姜黄素对肠黏膜屏障保护作用的实验研究
|
摘要
背景与目的
肠黏膜屏障功能是指正常肠道具有较为完善的功能隔离带,可防止致病性病原体及毒素的侵入。在严重创伤、大面积烧伤、出血性休克、手术、放化疗、长期应用广谱抗生素或免疫抑制剂、营养不良、重症胰腺炎等应激状态下或长期进行肠外营养的情况下,肠道的结构和功能可能受到严重损害,导致肠道屏障功能障碍,进而引起肠道细菌易位,甚至诱发全身炎性反应综合征(SIRS),进而导致多器官功能衰竭(MODS)而危及生命。如何及时正确评估肠道屏障功能对于判断患者的病情、估计其预后、并及时给予综合治疗具有非常重要的意义。但目前直接观察肠道屏障功能仍较困难,多通过间接的方法进行监测。
D-乳酸是胃肠道固有细菌的代谢终产物,肠道多种细菌均可产生。哺乳动物体内不具备将其快速代谢分解的酶系统。当肠黏膜生物屏障受损时,其通透性增加,肠道中细菌产生的D-乳酸通过受损肠黏膜经循环入血,由于哺乳动物没有D-乳酸脱氢酶,此时可见血中D-乳酸水平升高。因此检测其外周血水平即可反映肠黏膜损害程度和通透性的变化。二胺氧化酶(DAO)是人类和所有哺乳动物肠黏膜上层绒毛细胞胞浆中具有高度活性的细胞内酶,当肠黏膜上皮细胞受损、坏死后该酶释放入血,或随坏死脱落的肠黏膜细胞进入肠腔内,导致血浆和肠腔内DAO活性增高而肠黏膜DAO活性降低。由于DAO在外周血中活性稳定,因而可通过无创测定其在外周血中的变化,间接反映肠黏膜状态,但它主要反映的是肠黏膜上皮损伤与修复情况,而不是肠道通透性的改变。
白细胞的过度激活是炎性病理过程中的一个重要环节。白细胞是否停滞并以浸润方式向血管外渗依赖于炎症部位白细胞和内皮细胞膜表面的黏附分子(intercellular adhesion molecule,ICAM)的表达与功能。ICAM是指由细胞合成并组装于细胞表面或分泌至细胞外基质的可促进细胞与细胞或细胞与细胞外基质间黏附的一类糖蛋白。其中ICAM-1属于免疫球蛋白超家族,在炎性肠病发病中的作用近年来受到关注。正常肠组织ICAM-1通常低水平表达于血管内皮细胞、肠黏膜固有层和淋巴结中的单核巨噬细胞。髓过氧化物酶(MPO)是主要存在于中性粒细胞中的一种酶,其活性高低反应了中性粒细胞浸润程度。
超氧化物歧化酶(SOD)是内源性超氧阴离子自由基清除剂,其活性高低可部分反映机体清除自由基的能力。
现在研究发现,肠道损伤所涉及的缺血、炎症、凋亡等多个病理机制与丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPKs)信号道路的调节有关,p38在炎性反应起重要作用。
目前,对肠黏膜屏障功能的保护尚缺乏令人满意的有效手段,因此寻找自然界存在的对人类疾病有治疗作用的药物已成为医学界和药学界共同关注的课题。姜黄素是一种天然低毒的药用植物,被世界卫生组织/食品管理局批准为天然食品添加剂。国内外许多文献相继报道姜黄素具有抗感染、抗炎、抗氧化、清除自由基等作用。最近的研究表明:姜黄素能通过抑制NF-κB、p38的活性,减轻实验性大鼠结肠炎,证明姜黄素通过抑制NF-κB的活性而增加TNF-a诱导的Caco-2细胞的紧密连接;姜黄素通过抑制p38、JNK和NF-κB活性,减轻志贺样毒素与TNF-a诱导的HT29细胞株的结合,减少细胞的死亡。
本课题拟通过建立大鼠小肠炎模型及LPS刺激IEC-6细胞的应激反应,观察姜黄素对肠黏膜屏障的保护作用以及对MAPK信号传导通路、MKP-1、核转录因子NF-κB、炎症介质和化学介质的影响,探讨姜黄素对肠黏膜作用的分子机制。
材料与方法
一、姜黄素对大鼠肠黏膜的保护作用及机制的研究
采用腹腔内注射氨甲蝶呤(MTX)的方法制备SD大鼠小肠炎模型。将60只大鼠随机分为正常对照组(腹腔内注射生理盐水)、模型组(MTX,20mg/kg)、姜黄素组(curcumin 100mg/kg)和N-乙酰半胱氨酸组(NAC,150mg/kg),制模当天开始,每日按剂量灌胃一次,正常对照组和模型组灌生理盐水。制模过程中每天观察大鼠腹泻和肉眼血便情况,在实验第6d处死大鼠,取部分肠段置于多聚甲醛内固定、包埋、切片、HE染色,行一般组织病理学观察,30～50mg肠黏膜提取RNA、肠上皮细胞胞浆蛋白。
1,观察大鼠疾病活动指数(DAI)和小肠黏膜损伤指数(CMDI),光镜下组织学评分(HS)。
2,分光光度法检测血浆D-乳酸和DAO水平。
3,生化法检测大鼠小肠组织MPO和SOD活性。
4,运用ELISA测定大鼠血浆IL-10水平。
5,应用RT-PCR技术对大鼠肠上皮细胞内致炎细胞因子TNF-α、IL-1β、ICAM-1的mRNA表达水平进行检测。
6,Western blot分析胞质内IκB的降解、p38、ERK、JNK蛋白表达。
二、姜黄素对LPS诱导IEC-6细胞应激反应的保护作用及机制
应用大鼠肠上皮细胞株IEC-6细胞,以脂多糖作为刺激因素,将对数生长期的细胞随机分为正常对照组、脂多糖(LPS)组、姜黄素(curcumin pretreat,20μmol/l)预孵组、p38抑制剂SB203580预孵组(SB pretreat,20μmol/l)。正常对照组不加任何处理因素,其它3组均加入LPS,浓度为100ng/ml。姜黄素预孵组和p38抑制剂SB203580预孵组在加入LPS前60min分别加入浓度为20μmmol/l的姜黄素和SB203580。孵育30min后终止。
1,运用ELISA测定细胞上清液IL-10水平。
2,应用RT-PCR技术对IEC-6细胞TNF-α、IL-1β、ICAM-1的mRNA表达水平进行检测。
3,应用NF-κB激活—核转运检测试剂盒检测NF-κB(p65)在细胞核内的分布。
4,Western blot分析胞质内IκB的降解和MKP-1蛋白表达。
结果
一、姜黄素对大鼠肠黏膜的保护作用及机制的研究
1、姜黄素对大鼠粪便性状和肠黏膜组织学的影响
MTX组在腹腔内注射氨甲蝶呤后,第2d即出现腹泻,第3d部分出现肉眼血便,第6d全部出现肉眼血便。curcumin治疗组与NAC组症状无明显差别,于第3d开始出现腹泻、解黄色稀便等现象,但未出现血便,第6d也部分出现肉眼血便。组织切片观察发现:MTX组见黏膜及黏膜下层血管高度扩张充血,肠隐窝形态的丧失、绒毛萎缩、脱落,炎症细胞广泛浸润,呈典型炎症改变;curcumin治疗组与NAC组治疗组炎症明显减轻,肠隐窝形态丧失、绒毛萎缩、脱落现象较模型组明显减轻,炎症细胞浸润少。正常对照组无改变。以上结果说明:姜黄素对SD大鼠小肠炎有明显的治疗作用,对肠黏膜有保护作用,能够防止炎性细胞浸润,减弱炎症反应,减轻肠炎症状。
2、姜黄素对肠黏膜MPO、SOD活性的影响
模型组肠黏膜SOD、MPO活性分别明显高于正常组(P=0.000),姜黄素组和NAC组均可下调MPO活性,增强SOD活性。而且姜黄素可明显增强大鼠肠黏膜组织SOD表达,降低肠黏膜组织MPO活性。说明姜黄素具有抗氧化作用,同时可减轻炎细胞的浸润,对实验性大鼠小肠炎具有保护作用。
3、姜黄素可改善肠黏膜通透性
分光光度法检测血浆D-乳酸和DAO水平。结果显示:与正常组相比,MTX组D-乳酸和DAO水平明显升高(P=0.000)。curcumin组与NAC治疗组D-乳酸、DAO较MTX组有明显下降(P=0.000)。由此可见,姜黄素可以改善肠黏膜的通透性,对大鼠肠黏膜屏障具有保护作用。
4、姜黄素对大鼠肠上皮细胞内细胞炎症因子TNF-α、IL-1β、IL-10及ICAM-1表达的影响
运用ELISA测定大鼠血浆IL-10水平,应用RT-PCR技术对大鼠肠上皮细胞内致炎细胞因子TNF-α、IL-1β、ICAM-1的mRNA表达水平进行检测,结果显示:MTX组TNF-α、IL-1β、ICAM-1的mRNA表达水平均较正常对照组明显增高,Curcumin组与NAC治疗组TNF-α、IL-1β、ICAM-1的mRNA表达水平低于MTX组。但IL-10的表达高于MTX组(p=0.000),curcumin治疗组与NAC组治疗组之间差距无显著性(P=0.43)。上述结果表明:姜黄素对TNF-α、IL-1β、ICAM-1的mRNA表达有明显抑制作用,对IL-10的表达有增强的作用。
5、姜黄素对大鼠肠上皮细胞内IκB、MAPK信号转导通路的影响
实验显示MTX组胞质中IκB明显降解。但姜黄素和NAC组的IκB的降解较MTX组明显减少。MTX组磷酸化p38明显增加,均较正常对照组明显增高(p=0.000),但磷酸化的JNK1/JNK2和ERK1/ERK2蛋白表达无明显改变。结果表明:在MTX诱导的大鼠小肠炎肠上皮细胞的炎症反应中,磷酸化p38增加,姜黄素对磷酸化p38有明显的抑制作用(p=0.000)。
二、姜黄素对LPS诱导IEC-6细胞应激反应的保护作用及机制
1、姜黄素对LPS诱导IEC-6细胞MKP-1、IκB、p65的作用
LPS组磷酸化的MKP-1水平均较正常对照组明显增高(p=0.000),姜黄素和SB预孵组明显高于LPS组。LPS组胞质中IκB较正常对照组明显降解(p=0.000),但姜黄素和SB预孵组的IκB明显高于MTX组(p=0.000)。应用NF-κB激活—核转运检测试剂盒NF-κB(p65)在细胞核内的分布,通过免疫荧光染色检测发现:LPS组p65的核转运明显,核内红色荧光明显强于对照组,姜黄素和SB预孵组的红色荧光强于对照组,但弱于LPS组,说明LPS刺激的情况下,p65和IκB解聚,其核定位序列被暴露,从而被转运到细胞核内。但姜黄素能抑制p65的核转运。
2、姜黄素对IEC-6细胞内致炎细胞因子TNF-α、IL-1β,抑炎因子IL-10和ICAM-1 mRNA表达的影响
LPS组TNF-α、IL-1β、ICAM-1的表达水平均较正常对照组明显增高,IL-10明显减少。但姜黄素和SB预孵组的表达较LPS组表达明显减少,IL-10明显增高。上述结果表明:姜黄素对TNF-α、IL-1β、ICAM-1的mRNA表达有明显抑制作用,对IL-10表达有明显上调作用。
结论
一、姜黄素对MTX诱导的大鼠小肠炎肠黏膜的通透性有保护作用。姜黄素具有抗氧化作用,同时可减轻炎细胞的浸润。
二、姜黄素能够减少MTX诱导的大鼠小肠炎与LPS诱导的IEC-6细胞应激反应模型的TNF-α、IL-1β、ICAM-1 mRNA的表达,增强IL-10 mRNA的表达。
三、姜黄素对MTX致大鼠小肠炎模型的肠黏膜上皮NF-κB和p38信号转导通路有抑制作用,可以抑制NF-κB的激活和p38磷酸化。不影响对ERK、JNK蛋白的表达。
四、姜黄素增强了LPS诱导的IEC-6细胞的MKP-1的磷酸化作用,进而抑制了p38活性。并且对NF-κB的活性有抑制作用。
五、姜黄素对肠黏膜起保护作用的机制可能是通过增强MKP-1的磷酸化作用,抑制了NF-κB和p38活性,进而减少致炎细胞因子TNF-α、IL-1βmRNA和ICAM-1表达,增强抑炎因子IL-10的表达来实现的。
Background/Objective
The intestinal barrier functions a shielding band which can prevent the invasion of causative agents and toxin.Under the conditions of wound,opration,chemotherapy, radiotherapy,severe pancreatitis or long-term parenteral nutrition,the structure and the barrier function of intestinal mucous membrane may be seriously damaged,which may cause the intestinal mucosal barrier dysfunction,then causes the intestinal bacteria translocation,even induces systemic inflammation response syndrome (SIRS),at last result in multiple organs dysfunction syndrome(MODS ).But it is still difficult to detect the gut barrier function directly.
