修回日期: 2010-12-30
接受日期: 2011-01-21
在线出版日期: 2011-02-18
目的: 探讨c-Jun氨基末端激酶(JNK)选择性抑制剂SP600125对葡聚糖硫酸钠(DSS)诱导的急性期溃疡性结肠炎小鼠结肠组织内肿瘤坏死因(TNF)α的表达情况及血清白介素(IL)-6含量的影响.
方法: 40只♂C57BL/6小鼠随机分成5组, 每组8只. 正常对照组(A组)、DSS模型组(B组)、SP600125低剂量组(C组)、SP600125高剂量组(D组)、DMSO载体组(E组). A组小鼠饮用蒸馏水7 d; B-E组小鼠饮用3% DSS水溶液7 d. C组和D组小鼠分别以低、高剂量SP600125腹腔内注射, E组只给SP600125的载体DMSO, 给药方式同前. 造模及干预7 d后处死小鼠, 观察指标包括: 疾病活动指数(disease active index, DAI)、组织学损伤的评估(histological index, HI); 免疫组织化学法检测各组结肠黏膜p-JNK和TNF-α的变化, ELISA检测血清IL-6的水平.
结果: 造模第8天, B与E组之间的各项检测指标的差异均无统计学意义(均P>0.05); B组的DAI、HI都明显高于A组(8.00±1.41 vs 0.25±0.16; 5.88±0.99 vs 0.50±0.93, 均P<0.01), C组的DAI与B组相比无显著性差异(P>0.05), D组的DAI明显低于B组(4.38±1.51 vs 8.00±1.41, P<0.01), C、D组的HI都明显低于B组(3.88±1.46 vs 5.88±0.99; 2.63±0.74 vs 5.88±0.99, 均P<0.01); B组结肠黏膜的p-JNK、TNF-α及血清IL-6都明显高于A组(99.01±10.75 vs 116.41±10.46; 103.39±11.09 vs 120.24±10.67; 183.34±25.87 vs 75.91±20.27, 均P<0.01), 各项在C、D组中的水平都明显低于B组(105.94±10.93 vs 99.01±10.75; 110.21±11.05 vs 103.39±11.09; 140.37±32.07 vs 183.34±25.87; 114.52±11.06 vs 99.01±10.75; 117.87±11.00 vs 103.39±11.09; 108.61±20.34 vs 183.34±25.87, 均P<0.01).
结论: SP600125通过下调p-JNK表达, 降低小鼠结肠黏膜促炎因子TNF-α表达及血清IL-6的含量而发挥其干预作用.
引文著录: 赵翠娟, 王承党. c-Jun氨基末端激酶选择性抑制剂对葡聚糖硫酸钠诱导的小鼠结肠炎的影响. 世界华人消化杂志 2011; 19(5): 515-518
Revised: December 30, 2010
Accepted: January 21, 2011
Published online: February 18, 2011
AIM: To evaluate the influence of SP600125, a selective c-Jun N-terminal kinase (JNK) inhibitor, on the levels of tumor necrosis factor-α (TNF-α) in the intestinal mucosa and interleukin-6 (IL-6) in the serum of mice with dextran sulfate sodium (DSS)-induced ulcerative colitis (UC).
METHODS: Forty male C57BL/6 mice were randomly assigned to five groups: normal control group, model control group, low-dose SP600125 group, large-dose SP600125 group, and DMSO group. Except that the normal control group was given distilled water, animals of other groups were given 3% DSS in drinking water for 7 days to induce acute intestinal inflammation. Mice of the low- and large-dose SP600125 groups also received intraperitoneal injection of SP600125, while the DMSO group received DMSO. All animals were sacrificed on day 8 after treatment. The disease activity index (DAI) and histological index (HI) were calculated, the expression of p-JNK and TNF-α in the intestinal mucosa was measured by immunohistochemistry, and the level of IL-6 in the serum was measured by ELISA.
