溃结灵对原代大鼠结肠上皮细胞ITF表达及MAPK/ERK通路的影响
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摘要
一、研究背景
溃疡性结肠炎是一种病变部位主要位于结肠及直肠的慢性非特异性炎症反应。临床治疗多以抗炎、调节免疫为主。近年研究发现,肠道损伤后,除炎症损伤因素外,修复因素在病程的发展中同样具有非常重要的作用,两者的相互作用决定了UC病情的转归,许多生长因子参与肠黏膜修复过程。随着研究的发展,一些生长因子新制剂已逐渐应用于炎症损伤的治疗,并且取得不错的效果。肠三叶因子(ITF)是近年发现的一种由肠道杯状细胞分泌新型细胞生长因子,与其它肠道保护修复因子(MUC-2、EGF、TGF-α等)可一起共同作用,具有很强的细胞保护作用,可明显减轻多种损伤因子介导的肠黏膜损伤。因此,研究中药复方对这些特异保护因子的调节作用,有望为UC治疗提供新的思路。
经验方溃结灵以清热健脾活血为治则,临床研究表明其对UC疗效较好。前期研究采用三硝基苯磺酸灌肠复制UC大鼠模型,发现溃结灵可明显减轻UC大鼠肠黏膜炎症反应,可下调炎症因子如Toll样受体、IL-1β、TNF-α、NF-κB等的表达,改善肠黏膜超微结构,上调造模3、7、14天模型大鼠肠黏膜ITFmRNA、MUC-2蛋白的表达及14天TGF-α和pERK1/2的蛋白表达；对UC血清致Caco-2细胞损伤模型的研究发现,溃结灵含药血清可促进Caco-2细胞损伤模型细胞增殖,增加ITF基因、MUC-2蛋白的表达以及上调pERK、pMEK蛋白的表达。本研究拟继续从促黏膜修复角度入手,观察溃结灵含药血清对大鼠结肠上皮细胞ITF表达及其信号转导通路相关蛋白ERK、MEK磷酸化变化的影响,进一步探讨溃结灵治疗UC的作用机理,和前期的实验互为佐证。
二.研究方法与结果
1.原代大鼠结肠上皮细胞分离培养方法的建立
原代大鼠结肠上皮细胞来源于正常大鼠结肠组织,能真实反映机体生理和病理变化,然而分离培养技术是一个难题,限制了其应用。在参考国内外文献的基础上,本研究建立了大鼠原代结肠上皮细胞分离培养方法。可为结肠上皮形态及功能研究提供理想的细胞模型。
方法：采用6-15日龄乳鼠,取结肠剪碎、反复清洗后,弃上清,加10ml、0.1%Ⅰ型胶原酶和透明质酸酶联合配制的消化液,37℃消化25min,分离上皮细胞团；吹打消化液,取上清,加入培养基重悬反复离心3次后,细胞沉淀接种于含10%胎牛血清的完全培养基中,37℃、5%CO2细胞培养箱中培养。采用差速贴壁法及相差消化法去除成纤维细胞,细胞贴壁长满瓶80%-90%后用2.5g/L的胰酶消化液消化并传代。结果：成功分离结肠上皮细胞团且活力较高。24h后可见部分细胞团贴壁,贴壁细胞为多角形,4-8d逐渐融合成片,呈现明显的“铺路石”样。细胞经传代处理后成纤维细胞明显减少。第2代细胞在生长过程中逐渐生长成性似IEC-6上皮细胞,状态较好,增殖较快。细胞经免疫荧光染色及透射电镜观察鉴定为上皮细胞。冻存处理复苏后细胞状态较佳。
2.大鼠结肠上皮细胞ITF/EGF、MUC-2/TGF-α及pERK/pMEK表达的研究
胃肠黏膜保护及促修复因子ITF、MUC-2、EGF、TGF-α在肠黏膜损伤修复及肠黏膜屏障重建过程中具有重要的作用。本研究对结肠上皮细胞是否有以上生长因子的表达进行了检测。为后续开展药物对肠上皮细胞作用的指标选择提供基础及参考。
方法：采用荧光定量PCR (Real-Time PCR)检测结肠上皮细胞ITF、EGFmRNA的表达；采用Elisa的方法检测MUC-2/TGF-α蛋白表达；采用Western bolt方法检测pERK/pMEK蛋白表达。
结果：结肠上皮细胞表达ITF/EGFmRNA、MUC-2/TGF-α蛋白及pERK/pMEK蛋白。可用于肠黏膜损伤修复的相关实验研究。
3.溃结灵含药血清对UC模型血清致损的结肠上皮细胞超微结构的影响
本课题整体实验研究发现,溃结灵可明显减轻TNBS灌肠复制的UC大鼠的炎症反应,可促进肠道上皮杯状细胞的分泌,减轻结肠组织超微结构损伤,进而促进肠黏膜修复及重建。本实验在此基础上,观察溃结灵含药血清对UC大鼠血清致结肠上皮细胞损伤模型细胞超微结构的影响。进一步从细胞形态学方面探讨溃结灵的作用。
方法：三硝基苯磺酸灌肠法复制溃疡性结肠炎大鼠模型,3天后腹主动脉采血,分离获取血清,作为损伤剂作用结肠上皮细胞24h模拟细胞损伤模型,透射电镜法观察结肠上皮细胞超微结构的改变。