The D-lactate,which may be produced by many germs,is metabolic end product of bacteria in gastrointestinal tract.Without any D-lactate dehydrogenase in the body of mammalian,the D-lactate from enterbacterium will go into blood and the level of D-lactate will increase when intestinal mucosa damages or mucosa permeability changes.Therafore,the examination of D-lactate in peripheral blood may reflect the damage and permeability of intestinal mucosa.DAO is a kind of endocellular enzyme with high activitiy,which exists in the plasm of chorionic villi in intestinal stratum supravasculare in human and all other mammals.When intestinal epithelial cells are injured,the release of endocellular DAO increases and to enters the intestinal intercellular space,the lymph vessel and the blood stream,and finally causes DAO in blood plasma to elevate.The activitiy of DAO in peripheral blood is stable.When small intestinal mucosal barrier nonfunction,the intestinal mucosa membrane cell falls off into the enteric cavity,then DAO enters lymph vessel and the blood stream in the intestines intercellular space,causes blood DAO to elevate. The activity of blood DAO may reflect the extent of damage and repair in intestinal tract.Leukocytic's over-activation is a very important bit of the IBD patho process. Leukocytic staying and exosmoseing on inflammation position depend on ICAM,s expressin and function in the surface of WBC and endothelial cell.ICAMS are glycoproteinwhich are synthesized by the cell and then assembled in the cell surface or secrete to the extracellular matrix,involving in cell-cell or cell-extracellular matrix interaction.And ICAM-1,belonging to the immunoglobulin superfamily, received the attention in recent years in the process of IBD.The expression of ICAM-1 in the normal intestine tissue are usually low in the vascular endothelial cell,in the intestines mucosa lamina propria and in the monocyte-macrophages in the lymph nodes.Myeloperoxidase(MPO) is a major presence in the neutrophil of an enzyme,its level of activity reflects the extent of neutrophil infiltration.
Superoxide dismutase(SOD) is endogenous superoxide radical scavengers,their activity level can be reflected in part the ability of the body eliminate free radicals.
Nowadays study show,the mitogen-activated protein kinases(MAPKs) cascade,especially p38 play a critical role in intestinal damage such as ischemic, inflammation and apoptosis,
At present,it is still short of effective way to protect intestines mucosal barrier function.So it is a global topic today to search for natural medicines effective for various diseases,curcumin,as a low-toxitity natural meidcinal herb,has recently been documented at home and abroad to have anti-infection, anti-inflammation,anti-oxidization and free-radical removal effect.Curcumin is ratified as food additive by World Health Organization(WHO),Food and Drug Administration(FDA).Up-to-date study found that the widely used food additive curcumin is able to attenuate experimental colitis through a mechanism correlated with the inhibition of the activation of NF-κB and effects a reduction in the activity of p38 MAPK.Curcumin could prevent the increase in Caco-2 permeability,indicating that NF-κB activation was required for the TNF-α-induced increase in Caco-2 permeability.Curcumin decreases binding of Shiga-like toxin-1B on human intestinal epithelial cell line HT29 stimulated with TNF-αand IL-1β:suppression of p38, JNK and NF-κB p65 as potential targets.Curcumin significantly attenuated Stx-1 induced cell death.
In this study,by developing rat models of enteritis and of intestinal epithelial damage,we assessed the protective role of curcumin on intestinal mucosal barrier function,the activation of epithelial intra- and extra-cellular MAKP signaling transduction and nuclear transcription factors,and the release of the inflammation and chemical mediators,with the objecitve of clarifying the molecular mechanism of curcumin's role in the intestinal barrier function,through in vivo and in vitra experiment systems
Methods
ⅠThe protecitve role of curcumin on the intestinal mucosa of rats with methotrexate-induced enteritis
Enteritis was induced in rats through peritoneal injection of methotrexate(20 mg/kg).The rats were randomly divided into 4 groups:normal control group (peritoneal injection of normal saline only),model control group(MTX, 20mg/kg),curcumin group(100mg/kg)and N-Acetylcysteine(NAC,150mg/kg) positive control group(100mg/kg).From the first day that the models were made, the drugs were intragastrically administrated once a day for 7 days,the rats were perfused with the specified dosage,and the rats in the normal control group and the model control group were injected with saline.Every day for the whole duration of the model making,diarrhea and hematochezia diarrhea as observed with the naked eye were checked.On the 6~(th) day,the rats were killed,blood samples were collected from the hearts,intestinal segments were taken and immersed in paraffin for fixation,embedding,slicing and HE staining,and then underwent the general histopathological observation.Finally,30-50mg of the intestinal mucosa were collected for epithelial RNA,total proteins and ribonuclear proteins extraction.
1.The disease activity index(DAI),colonic mucosal damage index(CMDI) and histological score(HS) of the rats were observed and evaluated.
2.The activity of SOD and MPO in the tissues of the small intestines in the rats was determined by colorimetry.
3.The levels of plasma D-lactate and DAO of the small intestinal were detected by UV-spectrophotometry.
4.The levels of mRNA expressions of TNF-α,IL-1β,ICAM-1 were detected by a semi-quantatitive assay RT-PCR.
5.The level of mRNA expressions of IL-10 was detected by ELISA(enzyme linked immunosorbent assay).
6.The expressions of phosphorylated IκB,p38,ERK,JNK proteins were determined with antiphosphospecific antiboday by using western blot.ⅡStudy on the protective role of curcumin on LPS-induced stress reaction of IEC-6 cells
With lipopolysaccharide as the stimulator,IEC-6 cells in the logarithmic phase were divided into 4 groups at random:the normal control group,LPS group, curcumin pretreatment group(curcumin pretreatment) and p38 inhibitor(SB203580) pretreatment group(SB pretreatment).LPS(100ng/ml) was added to all groups except normal control group which was no any treatment;Sixty minutes before LPS was added to the Pre-curcumin and Pre-SB group,curcumin(20μmmol/1) and SB (20μmol/1) were added to the groups,respectively.Then the LEC-6 cells were incubated for 30 minutes.
1.The levels of mRNA expressions of IL-10 was detected by ELISA(enzyme linked immunosorbent assay).
2.The levels of mRNA expressions of TNF-α、IL-1β、ICAM-1 were detected by a semi-quantatitive assay RT-PCR.
3.NF-κB DNA binding activity was evaluated by NF-κB activation- tronslocation dectection kit.
4.Degradation of IκB in the cytoplasm was detected by Western blot.
5.The expressions of phosphorylated MKP-1 was determined with antiphosphospecific antiboday by using western blot.
Results
ⅠThe protecitve role of curcumin in the intestinal mucosa of rats with methotrexate-induced enteritis
1.Effects of curcumin on the rats' symptoms and histology With the rats in the MTX group,diarrhea was observed on the 2nd day after the injection of MTX; on the 3rd day,hematochezia diarrhea was observed with the naked eyes in some rats,and on the 6th day,in all of them.The group pretreated with curcumin and the NAC group exhibited no distinct differences:on the 3rd day,diarrhea was observed but hematochezia diarrhea was not.On the 6th day,hematochezia diarrhea was found in all the rats.Observation of the histological sections revealed a high expansion and hyperemia with the mucosal and submucosal blood vessels,loss of crypt foci shape of the colon,withering and depilation of the villus and a wide range of infiltration of inflammatory cells typical of inflammation,with the rats in MTX group.Compared with the model group,the inflammation,loss of crypt loci shape of the colon,withering and depilation of the villus were all markedly less intense, and the infiltration of inflammatory cells were fewer.No differences were observed with the normal control group.The results above illustrated that curcumin,helping stop the infiltration of inflammatory cells and alleviate the inflammation reaction and symptoms of enteritis,had a marked therapeutical effect on enteritis and a protective role for the intestinal mucosa of the rats.
2.Determination of MPO and SOD activity of the intestinal mucosa
It was revealed that the SOD and MPO activity of the intestinal mucosa in model group were significantly higher than in normal group during the same period (P=0.000) and that for both the Curcumin group and the NAC group,MPO activity could be lowered and SOD activity could be increased.Furthermore,it was demonstrated that curcumin markedly increased the intestinal mucosal SOD expression of the rats and lowered the MPO activity.The results illustrated that curcumin had anti-oxidization effect and could lessen the infiltration of the inflammatory cells,thus having a protective role for experimental rat enteritis.
3.The effect of curcumin on mucosal intestinal permeability
It was revealed that the two levels of the MTX group were distinctly higher than those of the normal group,and those of the Curcumin group and NAC group were markedly lower than those of the MTX group in the same peirod(P=0.000).This proved that curcumin could improve the permeability of the intestinal mucosa and had a protective role in the rat intestinal barrier function.
4.Influence of curcumin on the expression of TNF-α,IL-1β,ICAM-1 and IL-10.
