黄芪多糖对内毒素致小肠上皮细胞损伤的保护作用
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摘要
前言
肠上皮细胞是肠道粘膜屏障的重要组成部分,同时被认为在宿主粘膜表面的天然及获得性免疫系统中起中心调节作用,是宿主与病原微生物双向联系的第一道防御。一些肠道疾病和非肠道疾病均可引发肠功能障碍,各种病理因素如创伤、休克、严重感染等重症应激状态时,全身免疫功能低下,大量肠道内细菌和内毒素LPS侵入体循环及肠组织中,造成细菌移位和肠源性内毒素血症,从而进一步加剧肠上皮细胞的损伤,激活细胞内一系列的免疫反应,导致一些炎症介质如TNF-α、IL-6、IL-8、血小板激活因子等大量的产生和释放,引起全身炎症反应综合征(SIRS),启动并加速多系统器官功能衰竭(MSOF)。研究LPS介导的肠上皮细胞损伤已经成为一个广泛注意的研究领域。近年来已经取得很大进展。
NF-κB是肠道免疫功能相关的许多基因表达的关键调节因子,它为许多细胞因子(IL-1、IL-2、IL-6、IL-8、IL-12等)在淋巴细胞、上皮细胞、单核细胞表达中起着不可缺少的作用。目前已经明确,LPS作用下,肠道上皮细胞内NF-κB可被激活,高效诱导多种细胞因子(IL-1、IL-6、IL-8、TNF、粒细胞-巨噬细胞集落刺激因子),粘附分子等的基因表达增加,同时对参与炎症反应的放大与级联瀑布效应的多种酶的基因表达也具有重要的调控作用。由于NF-κB的激活是目前所认识的导致肠道炎症损伤的重要通路,因此以这条通路出发,研究阻断NF-κB激活途径以阻断调控炎症反应蛋白合成,达到抑制炎症反应的作用是治疗肠道损伤的一个很有前景的途径。
丝裂原活化蛋白激酶(MAPK)是一类细胞内广泛分布的丝氨酸/苏氨酸残基的蛋白激酶,是一族连接细胞膜表面受体与决定性基因表达之间的重要信号调节酶。在哺乳细胞至少已克隆了四个MAPK亚族。分别为细胞外信号调节激酶(ERK1/ERK2)、cJun氨基末端激酶(JNK1/JNK 2)、P38 MAPK(α、β)和ERK5。MAPK被激活后可停留在胞质中,激活一系列其它蛋白激酶,使细胞骨架成分磷酸化,亦可经核转位进入细胞核激活各自的核内转录因子,使其发生磷酸化,从而启动某些基因表达,促进有关蛋白质的合成和通道改变,完成对细胞外刺激的反应。Grishin等学者采用LPS刺激体外培养的IEC-6细胞发现,P38MAPK磷酸化及COX-2表达增强,并具有剂量依赖性。这项研究从体外实验角度证实了P38MAPK参与了LPS所致的肠道上皮细胞的损伤。MAPK通过多层次、多方面的信号调节,影响着基因表达、介质释放和细胞内其他信号通路,参与了肠道细胞的促炎因子释放等损伤过程,因此在信号通路水平阻断和调控MAPK信号分子的表达和活性将为治疗肠道损伤提供新的思路和途径。
黄芪为我国传统中药,因其低毒、副反应小且具有增强和恢复免疫功能等作用,目前国内已经将其应用于防治肿瘤、免疫功能低下和免疫缺陷等疾病,发挥其增强免疫和免疫调节剂的作用。黄芪多糖是黄芪发挥作用的一种重要的单体成分。王立新等给内毒素处理小鼠腹腔注射黄芪多糖,发现它能拮抗内毒素引起的肝匀浆中丙二醛(MDA)升高及还原型谷胱甘肽(GSH)降低,对内毒素处理小鼠肝脏线粒体结构的损伤有保护作用。路景涛研究证实黄芪多糖能够抑制细菌脂多糖诱导大鼠腹腔巨噬细胞释放的肿瘤坏死因子、白细胞介素和一氧化氮的分泌,故体内实验研究已经说明黄芪多糖可以通过抑制炎症因子的分泌从而减少组织细胞的损伤来发挥其对机体的免疫保护作用。
目前国内外关于黄芪多糖作用的机理研究较多,但尚无关于黄芪多糖是否对LPS导致的肠道损伤具有保护作用的研究,以及这种保护作用是否与信号转导通路有关。本研究即是以肠上皮细胞IEC-6为研究对象,研究LPS导致IEC-6细胞损伤后,黄芪多糖是否能有效的促进上皮细胞增殖从而起到肠粘膜屏障修复作用;是否通过抑制MAPK/NF-κB信号转导通路从而抑制炎症因子释放,发挥其肠道免疫保护作用。通过研究旨在探讨黄芪多糖发挥肠道保护作用的调节作用机制,从而为其临床应用提供理论依据。
实验方法
一、材料
IEC-6细胞株购自中国医学科学院肿瘤医院生物检测中心。将IEC-6细胞株复苏培养,用含10%胎牛血清的DMEM培养基,配以0.01mg/ml的胰岛素,在CO2培养箱(37℃,5%CO2)中培养;次日换液,至80%左右融合时,0.25%胰酶消化传代,5天传代一次,传代5次后的细胞用于实验分组。
将培养的细胞随机分为五大组:
1组:空白对照组:以DMEM培养液为空白对照;
2组:单纯LPS组:LPS浓度为10 ug/ml;
3组:单纯APS组:分为四亚组:①APS50 ug/ml;②APS100 ug/ml;③APS200ug/ml;④APS500 ug/ml;
4组:LPS与不同浓度APS组:分为四亚组;①LPS10 ug/ml+APS50ug/ml组;②LPS10 ug/ml+APS 100 ug/ml组;③LPS10 ug/ml+APS 200 ug/ml组;④LPS10ug/ml+APS 500 ug/ml组;
5组:APS与不同浓度LPS组:分为三亚组:①APS500 ug/ml+LPS 5 ug/ml;②APS500 ug/ml+LPS 10 ug/ml;③APS500 ug/ml+LPS 20 ug/ml。
二、方法
(一)MTT方法检测细胞增殖率
1、将正常IEC-6细胞置于CO2培养箱(37℃,5%CO2)中,给予不同浓度APS(50 ug/ml,100 ug/ml,200 ug/ml,500 ug/ml)分别培育24小时,48小时,72小时,MTT方法检测单纯APS对正常IEC-6细胞增殖率的影响;
2、将IEC-6细胞中加入LPS(10ug/ml)作用1小时后,在培养体系中加入不同浓度的APS(50 ug/ml,100 ug/ml,200 ug/ml,500 ug/ml)分别培育24小时,48小时,72小时,MTT方法检测APS对LPS损伤后的IEC-6细胞增殖作用;
3、将培养的IEC-6细胞用APS500ug/ml预处理24小时,然后加以不同浓度的LPS(分别为5ug/ml,10ug/ml,20ug/ml),观察1小时,MTT方法测定APS预处理后对LPS损伤IEC-6细胞增殖率的影响。
(二)RT-PCR方法检测TNF-α及IL-8mRNA
IEC-6细胞在DMEM培养基中加入不同浓度的APS(50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml)培育24小时。1
、给予LPS(10ug/ml)刺激1小时后,收集细胞,提取并检测总RNA,合成cDNA,PCR扩增TNF-α,IL-8和GAPDH,琼脂糖凝胶电泳分析APS对LPS损伤IEC-6分泌的TNF-α及IL-8mRNA的表达的影响;
2、分别给予LPS(10ug/ml)刺激1小时、4小时后收集细胞,提取并检测总RNA,合成cDNA,PCR扩增TNF-α,IL-8和GAPDH,琼脂糖凝胶电泳分析不同时间点的APS对LPS损伤IEC-6分泌TNF-α及IL-8mRNA的变化。
(三)Western-Blotting方法检测P-ERK1/2,P-JNK,P-P38及NF-κB,IκB-α蛋白的表达
IEC-6细胞在DMEM培养基中加入不同浓度的APS(50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml)培育24小时。
1、给予LPS(10ug/ml)刺激15分钟,收集细胞,提取细胞总蛋白并定量,SDS凝胶电泳,转膜并封闭后,抗原抗体杂交孵育,显色后凝胶成像仪分析APS对LPS损伤IEC-6分泌的IκB-α蛋白的表达影响;
2、给予LPS(10ug/ml)刺激30分钟,方法同前,提取核内蛋白并定量,检测APS对LPS损伤IEC-6分泌的NF-κB蛋白的表达影响;
3、给予LPS(10ug/ml)刺激1小时,方法同前,检测APS对LPS损伤IEC-6分泌的P-ERK1/2,P-JNK,P-P38蛋白的表达影响。
三、统计学分析
所有数据以均值±标准差((?)±s)表示,用SPSS11.5统计软件包采用单因素方差分析LSD法检验。P＜0.05有显著性差异。
