠匀浆MPO活性升高，同时结肠匀浆中Hcy水平明显增高，提示Hcy加重结肠炎症病理损伤(Fig 1, Tab1)。

2.2Hcy对结肠炎大鼠小肠黏膜组织中MLCK、p-MLCK蛋白及mRNA表达的影响与正常对照组比较，TNBS模型对照组大鼠小肠黏膜上皮细胞MLCK水平升高，MLCK和p-MLCK蛋白表达增加。与TNBS模型对照组相比，TNBS模型+Hcy注射组大鼠小肠黏膜上皮细胞MLCK水平，MLCK和p-MLCK蛋白表达明显增加(P<0.05)，提示Hcy可能参与影响了MLCK磷酸化过程(Tab2,Fig 2,Tab3)。

A: Normal group; B: Normal+Hcy group, C: TNBS/ethanol group, D: TNBS/ethanol+Hcy group

A: Normal group; B: Normal+Hcy group, C: TNBS/ethanol group, D: TNBS/ethanol+Hcy group.**P<0.01vsGroup A;##P<0.01vsGroup C

Note see Tab1

A: Normal group; B: Normal+Hcy group, C: TNBS/ethanol group, D: TNBS/ethanol+Hcy group

与正常对照组比较，TNBS模型对照组大鼠小肠黏膜上皮细胞MLCK mRNA表达增加。与TNBS模型对照组相比，TNBS模型+Hcy注射组大鼠小肠黏膜上皮细胞MLCK mRNA明显增加(P<0.05)(Fig 3)。

2.3Hcy对结肠炎大鼠小肠黏膜组织中MEK、ERK、p-ERK蛋白表达的影响与正常对照组相比，TNBS模型对照组大鼠小肠黏膜组织MEK、ERK、p-ERK蛋白表达增加。与TNBS模型对照组相比，TNBS模型+Hcy注射组大鼠小肠黏膜上皮细胞MEK、ERK、p-ERK表达明显增加，提示Hcy可能参与了MEK-ERK-MLCK蛋白磷酸化调控过程(Fig 4，Tab3)。

3讨论

IBD的发病机制目前尚不明确，肠黏膜通透性降低，引起肠黏膜屏障功能损害在IBD的病理生理过程中具有重要作用[14]。肠黏膜屏障损害可加重肠黏膜炎症和免疫反应，被认为是感染和免疫因素启动肠道炎症的关键环节。由肠黏膜上皮细胞、细胞间紧密连接等组成的机械屏障是肠黏膜屏障的重要结构。研究发现，皮下注射Hcy的结肠炎大鼠小肠组织EB含量明显增高，小肠黏膜上皮细胞间隙明显增宽，并且伴有紧密连接结构的破坏，抑制Claudin-1、Occludin和ZO-1表达，提示Hcy可能损伤小肠黏膜上皮细胞连接，增加肠黏膜通透性，进一步加重肠道炎症损伤过程。

A: Normal group; B: Normal+Hcy group, C: TNBS/ethanol group, D: TNBS/ethanol+Hcy group.*P<0.05vsTNBS control group after one-way ANOVA

A: Normal group; B: Normal+Hcy group, C: TNBS/ethanol group, D: TNBS/ethanol+Hcy group

MLCK介导的肌球蛋白轻链(myosin light chain，MLC)磷酸化信号通路是调控紧密连接功能的重要信号通路[4,15]。MLCK是一种Ca2+/钙调蛋白(calmodulin, Cam)依赖的蛋白激酶。Ca2+/CaM复合体能够与MLCK结合，解除MLCK的天然抑制序列，形成激活的p-MLCK[16-17]。p-MLCK能够使MLC的Ser19和Thr18发生磷酸化[18]，MLC空间构象发生改变[19]，促进肌动蛋白与肌球蛋白丝收缩，使上皮细胞TJ开放，调节上皮细胞黏膜通透性[20]。因此，MLCK在肠黏膜上皮细胞TJ损伤与修复过程中起重要作用。研究表明，MLCK基因过表达的小鼠肺血管上皮通透性增强[21]。MLCK特异性抑制剂能够降低细胞内p-MLC水平，恢复肠黏膜屏障功能[22-24]葡聚糖硫酸钠结肠炎小鼠肠黏膜中MLCK活性增高，导致肠黏膜通透性增加，加重了肠道炎症损伤[25]。ERK可以调控MLCK磷酸化，如MLCK激活引起内皮细胞收缩，细胞间隙增宽，脂质进入内皮下层，加速动脉粥样硬化改变[26]。

A: Normal group; B: Normal+Hcy group, C: TNBS/ethanol group, D: TNBS/ethanol+Hcy group.*P<0.05vsGroup C.