The levels of mRNA expressions of TNF-α,IL-1β,ICAM-1 were detected by a semi-quantatitive assay RT-PCR,and IL-10 were detected by ELISA(enzyme linked immunosorbent assay).The expressions of the above four were significantly higher in the MTX treated group than in the control group.The expression sof mRNA of TNF-α、IL-1β、ICAM- 1 of the curcumin group and NAC group were lower than those of the MTX group,but the expression of IL-10,higher.The expressions of all the four mentioned above exhibited no significant differences between the Curcumin group and NAC group.These data suggested that curcumin could inhibit the expression of TNF-α,IL-1βand ICAM-1,and up-regulate the expression of IL-10.
5.Modulation of cell signalling pathway and IκB by curcumin in intestinal mucosa
The expressions of phosphorylated p38,ERK and JNK proteins were determined with antiphosphospecific antiboday by using Western blot.After MTX were gaven,marked phosphorylation of p38 was observed,while in the curcumin group and SB group,the phosphorylation was distinctly less,no distinct changes were observed in the phosphorylated JNK and ERK protein.
Degradation of IκB in the cytoplasm was detected by Western blot.It was observed that the NF-κB was obviously activated,its expression was significantly stronger in the nuclei of MTX treated cells,and the IκB in the cytoplasm was markedly degraded.But the translocation of NF-κB and degradation of IκB were distinctly less in the curcumin group and NAC group than in the MTX group,and the activation of NF-κB in the curcumin group and NAC group,markedly less.ⅡStudy on the protective role and its mechanism of curcumin in LPS-induced stress reaction of IEC-6 cells
1.Modulation of cell signalling pathway by curcumin in vitro
The expressions of phosphorylated MKP-1 proteins were determined with antiphosphospecific antiboday by using western blot.After LPS stimulation,marked phosphorylation of MKP-1 was observed,while in the curcumin pretreat group and SB pretreat group,the phosphorylation was distinctly increase.
NF-κB DNA binding activity was evaluated by NF-κB activation-tronslocation dectection kit.Degradation of IκB in the cytoplasm was detected by Western blot.It was observed that the NF-κB was obviously activated,its expression was significantly stronger in the nuclei of LPS treated cells,and the IκB in the cytoplasm was markedly degraded.But the translocation of NF-κB and degradation of IκB were distinctly less in the curcumin pretreat group and SB pretreat group than in the LPS group,and the activation of NF-κB in the curcumin pretreat group and SB pretreat group,markedly less.
2.Influence of curcumin on the expression of TNF-α,IL-1β,IL-10 and ICAM-1.
The expressions of TNF-α,IL-1βand ICAM-1 were significantly higher in the LPS treated group than in the control group,and IL-10 was markedly lowerl.After pretreatment with curcumin and SB,the proinflammatory cytokines mRNA expressions were significantly decreased,and IL-10 was significantly increased.These data suggested that curcumin could inhibit the expression of TNF-α,IL-1βand ICAM-1,and markedly up-regulate the expression of IL-10.
ⅢStatistical analysis
All data are presented as mean±S.D.of three independent experiments.Comparision of the effects of various treatments was performed using factor analysis,one-way ANOVA analysis of variance and a two-tailed t-test. Difference with a p value of＜0.05 were considered statistically significant. Conclusion
1.Curcumin can protecte in the intestinal mucosa of rats with methotrexate-induced enteritis of small intestines,curcumin possessed the anti-oxidization effefct and could lessen the infiltration of the inflammatory cells.
2.Curcumin could inhibit the expression of TNF-α,IL-1βand ICAM-1,and up-regulate the expression of IL-10 in methotrexate-induced enteritis of small intestines of rats and LPS-induced stress reaction of IEC-6 cells.
3.Curcumin is able to inhibite the activation of NF-κB and effect a reduction in the activity of p38 MAPK in methotrexate-induced enteritis of small intestines of rats,but no effects on the expression of ERK and JNK protein.
4.LPS alone enhances phosphorylation of MKP-1 and that curcumin in combination with LPS further enhances the levels of MKP-1 phosphorylation, compared to those observed in the presence of LPS alone.we also examined that curcumin could activate MAP-1 and subsequently diminish p38 phosphorylation and inhibits translocation of NF-κB in LPS -induced IEC-6.
5.Curcumin is able to protecte the intestinal mucosa through a mechanism correlated with the enhances the levels of MKP-1 phosphorylation,compared and inhibition of the activation of NF-κB and effects a reduction in the activity of p38 MAPK,the subsequent reduction of the mRNA and ICAM-1 expressions of TNF-αand IL-1β,and the reinforcement of IL-10.
肠黏膜屏障功能是指正常肠道具有较为完善的功能隔离带,可防止致病性病原体及毒素的侵入。在严重创伤、大面积烧伤、出血性休克、手术、放化疗、长期应用广谱抗生素或免疫抑制剂、营养不良、重症胰腺炎等应激状态下或长期进行肠外营养的情况下,肠道的结构和功能可能受到严重损害,导致肠道屏障功能障碍,进而引起肠道细菌易位,甚至诱发全身炎性反应综合征(SIRS),进而导致多器官功能衰竭(MODS)而危及生命。如何及时正确评估肠道屏障功能对于判断患者的病情、估计其预后、并及时给予综合治疗具有非常重要的意义。但目前直接观察肠道屏障功能仍较困难,多通过间接的方法进行监测。
D-乳酸是胃肠道固有细菌的代谢终产物,肠道多种细菌均可产生。哺乳动物体内不具备将其快速代谢分解的酶系统。当肠黏膜生物屏障受损时,其通透性增加,肠道中细菌产生的D-乳酸通过受损肠黏膜经循环入血,由于哺乳动物没有D-乳酸脱氢酶,此时可见血中D-乳酸水平升高。因此检测其外周血水平即可反映肠黏膜损害程度和通透性的变化。二胺氧化酶(DAO)是人类和所有哺乳动物肠黏膜上层绒毛细胞胞浆中具有高度活性的细胞内酶,当肠黏膜上皮细胞受损、坏死后该酶释放入血,或随坏死脱落的肠黏膜细胞进入肠腔内,导致血浆和肠腔内DAO活性增高而肠黏膜DAO活性降低。由于DAO在外周血中活性稳定,因而可通过无创测定其在外周血中的变化,间接反映肠黏膜状态,但它主要反映的是肠黏膜上皮损伤与修复情况,而不是肠道通透性的改变。
白细胞的过度激活是炎性病理过程中的一个重要环节。白细胞是否停滞并以浸润方式向血管外渗依赖于炎症部位白细胞和内皮细胞膜表面的黏附分子(intercellular adhesion molecule,ICAM)的表达与功能。ICAM是指由细胞合成并组装于细胞表面或分泌至细胞外基质的可促进细胞与细胞或细胞与细胞外基质间黏附的一类糖蛋白。其中ICAM-1属于免疫球蛋白超家族,在炎性肠病发病中的作用近年来受到关注。正常肠组织ICAM-1通常低水平表达于血管内皮细胞、肠黏膜固有层和淋巴结中的单核巨噬细胞。髓过氧化物酶(MPO)是主要存在于中性粒细胞中的一种酶,其活性高低反应了中性粒细胞浸润程度。
超氧化物歧化酶(SOD)是内源性超氧阴离子自由基清除剂,其活性高低可部分反映机体清除自由基的能力。
现在研究发现,肠道损伤所涉及的缺血、炎症、凋亡等多个病理机制与丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPKs)信号道路的调节有关,p38在炎性反应起重要作用。
目前,对肠黏膜屏障功能的保护尚缺乏令人满意的有效手段,因此寻找自然界存在的对人类疾病有治疗作用的药物已成为医学界和药学界共同关注的课题。姜黄素是一种天然低毒的药用植物,被世界卫生组织/食品管理局批准为天然食品添加剂。国内外许多文献相继报道姜黄素具有抗感染、抗炎、抗氧化、清除自由基等作用。最近的研究表明:姜黄素能通过抑制NF-κB、p38的活性,减轻实验性大鼠结肠炎,证明姜黄素通过抑制NF-κB的活性而增加TNF-a诱导的Caco-2细胞的紧密连接;姜黄素通过抑制p38、JNK和NF-κB活性,减轻志贺样毒素与TNF-a诱导的HT29细胞株的结合,减少细胞的死亡。
本课题拟通过建立大鼠小肠炎模型及LPS刺激IEC-6细胞的应激反应,观察姜黄素对肠黏膜屏障的保护作用以及对MAPK信号传导通路、MKP-1、核转录因子NF-κB、炎症介质和化学介质的影响,探讨姜黄素对肠黏膜作用的分子机制。
材料与方法
一、姜黄素对大鼠肠黏膜的保护作用及机制的研究
采用腹腔内注射氨甲蝶呤(MTX)的方法制备SD大鼠小肠炎模型。将60只大鼠随机分为正常对照组(腹腔内注射生理盐水)、模型组(MTX,20mg/kg)、姜黄素组(curcumin 100mg/kg)和N-乙酰半胱氨酸组(NAC,150mg/kg),制模当天开始,每日按剂量灌胃一次,正常对照组和模型组灌生理盐水。制模过程中每天观察大鼠腹泻和肉眼血便情况,在实验第6d处死大鼠,取部分肠段置于多聚甲醛内固定、包埋、切片、HE染色,行一般组织病理学观察,30～50mg肠黏膜提取RNA、肠上皮细胞胞浆蛋白。
1,观察大鼠疾病活动指数(DAI)和小肠黏膜损伤指数(CMDI),光镜下组织学评分(HS)。
2,分光光度法检测血浆D-乳酸和DAO水平。
3,生化法检测大鼠小肠组织MPO和SOD活性。
4,运用ELISA测定大鼠血浆IL-10水平。
5,应用RT-PCR技术对大鼠肠上皮细胞内致炎细胞因子TNF-α、IL-1β、ICAM-1的mRNA表达水平进行检测。
6,Western blot分析胞质内IκB的降解、p38、ERK、JNK蛋白表达。
二、姜黄素对LPS诱导IEC-6细胞应激反应的保护作用及机制
应用大鼠肠上皮细胞株IEC-6细胞,以脂多糖作为刺激因素,将对数生长期的细胞随机分为正常对照组、脂多糖(LPS)组、姜黄素(curcumin pretreat,20μmol/l)预孵组、p38抑制剂SB203580预孵组(SB pretreat,20μmol/l)。正常对照组不加任何处理因素,其它3组均加入LPS,浓度为100ng/ml。姜黄素预孵组和p38抑制剂SB203580预孵组在加入LPS前60min分别加入浓度为20μmmol/l的姜黄素和SB203580。孵育30min后终止。
1,运用ELISA测定细胞上清液IL-10水平。
2,应用RT-PCR技术对IEC-6细胞TNF-α、IL-1β、ICAM-1的mRNA表达水平进行检测。
3,应用NF-κB激活—核转运检测试剂盒检测NF-κB(p65)在细胞核内的分布。
4,Western blot分析胞质内IκB的降解和MKP-1蛋白表达。
结果
一、姜黄素对大鼠肠黏膜的保护作用及机制的研究
1、姜黄素对大鼠粪便性状和肠黏膜组织学的影响
MTX组在腹腔内注射氨甲蝶呤后,第2d即出现腹泻,第3d部分出现肉眼血便,第6d全部出现肉眼血便。curcumin治疗组与NAC组症状无明显差别,于第3d开始出现腹泻、解黄色稀便等现象,但未出现血便,第6d也部分出现肉眼血便。组织切片观察发现:MTX组见黏膜及黏膜下层血管高度扩张充血,肠隐窝形态的丧失、绒毛萎缩、脱落,炎症细胞广泛浸润,呈典型炎症改变;curcumin治疗组与NAC组治疗组炎症明显减轻,肠隐窝形态丧失、绒毛萎缩、脱落现象较模型组明显减轻,炎症细胞浸润少。正常对照组无改变。以上结果说明:姜黄素对SD大鼠小肠炎有明显的治疗作用,对肠黏膜有保护作用,能够防止炎性细胞浸润,减弱炎症反应,减轻肠炎症状。
2、姜黄素对肠黏膜MPO、SOD活性的影响