实验结果
一、LPS作用下IEC-6形态学改变
正常小肠上皮细胞由均匀的上皮样细胞群体组成,呈铺路石镶嵌排列,互不重叠,为典型单层,细胞为不规则多角形,边界清楚,细胞核较大,呈现卵圆形,细胞间相互连接,呈现旺盛的增殖活性。LPS损伤的小肠上皮细胞呈现细胞边界模糊,细胞由多角形转变为圆形,细胞胞浆内出现大量颗粒样物质,部分细胞膜破裂,细胞形态不完整。
二、APS对LPS损伤IEC-6细胞增殖作用的影响
1、APS对正常IEC-6细胞具有促增殖作用。MTT结果分析表明:APS在不同时间对细胞增殖作用不同,且不同浓度的APS处理后对细胞的增殖作用有显著性差异(P＜0.05)。APS对IEC-6细胞呈现剂量依赖性促增殖作用,随着处理时间的延长逐渐减弱,表现为第24小时APS50ug/ml即显示出促增殖作用,而在第72小时,APS浓度为200ug/ml时才表现出显著的促增殖效应。
2、APS对LPS损伤IEC-6细胞后不具有显著的促细胞增殖作用。MTT结果发现:受LPS作用的细胞仅在第24小时APS浓度为200ug/ml和500ug/ml时表现有促增殖作用,而在48小时及72小时检测时间点及不同浓度APS组均未见到显著促增殖效应(P＞0.05)。故当IEC-6细胞受到LPS损伤时,APS未能显现出显著的促增殖作用。
3、APS预处理能增强LPS损伤后IEC-6细胞的增殖活性。MTT结果发现:在受LPS作用5ug/ml时,与对照组比较,APS预处理组没有促增殖作用(P＞0.05)。在受LPS10ug/ml以及20ug/ml作用时,与对照组比较,APS预处理组有显著的促增殖作用(P＜0.01)。
三、APS对LPS刺激IEC-6细胞分泌产生的TNF-α、IL-8 mRNA的影响
1、RT-PCR检测结果显示,当LPS刺激IEC-6细胞时,TNF-α、IL-8 mRNA被诱导分泌具有显著意义的增加(P＜0.01),而黄芪多糖(APS)可以抑制这种分泌的增加,并且具有剂量依赖性:50ug/ml黄芪多糖可以部分抑制LPS刺激IEC-6产生的TNF-α及IL-8 mRNA水平,而200ug/ml及500ug/ml黄芪多糖随着浓度的增加,其抑制TNF-α及IL-8 mRNA的水平逐渐增加(P＜0.01)。
2、RT-PCR检测结果同样显示:黄芪多糖(APS)抑制TNF-α、IL-8 mRNA分泌增加具有时间依赖性:LPS诱导TNF-αmRNA表达被黄芪多糖有效的抑制,1小时和4小时的抑制率分别为10.3%和25.5%,LPS诱导IL-8 mRNA表达同样被黄芪多糖有效的抑制:1小时和4小时的抑制率分别为15.3%和18.8%。
四、APS对LPS诱导IEC-6细胞产生的NF-κB蛋白的表达影响分析
Western-Blotting方法检测结果显示,当LPS刺激IEC-6细胞时,NF-κB的抑制蛋白IκB-a表达降低,NF-κB蛋白表达具有显著意义的增加(P＜0.01),黄芪多糖(APS)显著抑制这种作用,并且具有剂量依赖性,随着浓度的增加,500ug/mlAPS表现出更强的抑制NF-κB蛋白的表达作用(P＜0.01)。
五、APS对LPS刺激IEC-6细胞分泌的P-ERK1/2,P-JNK,P-P38蛋白作用的表达分析
通过检测APS对LPS所致的IEC-6细胞分泌P-P38在蛋白水平上的影响研究发现,当LPS刺激IEC-6细胞时,P38蛋白磷酸化增强,被诱导分泌具有显著意义的增加(P＜0.01),APS抑制P38蛋白磷酸化,随着APS浓度的增加,其抑制P38蛋白磷酸化作用逐渐增强,其中APS浓度在500ug/ml时抑制作用最强。同样,LPS导致的IEC-6细胞分泌的ERK1/2及JNK蛋白磷酸化也有明显表达,且差异具有统计学意义(P＜0.05),但是APS对LPS诱导引起的ERK1/2及JNK蛋白磷酸化表达却无显著意义影响(P＞0.05)。
结论
1、黄芪多糖对正常IEC-6细胞具有促进细胞增殖的作用;当细胞受到LPS损伤时,黄芪多糖却不能显现出显著的促增殖修复作用,提示黄芪多糖的促细胞增殖作用有赖于细胞处于正常的生理状态;黄芪多糖预处理能增强LPS损伤后IEC-6细胞的增殖活性,说明黄芪多糖早期干预能够降低LPS对IEC-6细胞的损伤而显现出对细胞的保护作用,APS的治疗效果也取决于LPS的损伤程度的轻重。
2、LPS作用于小肠上皮细胞后,细胞因子TNF-α和IL-8 mRNA大量表达,而APS可以抑制LPS刺激细胞分泌的TNF-α和IL-8 mRNA的过量产生,且APS对这种抑制作用具有浓度及时间依赖性。说明黄芪多糖对小肠上皮细胞的免疫保护作用可能是通过抑制细胞因子TNF-α、IL-8等的过量表达,从而减少其对组织细胞的炎性损伤。
3、LPS刺激IEC-6时,IκB-a蛋白表达降低,细胞核内NF-κB蛋白表达增加,而APS能够有效抑制NF-κB蛋白表达的增长及IκB-a表达的降低,且随着黄芪多糖浓度的增加,抑制NF-κB蛋白的作用逐渐增强。说明黄芪多糖抑制细胞因子的过量表达可能是通过抑制核因子NF-κB信号通路来实现的。
4、LPS作用于小肠上皮细胞后,其分泌的P38MAPK蛋白磷酸化增强,APS可以显著抑制这种分泌的增加,并具有剂量依赖性,说明APS对LPS所致的IEC-6细胞的免疫保护作用也可能是通过P38MAPK信号通路实现的。LPS作用于小肠上皮细胞后,其分泌的ERK1/2及JNK蛋白磷酸化同样增强,但是APS对ERK1/2及JNK蛋白磷酸化没有显著意义影响。说明APS对LPS所致的IEC-6细胞的免疫保护作用不是通过ERK1/2及JNK信号通路实现的。
Objective
Intestinal epithelial cell is an important component of intestinal mucosal barrier.It is the first mucosal surface of the body and considered as acquired immune systems of the host which plays a central role in regulating.It is also the first two-way connection defense between pathogenic microorganisms and the host.Some non-intestinal diseases and gastrointestinal diseases can cause intestinal dysfunction.When severe stress state, the systemic immune function is low.A large number of intestinal bacteria and endotoxin intruse circulation and cause intestinal bacterial translocation and endotoxemia,which will further aggravate the intestinal epithelial cell injury,activate a series of immune response,result in a number of inflammatory mediators such as TNF-α、IL-6、IL-8、platelet activating factor.A large number of production can cause systemic inflammatory response syndrome(SIRS),activate and accelerate the multi-system organ failure(MSOF).Research on LPS-mediated intestinal epithelial cell injury has become a widespread area of study.Much progress has been made in recent years.