Hcy通过促进TNF-α、IL-6、IFN- γ等炎症因子表达[27]，激活NADPH、p38MAPK[28-29]等途径引起氧化损伤，是多种心脑血管疾病的独立危险因素[30]。研究发现，Hcy可通过ERK-MLCK信号调控通路促进MMP-9合成，参与内皮细胞通透性下调过程，引起脑血管重塑。

既往研究表明，IBD病人血浆以及肠黏膜组织Hcy水平明显增高，提示Hcy可能影响IBD患者肠黏膜通透性[31]。本研究发现，Hcy增加实验性结肠炎大鼠小肠组织中MLCK、p-MLCK、ERK、MEK、p-ERK的mRNA和蛋白表达，提示Hcy可能通过促进MEK-ERK-MLCK蛋白磷酸化过程，损伤肠上皮细胞紧密连接，引起肠黏膜通透性增加，加重肠道炎症过程，为阐明IBD中Hcy影响肠黏膜通透性的作用机制提供实验依据。

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Effect of homocysteine on the intestinal permeability by regulating MEK-ERK-MLCK signal transduction in experimental colitis rats

DING Shao-zhen, DING Hao, MEI Qiao, LIU Xiao-chang, HU Jing, HU Yong-mei, XU Jian-ming

(DeptofGastroenterology,theFirstAffiliatedHospitalofAnhuiMedicalUniversity,theKeyLaboratoryofDigestiveDiseasesofAnhuiProvince,Hefei230022,China)

Key words:homocysteine; inflammatory bowel disease; permeability; tight junction; myosin light chain kinase; extracellular regulated protein kinase

Abstract:AimTo investigate whether Hcy influenced the intestinal mucosal permeability by regulating MEK-ERK-MLCK pathway.MethodsSD rats were divided into 4 groups: normal group, normal+Hcy group, TNBS/ethanol group, TNBS/ethanol+Hcy group.Experimental colitis model with hyperhomocystinemia was established in rats with intracolonic administration of TNBS and subcutaneous injection of Hcy. The colonic mucosal tissue was collected for histopathological examination and activity of myeloperoxidase(MPO). The protein expression of MLCK, p-MLCK, MEK, ERK and p-ERK in intestinal mucosal tissues was examined by Western blot method. The mRNA expression of MLCK was examined by RT-qPCR method.ResultCompared with the normal group and TNBS group, the DAI and HI scores and the MPO activity were increased in TNBS/ethanol+Hcy group(P<0.01). Western blot and RT-qPCR showed that expression of MLCK, p-MLCK, MEK, ERK and p-ERK increased in small intestine in TNBS/ethanol+Hcy group. ConclusionHcy can increase intestinal permeability in TNBS-induced colitis rats by regulating the expression of MEK-ERK-MLCK signal pathway.

收稿日期:2015-11-30修回日期：2016-02-08

基金项目：国家自然科学基金资助项目(No 81470809)；杨森科学研究委员会中国分会研究基金(No 2012JRCC 消化 02)

作者简介：丁少桢(1991-)，男，硕士，研究方向：炎症性肠病，E-mail:magthoridon@sina.com; 梅俏(1971-)，男，博士，副教授，研究方向:炎症性肠病，通讯作者，E-mail:meiqiao@hotmail.com

doi:10.3969/j.issn.1001-1978.2016.04.012

文献标志码：A

文章编号：1001-1978(2016)04-0498-05

中国图书分类号：R-322; R322.45；R345.57；R574.62；R977.3；R977.4

网络出版时间：2016-3-18 11:22网络出版地址：http://www.cnki.net/kcms/detail/34.1086.R.20160318.1122.024.html