模型组肠黏膜SOD、MPO活性分别明显高于正常组(P=0.000),姜黄素组和NAC组均可下调MPO活性,增强SOD活性。而且姜黄素可明显增强大鼠肠黏膜组织SOD表达,降低肠黏膜组织MPO活性。说明姜黄素具有抗氧化作用,同时可减轻炎细胞的浸润,对实验性大鼠小肠炎具有保护作用。
3、姜黄素可改善肠黏膜通透性
分光光度法检测血浆D-乳酸和DAO水平。结果显示:与正常组相比,MTX组D-乳酸和DAO水平明显升高(P=0.000)。curcumin组与NAC治疗组D-乳酸、DAO较MTX组有明显下降(P=0.000)。由此可见,姜黄素可以改善肠黏膜的通透性,对大鼠肠黏膜屏障具有保护作用。
4、姜黄素对大鼠肠上皮细胞内细胞炎症因子TNF-α、IL-1β、IL-10及ICAM-1表达的影响
运用ELISA测定大鼠血浆IL-10水平,应用RT-PCR技术对大鼠肠上皮细胞内致炎细胞因子TNF-α、IL-1β、ICAM-1的mRNA表达水平进行检测,结果显示:MTX组TNF-α、IL-1β、ICAM-1的mRNA表达水平均较正常对照组明显增高,Curcumin组与NAC治疗组TNF-α、IL-1β、ICAM-1的mRNA表达水平低于MTX组。但IL-10的表达高于MTX组(p=0.000),curcumin治疗组与NAC组治疗组之间差距无显著性(P=0.43)。上述结果表明:姜黄素对TNF-α、IL-1β、ICAM-1的mRNA表达有明显抑制作用,对IL-10的表达有增强的作用。
5、姜黄素对大鼠肠上皮细胞内IκB、MAPK信号转导通路的影响
实验显示MTX组胞质中IκB明显降解。但姜黄素和NAC组的IκB的降解较MTX组明显减少。MTX组磷酸化p38明显增加,均较正常对照组明显增高(p=0.000),但磷酸化的JNK1/JNK2和ERK1/ERK2蛋白表达无明显改变。结果表明:在MTX诱导的大鼠小肠炎肠上皮细胞的炎症反应中,磷酸化p38增加,姜黄素对磷酸化p38有明显的抑制作用(p=0.000)。
二、姜黄素对LPS诱导IEC-6细胞应激反应的保护作用及机制
1、姜黄素对LPS诱导IEC-6细胞MKP-1、IκB、p65的作用
LPS组磷酸化的MKP-1水平均较正常对照组明显增高(p=0.000),姜黄素和SB预孵组明显高于LPS组。LPS组胞质中IκB较正常对照组明显降解(p=0.000),但姜黄素和SB预孵组的IκB明显高于MTX组(p=0.000)。应用NF-κB激活—核转运检测试剂盒NF-κB(p65)在细胞核内的分布,通过免疫荧光染色检测发现:LPS组p65的核转运明显,核内红色荧光明显强于对照组,姜黄素和SB预孵组的红色荧光强于对照组,但弱于LPS组,说明LPS刺激的情况下,p65和IκB解聚,其核定位序列被暴露,从而被转运到细胞核内。但姜黄素能抑制p65的核转运。
2、姜黄素对IEC-6细胞内致炎细胞因子TNF-α、IL-1β,抑炎因子IL-10和ICAM-1 mRNA表达的影响
LPS组TNF-α、IL-1β、ICAM-1的表达水平均较正常对照组明显增高,IL-10明显减少。但姜黄素和SB预孵组的表达较LPS组表达明显减少,IL-10明显增高。上述结果表明:姜黄素对TNF-α、IL-1β、ICAM-1的mRNA表达有明显抑制作用,对IL-10表达有明显上调作用。
结论
一、姜黄素对MTX诱导的大鼠小肠炎肠黏膜的通透性有保护作用。姜黄素具有抗氧化作用,同时可减轻炎细胞的浸润。
二、姜黄素能够减少MTX诱导的大鼠小肠炎与LPS诱导的IEC-6细胞应激反应模型的TNF-α、IL-1β、ICAM-1 mRNA的表达,增强IL-10 mRNA的表达。
三、姜黄素对MTX致大鼠小肠炎模型的肠黏膜上皮NF-κB和p38信号转导通路有抑制作用,可以抑制NF-κB的激活和p38磷酸化。不影响对ERK、JNK蛋白的表达。
四、姜黄素增强了LPS诱导的IEC-6细胞的MKP-1的磷酸化作用,进而抑制了p38活性。并且对NF-κB的活性有抑制作用。
五、姜黄素对肠黏膜起保护作用的机制可能是通过增强MKP-1的磷酸化作用,抑制了NF-κB和p38活性,进而减少致炎细胞因子TNF-α、IL-1βmRNA和ICAM-1表达,增强抑炎因子IL-10的表达来实现的。
Background/Objective
The intestinal barrier functions a shielding band which can prevent the invasion of causative agents and toxin.Under the conditions of wound,opration,chemotherapy, radiotherapy,severe pancreatitis or long-term parenteral nutrition,the structure and the barrier function of intestinal mucous membrane may be seriously damaged,which may cause the intestinal mucosal barrier dysfunction,then causes the intestinal bacteria translocation,even induces systemic inflammation response syndrome (SIRS),at last result in multiple organs dysfunction syndrome(MODS ).But it is still difficult to detect the gut barrier function directly.
The D-lactate,which may be produced by many germs,is metabolic end product of bacteria in gastrointestinal tract.Without any D-lactate dehydrogenase in the body of mammalian,the D-lactate from enterbacterium will go into blood and the level of D-lactate will increase when intestinal mucosa damages or mucosa permeability changes.Therafore,the examination of D-lactate in peripheral blood may reflect the damage and permeability of intestinal mucosa.DAO is a kind of endocellular enzyme with high activitiy,which exists in the plasm of chorionic villi in intestinal stratum supravasculare in human and all other mammals.When intestinal epithelial cells are injured,the release of endocellular DAO increases and to enters the intestinal intercellular space,the lymph vessel and the blood stream,and finally causes DAO in blood plasma to elevate.The activitiy of DAO in peripheral blood is stable.When small intestinal mucosal barrier nonfunction,the intestinal mucosa membrane cell falls off into the enteric cavity,then DAO enters lymph vessel and the blood stream in the intestines intercellular space,causes blood DAO to elevate. The activity of blood DAO may reflect the extent of damage and repair in intestinal tract.Leukocytic's over-activation is a very important bit of the IBD patho process. Leukocytic staying and exosmoseing on inflammation position depend on ICAM,s expressin and function in the surface of WBC and endothelial cell.ICAMS are glycoproteinwhich are synthesized by the cell and then assembled in the cell surface or secrete to the extracellular matrix,involving in cell-cell or cell-extracellular matrix interaction.And ICAM-1,belonging to the immunoglobulin superfamily, received the attention in recent years in the process of IBD.The expression of ICAM-1 in the normal intestine tissue are usually low in the vascular endothelial cell,in the intestines mucosa lamina propria and in the monocyte-macrophages in the lymph nodes.Myeloperoxidase(MPO) is a major presence in the neutrophil of an enzyme,its level of activity reflects the extent of neutrophil infiltration.
Superoxide dismutase(SOD) is endogenous superoxide radical scavengers,their activity level can be reflected in part the ability of the body eliminate free radicals.
Nowadays study show,the mitogen-activated protein kinases(MAPKs) cascade,especially p38 play a critical role in intestinal damage such as ischemic, inflammation and apoptosis,
At present,it is still short of effective way to protect intestines mucosal barrier function.So it is a global topic today to search for natural medicines effective for various diseases,curcumin,as a low-toxitity natural meidcinal herb,has recently been documented at home and abroad to have anti-infection, anti-inflammation,anti-oxidization and free-radical removal effect.Curcumin is ratified as food additive by World Health Organization(WHO),Food and Drug Administration(FDA).Up-to-date study found that the widely used food additive curcumin is able to attenuate experimental colitis through a mechanism correlated with the inhibition of the activation of NF-κB and effects a reduction in the activity of p38 MAPK.Curcumin could prevent the increase in Caco-2 permeability,indicating that NF-κB activation was required for the TNF-α-induced increase in Caco-2 permeability.Curcumin decreases binding of Shiga-like toxin-1B on human intestinal epithelial cell line HT29 stimulated with TNF-αand IL-1β:suppression of p38, JNK and NF-κB p65 as potential targets.Curcumin significantly attenuated Stx-1 induced cell death.
In this study,by developing rat models of enteritis and of intestinal epithelial damage,we assessed the protective role of curcumin on intestinal mucosal barrier function,the activation of epithelial intra- and extra-cellular MAKP signaling transduction and nuclear transcription factors,and the release of the inflammation and chemical mediators,with the objecitve of clarifying the molecular mechanism of curcumin's role in the intestinal barrier function,through in vivo and in vitra experiment systems
Methods
ⅠThe protecitve role of curcumin on the intestinal mucosa of rats with methotrexate-induced enteritis
Enteritis was induced in rats through peritoneal injection of methotrexate(20 mg/kg).The rats were randomly divided into 4 groups:normal control group (peritoneal injection of normal saline only),model control group(MTX, 20mg/kg),curcumin group(100mg/kg)and N-Acetylcysteine(NAC,150mg/kg) positive control group(100mg/kg).From the first day that the models were made, the drugs were intragastrically administrated once a day for 7 days,the rats were perfused with the specified dosage,and the rats in the normal control group and the model control group were injected with saline.Every day for the whole duration of the model making,diarrhea and hematochezia diarrhea as observed with the naked eye were checked.On the 6~(th) day,the rats were killed,blood samples were collected from the hearts,intestinal segments were taken and immersed in paraffin for fixation,embedding,slicing and HE staining,and then underwent the general histopathological observation.Finally,30-50mg of the intestinal mucosa were collected for epithelial RNA,total proteins and ribonuclear proteins extraction.