NF-κB is the key factor of the expression of many genes regulated the intestinal immune function,which play an indispensable role for many cytokines(IL-1、IL-2、IL-6、IL-8、IL-12) expressed in lymphocytes,epithelial cells and mononuclear cells.Now clear that,NF-κB in intestinal epithelial cells may be activated when stimulated by LPS.NF-κB can induce the increase in gene expression of multi-cytokine (IL-1、IL-6、IL-8、TNF),adhesion molecules,chemokines factor and the acute phase protein.Therefore,the NF-κB,as a transcription factor of the continue transformation in the gastrointestinal tract,regulate the expression of inflammatory genes,trigger and expand the inflammatory response,result in the occurrence of disease.Since the activation of NF-κB is known as an important pathway leading to intestinal epithelial cell injury,the study on blocking NF-κB activation in order to control and inhibit inflammatory response is a promising way in the treatment of gastro-intestinal injury.
Mitogen-activated protein kinase(MAPK) is the serine/threonine residues of the protein kinase widely distributed within the cells.It is also an important signal regulatory enzymes connected the cell surface receptor with its decisive gene expression.In mammalian cells,it have been cloned at least four MAPK family: respectively extracellular signal- regulated kinase(ERK1/ERK2),cJun N-terminal kinase(JNK1/JNK 2),P38 MAPK(α,β) and ERK5.The activation of MAPK can stay in the cytoplasm to range other protein kinase.As a result,cytoskeletal components phosphorylate.MAPK is activated by phosphorylation,translocated to the nucleus, which trigger its transcription factor gene expression and promote the protein synthesis.Grishin and other scholars found that P38MAPK and COX-2 phosphorylation expression increased when IEC-6 cells stimulated by LPS in vitro.The phosphorylation of P38 induced by LPS in IEC-6 with a dose-dependent manner.The study had confirmed that P38MAPK involved in the inflammation of the intestinal epithelial cells induced by the LPS in vitro.MAPK affect gene expression,the media release and other cell signaling pathways through a multi-level,multi-faceted signal conditioning.It involved the release of pro-inflammatory cytokines and apoptosis injury in the intestinal cells.It can be believed that blocking and controlling MAPK signaling molecule expression and activity of intestinal injury will provide new ideas and approaches.
Astragalus is our traditional Chinese medicine.Research shows that the Astragalus can promote normal development of immune organs and impaired immune organ recovery,enhance and restore immune function.It has been used to control tumor,immunocompromised and immunodeficiency diseases in China.Astragalus mongholicus polysaccharides(APS) is an important component of Astragalus.Many studies have confirmed that the APS regulate immune and stimulate the hematopoietic function.Wang Li xin found that APS antagonized the drug from the liver homogenate index of malondialdehyde(MDA) levels and reduced glutathione(GSH).Lu Jing Tao has confirmed the APS can inhibit lipopolysaccharide-induced rat peritoneal macrophages releasing of tumor necrosis factor,interleukin-1 and the secretion of nitric oxide.It showed that APS can inhibit the secretion of inflammatory cytokines thereby reducing tissue injury to play its role in immune protection.
At home and abroad,no study was done on whether the APS has a protective effect in intestinal injury caused by LPS,as well as whether the protective effect of APS related with some signal transduction pathway.After the IEC-6 cells were stimulated by LPS,We studied whether the APS can effectively promote cell proliferation,whether APS plays the role in the protection of intestinal realated with the signal of MAPK/NF-κB transduction pathway.Through the study,we explore the mechanism of APS in intestinal protection so as to provide a theoretical basis for clinical application.
Material and methods
1.Materials
IEC-6 cells were purchased from the Test Center of Chinese Academy of Medical Sciences.IEC-6 cells were cultured with DMEM medium supplemented with 10%fetal bovine serum and 0.01 mg/ml insulin.IEC-6 ceils were cultured in the CO_2 incubator(37℃,5%CO_2).It is soluted for the next day to about 80 per cent covered, then incubated with trypsin(0.25%).Cells from passage 5 were used for study.
The cultured cells will be divided into 5 groups:Group 1:the control group,add DMEM only for control;Group 2:LPS group,the cultured cells by adding LPS(10 ug/ ml);Group 3:APS group:it divide into 4 sub-group:①APS50 ug/ml;②APS100 ug/ml;③APS200 ug/ml;④APSS00 ug/ml;Group 4:LPS and different concentration APS group:it divide into 4 sub-group:①LPS10 ug/ml+APS50ug/ml;②LPS10 ug/ml +APS 100 ug/ml;③LPS10 ug/ml+APS 200 ug/ml;④LPS10 ug/ml+APS 500 ug/ml; Group 5:APS and different concentration LPS group:it divide into 3 sub-group:①APS500 ug/ml+LPS 5 ug/ml;②APS500 ug/ml+LPS 10 ug/ml;③APS500 ug/ml+LPS 20 ug/ml.
2.Methods
The rat small intestinal cell line IEC-6 was grown in Dulbecco's modified.
Eagle's medium(DMEM) supplemented with 10%FBS and 0.01mg/ml insulin.IEC-6 cells were cultured at 37℃in a humidified atmosphere containing 5%CO2 with various concentration of APS(50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for 24h,48h,72h.After incubation,Cell proliferation was measured by MTT assay to observe the effects of APS on normal IEC-6.The IEC-6 cells were stimulated with LPS(10ug/ml) for 1h,then added with various concentration of APS (50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for 24h,48h,72h.Cell proliferation was measured by MTT assay to observe the effects of APS on LPS- stimulated IEC-6.The IEC-6 cells were pretreated with APS(500ug/ml) for 24h,then added with various concentration of LPS(5ug/ml,10ug/ml,20ug/ml) for 1h.After incubation,Cell proliferation was measured by MTT assay to observe the effects of pretreated APS on LPS-stimulated IEC-6.
Reverse Transcriptase-Mediated PCR(RT-PCR) Analysis detect TNF-αmRNA and IL-8 mRNA.
IEC-6 cells were culture in DMEM with various concentration of APS(50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for24h,then added LPS(10ug/ml) for 1h and 4h to detect TNF-αmRNA and IL-8 mRNA.
Western-Blotting assay to detect protein of P-ERK1/2,P-JNK,P-P38,NF-κB and IκB-α.
The cells were cultured in DMEM medium with various concentrations of APS (50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for 24h,then added LPS for 15 min to detect IκB-α,for 30 min to detect NF-κB,and for 1h to detect phosphorylated-P38, phosphorylated-ERK1/2,phosphorylated-JNK.
3.Statistical analysis
SPSS11.5 was used to perform statistical analysis,with all data expressed as mean±SEM.Ststistically significantdifferences in the values were analyzed with ANOVA for inter-group comparion.P-value＜0.05 were considered statistically significant.
Results
1.Morphology change in IEC-6
Normal intestinal epithelial cells were embedded as paving stones,not overlapping. A typical single-layer cell was seen clearly with larger nucleus.Intestinal epithelial cells stimulated by LPS were showed fuzzy boundary cells.A large number of cells membrane rupture and cell morphology was not integrity.
2.The proliferation impact of APS on IEC-6 cells stimulated by LPS.
APS can promote normal IEC-6 cell proliferation.