结果：正常对照组及空白对照组细胞可见丰富的微绒毛,有的呈小片状。细胞内可见丰富的线粒体,还可见核糖体、内质网及高、中、低等电子密度分泌颗粒；UC模型血清造模组可见较少微绒毛,胞质内可见粗面内质网扩张,少量线粒体及线粒体肿胀,空泡样变甚至溶解,有大量的胞质内空泡形成,微绒毛较少或消失,分泌颗粒较少,为中低等电子密度。给予溃结灵含药血清组细胞形态较好,细胞微绒毛及线粒体等细胞器丰富,可见较多高、中、低等电子密度分泌颗粒。
4.溃结灵含药血清对正常大鼠结肠上皮细胞增殖的影响
结肠上皮细胞在肠黏膜损伤修复中具有重要的作用,UC发病时,促进结肠上皮细胞的增殖、移行、分化,可加速受损黏膜的修复以及肠黏膜屏障功能的恢复。本实验采用溃结灵含药血清干预结肠上皮细胞,探讨溃结灵含药血清对结肠上皮细胞增殖的影响。
方法：采用血清药理学方法制备溃结灵含药血清；MTT法测定溃结灵含药血清干预大鼠结肠上皮细胞12、24、48h后细胞增殖情况。
结果：空白大鼠血清对大鼠结肠上皮细胞(CEC)生长无影响,溃结灵中剂量(5%含药血清)作用24h、48h均明显促进CEC增殖(p<0.05)
5.溃结灵含药血清对TNF-α致结肠上皮细胞损伤模型细胞增殖的影响
TNF-α为公认的UC致病因子,是肠道上皮细胞增殖和凋亡的主要促炎因子,UC发病时,TNF-α大量分泌,可使结肠上皮细胞凋亡,肠黏膜屏障的通透性增加,NF-κB被激活,进一步扩发炎症反应,损伤肠黏膜。本实验观察不同浓度TNF-α对大鼠上皮细胞增殖的影响,以确定TNF-α合适的作用浓度及作用时间建立结肠上皮细胞损伤模型,并测定溃结灵含药血清对TNF-α干预下结肠上皮细胞增殖的情况,从细胞增殖的方面,进一步探讨溃结灵对受损结肠上皮细胞的保护作用。
方法：以25,50,100,150,200ng/ml的TNF-α干预结肠上皮细胞,MTT法测定不同干预时间(3h,6h,24h)细胞的增殖情况,同法测定预先给予溃结灵含药血清对50ng/mlTNF-α干预结肠上皮细胞6h细胞增殖的影响。
结果：①50ng/ml TNF-α干预结肠上皮细胞6h后可明显抑制细胞增殖(p<0.05)；②24h时,各浓度TNF-α均明显抑制结肠上皮细胞增殖(p<0.01)③与模型组比较,溃结灵含药血清各剂量组均有一定的促进TNF-α干预下结肠上皮细胞增殖的作用,但无统计学意义(p>0.05)。
6.溃结灵含药血清对TNF-α干预下大鼠结肠上皮细胞ITF、EGFmRNA表达的影响
ITF、EGF在胃肠道上皮保护和促进黏膜愈合方面发挥着重要的作用,与UC的修复愈合关系密切。本研究观察溃结灵含药血清对TNF-α干预下结肠上皮细胞EGF及ITFmRNA表达的影响,以进一步在离体细胞水平探讨溃结灵促进肠黏膜损伤修复的作用靶标。
方法：以50ng/mlTNF-α干预结肠上皮细胞6h,造成细胞损伤模型,采用荧光定量PCR (Real-Time PCR)法检测不同浓度溃结灵含药血清对此细胞损伤模型ITF及EGFmRNA的表达的影响。
结果：①模型组EGF、ITFmRNA表达较空白对照组减低；②溃结灵各剂量组(?)GFmRNA相对表达量与模型组相比,无统计学意义(p>0.05)；③溃结灵高(10%含药血清)、中剂量组(5%含药血清)ITFmRNA表达量明显高于模型组(p<0.05,p<0.01)。
7.溃结灵含药血清对TNF-α干预下大鼠结肠上皮细胞MUC-2、TGF-α蛋白表达的影响
MUC-2、TGF-α在黏膜保护和肠道疾病修复机制中占有重有地位,且在肠道黏膜修复中MUC-2、TGF-α、EGF、与ITF具有协同作用,保护和促进黏膜修复。本研究进一步观察了溃结灵含药血清对TNF-α干预下结肠上皮细胞MUC-2及TGF-α蛋白表达的影响,探讨溃结灵促进肠黏膜损伤修复的又一可能作用靶标,以进一步揭示溃结灵治疗UC的分子作用机理。
方法：TNF-α(50ng/ml)干预结肠上皮细胞生长6h造成结肠上皮细胞损伤模型,采用Elisa方法检测不同浓度溃结灵含药血清对此细胞损伤模型MUC-2及TGF-α蛋白含量的影响。
结果：①TNF-α作用于细胞后,与空白对照组比较,模型对照组MUC-2含量增加(p<0.01), TGF-α含量降低,但无统计学差异；②与模型对照组比较,溃结灵含药血清高剂量组MUC-2含量增加(p<0.05),溃结灵含药血清高剂量组TGF-α含量显著升高(p<0.01)。
8.溃结灵含药血清对TNF-α干预下大鼠结肠上皮细胞pERKl/2、pMEK1/2蛋白表达的影响