RESULTS: On day 8 after DSS exposure, DAI and HI showed no significant difference between the model control group and DMSO group E (both P > 0.05) but were significantly higher in the model control group than in the normal control group (8.00 ± 1.41 vs 0.25 ± 0.16; 5.88 ± 0.99 vs 0.50 ± 0.93, both P < 0.01). Although there was no significant difference in DAI between the low-dose SP600125 group and model control group (P > 0.05), DAI was significantly lower in the large-dose SP600125 group than in the model control group (4.38 ± 1.51 vs 8.00 ± 1.41, P < 0.01). HI in the two SP600125 groups were significantly lower than those in the model control group (3.88 ± 1.46 vs 5.88 ± 0.99; 2.63 ± 0.74 vs 5.88 ± 0.99, both P < 0.01). The levels of p-JNK and TNF-α in the intestinal mucosa and that of IL-6 in the serum were significantly higher in the model control than in normal control group (99.01 ± 10.75 vs 116.41 ± 10.46; 103.39 ± 11.09 vs 120.24 ± 10.67; 183.34 ± 25.87 vs 75.91 ± 20.27, all P < 0.01), but were significantly lower in the two SP600125 groups than in the model control group (105.94 ± 10.93 vs 99.01 ± 10.75; 110.21 ± 11.05 vs 103.39 ± 11.09; 140.37 ± 32.07 vs 183.34 ± 25.87; 114.52 ± 11.06 vs 99.01 ± 10.75; 117.87 ± 11.00 vs 103.39 ± 11.09; 108.61 ± 20.34 vs 183.34 ± 25.87, all P < 0.01).
CONCLUSION: Treatment with SP600125 can down-regulate the expression of p-JNK and TNF-α in the intestinal mucosa and IL-6 in the serum of mice with experimental UC.
- Citation: Zhao CJ, Wang CD. Treatment with SP600125, a selective JNK inhibitor, down-regulates the levels of TNF-α in the intestinal mucosa and IL-6 in the serum of mice with DSS-induced ulcerative colitis. Shijie Huaren Xiaohua Zazhi 2011; 19(5): 515-518
- URL: https://www.wjgnet.com/1009-3079/full/v19/i5/515.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v19.i5.515
溃疡性结肠炎(ulcerative colitis, UC)是以结肠黏膜溃疡为主的慢性非特异性炎性肠病, 异常免疫应答反应是其主要的病理生理学机制, 而细胞因子是介导活化的免疫细胞和非免疫细胞间相互作用的必需因子[1]. c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)被磷酸化后可以使许多参与炎症反应的细胞因子的基因表达[2], 且在调节Thl/Th2免疫细胞的分化中的起到重要作用[3], 可能是UC发病过程的重要环节[4]. 本研究采用JNK选择性抑制剂SP600125抑制JNK的活化, 并检测小鼠结肠黏膜内的P-JNK以及促炎因子肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)和血清白介素-6(interferon-6, IL-6)的改变情况, 探讨SP600125对UC的干预作用及其可能机制.
♂C57BL/6小鼠40只(体质量l8-22 g)购自上海斯莱克实验动物有限责任公司. 葡聚糖硫酸钠(dextran sodium sulphate, DSS)为美国MP Biomedicals公司产品; c-Jun氨基末端激酶选择性抑制剂SP600125为Sigma公司产品; p-JNK小鼠抗小鼠单克隆抗体(sc-6254)为美国Santa公司产品; TNF-α兔抗小鼠多克隆抗体(PR-0078)为美国Proteintech pTGLAB公司产品. 非生物素二步法免疫组织化学检测试剂盒(抗鼠、抗兔)、小鼠IL-6 ELISA试剂盒等为北京中杉公司产品.