1.The disease activity index(DAI),colonic mucosal damage index(CMDI) and histological score(HS) of the rats were observed and evaluated.
2.The activity of SOD and MPO in the tissues of the small intestines in the rats was determined by colorimetry.
3.The levels of plasma D-lactate and DAO of the small intestinal were detected by UV-spectrophotometry.
4.The levels of mRNA expressions of TNF-α,IL-1β,ICAM-1 were detected by a semi-quantatitive assay RT-PCR.
5.The level of mRNA expressions of IL-10 was detected by ELISA(enzyme linked immunosorbent assay).
6.The expressions of phosphorylated IκB,p38,ERK,JNK proteins were determined with antiphosphospecific antiboday by using western blot.ⅡStudy on the protective role of curcumin on LPS-induced stress reaction of IEC-6 cells
With lipopolysaccharide as the stimulator,IEC-6 cells in the logarithmic phase were divided into 4 groups at random:the normal control group,LPS group, curcumin pretreatment group(curcumin pretreatment) and p38 inhibitor(SB203580) pretreatment group(SB pretreatment).LPS(100ng/ml) was added to all groups except normal control group which was no any treatment;Sixty minutes before LPS was added to the Pre-curcumin and Pre-SB group,curcumin(20μmmol/1) and SB (20μmol/1) were added to the groups,respectively.Then the LEC-6 cells were incubated for 30 minutes.
1.The levels of mRNA expressions of IL-10 was detected by ELISA(enzyme linked immunosorbent assay).
2.The levels of mRNA expressions of TNF-α、IL-1β、ICAM-1 were detected by a semi-quantatitive assay RT-PCR.
3.NF-κB DNA binding activity was evaluated by NF-κB activation- tronslocation dectection kit.
4.Degradation of IκB in the cytoplasm was detected by Western blot.
5.The expressions of phosphorylated MKP-1 was determined with antiphosphospecific antiboday by using western blot.
Results
ⅠThe protecitve role of curcumin in the intestinal mucosa of rats with methotrexate-induced enteritis
1.Effects of curcumin on the rats' symptoms and histology With the rats in the MTX group,diarrhea was observed on the 2nd day after the injection of MTX; on the 3rd day,hematochezia diarrhea was observed with the naked eyes in some rats,and on the 6th day,in all of them.The group pretreated with curcumin and the NAC group exhibited no distinct differences:on the 3rd day,diarrhea was observed but hematochezia diarrhea was not.On the 6th day,hematochezia diarrhea was found in all the rats.Observation of the histological sections revealed a high expansion and hyperemia with the mucosal and submucosal blood vessels,loss of crypt foci shape of the colon,withering and depilation of the villus and a wide range of infiltration of inflammatory cells typical of inflammation,with the rats in MTX group.Compared with the model group,the inflammation,loss of crypt loci shape of the colon,withering and depilation of the villus were all markedly less intense, and the infiltration of inflammatory cells were fewer.No differences were observed with the normal control group.The results above illustrated that curcumin,helping stop the infiltration of inflammatory cells and alleviate the inflammation reaction and symptoms of enteritis,had a marked therapeutical effect on enteritis and a protective role for the intestinal mucosa of the rats.
2.Determination of MPO and SOD activity of the intestinal mucosa
It was revealed that the SOD and MPO activity of the intestinal mucosa in model group were significantly higher than in normal group during the same period (P=0.000) and that for both the Curcumin group and the NAC group,MPO activity could be lowered and SOD activity could be increased.Furthermore,it was demonstrated that curcumin markedly increased the intestinal mucosal SOD expression of the rats and lowered the MPO activity.The results illustrated that curcumin had anti-oxidization effect and could lessen the infiltration of the inflammatory cells,thus having a protective role for experimental rat enteritis.
3.The effect of curcumin on mucosal intestinal permeability
It was revealed that the two levels of the MTX group were distinctly higher than those of the normal group,and those of the Curcumin group and NAC group were markedly lower than those of the MTX group in the same peirod(P=0.000).This proved that curcumin could improve the permeability of the intestinal mucosa and had a protective role in the rat intestinal barrier function.
4.Influence of curcumin on the expression of TNF-α,IL-1β,ICAM-1 and IL-10.
The levels of mRNA expressions of TNF-α,IL-1β,ICAM-1 were detected by a semi-quantatitive assay RT-PCR,and IL-10 were detected by ELISA(enzyme linked immunosorbent assay).The expressions of the above four were significantly higher in the MTX treated group than in the control group.The expression sof mRNA of TNF-α、IL-1β、ICAM- 1 of the curcumin group and NAC group were lower than those of the MTX group,but the expression of IL-10,higher.The expressions of all the four mentioned above exhibited no significant differences between the Curcumin group and NAC group.These data suggested that curcumin could inhibit the expression of TNF-α,IL-1βand ICAM-1,and up-regulate the expression of IL-10.
5.Modulation of cell signalling pathway and IκB by curcumin in intestinal mucosa
The expressions of phosphorylated p38,ERK and JNK proteins were determined with antiphosphospecific antiboday by using Western blot.After MTX were gaven,marked phosphorylation of p38 was observed,while in the curcumin group and SB group,the phosphorylation was distinctly less,no distinct changes were observed in the phosphorylated JNK and ERK protein.
Degradation of IκB in the cytoplasm was detected by Western blot.It was observed that the NF-κB was obviously activated,its expression was significantly stronger in the nuclei of MTX treated cells,and the IκB in the cytoplasm was markedly degraded.But the translocation of NF-κB and degradation of IκB were distinctly less in the curcumin group and NAC group than in the MTX group,and the activation of NF-κB in the curcumin group and NAC group,markedly less.ⅡStudy on the protective role and its mechanism of curcumin in LPS-induced stress reaction of IEC-6 cells
1.Modulation of cell signalling pathway by curcumin in vitro
The expressions of phosphorylated MKP-1 proteins were determined with antiphosphospecific antiboday by using western blot.After LPS stimulation,marked phosphorylation of MKP-1 was observed,while in the curcumin pretreat group and SB pretreat group,the phosphorylation was distinctly increase.
NF-κB DNA binding activity was evaluated by NF-κB activation-tronslocation dectection kit.Degradation of IκB in the cytoplasm was detected by Western blot.It was observed that the NF-κB was obviously activated,its expression was significantly stronger in the nuclei of LPS treated cells,and the IκB in the cytoplasm was markedly degraded.But the translocation of NF-κB and degradation of IκB were distinctly less in the curcumin pretreat group and SB pretreat group than in the LPS group,and the activation of NF-κB in the curcumin pretreat group and SB pretreat group,markedly less.
2.Influence of curcumin on the expression of TNF-α,IL-1β,IL-10 and ICAM-1.
The expressions of TNF-α,IL-1βand ICAM-1 were significantly higher in the LPS treated group than in the control group,and IL-10 was markedly lowerl.After pretreatment with curcumin and SB,the proinflammatory cytokines mRNA expressions were significantly decreased,and IL-10 was significantly increased.These data suggested that curcumin could inhibit the expression of TNF-α,IL-1βand ICAM-1,and markedly up-regulate the expression of IL-10.
ⅢStatistical analysis
All data are presented as mean±S.D.of three independent experiments.Comparision of the effects of various treatments was performed using factor analysis,one-way ANOVA analysis of variance and a two-tailed t-test. Difference with a p value of＜0.05 were considered statistically significant. Conclusion
1.Curcumin can protecte in the intestinal mucosa of rats with methotrexate-induced enteritis of small intestines,curcumin possessed the anti-oxidization effefct and could lessen the infiltration of the inflammatory cells.
2.Curcumin could inhibit the expression of TNF-α,IL-1βand ICAM-1,and up-regulate the expression of IL-10 in methotrexate-induced enteritis of small intestines of rats and LPS-induced stress reaction of IEC-6 cells.
3.Curcumin is able to inhibite the activation of NF-κB and effect a reduction in the activity of p38 MAPK in methotrexate-induced enteritis of small intestines of rats,but no effects on the expression of ERK and JNK protein.
4.LPS alone enhances phosphorylation of MKP-1 and that curcumin in combination with LPS further enhances the levels of MKP-1 phosphorylation, compared to those observed in the presence of LPS alone.we also examined that curcumin could activate MAP-1 and subsequently diminish p38 phosphorylation and inhibits translocation of NF-κB in LPS -induced IEC-6.
5.Curcumin is able to protecte the intestinal mucosa through a mechanism correlated with the enhances the levels of MKP-1 phosphorylation,compared and inhibition of the activation of NF-κB and effects a reduction in the activity of p38 MAPK,the subsequent reduction of the mRNA and ICAM-1 expressions of TNF-αand IL-1β,and the reinforcement of IL-10.
引文
[1]Van,t Land B,van Beek NM,van den Berg JJ,M!Rabet L.Lact ferrin reduces methotrexate-induced small intestinal damage,possibly through inhibition of GLP-2-mediated epithelial cell proliferation[J].Dig Dis Sci,2004,49(3):425-33
[2]Howarth GS,Francis GL,Cool JC et al.Milk growth factors enriched from cheese whey ameliorate intestinal damage by methotrexate when administered orally to rats[J].Nutr,1996,126:2519-30.
[3]Yagci G,Gul H,Simsek A,et al.Beneficial effects of N-acetyl-cysteine on sodium taurocholate-induced pancreatitis in rats[J].Gastroenterol,2004,39(3):268-76
[4]费保莹,邓长生,朱尤庆.细胞黏附分子与炎症性肠病.国外医学消化系疾病分册,1999,19:15-17
[5]SunZ,WangX,LassonA,et al.Effects of inhibition of PAF,ICAM-1 and PECAM-1 on gut barrier failure caused by intestinal ischemia and reperfusion[J].Scand J Gastroenterol,2001,36(1):55-65
[6]Gao F,Ueda S,Horie T.Effect of a synthetic analog of prostaglandin E1 on the intestinal mucosa of methotrexate-treated rats[J].Anticancer Res,2001,21(3B):1913-17
[7]Zafarullah M,Li W Q,Sylvester J,et al.Molecular mechanisms of Nacetylcysteine actions[J].Cell Mol Life Sci,2003,60(1):6-20.
[8]Xian CJ,Couper R,Howarth GS,et al.Increased exprassion of HGF and c-met in rat small intcstinal during rcovcry from methotrexate induced muositis[J].Br J Cancer,2000,82(4):951-4.
[9]Steidler L,Hans W,Schotte L,et al.Treatment of murine colitis by Lactococcus lactis secreting interleukin-10[J].Science,2000,289:1352-5.
[10]Sandborn WJ,Strategies for targeting tumour necrosis factor in IBD[J].Best Pract Res Clin Gastroenterol, 2003,17(1):105-17.
[11] Steidler L, Hans W, Schotte L, et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10[J]. Science, 2000, 289: 1352-5.
[12] Sandborn WJ, Strategies for targeting tumour necrosis factor in IBD[J]. Best Pract Res Clin Gastroenterol,2003,17(1):105-17.