MTT result showed:Different concentrations of APS showed a significant role in promoting IEC-6 cell proliferation(P＜0.05).APS on the IEC-6 cells has a dose-dependent role in promoting proliferation.With the extension of time,the effect of promoting cell proliferation gradually weakened.For the first 24- hour,the concentration of 50 ug/ ml APS can promote the cell proliferation significantly.However,in the 72- hour,the concentration of 200 ug/ ml APS displayed a remarkable effect of promoting cell proliferation.
APS cannot promote IEC-6 cell proliferation which stimulated by LPS.
MTT result showed:APS displayed a nonremarkable effect of promoting cell proliferation on IEC-6 cells stimulated by LPS.Only in the first 24- hour,the concentration of 200 ug/ml and 500 ug/ml APS showed significant role in promoting cell proliferation.In the 48-hour and 72-hour,with the different concentrations of APS did not have effect of promoting cell proliferation(P＞0.05).
Pretreated APS can promote IEC-6 cell proliferation which stimulated by LPS.
MTT result showed:When IEC-6 cells were stimulated by the concentration of 5 ug/ml LPS,Pretreated APS do not promote cell proliferation on IEC-6 compared with the control group(P＞0.05).But when IEC-6 cells were stimulated by the concentration of 10 ug/ml and 20 ug/ml LPS,Pretreated APS group promote cell proliferation on IEC-6 compared with the control group(P＜0.01).The results indicate that, pretreatment with APS can reduce IEC-6 cell injury induced by LPS.
3.APS inhibited the secretion of TNF-α,IL-8 mRNA production in IEC-6 cells stimulated by LPS.
APS abrogates LPS-induced TNF-αmRNA expression in IEC-6.
We first evaluted the effects of APS on LPS-induced TNF-αgene expression in intestinal cell line IEC-6.Stimulation of IEC-6 by LPS markedly increased the production of TNF-α.We examined the effect of APS on the mRNA level of TNF-αfrom IEC-6 when stimulated with LPS.Cells were pretreated with various concentrations of APS(50ug/ml,100ug/ml,200ug/ml,500ug/ml) for 24h,stimulated with LPS(10ug/ml)for 1h and TNF-αgene expression was measured by RT-PCR assay.RT-PCR analysis showed that TNF-αmRNA is induced readily from IEC-6 by LPS.However,this induction was inhibited by APS in concentration- dependent manner:50ug/ml of APS partially supressed TNF-αmRNA from LPS activated IEC-6,and 500ug/ml of APS significantly inhibited the amount of TNF-αmRNA.(P＜0.01 compared with LPS stimulation in the absence of APS).
APS abrogates LPS-induced IL-8 mRNA expression in IEC-6.
We then examined the effect of APS in the production of IL-8 from IEC-6 by LPS.IL-8 mRNA was induced readily from IEC-6 by LPS.However,the mRNA level of IL-8 was inhibited by APS in a concentration- dependent manner:addition of APS partially supressed IL-8 level at 50ug/ml and 100ug/ml,respectively,and 500ug/ml of APS significantly inhibited IL-8 at mRNA level.(P＜0.01 compared with LPS stimulation in the absence of APS).
APS Inhibited both TNF-αand IL-8 Production from LPS-Activated IEC-6 in a time-dependent manner.
Next,IEC-6 cells were pretreated with APS(500ug/ml) for 24h,stimulated with LPS(10ug/ml) for 1h to 4h and TNF-α,IL-8 gene expression was measured by RT-PCR.LPS-induced TNF-αmRNA expression is inhibited by APS treatment(10.3% and 25.5%inhibition at 1h and 4h poststimulation,respectively).LPS-induced IL-8 mRNA expression is inhibited by APS treatment(15.3%and 18.8%inhibition at 1h and 4h poststimulation,respectively).
4.APS inhibited NF-κB protein expression in IEC-6 cells induced by LPS.
It has been reported that APS abrogates LPS-induced pro-inflammatory cytokine production in IEC-6.A critical downstream effector pathway induced by LPS is the NF-κB transcriptional system.NF-κB is usually held in an inactivated state in the cytoplasm of unstimulated cells through interaction with IκB-α(NF-κB inhibitory protein).After stimulation with LPS,the IκB-αis degraded and release the NF-κB to translocate into nucleus,where NF-κB drive the expression of pro-inflammatory cytokines.Thus we next assessed whether APS abrogates LPS-induced NF-κB transcriptional activity.To do this,we examined whether APS blocks NF-κB translocation into nucleus,since nuclear translocation is required for NF-κB-Mediated transcriptional activation.We examined the levels of NF-κB in the nucleus of LPS-stimulated IEC-6 cells in the presence or absence of APS.LPS stimulation resulted in an increase in protein level in the nucleus.Treatment with APS abolished this effect.To dissect the effect of APS on LPS-induced NF-κB signal,we next determined IκB-αprotein involved in NF-κB pathway using Western blot analysis.It shows that LPS decrease IκB-αin IEC-6cells,which are blocked in APS-pretreated cells,suggesting that APS inhibits the translocation of NF-κB into the nucleus,and may subsequently suppress LPS-induced NF-κB activation.
5.The analysis of the expression of MAPK protein in IEC-6 cells induced by APS.
It is reported that the activation of the MAPKS P38 is important signaling pathways responsible for the pro-duction of pro-inflammatory cytokines when IEC-6 is activated.In order to further understand the mechanisms of APS-mediated anti-inflammation in IEC-6,we examined whether APS inhibits LPS-triggered activation of MAPK signalling incluing P38,ERK1/2 and JNK.The active phosphorylated levels of P38,ERK1/2 and JNK were analyzed in LPS-stimulated IEC-6 cells following treatment with or without APS.Results indicated that P38,ERK1/2 and JNK were strongly activated in IEC-6 when stimulated with LPS.P38,ERK1/2 and JNK were all activated in IEC-6 in lh after LPS treatment.However,treatment with APS results in a reduction in LPS-induced P38 phosphorylation.And LPS-induced phosphorylation of P38 is inhibited in a concentration-dependent manner.When 50ug/ml of APS is added,LPS-induced P38 was partially blocked in IEC-6,and 500ug/ml of APS significantly inhibited P38 phosphorylation in LPS-stimulated IEC-6.However,APS did not alter the activation of the protein levels of ERK1/2 and JNK.The observations indicate that APS may inhibit the activation of P38 kinases but not the ERK1/2 and JNK in IEC-6 stimulated by LPS.
Conclusions
1.APS has a role of promoting the normal IEC-6 cell proliferation.APS displayed a nonremarkable effect of promoting cell proliferation on IEC-6 cells stimulated by LPS.When IEC-6 cells were pretreated with APS,APS can promote cell proliferation compared with the control group.The results indicate that pretreatment with APS can reduce IEC-6 cell injury induced by LPS.
2.APS abrogates LPS-induced TNF-αand IL-8 mRNA expression in IEC-6. TNF-αand IL-8 mRNA is induced readily from IEC-6 by LPS.However,this induction was inhibited by APS in concentration- dependent manner.We also confirm APS innhibited both TNF-αand IL-8 production from LPS-Activated IEC-6 in a time-dependent manner.
3.APS inhibited NF-κB protein expression in IEC-6 cells induced by LPS.LPS stimulation resulted in an increase in NF-κB protein level in the nucleus.Treatment with APS abolished this effect.LPS decrease IκB-αin IEC-6 cells,which are blocked in APS-pretreated cells,suggesting that APS inhibits the translocation of NF-κB into the nucleus,and may subsequently suppress LPS-induced NF-κB activation.
4.P38,ERK1/2 and JNK were strongly activated in IEC-6 when stimulated with LPS.However,treatment with APS results in a reduction in LPS-induced P38 phosphorylation.And LPS-induced phosphorylation of P38 is inhibited in a concentration-dependent manner.APS did not alter the activation of the protein levels of ERK1/2 and JNK phosphorylation.APS may inhibit the activation of P38 kinases but not the ERK1/2 and JNK in IEC-6 stimulated by LPS.