ERK信号转导通路参与调节细胞的增生、分化、凋亡等过程。其在真核细胞内广泛存在,许多因子通过ERK通路发挥作用。而细胞信号中传递丝裂原信号的关键激酶是胞外信号调节蛋白激酶(ERK1/2), ERK被激活后,可诱导细胞产生分裂、增殖等效应。本研究观察溃结灵含药血清对结肠上皮细胞损伤模型pERK1/2、pMEK1/2蛋白表达的影响。以期进一步揭示溃结灵治疗UC的作用机理。
方法：TNF-α(50ng/ml)干预结肠上皮细胞生长6h造成结肠上皮细胞损伤模型,提取细胞全蛋白,采用WesternBlot法检测溃结灵含药血清对此上皮细胞损伤模型pMEK1/2, pERK1/2蛋白表达水平的影响,内参蛋白为β-actin,目的蛋白的相对含量为目的蛋白与β-actin密度的比值,结果进行组间比较分析。
结果：①50ng/ml TNF-α作用于结肠上皮细胞6h后,细胞内pMEK1/2、pERK1/2蛋白表达均有增加；②溃结灵高剂量(10%含药血清)、中剂量(5%含药血清)明显促进pMEK1/2蛋白表达(p<0.05),溃结灵中剂量(5%含药血清)明显促进pERK1/2蛋白表达(p<0.05)。提示溃结灵含药血清对TNF-α法干预下结肠大鼠上皮细胞pERK1/2、 pMEK1/2蛋白表达有上调作用,ERK信号通路可能是溃结灵保护及修复肠黏膜损伤的转导途径之一
三、研究结论
1.本研究建立了大鼠结肠上皮细胞原代培养体系,为结肠上皮生理、病理及药物作用机制提供了体外研究平台。对研究肠上皮细胞与炎症性肠病病因学的相关性及其中药对炎症性肠病的防治具有重要的意义。
2、本研究结果显示,结肠上皮细胞表达ITFmRNA, EGFmRNA, MUC-2、TGF-α蛋白,ITF,MUC-2由肠道杯状细胞分泌,证明,此细胞可用于肠黏膜损伤修复的相关实验研究。
3.溃结灵含药血清能够促进正常大鼠结肠上皮细胞的增殖,改善UC大鼠血清致结肠上皮细胞损伤模型的超微结构,对TNF-α干预下的结肠上皮细胞具有一定的促增殖作用。提示,促进结肠上皮细胞增殖是该方的疗效机理之一
4.溃结灵含药血清能上调TNF-α干预下的结肠上皮细胞ITF基因、MUC-2、TGF-α蛋白表达以及pERK1/2、pMEK1/2蛋白表达。提示,溃结灵可通过激活ERK通路,促进上皮细胞的增殖移行,进而加速肠黏膜损伤修复。也可能是其作用的分子机制之一
Backgrouds
Ulcerative colitis (UC) is a chronic and non-specific inflammatory disease of the colorectal mucosa. Anti-inflammatory and regulating immunity are the main clinical treatment of UC. Recent studies have found that after the damage of intestinal tract, in addition to inflammatory and injury factors, the repair factors also play an equally important role in the development of disease, the prognosis of disease is determined by the interaction between them, and many growth factors participate in the whole repair process. With the development of research, some growth factors have been gradually applied to cure inflammatory injury and obtained good results. Intestinal Trefoil Factor (ITF) is a new cell growth factor found recently,which is secreted by the goblet cells and mainly distributes in the intestinal tract. It has a strong cell protecting effect, synergizing with other intestinal repair factors (MUC-2, EGF, TGF-a,etc.) to reduce the multiple factor-mediated injury of intestinal mucosa significantly. Therefore, the study of formula on the regulation of specific protective factors may help to develop a new way to treat UC. Kui jie ling decoction (KD)was frequently used to treat UC patients. The therapeutic principle of KD is clearing away heat, invigorating the spleen and activating blood.It showed affirmative therapeutic effect on patients. Some experiments about effect of KD on UC model rats induced by TNBS were observed before. The observation showed that the number and area of ulcer in model rats were significantly reduced with the administration of KD. Pathological changes such as inflammatory cell infiltration and edema were improved. The expression of imflammatory factors such as Toll-like receptor, IL-1β, TNF-α, NF-κB were reduced. At the same time KD formula could improve ultrstructual changes, increasse gene expression of ITF and protein expression of MUC-2on the3th,7th and14th, raise gene expression of EGF, protein expression of TGF-α and pERK1/2on the14th in colonic mucosa of UC model rats. And in the reaserch of Caco-2cells injuried model induced by Serum of UC Model Rats have showed that KD medicated serum could promote Caco-2cells prolifiration, enhance mRNA expression of ITF and protein expression of MUC-2, pER、pMEK. In short, KD may regulate and enhance the expression of ITF and MUC-2, The interaction of ITF and MUC-2may reinforce the effect of protecting colonic mucosa and Caco2cells injuried model induced by Serum of UC Model Rats. In this article, the gene and the key elements of MEK/ERK pathway such as the activation of MEK and ERK in rat colonic epithelial cells were observed to evaluate the interventional effect of KD medicated serum, which would help to further explore mechanism of KD in treatment of UC and provide evidence for the previous reaserch.