[13] Farhadi A, Banan A , Fields J, etal Intestinal barrier: an intelface between health and disease [J ].Gastrpenterol Hepatd ,2003,18(5):479-97
[14] Tibbie JA. Sigthorsson G.Bridger S. et al. Surrogate markers of intestinal inflammation are redictive of relapse in patients with inflammatary bowel disease [J]. Gastroenterology, 2000,119(3): 15-22
[15] Kalff JC, Hierholzer C, Tsukada K, et al. Hemorrhagic shock results in intestinal muscularis intercellular adhesion molecule (ICAM-1) expression, neutrophil infiltration, and smooth muscle dysfunction[J]. Arch Orthop Trauma Surg, 1999,119(1-2):89-93.
[16] Olanders K, Sun Z, Borjesson A, et al. The effect of intestinal ischemia and reperfusion injury on ICAM-1 expression, endothelial barrier function, neutrophil tissue influx, and protease inhibitor levels in rats. Shock[J]. 2002, 18(1):86-92
[17] Chretien A, Dierick JF, Delaive E, Role of TGF-betal-independent changes in protein neosynthesis, p38alpha(MAPK), and cdc42 in hydrogen peroxide- induced senescence-like morphogenesis[J]. Free Radic Biol Med, 2008,44(9): 1732-51.
[18] Parlesak A, Schaeckeler S, Moser L, et al .Conjugated primary bile salts reduce permeability of endotoxin through intestinal epithelial cells and synergize with phosphatidylcholine in suppression of inflammatory cytokine production [J]. Crit Care Med ,2007,35(10):2367-74.
[19] Zhang H, Ma H, Lu B, et al .Inhibitory effect of dietary n-3 polyunsaturated fatty acids to intestinal IL-15 expression is associated with reduction of TCRalphabeta+CD8alpha+CD8beta- intestinal intraepithelial lymphocytes[J]. Nutr Biochem, 2007,( 11) 367-71
[20] Werner T , Haller D. Intestinal epithelial cell signalling and chronic inflammation: From the proteome to specific molecular mechanisms[J]. Mutat Res, 2007,(22)42-57
[21] Sydora BC, Macfarlane SM, Walker JW, et al. Epithelial barrier disruption allows nondisease-causing bacteria to initiate and sustain IBD in the IL-10 gene-deficient mouse[J]. Inflamm Bowel Dis, 2007,13(8):947-54
[22] Laharie D, Menard S, Asencio C, et al .Effect of rebamipide on the colonic barrier in interleukin-10-deficient mice[J]. Dig Dis Sci, 2007,52(1 ):84-92.
[23] Hale LP, Gottfried MR, Swidsinski A, et al. Piroxicam treatment of IL-10-deficient mice enhances colonic epithelial apoptosis and mucosal exposure to intestinal bacteria[J].Inflamm Bowel Dis, 2005,11 (12): 1060-9.
[24] Campbell J, Ciesielski CJ, Hunt AE, et al. A novel mechanism for TNF-alpha regulation by p38 MAPK: involvement of NF-kappa B with implications for therapy in rheumatoid arthritis[J]. Immunol, 2004 ,173(11):6928-37.
[25] Lappas M, Permezel M, Georgiou HM, et al. Nuclear factor kappa B regulation of proinflammatory cytokines in human gestational tissues in vitro. Biol Reprod[J]. 2002,67(2):668-73.
[26] Weaver SA, Russo MP, Wright KL, et al. Regulatory role of phosphati- dylinositol 3-kinase on TNF-alpha-induced cyclooxygenase 2 expression in colonic epithelial cells[J]. Gastroenterology, 2001 ,120(5):1117-27.
[27] Jeong HJ, Koo HN, Na HJ, et al. Inhibition of TNF-alpha and IL-6 production by Aucubin through blockade of NF-kappaB activation RBL-2H3 mast cells[J]. Cytokine, 2002 ,18(5):252-9.
[28] Zingarelli B, Yang Z, Hake P W , et al.A bsence of endogenous interleuk -10 enhances early stress response during postischaemic injury in mice intestine[J]. Gut, 2001,48 (5): 610-21
[29] W aetzig G H, Seegert D, Ro senstiel P, et al. p38 m itogen activated protein kinase is activated and linked to TNF-alpha signaling in inflammato ry bowel disease[J]. Immunol,2002,168 (10 ) :5342 -51
[30] Tho rpe C M , Hurley B P, L incicome L L , et al. Sh iga toxins stimulate secretion of interleuk in 8 from intestinal ep ithelial cells[J]. Infect Immun, 1999, 67(11) :5985-91
[31] Kong PJ, Lee HJ, Lee SH, et al. c-Jun N-terminal kinase and p38 mitogen-activated protein kinase mediate double-strand RNA-induced inducible nitric oxide synthase expression in microglial cells[J].Neurosci Lett, 2008 ,433(3):215-8.
[1]Souza KL,Gurgul-Convey E,Elsner M,et al..Interaction between proinflammatory and anti-inflammatory cytokines in insulin-producing cells[J].Endocrinol,2008,197(1):139-50
[2]Kramer F,Torzewski J,Kamenz J,et al.Interleukin-lbeta stimulates acute phase response and C-reactive protein synthesis by inducing an NFkappaB- and C/EBPbeta-dependent autocrine interleukin-6 loop[J].Mol Immunol.2008,45(9):2678-89.
[3]Guo W,Wise ML,Collins FW,et al.Avenanthramides,polyphenols from oats,inhibit IL-1 beta-induced NF-kappaB activation in endothelial cells[J].Free Radic Biol Med,2008,44(3):415-29.
[4]Adler HS,Steinbrink K,et al.MAP kinase p38 and its relation to T cell anergy and suppressor function of regulatory T cells[J].Cell Cycle,2008,7(2):169-70.
[5]Son YH,Jeong YT,Lee KA,et al.Roles of MAPK and NF-kappaB in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells.J Cardiovasc Pharmacol[J].2008,51(1):71-7
[6]Rajapakse N,Kim MM,Mendis E,et al.Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW264.7cells by carboxybutyrylated glucosamine takes place via down-regulation of mitogen-activated protein kinase-mediated nuclear factor-kappaB signaling[J]. Immunology. 2008,123(3):348-57.
[7] Li Y, Chu N, Hu A, et al .Inducible IL-23p 19 expression in human microglia via p38 MAPK and NF-kappaB signal pathways[J]. Exp Mol Pathol,2008, 84(1): 1-8.
[8] Fitzgerald DC, Meade KG, McEvoy AN, et al .Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells[J]. Vet Immunol Immunopathol, 2007,116(1-2):59-68.
[9] Lahat G, Halperin D, Barazovsky E, et al .Immunomodulatory effects of ciprofloxacin in TNBS-induced colitis in mice[J]. Inflamm Bowel Dis, 2007, 13(5):557-65
[10] Abke S, Neumeier M, Weigert J, et al . Adiponectin-induced secretion of interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1,CCL2) and interleukin-8 (IL-8, CXCL8) is impaired in monocytes from patients with type I diabetes[J]. Cardiovasc Diabetol, 2006 ,5-17.
[11] Shalit I, Halperin D, Haite D, et al. Anti-inflammatory effects of moxifloxacin on IL-8, IL-1beta and TNF-alpha secretion and NFkappaB and MAP-kinase activation in human monocytes stimulated with Aspergillus fumigatus[J]. Antimicrob Chemother, 2006,57(2):230-5.
[12] Yeh CH, Chen TP, Wu YC, et al .Inhibition of NFkappaB activation with curcumin attenuates plasma inflammatory cytokines surge and cardiomyocytic apoptosis following cardiac ischemia/reperfusion[J]. Surg Res, 2005,125(1): 109-16
[13] ShishodiaS, KoulD, AggarwalBB. et al .Cyclooxygenase(COX) 2 inhibitor celecoxibabrogates TNF induced NF— kappaB activation through inhibition of activation of IkappaBalpha kinase and Aktinhumannon small lcelllung- rcinoma: correlation with suppres sionof COX 2synthesis[J]. Immunol, 2004,73(3):2011-22.
[14] De Plaen IG , Han XB , Liu X , et al .Lipopolysaccharide induces CXCL2/macrophage inflammatory protein-2 gene expression in enterocytes via NF-kappaB activation: independence from endogenous TNF-alpha and platelet-activating factor[J]. Immunology, 2006 ,118(2): 153-63.
[15] Grishin AV, Wang J, Potoka DA, et al .Lipopolysaccharide induces cyclooxygenase-2 in intestinal epithelium via a noncanonical p38 MAPK pathway[J]. Immunol, 2006 ,176(1):580-8.
[16] Kim JY, Park SJ, Yun KJ, et al .Isoliquiritigenin isolated from the roots of Glycyrrhiza uralensis inhibits LPS-induced iNOS and COX-2 expression via the attenuation of NF-kappaB in RAW 264.7 macrophages[J]. Eur J Pharmacol, 2008,584(1):175-84.
[17] Liu S, Feng G, Wang GL, et al. p38 inhibition attenuates LPS-induced acute lung injury involvement of NF-kappaB pathway [J]. Eur J Pharmacol. 2008,584(1):159-65.
[18] Wu LC, Fan NC, Lin MH , et al .Anti-inflammatory Effect of Spilanthol from Spilanthes acmella on Murine Macrophage by Down-Regulating LPS-induced Inflammatory Mediators[J]. Agric Food Chem, 2008,56(7):2341-9.
[19] Son YH, Jeong YT, Lee KA, et al .Roles of MAPK and NF-kappaB in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells[J]. Cardiovasc Pharmacol, 2008,51(1):71 -7
[20] Kim BH, Roh E, Lee HY, Lee IJ, et al . Benzoxathiole derivative blocks lipopolysaccharide-induced nuclear factor-kappaB activation and nuclear factor-kappaB-regulated gene transcription through inactivating inhibitory kappaB kinase beta[J]. Mol Pharmacol, 2008 ,73(4):1309-18.
[21] Tanaka T, Oh-Hashi K, Shitara H, et al .NF-kappaB independent signaling pathway is responsible for LPS-induced GDNF gene expression in primary rat glial cultures[J]. Neurosci Lett, 2008,431(3):262-7.
[22] Jung HW, Chung YS, Kim YS, et al. Celastrol inhibits production of nitric oxide and proinflammatory cytokines through MAPK signal transduction and NF-kappaB in LPS-stimulated BV-2 microglial cells[J]. Exp Mol Med, 2007,39(6):715-21.
[23] Jones E, Adcock IM, Ahmed BY , et al .Modulation of LPS stimulated NF-kappaB mediated Nitric Oxide production by PKCepsilon and JAK2 in RAW macrophages[J]. Inflamm (Lond), 2007, (24):4:23
[24] Xu Y, Liu XD, Gong X, Eissa NT , et al .Signaling pathway of autophagy associated with innate immunity[J]. Autophagy, 2008,4(1 ):110-2.