肠上皮细胞是肠道粘膜屏障的重要组成部分,同时被认为在宿主粘膜表面的天然及获得性免疫系统中起中心调节作用,是宿主与病原微生物双向联系的第一道防御。一些肠道疾病和非肠道疾病均可引发肠功能障碍,各种病理因素如创伤、休克、严重感染等重症应激状态时,全身免疫功能低下,大量肠道内细菌和内毒素LPS侵入体循环及肠组织中,造成细菌移位和肠源性内毒素血症,从而进一步加剧肠上皮细胞的损伤,激活细胞内一系列的免疫反应,导致一些炎症介质如TNF-α、IL-6、IL-8、血小板激活因子等大量的产生和释放,引起全身炎症反应综合征(SIRS),启动并加速多系统器官功能衰竭(MSOF)。研究LPS介导的肠上皮细胞损伤已经成为一个广泛注意的研究领域。近年来已经取得很大进展。
NF-κB是肠道免疫功能相关的许多基因表达的关键调节因子,它为许多细胞因子(IL-1、IL-2、IL-6、IL-8、IL-12等)在淋巴细胞、上皮细胞、单核细胞表达中起着不可缺少的作用。目前已经明确,LPS作用下,肠道上皮细胞内NF-κB可被激活,高效诱导多种细胞因子(IL-1、IL-6、IL-8、TNF、粒细胞-巨噬细胞集落刺激因子),粘附分子等的基因表达增加,同时对参与炎症反应的放大与级联瀑布效应的多种酶的基因表达也具有重要的调控作用。由于NF-κB的激活是目前所认识的导致肠道炎症损伤的重要通路,因此以这条通路出发,研究阻断NF-κB激活途径以阻断调控炎症反应蛋白合成,达到抑制炎症反应的作用是治疗肠道损伤的一个很有前景的途径。
丝裂原活化蛋白激酶(MAPK)是一类细胞内广泛分布的丝氨酸/苏氨酸残基的蛋白激酶,是一族连接细胞膜表面受体与决定性基因表达之间的重要信号调节酶。在哺乳细胞至少已克隆了四个MAPK亚族。分别为细胞外信号调节激酶(ERK1/ERK2)、cJun氨基末端激酶(JNK1/JNK 2)、P38 MAPK(α、β)和ERK5。MAPK被激活后可停留在胞质中,激活一系列其它蛋白激酶,使细胞骨架成分磷酸化,亦可经核转位进入细胞核激活各自的核内转录因子,使其发生磷酸化,从而启动某些基因表达,促进有关蛋白质的合成和通道改变,完成对细胞外刺激的反应。Grishin等学者采用LPS刺激体外培养的IEC-6细胞发现,P38MAPK磷酸化及COX-2表达增强,并具有剂量依赖性。这项研究从体外实验角度证实了P38MAPK参与了LPS所致的肠道上皮细胞的损伤。MAPK通过多层次、多方面的信号调节,影响着基因表达、介质释放和细胞内其他信号通路,参与了肠道细胞的促炎因子释放等损伤过程,因此在信号通路水平阻断和调控MAPK信号分子的表达和活性将为治疗肠道损伤提供新的思路和途径。
黄芪为我国传统中药,因其低毒、副反应小且具有增强和恢复免疫功能等作用,目前国内已经将其应用于防治肿瘤、免疫功能低下和免疫缺陷等疾病,发挥其增强免疫和免疫调节剂的作用。黄芪多糖是黄芪发挥作用的一种重要的单体成分。王立新等给内毒素处理小鼠腹腔注射黄芪多糖,发现它能拮抗内毒素引起的肝匀浆中丙二醛(MDA)升高及还原型谷胱甘肽(GSH)降低,对内毒素处理小鼠肝脏线粒体结构的损伤有保护作用。路景涛研究证实黄芪多糖能够抑制细菌脂多糖诱导大鼠腹腔巨噬细胞释放的肿瘤坏死因子、白细胞介素和一氧化氮的分泌,故体内实验研究已经说明黄芪多糖可以通过抑制炎症因子的分泌从而减少组织细胞的损伤来发挥其对机体的免疫保护作用。
目前国内外关于黄芪多糖作用的机理研究较多,但尚无关于黄芪多糖是否对LPS导致的肠道损伤具有保护作用的研究,以及这种保护作用是否与信号转导通路有关。本研究即是以肠上皮细胞IEC-6为研究对象,研究LPS导致IEC-6细胞损伤后,黄芪多糖是否能有效的促进上皮细胞增殖从而起到肠粘膜屏障修复作用;是否通过抑制MAPK/NF-κB信号转导通路从而抑制炎症因子释放,发挥其肠道免疫保护作用。通过研究旨在探讨黄芪多糖发挥肠道保护作用的调节作用机制,从而为其临床应用提供理论依据。
实验方法
一、材料
IEC-6细胞株购自中国医学科学院肿瘤医院生物检测中心。将IEC-6细胞株复苏培养,用含10%胎牛血清的DMEM培养基,配以0.01mg/ml的胰岛素,在CO2培养箱(37℃,5%CO2)中培养;次日换液,至80%左右融合时,0.25%胰酶消化传代,5天传代一次,传代5次后的细胞用于实验分组。
将培养的细胞随机分为五大组:
1组:空白对照组:以DMEM培养液为空白对照;
2组:单纯LPS组:LPS浓度为10 ug/ml;
3组:单纯APS组:分为四亚组:①APS50 ug/ml;②APS100 ug/ml;③APS200ug/ml;④APS500 ug/ml;
4组:LPS与不同浓度APS组:分为四亚组;①LPS10 ug/ml+APS50ug/ml组;②LPS10 ug/ml+APS 100 ug/ml组;③LPS10 ug/ml+APS 200 ug/ml组;④LPS10ug/ml+APS 500 ug/ml组;
5组:APS与不同浓度LPS组:分为三亚组:①APS500 ug/ml+LPS 5 ug/ml;②APS500 ug/ml+LPS 10 ug/ml;③APS500 ug/ml+LPS 20 ug/ml。
二、方法
(一)MTT方法检测细胞增殖率
1、将正常IEC-6细胞置于CO2培养箱(37℃,5%CO2)中,给予不同浓度APS(50 ug/ml,100 ug/ml,200 ug/ml,500 ug/ml)分别培育24小时,48小时,72小时,MTT方法检测单纯APS对正常IEC-6细胞增殖率的影响;
2、将IEC-6细胞中加入LPS(10ug/ml)作用1小时后,在培养体系中加入不同浓度的APS(50 ug/ml,100 ug/ml,200 ug/ml,500 ug/ml)分别培育24小时,48小时,72小时,MTT方法检测APS对LPS损伤后的IEC-6细胞增殖作用;
3、将培养的IEC-6细胞用APS500ug/ml预处理24小时,然后加以不同浓度的LPS(分别为5ug/ml,10ug/ml,20ug/ml),观察1小时,MTT方法测定APS预处理后对LPS损伤IEC-6细胞增殖率的影响。
(二)RT-PCR方法检测TNF-α及IL-8mRNA
IEC-6细胞在DMEM培养基中加入不同浓度的APS(50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml)培育24小时。1
、给予LPS(10ug/ml)刺激1小时后,收集细胞,提取并检测总RNA,合成cDNA,PCR扩增TNF-α,IL-8和GAPDH,琼脂糖凝胶电泳分析APS对LPS损伤IEC-6分泌的TNF-α及IL-8mRNA的表达的影响;
2、分别给予LPS(10ug/ml)刺激1小时、4小时后收集细胞,提取并检测总RNA,合成cDNA,PCR扩增TNF-α,IL-8和GAPDH,琼脂糖凝胶电泳分析不同时间点的APS对LPS损伤IEC-6分泌TNF-α及IL-8mRNA的变化。
(三)Western-Blotting方法检测P-ERK1/2,P-JNK,P-P38及NF-κB,IκB-α蛋白的表达
IEC-6细胞在DMEM培养基中加入不同浓度的APS(50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml)培育24小时。
1、给予LPS(10ug/ml)刺激15分钟,收集细胞,提取细胞总蛋白并定量,SDS凝胶电泳,转膜并封闭后,抗原抗体杂交孵育,显色后凝胶成像仪分析APS对LPS损伤IEC-6分泌的IκB-α蛋白的表达影响;
2、给予LPS(10ug/ml)刺激30分钟,方法同前,提取核内蛋白并定量,检测APS对LPS损伤IEC-6分泌的NF-κB蛋白的表达影响;
3、给予LPS(10ug/ml)刺激1小时,方法同前,检测APS对LPS损伤IEC-6分泌的P-ERK1/2,P-JNK,P-P38蛋白的表达影响。
三、统计学分析
所有数据以均值±标准差((?)±s)表示,用SPSS11.5统计软件包采用单因素方差分析LSD法检验。P＜0.05有显著性差异。
实验结果
一、LPS作用下IEC-6形态学改变