Methods and Results
1. Establishment of the method for primary culture of colonic epithelial cell
The primary Colonic epithelial cell originated from normal colon tissue in rats, which can really reflect physiological and pathological changes of colon. But isolation and culture technology of CEC is a problem, which limited its application. After researched on domestic and foreign literatures, the method for primary culture of colonic epithelial cell was established in this study, which could provide a research platform in vitro for the study on physiological, physiological and medicine function mechanism of colonic epithelial cell.
Methods:Colons obtained from suckling rats (6-15d) were cut into small pieces and washed. Colonic epithelial cell cluster were separated by10ml0.1%collagenase I and hyaluronidase digestion for25min at37℃. After pipetting digestion, the supernant was transfered into another new tube and DMEM solution was added in to centrifuge for3times. Then cells were cultured in DMEM solution containing10%fetal bovine serum in a CO2incubator with a saturated humidity at37℃. Fibroblasts were removed by using the phase difference digestion and adherence. When80%-90%of the cells were adherent to the culture plat passaged with2.5g/L pancreatin digestion.
Results:The colonic epithelial cell clusters were successfully obtained which showed high viability and became adherent after24hours culture, the cells represented polygonal shape, and grew into pavestone-like monola yer gradually in4-8days,which showed an excellent proliferate ability. Fibroblasts were significantly decreased after passage. During cell growth, passage2cells grew into polygonal shape just like the shape of IEC-6. The colonic epithelial cells were identified by the ways of immunofluores cence staining and TEM observation. The cells were in good condition after being frozen and thawed.
2. Study on expression of ITF/EGF、MUC-2/TGF-αand pERK/pMEK in colonic epithelial cells
Protective and repair factors in gastrointestinal mucosa such as ITF、MUC-2、 EGF and TGF-α play an important role in the process of repair and reconstruction of intestinal mucosal barrier. In this research, the expression of ITF and other repaire factors were detected to provide basic and reference for index selection in the preceeding research on colonic epithelial cells.
Methods:real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of ITF、EGFmRNA;The protein expression of MUC-2/TGF-α were determined by the method of Elisa kit;And western blot technique was used to detect protein levels of pERK1/2and pMEK1/2.
Results:the Gene expression of ITF/EGF and the protein expression of MUC-2/TGF-α、pERK/pMEK in CECs were dected.
3. Effect of KD medicated serum on the rat colonic epithelial cell ultramicrostructure in cell damage model induced by serum of UC model rats
The previous reaserch found that KD could improve the morph and function of globlet cells and alleviate the ultrastructure damage in colonic mucosa of UC Model Rats. Based on that, the effect of KD medicated serum on epithelial cell ultrastructures damaged by serum of UC model rats was further studied in this study to explore effect of KD from the aspect of cytomorphology
Methods:Rats received enteroclysis of trinitro-benzene-sulfonic acid (TNBS) to induce ulcerative colitis models for3days. Then, blood samples were collected from abdominal aorta to get serum, rat colonic epithelial cell damage model was made by intervened with the serum for24h. Then, rat colonic epithelial cell ultramicrostructure were observed by transmission electron microscopy.
Results:Ultrastructures of colonic epithelial cell in normal and blank control group were abundant microvilli、organelles and secretroy granules with high、moderate and low electrondensity;In model group with the appearance of a few microvilli, expand of rough, cytoplasmic vacuolization, a small number of mitochondria, a few secretory granule, many vacuoles in the cytoplasm;While in the group of10%KD medicated serum showed that abundant microvilli exist, mitochondrions and other organelles were abundant, secretroy granules with high、moderate and low electrondensity could be seen.