[25] Kita T, Ogawa M, Sato H, et al .Role of p38 mitogen-activated protein kinase pathway on heart failure in the infant rat after burn injury[J]. Int J Exp Pathol, 2008,89(1):55-63.
[26] Huh JE, Yim JH, Lee HK, et al. Prodigiosin isolated from Hahella chejuensis suppresses lipopolysaccharide-induced NO production by inhibiting p38 MAPK, JNK and NF-kappaB activation in murine peritoneal macrophages[J]. Int Immunopharmacol, 2007 ,7(13):1825-33.
[27] Son YH, Jeong YT, Lee KA , et al .Roles of MAPK and NF-kappaB in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells[J]. J Cardiovasc Pharmacol, 2008,51(1):71-7
[28] Usluoglu N,Pavlovic J, Moelling K , et al. RIP2 mediates LPS-induced p38 and IkappaBalpha signaling including IL-12 p40 expression in human monocyte-derived dendritic cells[J]. Eur J Immunol, 2007,37(8):2317-25.
[29] Lappas M, Permezel M, Rice GE , et al. Mitogen-activated protein kinase proteins regulate LPS-stimulated release of pro-inflammatory cytokines and prostaglandins from human gestational tissues[J].Placenta. 2007,28(8-9): 936-45.
[30] Lin WN, Luo SF, Lee CW, et al. volvement of MAPKs and NF-kappaB in LPS-induced VCAM-1 expression in human tracheal smooth muscle cells[J]. Cell Signa,2007,19(6):1258-67.
[31] Pladzyk A, Reddy AB, Yadav UC , et al . Inhibition of aldose reductase prevents lipopolysaccharide-induced inflammatory response in human lens epithelial cells[J]. Invest Ophthalmol Vis Sci, 2006,47(12):5395-403
[32] Emre Y, Hurtaud C, Nubel T , et al. Mitochondria contribute to LPS-induced MAPK activation via uncoupling protein UCP2 in macrophages[J]. Biochem J, 2007 ,402(2):271-8
[33] Jung KK, Lee HS, Cho JY, et al .Inhibitory effect of curcumin on nitric oxide production from lipopolysaccharide-activated primary microglia[J]. Life Sci, 2006,79(21):2022-31.
[34] CarlVS, Brown SteinkeK, NicklinMJ, et.al. oil like receptor and (TLR2 and TLR)agenists differentially regulate secretoryinterleuk in lrecepto- rant agonist gene expression in macrophages[J].BiolChem, 2002,77(20): 17448- 56.
[35] Elass E, Coddeville B, Kremer L, et.al. Mycobacterial lipomannan induces MAP kinase phosphatase-1 expression in macrophages[J]. FEBS Lett, 2008, 582(3): 45-50.
[36] Hou N, Torii S, Saito N, et.al. Reactive Oxygen Species-Mediated Pancreatic {beta}-Cell Death Is Regulated by Interactions between Stress-Activated Protein Kinases, p38 and c-Jun N-Terminal Kinase, and Mitogen-Activated Protein Kinase Phosphatases[J]. Endocrinology, 2008,149(4):1654-65.
[37] Kinney CM, Chandrasekharan UM, Mavrakis L, et.al. VEGF and thrombin induce MKP-1 through distinct signaling pathways: role for MKP-1 in endothelial cell migration[J]. Am J Physiol Cell Physiol, 2008,294(1):241-50.
[38] Venugopal SK, Chen J, Zhang Y, et.al. Role of MAPK phosphatase-1 in sustained activation of JNK during ethanol-induced apoptosis in hepatocyte-like VL-17A cells[J]. Biol Chem. 2007,282(44):31900-8.
[39] Jang BC, Lim KJ, Suh MH, et.al. Dexamethasone suppresses interleukin-1 beta induced human beta-defensin mRNA expression: involvement of p38 MAPK, JNK, MKP-1, and NF-kappaB transcnptional factor in A549 cells[J]. FEMS Immunol Med Microbiol, 2007 ,51(1):171-84.
[40] Ito A, Suganami T, Miyamoto Y, et.al. Role of MAPK phosphatase-1 in the induction of monocyte chemoattractant protein-1 during the course of adipocyte hypertrophy [J]. J Biol Chem, 2007 ,282(35):25445-52.
[41] Kondoh K, Sunadome K, Nishida E. Notch signaling suppresses p38 MAPK activity via induction of MKP-1 in myogenesis[J]. Biol Chem. 2007(2), 3058-65.
[42] Nimah M., Zhao Denenberg, A.G Bueno, et.al.ContributionofMKP-1 regulation of p38 to endotox into lerance[J]. hock, et.al.2005,23:80-87
[2]Howarth GS,Francis GL,Cool JC et al.Milk growth factors enriched from cheese whey ameliorate intestinal damage by methotrexate when administered orally to rats[J].Nutr,1996,126:2519-30.
[3]Yagci G,Gul H,Simsek A,et al.Beneficial effects of N-acetyl-cysteine on sodium taurocholate-induced pancreatitis in rats[J].Gastroenterol,2004,39(3):268-76
[4]费保莹,邓长生,朱尤庆.细胞黏附分子与炎症性肠病.国外医学消化系疾病分册,1999,19:15-17
[5]SunZ,WangX,LassonA,et al.Effects of inhibition of PAF,ICAM-1 and PECAM-1 on gut barrier failure caused by intestinal ischemia and reperfusion[J].Scand J Gastroenterol,2001,36(1):55-65
[6]Gao F,Ueda S,Horie T.Effect of a synthetic analog of prostaglandin E1 on the intestinal mucosa of methotrexate-treated rats[J].Anticancer Res,2001,21(3B):1913-17
[7]Zafarullah M,Li W Q,Sylvester J,et al.Molecular mechanisms of Nacetylcysteine actions[J].Cell Mol Life Sci,2003,60(1):6-20.
[8]Xian CJ,Couper R,Howarth GS,et al.Increased exprassion of HGF and c-met in rat small intcstinal during rcovcry from methotrexate induced muositis[J].Br J Cancer,2000,82(4):951-4.
[9]Steidler L,Hans W,Schotte L,et al.Treatment of murine colitis by Lactococcus lactis secreting interleukin-10[J].Science,2000,289:1352-5.
[10]Sandborn WJ,Strategies for targeting tumour necrosis factor in IBD[J].Best Pract Res Clin Gastroenterol, 2003,17(1):105-17.
[11] Steidler L, Hans W, Schotte L, et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10[J]. Science, 2000, 289: 1352-5.
[12] Sandborn WJ, Strategies for targeting tumour necrosis factor in IBD[J]. Best Pract Res Clin Gastroenterol,2003,17(1):105-17.
[13] Farhadi A, Banan A , Fields J, etal Intestinal barrier: an intelface between health and disease [J ].Gastrpenterol Hepatd ,2003,18(5):479-97
[14] Tibbie JA. Sigthorsson G.Bridger S. et al. Surrogate markers of intestinal inflammation are redictive of relapse in patients with inflammatary bowel disease [J]. Gastroenterology, 2000,119(3): 15-22
[15] Kalff JC, Hierholzer C, Tsukada K, et al. Hemorrhagic shock results in intestinal muscularis intercellular adhesion molecule (ICAM-1) expression, neutrophil infiltration, and smooth muscle dysfunction[J]. Arch Orthop Trauma Surg, 1999,119(1-2):89-93.
[16] Olanders K, Sun Z, Borjesson A, et al. The effect of intestinal ischemia and reperfusion injury on ICAM-1 expression, endothelial barrier function, neutrophil tissue influx, and protease inhibitor levels in rats. Shock[J]. 2002, 18(1):86-92
[17] Chretien A, Dierick JF, Delaive E, Role of TGF-betal-independent changes in protein neosynthesis, p38alpha(MAPK), and cdc42 in hydrogen peroxide- induced senescence-like morphogenesis[J]. Free Radic Biol Med, 2008,44(9): 1732-51.
[18] Parlesak A, Schaeckeler S, Moser L, et al .Conjugated primary bile salts reduce permeability of endotoxin through intestinal epithelial cells and synergize with phosphatidylcholine in suppression of inflammatory cytokine production [J]. Crit Care Med ,2007,35(10):2367-74.
[19] Zhang H, Ma H, Lu B, et al .Inhibitory effect of dietary n-3 polyunsaturated fatty acids to intestinal IL-15 expression is associated with reduction of TCRalphabeta+CD8alpha+CD8beta- intestinal intraepithelial lymphocytes[J]. Nutr Biochem, 2007,( 11) 367-71
[20] Werner T , Haller D. Intestinal epithelial cell signalling and chronic inflammation: From the proteome to specific molecular mechanisms[J]. Mutat Res, 2007,(22)42-57
[21] Sydora BC, Macfarlane SM, Walker JW, et al. Epithelial barrier disruption allows nondisease-causing bacteria to initiate and sustain IBD in the IL-10 gene-deficient mouse[J]. Inflamm Bowel Dis, 2007,13(8):947-54
[22] Laharie D, Menard S, Asencio C, et al .Effect of rebamipide on the colonic barrier in interleukin-10-deficient mice[J]. Dig Dis Sci, 2007,52(1 ):84-92.
[23] Hale LP, Gottfried MR, Swidsinski A, et al. Piroxicam treatment of IL-10-deficient mice enhances colonic epithelial apoptosis and mucosal exposure to intestinal bacteria[J].Inflamm Bowel Dis, 2005,11 (12): 1060-9.
[24] Campbell J, Ciesielski CJ, Hunt AE, et al. A novel mechanism for TNF-alpha regulation by p38 MAPK: involvement of NF-kappa B with implications for therapy in rheumatoid arthritis[J]. Immunol, 2004 ,173(11):6928-37.
[25] Lappas M, Permezel M, Georgiou HM, et al. Nuclear factor kappa B regulation of proinflammatory cytokines in human gestational tissues in vitro. Biol Reprod[J]. 2002,67(2):668-73.
[26] Weaver SA, Russo MP, Wright KL, et al. Regulatory role of phosphati- dylinositol 3-kinase on TNF-alpha-induced cyclooxygenase 2 expression in colonic epithelial cells[J]. Gastroenterology, 2001 ,120(5):1117-27.
[27] Jeong HJ, Koo HN, Na HJ, et al. Inhibition of TNF-alpha and IL-6 production by Aucubin through blockade of NF-kappaB activation RBL-2H3 mast cells[J]. Cytokine, 2002 ,18(5):252-9.
[28] Zingarelli B, Yang Z, Hake P W , et al.A bsence of endogenous interleuk -10 enhances early stress response during postischaemic injury in mice intestine[J]. Gut, 2001,48 (5): 610-21
[29] W aetzig G H, Seegert D, Ro senstiel P, et al. p38 m itogen activated protein kinase is activated and linked to TNF-alpha signaling in inflammato ry bowel disease[J]. Immunol,2002,168 (10 ) :5342 -51
[30] Tho rpe C M , Hurley B P, L incicome L L , et al. Sh iga toxins stimulate secretion of interleuk in 8 from intestinal ep ithelial cells[J]. Infect Immun, 1999, 67(11) :5985-91
[31] Kong PJ, Lee HJ, Lee SH, et al. c-Jun N-terminal kinase and p38 mitogen-activated protein kinase mediate double-strand RNA-induced inducible nitric oxide synthase expression in microglial cells[J].Neurosci Lett, 2008 ,433(3):215-8.