正常小肠上皮细胞由均匀的上皮样细胞群体组成,呈铺路石镶嵌排列,互不重叠,为典型单层,细胞为不规则多角形,边界清楚,细胞核较大,呈现卵圆形,细胞间相互连接,呈现旺盛的增殖活性。LPS损伤的小肠上皮细胞呈现细胞边界模糊,细胞由多角形转变为圆形,细胞胞浆内出现大量颗粒样物质,部分细胞膜破裂,细胞形态不完整。
二、APS对LPS损伤IEC-6细胞增殖作用的影响
1、APS对正常IEC-6细胞具有促增殖作用。MTT结果分析表明:APS在不同时间对细胞增殖作用不同,且不同浓度的APS处理后对细胞的增殖作用有显著性差异(P＜0.05)。APS对IEC-6细胞呈现剂量依赖性促增殖作用,随着处理时间的延长逐渐减弱,表现为第24小时APS50ug/ml即显示出促增殖作用,而在第72小时,APS浓度为200ug/ml时才表现出显著的促增殖效应。
2、APS对LPS损伤IEC-6细胞后不具有显著的促细胞增殖作用。MTT结果发现:受LPS作用的细胞仅在第24小时APS浓度为200ug/ml和500ug/ml时表现有促增殖作用,而在48小时及72小时检测时间点及不同浓度APS组均未见到显著促增殖效应(P＞0.05)。故当IEC-6细胞受到LPS损伤时,APS未能显现出显著的促增殖作用。
3、APS预处理能增强LPS损伤后IEC-6细胞的增殖活性。MTT结果发现:在受LPS作用5ug/ml时,与对照组比较,APS预处理组没有促增殖作用(P＞0.05)。在受LPS10ug/ml以及20ug/ml作用时,与对照组比较,APS预处理组有显著的促增殖作用(P＜0.01)。
三、APS对LPS刺激IEC-6细胞分泌产生的TNF-α、IL-8 mRNA的影响
1、RT-PCR检测结果显示,当LPS刺激IEC-6细胞时,TNF-α、IL-8 mRNA被诱导分泌具有显著意义的增加(P＜0.01),而黄芪多糖(APS)可以抑制这种分泌的增加,并且具有剂量依赖性:50ug/ml黄芪多糖可以部分抑制LPS刺激IEC-6产生的TNF-α及IL-8 mRNA水平,而200ug/ml及500ug/ml黄芪多糖随着浓度的增加,其抑制TNF-α及IL-8 mRNA的水平逐渐增加(P＜0.01)。
2、RT-PCR检测结果同样显示:黄芪多糖(APS)抑制TNF-α、IL-8 mRNA分泌增加具有时间依赖性:LPS诱导TNF-αmRNA表达被黄芪多糖有效的抑制,1小时和4小时的抑制率分别为10.3%和25.5%,LPS诱导IL-8 mRNA表达同样被黄芪多糖有效的抑制:1小时和4小时的抑制率分别为15.3%和18.8%。
四、APS对LPS诱导IEC-6细胞产生的NF-κB蛋白的表达影响分析
Western-Blotting方法检测结果显示,当LPS刺激IEC-6细胞时,NF-κB的抑制蛋白IκB-a表达降低,NF-κB蛋白表达具有显著意义的增加(P＜0.01),黄芪多糖(APS)显著抑制这种作用,并且具有剂量依赖性,随着浓度的增加,500ug/mlAPS表现出更强的抑制NF-κB蛋白的表达作用(P＜0.01)。
五、APS对LPS刺激IEC-6细胞分泌的P-ERK1/2,P-JNK,P-P38蛋白作用的表达分析
通过检测APS对LPS所致的IEC-6细胞分泌P-P38在蛋白水平上的影响研究发现,当LPS刺激IEC-6细胞时,P38蛋白磷酸化增强,被诱导分泌具有显著意义的增加(P＜0.01),APS抑制P38蛋白磷酸化,随着APS浓度的增加,其抑制P38蛋白磷酸化作用逐渐增强,其中APS浓度在500ug/ml时抑制作用最强。同样,LPS导致的IEC-6细胞分泌的ERK1/2及JNK蛋白磷酸化也有明显表达,且差异具有统计学意义(P＜0.05),但是APS对LPS诱导引起的ERK1/2及JNK蛋白磷酸化表达却无显著意义影响(P＞0.05)。
结论
1、黄芪多糖对正常IEC-6细胞具有促进细胞增殖的作用;当细胞受到LPS损伤时,黄芪多糖却不能显现出显著的促增殖修复作用,提示黄芪多糖的促细胞增殖作用有赖于细胞处于正常的生理状态;黄芪多糖预处理能增强LPS损伤后IEC-6细胞的增殖活性,说明黄芪多糖早期干预能够降低LPS对IEC-6细胞的损伤而显现出对细胞的保护作用,APS的治疗效果也取决于LPS的损伤程度的轻重。
2、LPS作用于小肠上皮细胞后,细胞因子TNF-α和IL-8 mRNA大量表达,而APS可以抑制LPS刺激细胞分泌的TNF-α和IL-8 mRNA的过量产生,且APS对这种抑制作用具有浓度及时间依赖性。说明黄芪多糖对小肠上皮细胞的免疫保护作用可能是通过抑制细胞因子TNF-α、IL-8等的过量表达,从而减少其对组织细胞的炎性损伤。
3、LPS刺激IEC-6时,IκB-a蛋白表达降低,细胞核内NF-κB蛋白表达增加,而APS能够有效抑制NF-κB蛋白表达的增长及IκB-a表达的降低,且随着黄芪多糖浓度的增加,抑制NF-κB蛋白的作用逐渐增强。说明黄芪多糖抑制细胞因子的过量表达可能是通过抑制核因子NF-κB信号通路来实现的。
4、LPS作用于小肠上皮细胞后,其分泌的P38MAPK蛋白磷酸化增强,APS可以显著抑制这种分泌的增加,并具有剂量依赖性,说明APS对LPS所致的IEC-6细胞的免疫保护作用也可能是通过P38MAPK信号通路实现的。LPS作用于小肠上皮细胞后,其分泌的ERK1/2及JNK蛋白磷酸化同样增强,但是APS对ERK1/2及JNK蛋白磷酸化没有显著意义影响。说明APS对LPS所致的IEC-6细胞的免疫保护作用不是通过ERK1/2及JNK信号通路实现的。
Objective
Intestinal epithelial cell is an important component of intestinal mucosal barrier.It is the first mucosal surface of the body and considered as acquired immune systems of the host which plays a central role in regulating.It is also the first two-way connection defense between pathogenic microorganisms and the host.Some non-intestinal diseases and gastrointestinal diseases can cause intestinal dysfunction.When severe stress state, the systemic immune function is low.A large number of intestinal bacteria and endotoxin intruse circulation and cause intestinal bacterial translocation and endotoxemia,which will further aggravate the intestinal epithelial cell injury,activate a series of immune response,result in a number of inflammatory mediators such as TNF-α、IL-6、IL-8、platelet activating factor.A large number of production can cause systemic inflammatory response syndrome(SIRS),activate and accelerate the multi-system organ failure(MSOF).Research on LPS-mediated intestinal epithelial cell injury has become a widespread area of study.Much progress has been made in recent years.