将小鼠按照体质量随机分为5组: 正常对照组(A组)、DSS模型组(B组)、低剂量SP组(C组)、高剂量SP组(D组)、DMSO组(E组). B-E组小鼠连续自由饮用3%DSS溶液7 d, A组小鼠自由饮用蒸馏水. 在给予3%DSS之前2 h开始, C组和D组小鼠腹腔内注射SP600125溶液(剂量分别为10 mg/kg、20 mg/kg), A组和B组腹腔内注射0.9%氯化钠溶液, E组腹腔内注射DMSO. 第7天时处死小鼠, 取心脏血分离血清, ELISA法测定血清IL-6浓度; 参照Murano等[5]方法获得疾病活动指数(disease active index, DAI); 在结肠病变最严重处取组织标本作冰冻切片, 进行p-JNK和TNF-α免疫组织化学染色, 按说明书程序进行, 设阴性对照; 并取组织作常规石蜡包埋、切片、HE染色, 参照Dieleman等[6]方法计算组织学损伤的评估(histological index, HI).
统计学处理 数据以mean±SD表示, Levene法方差齐性检验, 然后行单因素方差分析(ANOVA), 组间两两比较采用LSD-t(方差齐)或Tamhane's T2(方差不齐)法. 设α = 0.05, 以P<0.05为有显著性意义. 采用SPSS13.0统计软件.
实验期间无动物死亡. A组动物结肠黏膜正常; B组和E组黏膜结构损伤明显, 结肠黏膜不完整, 广泛黏膜糜烂、隐窝炎及脓肿形成, 大部分腺体被破坏, 腺管排列紊乱, 大量中性粒细胞浸润; C组和D组动物结肠黏膜改变相对较轻. A组-E组的DAI差异有显著性意义(P = 0.000), 其中B-E组高于A组(均P<0.01), C、D组低于B组(均P<0.01), 而B组与E组间(P = 1.000)、C组与D组间(P = 0.865)的差异无统计学意义(表1). A组-B组的HI差异有统计学意义(P = 0.000), 其中B组-E组HI均高于A组(均P<0.01), C组和D组的HI均低于B组(均P<0.01), 而B组与E组间(P = 0.805)、C组与D组间的差异无统计学意义(P = 0.389, 表1).
A组-E组动物的血清IL-6浓度分别是: 75.91 μg/L±20.27 μg/L、183.34 μg/L±25.87 μg/L、140.37 μg/L±32.07 μg/L、108.61 μg/L±20.34 μg/L、182.40 μg/L±33.42 μg/L, 各组间差异有显著性意义(P = 0.000), 其中B组和E组的IL-6浓度分别高于A组(均P = 0.000), C组和D组分别低于B组(P = 0.003, 0.000), 而B组和E组间、C组和D组间的差异无统计学意义(P = 0.945, 0.679).
阳性染色均为细胞质或细胞膜棕黄色染色, 用JD801形态学显微图像分析系统计算灰度值(IA), IA越高则表示其表达水平越低. A组-E组间的p-JNK的平均IA差异有统计学意义(P = 0.000). B组和E组的p-JNK的平均IA低于A组(均P = 0.000); C组和D组的平均IA则高于B组(均P = 0.000); A组、C组和D组3组之间, 以及B组与E组间的IA差异无统计学差异(P = 0.897, 0.963). 各组结肠黏膜TNF-α的IA变化与p-JNK的变化相一致(表2).
本实验发现, 与A组相比, B组和E组的小鼠结肠黏膜内p-JNK的表达显著增高, 同时相应部位TNF-α表达及血清内IL-6浓度均明显增高; SP600125可以降低结肠黏膜内p-JNK的表达, 同时使结肠高表达的TNF-α及血清内浓度增高的IL-6都显著降低, DAI和病理炎症减轻.
大量实验证明细胞因子的调控和释放与JNK信号传导通路密切相关, 许多种胞外刺激因素磷酸化和活化JNK, 包括脂多糖(lipopolysaccharides, LPS)、细胞因子和生长因子等, 活化后的JNK可以磷酸化c-Jun、c-fos、ATF2和ATF3等转录因子, 进而可以使许多参与炎症反应的细胞因子的基因表达, 如TNF-α、IL-1、IL-6、IL-8等[2,7]. Swantek等[8]研究发现利用JNK缺失突变手段可以阻止TNF-α的翻译过程; 同样利用SP600125干预CD14+细胞后出现TNF-α mRNA半衰期明显缩短; 另外, 在细胞实验中, SP600125抑制JNK下游转录因子c-Jun的磷酸化后, 基因表达受到抑制的细胞因子还有COX-2、IFN-γ、ICAM-1、IL-1β、IL-2、IL-6和IL-8等[9,10].