[1]Souza KL,Gurgul-Convey E,Elsner M,et al..Interaction between proinflammatory and anti-inflammatory cytokines in insulin-producing cells[J].Endocrinol,2008,197(1):139-50
[2]Kramer F,Torzewski J,Kamenz J,et al.Interleukin-lbeta stimulates acute phase response and C-reactive protein synthesis by inducing an NFkappaB- and C/EBPbeta-dependent autocrine interleukin-6 loop[J].Mol Immunol.2008,45(9):2678-89.
[3]Guo W,Wise ML,Collins FW,et al.Avenanthramides,polyphenols from oats,inhibit IL-1 beta-induced NF-kappaB activation in endothelial cells[J].Free Radic Biol Med,2008,44(3):415-29.
[4]Adler HS,Steinbrink K,et al.MAP kinase p38 and its relation to T cell anergy and suppressor function of regulatory T cells[J].Cell Cycle,2008,7(2):169-70.
[5]Son YH,Jeong YT,Lee KA,et al.Roles of MAPK and NF-kappaB in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells.J Cardiovasc Pharmacol[J].2008,51(1):71-7
[6]Rajapakse N,Kim MM,Mendis E,et al.Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW264.7cells by carboxybutyrylated glucosamine takes place via down-regulation of mitogen-activated protein kinase-mediated nuclear factor-kappaB signaling[J]. Immunology. 2008,123(3):348-57.
[7] Li Y, Chu N, Hu A, et al .Inducible IL-23p 19 expression in human microglia via p38 MAPK and NF-kappaB signal pathways[J]. Exp Mol Pathol,2008, 84(1): 1-8.
[8] Fitzgerald DC, Meade KG, McEvoy AN, et al .Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells[J]. Vet Immunol Immunopathol, 2007,116(1-2):59-68.
[9] Lahat G, Halperin D, Barazovsky E, et al .Immunomodulatory effects of ciprofloxacin in TNBS-induced colitis in mice[J]. Inflamm Bowel Dis, 2007, 13(5):557-65
[10] Abke S, Neumeier M, Weigert J, et al . Adiponectin-induced secretion of interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1,CCL2) and interleukin-8 (IL-8, CXCL8) is impaired in monocytes from patients with type I diabetes[J]. Cardiovasc Diabetol, 2006 ,5-17.
[11] Shalit I, Halperin D, Haite D, et al. Anti-inflammatory effects of moxifloxacin on IL-8, IL-1beta and TNF-alpha secretion and NFkappaB and MAP-kinase activation in human monocytes stimulated with Aspergillus fumigatus[J]. Antimicrob Chemother, 2006,57(2):230-5.
[12] Yeh CH, Chen TP, Wu YC, et al .Inhibition of NFkappaB activation with curcumin attenuates plasma inflammatory cytokines surge and cardiomyocytic apoptosis following cardiac ischemia/reperfusion[J]. Surg Res, 2005,125(1): 109-16
[13] ShishodiaS, KoulD, AggarwalBB. et al .Cyclooxygenase(COX) 2 inhibitor celecoxibabrogates TNF induced NF— kappaB activation through inhibition of activation of IkappaBalpha kinase and Aktinhumannon small lcelllung- rcinoma: correlation with suppres sionof COX 2synthesis[J]. Immunol, 2004,73(3):2011-22.
[14] De Plaen IG , Han XB , Liu X , et al .Lipopolysaccharide induces CXCL2/macrophage inflammatory protein-2 gene expression in enterocytes via NF-kappaB activation: independence from endogenous TNF-alpha and platelet-activating factor[J]. Immunology, 2006 ,118(2): 153-63.
[15] Grishin AV, Wang J, Potoka DA, et al .Lipopolysaccharide induces cyclooxygenase-2 in intestinal epithelium via a noncanonical p38 MAPK pathway[J]. Immunol, 2006 ,176(1):580-8.
[16] Kim JY, Park SJ, Yun KJ, et al .Isoliquiritigenin isolated from the roots of Glycyrrhiza uralensis inhibits LPS-induced iNOS and COX-2 expression via the attenuation of NF-kappaB in RAW 264.7 macrophages[J]. Eur J Pharmacol, 2008,584(1):175-84.
[17] Liu S, Feng G, Wang GL, et al. p38 inhibition attenuates LPS-induced acute lung injury involvement of NF-kappaB pathway [J]. Eur J Pharmacol. 2008,584(1):159-65.
[18] Wu LC, Fan NC, Lin MH , et al .Anti-inflammatory Effect of Spilanthol from Spilanthes acmella on Murine Macrophage by Down-Regulating LPS-induced Inflammatory Mediators[J]. Agric Food Chem, 2008,56(7):2341-9.
[19] Son YH, Jeong YT, Lee KA, et al .Roles of MAPK and NF-kappaB in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells[J]. Cardiovasc Pharmacol, 2008,51(1):71 -7
[20] Kim BH, Roh E, Lee HY, Lee IJ, et al . Benzoxathiole derivative blocks lipopolysaccharide-induced nuclear factor-kappaB activation and nuclear factor-kappaB-regulated gene transcription through inactivating inhibitory kappaB kinase beta[J]. Mol Pharmacol, 2008 ,73(4):1309-18.
[21] Tanaka T, Oh-Hashi K, Shitara H, et al .NF-kappaB independent signaling pathway is responsible for LPS-induced GDNF gene expression in primary rat glial cultures[J]. Neurosci Lett, 2008,431(3):262-7.
[22] Jung HW, Chung YS, Kim YS, et al. Celastrol inhibits production of nitric oxide and proinflammatory cytokines through MAPK signal transduction and NF-kappaB in LPS-stimulated BV-2 microglial cells[J]. Exp Mol Med, 2007,39(6):715-21.
[23] Jones E, Adcock IM, Ahmed BY , et al .Modulation of LPS stimulated NF-kappaB mediated Nitric Oxide production by PKCepsilon and JAK2 in RAW macrophages[J]. Inflamm (Lond), 2007, (24):4:23
[24] Xu Y, Liu XD, Gong X, Eissa NT , et al .Signaling pathway of autophagy associated with innate immunity[J]. Autophagy, 2008,4(1 ):110-2.
[25] Kita T, Ogawa M, Sato H, et al .Role of p38 mitogen-activated protein kinase pathway on heart failure in the infant rat after burn injury[J]. Int J Exp Pathol, 2008,89(1):55-63.
[26] Huh JE, Yim JH, Lee HK, et al. Prodigiosin isolated from Hahella chejuensis suppresses lipopolysaccharide-induced NO production by inhibiting p38 MAPK, JNK and NF-kappaB activation in murine peritoneal macrophages[J]. Int Immunopharmacol, 2007 ,7(13):1825-33.
[27] Son YH, Jeong YT, Lee KA , et al .Roles of MAPK and NF-kappaB in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells[J]. J Cardiovasc Pharmacol, 2008,51(1):71-7
[28] Usluoglu N,Pavlovic J, Moelling K , et al. RIP2 mediates LPS-induced p38 and IkappaBalpha signaling including IL-12 p40 expression in human monocyte-derived dendritic cells[J]. Eur J Immunol, 2007,37(8):2317-25.
[29] Lappas M, Permezel M, Rice GE , et al. Mitogen-activated protein kinase proteins regulate LPS-stimulated release of pro-inflammatory cytokines and prostaglandins from human gestational tissues[J].Placenta. 2007,28(8-9): 936-45.
[30] Lin WN, Luo SF, Lee CW, et al. volvement of MAPKs and NF-kappaB in LPS-induced VCAM-1 expression in human tracheal smooth muscle cells[J]. Cell Signa,2007,19(6):1258-67.
[31] Pladzyk A, Reddy AB, Yadav UC , et al . Inhibition of aldose reductase prevents lipopolysaccharide-induced inflammatory response in human lens epithelial cells[J]. Invest Ophthalmol Vis Sci, 2006,47(12):5395-403
[32] Emre Y, Hurtaud C, Nubel T , et al. Mitochondria contribute to LPS-induced MAPK activation via uncoupling protein UCP2 in macrophages[J]. Biochem J, 2007 ,402(2):271-8
[33] Jung KK, Lee HS, Cho JY, et al .Inhibitory effect of curcumin on nitric oxide production from lipopolysaccharide-activated primary microglia[J]. Life Sci, 2006,79(21):2022-31.
[34] CarlVS, Brown SteinkeK, NicklinMJ, et.al. oil like receptor and (TLR2 and TLR)agenists differentially regulate secretoryinterleuk in lrecepto- rant agonist gene expression in macrophages[J].BiolChem, 2002,77(20): 17448- 56.
[35] Elass E, Coddeville B, Kremer L, et.al. Mycobacterial lipomannan induces MAP kinase phosphatase-1 expression in macrophages[J]. FEBS Lett, 2008, 582(3): 45-50.
[36] Hou N, Torii S, Saito N, et.al. Reactive Oxygen Species-Mediated Pancreatic {beta}-Cell Death Is Regulated by Interactions between Stress-Activated Protein Kinases, p38 and c-Jun N-Terminal Kinase, and Mitogen-Activated Protein Kinase Phosphatases[J]. Endocrinology, 2008,149(4):1654-65.
[37] Kinney CM, Chandrasekharan UM, Mavrakis L, et.al. VEGF and thrombin induce MKP-1 through distinct signaling pathways: role for MKP-1 in endothelial cell migration[J]. Am J Physiol Cell Physiol, 2008,294(1):241-50.
[38] Venugopal SK, Chen J, Zhang Y, et.al. Role of MAPK phosphatase-1 in sustained activation of JNK during ethanol-induced apoptosis in hepatocyte-like VL-17A cells[J]. Biol Chem. 2007,282(44):31900-8.
[39] Jang BC, Lim KJ, Suh MH, et.al. Dexamethasone suppresses interleukin-1 beta induced human beta-defensin mRNA expression: involvement of p38 MAPK, JNK, MKP-1, and NF-kappaB transcnptional factor in A549 cells[J]. FEMS Immunol Med Microbiol, 2007 ,51(1):171-84.
[40] Ito A, Suganami T, Miyamoto Y, et.al. Role of MAPK phosphatase-1 in the induction of monocyte chemoattractant protein-1 during the course of adipocyte hypertrophy [J]. J Biol Chem, 2007 ,282(35):25445-52.
[41] Kondoh K, Sunadome K, Nishida E. Notch signaling suppresses p38 MAPK activity via induction of MKP-1 in myogenesis[J]. Biol Chem. 2007(2), 3058-65.
[42] Nimah M., Zhao Denenberg, A.G Bueno, et.al.ContributionofMKP-1 regulation of p38 to endotox into lerance[J]. hock, et.al.2005,23:80-87