NF-κB is the key factor of the expression of many genes regulated the intestinal immune function,which play an indispensable role for many cytokines(IL-1、IL-2、IL-6、IL-8、IL-12) expressed in lymphocytes,epithelial cells and mononuclear cells.Now clear that,NF-κB in intestinal epithelial cells may be activated when stimulated by LPS.NF-κB can induce the increase in gene expression of multi-cytokine (IL-1、IL-6、IL-8、TNF),adhesion molecules,chemokines factor and the acute phase protein.Therefore,the NF-κB,as a transcription factor of the continue transformation in the gastrointestinal tract,regulate the expression of inflammatory genes,trigger and expand the inflammatory response,result in the occurrence of disease.Since the activation of NF-κB is known as an important pathway leading to intestinal epithelial cell injury,the study on blocking NF-κB activation in order to control and inhibit inflammatory response is a promising way in the treatment of gastro-intestinal injury.
Mitogen-activated protein kinase(MAPK) is the serine/threonine residues of the protein kinase widely distributed within the cells.It is also an important signal regulatory enzymes connected the cell surface receptor with its decisive gene expression.In mammalian cells,it have been cloned at least four MAPK family: respectively extracellular signal- regulated kinase(ERK1/ERK2),cJun N-terminal kinase(JNK1/JNK 2),P38 MAPK(α,β) and ERK5.The activation of MAPK can stay in the cytoplasm to range other protein kinase.As a result,cytoskeletal components phosphorylate.MAPK is activated by phosphorylation,translocated to the nucleus, which trigger its transcription factor gene expression and promote the protein synthesis.Grishin and other scholars found that P38MAPK and COX-2 phosphorylation expression increased when IEC-6 cells stimulated by LPS in vitro.The phosphorylation of P38 induced by LPS in IEC-6 with a dose-dependent manner.The study had confirmed that P38MAPK involved in the inflammation of the intestinal epithelial cells induced by the LPS in vitro.MAPK affect gene expression,the media release and other cell signaling pathways through a multi-level,multi-faceted signal conditioning.It involved the release of pro-inflammatory cytokines and apoptosis injury in the intestinal cells.It can be believed that blocking and controlling MAPK signaling molecule expression and activity of intestinal injury will provide new ideas and approaches.
Astragalus is our traditional Chinese medicine.Research shows that the Astragalus can promote normal development of immune organs and impaired immune organ recovery,enhance and restore immune function.It has been used to control tumor,immunocompromised and immunodeficiency diseases in China.Astragalus mongholicus polysaccharides(APS) is an important component of Astragalus.Many studies have confirmed that the APS regulate immune and stimulate the hematopoietic function.Wang Li xin found that APS antagonized the drug from the liver homogenate index of malondialdehyde(MDA) levels and reduced glutathione(GSH).Lu Jing Tao has confirmed the APS can inhibit lipopolysaccharide-induced rat peritoneal macrophages releasing of tumor necrosis factor,interleukin-1 and the secretion of nitric oxide.It showed that APS can inhibit the secretion of inflammatory cytokines thereby reducing tissue injury to play its role in immune protection.
At home and abroad,no study was done on whether the APS has a protective effect in intestinal injury caused by LPS,as well as whether the protective effect of APS related with some signal transduction pathway.After the IEC-6 cells were stimulated by LPS,We studied whether the APS can effectively promote cell proliferation,whether APS plays the role in the protection of intestinal realated with the signal of MAPK/NF-κB transduction pathway.Through the study,we explore the mechanism of APS in intestinal protection so as to provide a theoretical basis for clinical application.
Material and methods
1.Materials
IEC-6 cells were purchased from the Test Center of Chinese Academy of Medical Sciences.IEC-6 cells were cultured with DMEM medium supplemented with 10%fetal bovine serum and 0.01 mg/ml insulin.IEC-6 ceils were cultured in the CO_2 incubator(37℃,5%CO_2).It is soluted for the next day to about 80 per cent covered, then incubated with trypsin(0.25%).Cells from passage 5 were used for study.
The cultured cells will be divided into 5 groups:Group 1:the control group,add DMEM only for control;Group 2:LPS group,the cultured cells by adding LPS(10 ug/ ml);Group 3:APS group:it divide into 4 sub-group:①APS50 ug/ml;②APS100 ug/ml;③APS200 ug/ml;④APSS00 ug/ml;Group 4:LPS and different concentration APS group:it divide into 4 sub-group:①LPS10 ug/ml+APS50ug/ml;②LPS10 ug/ml +APS 100 ug/ml;③LPS10 ug/ml+APS 200 ug/ml;④LPS10 ug/ml+APS 500 ug/ml; Group 5:APS and different concentration LPS group:it divide into 3 sub-group:①APS500 ug/ml+LPS 5 ug/ml;②APS500 ug/ml+LPS 10 ug/ml;③APS500 ug/ml+LPS 20 ug/ml.
2.Methods
The rat small intestinal cell line IEC-6 was grown in Dulbecco's modified.
Eagle's medium(DMEM) supplemented with 10%FBS and 0.01mg/ml insulin.IEC-6 cells were cultured at 37℃in a humidified atmosphere containing 5%CO2 with various concentration of APS(50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for 24h,48h,72h.After incubation,Cell proliferation was measured by MTT assay to observe the effects of APS on normal IEC-6.The IEC-6 cells were stimulated with LPS(10ug/ml) for 1h,then added with various concentration of APS (50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for 24h,48h,72h.Cell proliferation was measured by MTT assay to observe the effects of APS on LPS- stimulated IEC-6.The IEC-6 cells were pretreated with APS(500ug/ml) for 24h,then added with various concentration of LPS(5ug/ml,10ug/ml,20ug/ml) for 1h.After incubation,Cell proliferation was measured by MTT assay to observe the effects of pretreated APS on LPS-stimulated IEC-6.
Reverse Transcriptase-Mediated PCR(RT-PCR) Analysis detect TNF-αmRNA and IL-8 mRNA.
IEC-6 cells were culture in DMEM with various concentration of APS(50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for24h,then added LPS(10ug/ml) for 1h and 4h to detect TNF-αmRNA and IL-8 mRNA.
Western-Blotting assay to detect protein of P-ERK1/2,P-JNK,P-P38,NF-κB and IκB-α.
The cells were cultured in DMEM medium with various concentrations of APS (50 ug/ml;100 ug/ml;200 ug/ml;500 ug/ml) for 24h,then added LPS for 15 min to detect IκB-α,for 30 min to detect NF-κB,and for 1h to detect phosphorylated-P38, phosphorylated-ERK1/2,phosphorylated-JNK.
3.Statistical analysis
SPSS11.5 was used to perform statistical analysis,with all data expressed as mean±SEM.Ststistically significantdifferences in the values were analyzed with ANOVA for inter-group comparion.P-value＜0.05 were considered statistically significant.
Results
1.Morphology change in IEC-6
Normal intestinal epithelial cells were embedded as paving stones,not overlapping. A typical single-layer cell was seen clearly with larger nucleus.Intestinal epithelial cells stimulated by LPS were showed fuzzy boundary cells.A large number of cells membrane rupture and cell morphology was not integrity.
2.The proliferation impact of APS on IEC-6 cells stimulated by LPS.
APS can promote normal IEC-6 cell proliferation.