最近不断有研究表明JNK及其下游蛋白在一些慢性炎症情况中起到重要作用, 例如类风湿性关节炎[11], 也有研究证明在UC患者以及DSS诱导的SD大鼠结肠炎模型的炎症性结肠组织内出现JNK的显著活化, 而且SP600125对于体外培养的UC患者结肠组织细胞及粒细胞中炎症因子的产生明显的抑制作用[12].
细胞因子可以促进细胞间的相互作用, 刺激抗原特异效应细胞的增殖, 介导局部和系统炎症反应, 是UC在对外界刺激的免疫性炎症反应中一个重要因素[13]. UC患者肠道黏膜中大量分泌的TNF-α, 能激活多种免疫和炎症细胞而促进炎症形成; 还可以促进其他细胞因子的释放, 形成细胞因子网络, 扩大炎症连锁反应, 诱导结肠上皮细胞广泛凋亡、坏死脱落[14]; 血清及肠道黏膜明显增高的IL-6与病变范围和病变严重程度呈正相关[15]. 本次实验中使用DSS建立的小鼠结肠炎模型中也出现结肠黏膜内高表达的TNF-α以及血清IL-6浓度增高的表现. 而在使用SP600125, 一种选择性JNK抑制剂, 其显著降低小鼠结肠炎模型的结肠黏膜内p-JNK的表达后, 结肠黏膜内高表达的TNF-α以及血清内IL-6的表达都显著降低. 这表明小鼠结肠炎模型的肠黏膜组织中存在JNK的激活, 阻断其可以减少细胞因子的产生.
总之, SP600125可以缓解DSS致急性UC小鼠模型肠黏膜组织病理炎性改变以及明显降低小鼠的疾病活动度, 其机制可能与SP600125组断JNK的活化后, 从上游抑制了促炎因子TNF-α、IL-6的表达, 说明JNK通过调控和释放促炎因子在UC的发病中起着重要的作用. 而该信号传导通路在中其他成员在UC中的作用和地位将是我们进一步研究的.
溃疡性结肠炎(UC)主要是以侵及结肠黏膜且以溃疡为主的慢性非特异性炎症性疾病. 多数学者认为, UC是由环境因素和微生物因素之间复杂地相互作用于遗传易感人群, 引起肠道免疫反应过度亢进, 导致结肠黏膜损伤所致. 抑制促炎症性细胞因子的产生已成为目前治疗UC的一条新思路.
张筱茵, 副教授, 中国人民解放军第四军医大学西京医院消化疾病研究所
Swantek等研究发现利用JNK缺失突变手段可以阻止TNF-α的翻译过程.
本文利用DSS成功建立UC模型后, 既检测了黏膜组织中的促炎因子(TNF-α), 也检测了血清中的促炎症因子(IL-6), 并早期应用JNK选择性抑制剂SP600125干预小鼠JNK的活化, 检测小鼠结肠黏膜内的P-JNK以及促炎细胞因子TNF-α和血清IL-6 的改变情况.
JNK可能通过增加TNF-α、IL-6的表达而参与UC的发病过程, 早期应用JNK抑制剂SP600125可以通过对TNF-α、IL-6表达的抑制作用, 进而改善急性UC模型小鼠结肠组织损伤程度及临床表现, 因此JNK抑制剂SP600125具有潜在的临床应用价值.
JNKs: MAPK超家族成员之一, 属于进化上保守的丝氨酸/苏氨酸蛋白激酶, 也称作应激活化蛋白激酶(SAPKs).
本文设计合理, 具有一定的临床借鉴意义.
编辑:李军亮 电编:李薇
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