MTT result showed:Different concentrations of APS showed a significant role in promoting IEC-6 cell proliferation(P＜0.05).APS on the IEC-6 cells has a dose-dependent role in promoting proliferation.With the extension of time,the effect of promoting cell proliferation gradually weakened.For the first 24- hour,the concentration of 50 ug/ ml APS can promote the cell proliferation significantly.However,in the 72- hour,the concentration of 200 ug/ ml APS displayed a remarkable effect of promoting cell proliferation.
APS cannot promote IEC-6 cell proliferation which stimulated by LPS.
MTT result showed:APS displayed a nonremarkable effect of promoting cell proliferation on IEC-6 cells stimulated by LPS.Only in the first 24- hour,the concentration of 200 ug/ml and 500 ug/ml APS showed significant role in promoting cell proliferation.In the 48-hour and 72-hour,with the different concentrations of APS did not have effect of promoting cell proliferation(P＞0.05).
Pretreated APS can promote IEC-6 cell proliferation which stimulated by LPS.
MTT result showed:When IEC-6 cells were stimulated by the concentration of 5 ug/ml LPS,Pretreated APS do not promote cell proliferation on IEC-6 compared with the control group(P＞0.05).But when IEC-6 cells were stimulated by the concentration of 10 ug/ml and 20 ug/ml LPS,Pretreated APS group promote cell proliferation on IEC-6 compared with the control group(P＜0.01).The results indicate that, pretreatment with APS can reduce IEC-6 cell injury induced by LPS.
3.APS inhibited the secretion of TNF-α,IL-8 mRNA production in IEC-6 cells stimulated by LPS.
APS abrogates LPS-induced TNF-αmRNA expression in IEC-6.
We first evaluted the effects of APS on LPS-induced TNF-αgene expression in intestinal cell line IEC-6.Stimulation of IEC-6 by LPS markedly increased the production of TNF-α.We examined the effect of APS on the mRNA level of TNF-αfrom IEC-6 when stimulated with LPS.Cells were pretreated with various concentrations of APS(50ug/ml,100ug/ml,200ug/ml,500ug/ml) for 24h,stimulated with LPS(10ug/ml)for 1h and TNF-αgene expression was measured by RT-PCR assay.RT-PCR analysis showed that TNF-αmRNA is induced readily from IEC-6 by LPS.However,this induction was inhibited by APS in concentration- dependent manner:50ug/ml of APS partially supressed TNF-αmRNA from LPS activated IEC-6,and 500ug/ml of APS significantly inhibited the amount of TNF-αmRNA.(P＜0.01 compared with LPS stimulation in the absence of APS).
APS abrogates LPS-induced IL-8 mRNA expression in IEC-6.
We then examined the effect of APS in the production of IL-8 from IEC-6 by LPS.IL-8 mRNA was induced readily from IEC-6 by LPS.However,the mRNA level of IL-8 was inhibited by APS in a concentration- dependent manner:addition of APS partially supressed IL-8 level at 50ug/ml and 100ug/ml,respectively,and 500ug/ml of APS significantly inhibited IL-8 at mRNA level.(P＜0.01 compared with LPS stimulation in the absence of APS).
APS Inhibited both TNF-αand IL-8 Production from LPS-Activated IEC-6 in a time-dependent manner.
Next,IEC-6 cells were pretreated with APS(500ug/ml) for 24h,stimulated with LPS(10ug/ml) for 1h to 4h and TNF-α,IL-8 gene expression was measured by RT-PCR.LPS-induced TNF-αmRNA expression is inhibited by APS treatment(10.3% and 25.5%inhibition at 1h and 4h poststimulation,respectively).LPS-induced IL-8 mRNA expression is inhibited by APS treatment(15.3%and 18.8%inhibition at 1h and 4h poststimulation,respectively).
4.APS inhibited NF-κB protein expression in IEC-6 cells induced by LPS.
It has been reported that APS abrogates LPS-induced pro-inflammatory cytokine production in IEC-6.A critical downstream effector pathway induced by LPS is the NF-κB transcriptional system.NF-κB is usually held in an inactivated state in the cytoplasm of unstimulated cells through interaction with IκB-α(NF-κB inhibitory protein).After stimulation with LPS,the IκB-αis degraded and release the NF-κB to translocate into nucleus,where NF-κB drive the expression of pro-inflammatory cytokines.Thus we next assessed whether APS abrogates LPS-induced NF-κB transcriptional activity.To do this,we examined whether APS blocks NF-κB translocation into nucleus,since nuclear translocation is required for NF-κB-Mediated transcriptional activation.We examined the levels of NF-κB in the nucleus of LPS-stimulated IEC-6 cells in the presence or absence of APS.LPS stimulation resulted in an increase in protein level in the nucleus.Treatment with APS abolished this effect.To dissect the effect of APS on LPS-induced NF-κB signal,we next determined IκB-αprotein involved in NF-κB pathway using Western blot analysis.It shows that LPS decrease IκB-αin IEC-6cells,which are blocked in APS-pretreated cells,suggesting that APS inhibits the translocation of NF-κB into the nucleus,and may subsequently suppress LPS-induced NF-κB activation.
5.The analysis of the expression of MAPK protein in IEC-6 cells induced by APS.
It is reported that the activation of the MAPKS P38 is important signaling pathways responsible for the pro-duction of pro-inflammatory cytokines when IEC-6 is activated.In order to further understand the mechanisms of APS-mediated anti-inflammation in IEC-6,we examined whether APS inhibits LPS-triggered activation of MAPK signalling incluing P38,ERK1/2 and JNK.The active phosphorylated levels of P38,ERK1/2 and JNK were analyzed in LPS-stimulated IEC-6 cells following treatment with or without APS.Results indicated that P38,ERK1/2 and JNK were strongly activated in IEC-6 when stimulated with LPS.P38,ERK1/2 and JNK were all activated in IEC-6 in lh after LPS treatment.However,treatment with APS results in a reduction in LPS-induced P38 phosphorylation.And LPS-induced phosphorylation of P38 is inhibited in a concentration-dependent manner.When 50ug/ml of APS is added,LPS-induced P38 was partially blocked in IEC-6,and 500ug/ml of APS significantly inhibited P38 phosphorylation in LPS-stimulated IEC-6.However,APS did not alter the activation of the protein levels of ERK1/2 and JNK.The observations indicate that APS may inhibit the activation of P38 kinases but not the ERK1/2 and JNK in IEC-6 stimulated by LPS.
Conclusions
1.APS has a role of promoting the normal IEC-6 cell proliferation.APS displayed a nonremarkable effect of promoting cell proliferation on IEC-6 cells stimulated by LPS.When IEC-6 cells were pretreated with APS,APS can promote cell proliferation compared with the control group.The results indicate that pretreatment with APS can reduce IEC-6 cell injury induced by LPS.
2.APS abrogates LPS-induced TNF-αand IL-8 mRNA expression in IEC-6. TNF-αand IL-8 mRNA is induced readily from IEC-6 by LPS.However,this induction was inhibited by APS in concentration- dependent manner.We also confirm APS innhibited both TNF-αand IL-8 production from LPS-Activated IEC-6 in a time-dependent manner.
3.APS inhibited NF-κB protein expression in IEC-6 cells induced by LPS.LPS stimulation resulted in an increase in NF-κB protein level in the nucleus.Treatment with APS abolished this effect.LPS decrease IκB-αin IEC-6 cells,which are blocked in APS-pretreated cells,suggesting that APS inhibits the translocation of NF-κB into the nucleus,and may subsequently suppress LPS-induced NF-κB activation.
4.P38,ERK1/2 and JNK were strongly activated in IEC-6 when stimulated with LPS.However,treatment with APS results in a reduction in LPS-induced P38 phosphorylation.And LPS-induced phosphorylation of P38 is inhibited in a concentration-dependent manner.APS did not alter the activation of the protein levels of ERK1/2 and JNK phosphorylation.APS may inhibit the activation of P38 kinases but not the ERK1/2 and JNK in IEC-6 stimulated by LPS.
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