4. Effect of KD medicated serum on the proliferation of normal rat colonic epithelial cell
colonic epithelial cells play an important role in intestinal mucosal microstructural damage and reparation. Stimulation of colon epithelial cell proliferation, migration and differentiation could promote restoration of damaged intestinal mucosa and intestine mucosal barrier function with UC. In this study, KD medicated serum was used to intervene CECs growth to explore the effects of KD medicated serum on the proliferation of CECs.
Methods:Prepared KD medicated serum with Serum Pharmacological. MTT assay was used to determine cell growth of CECs under the effects of Kuijieling Decoction medicated serum for12、24、48h.
Results:There was no effect on the CECs proliferation with rat serum without medicine. And the CECs proliferation was promoted significantly after intervened with5%KD medicated serum for24h and48h (p<0.05)
5. Effect of KD medicated serum on the proliferation of damaged rat colonic epithelial cell induced by TNF-α
TNF-α is a generally accepted pathogenic factor in UC, and its also an inflammatory factors in the process of hyperplasia and apoptosis of intestinal epithelial cell In UC, the increased secretion of TNF-α can induce CEC apoptosis, increase Permeability of intestinal mucosal barrier, activate NF-κB pathway to further enlarge response of inflammation and damage intestinal mucosa. In this reaserch the effect of different concentrations of TNF-α on the proliferation of rat CECs was dected to determine the appropriate concentrations and action time of TNF-α to establish cell damage model, and then effect of KD medicated serum on the proliferation of damaged rat CECs induced by TNF-α was measured to explore cell protection effect of KD on CECs.
Method:CECs were treated with different concentration of TNF-α (25,50,100,150,200ng/mL)for3h,6h and24h. And then the proliferous activity in CECs was measured by MTT assays, and the same method was used to detect cell prolifiration treated with50ng/mlTNF-α for6h under the protect effect of KD medicated serum.
Results:①The CECs proliferation was inhibited significantly after intervened with50ng/ml TNF-α for6h (p<0.05);②All concentrations of TNF-a could inhibit CECs proliferation obviously after intervened with TNF-α for24h(p<0.01);③Different concentration of KD medicated serum could stimulate colonic epithelial cells to reproduce in a certain degree after damaged by TNF-α, but there was no significantly difference compaired to model group(p>0.05).
6. Effect of KD medicated serums on the mRNA expression of ITF and EGF in damaged rat colonic epithelial cell induced by TNF-α.
ITF and EGF are both cell protecting factors.They play an important role in epithelial protection of gastrointestinal tract and promoting mucosal healing which are closely related to UC repair. In this article the changes of ITF、EGFmRNA in CECs intervened with TNF-α under the effect of KD medicated serum were studied to evaluate the treatment mechanism of KD.
Method:The CECs were cultured in vitro and treated with50ng/ml TNF-α for6h, and cell damage model was made by this method, Real Time PCR was used to detect the expression of ITF、EGFmRNA under the effects of differrent concentration of Kuijieling Decoction medicated serum.
Results:①The mRNA expression of EGF and ITF in model group induced by TNF-α was lower than that in the blank control group;②The mRNA expression EGF in all KD groups had no significantly difference compaired to model group(p>0.05);③The expression of ITFmRNA in high-dosage group(10%KD medicated serum) and medium-dosage group(5%KD medicated serum) were significantly higher than that in the model group(p<0.05, p<0.01).
7. Effect of KD medicated serums on the protein expression of MUC-2and TGF-a in damaged rat colonic epithelial cell induced by TNF-α
TGF-α and MUC-2play an important role in mucosal protection and repair mechanisms of intestinal disease. And they have a good synergism effect with ITF and EGF to promte damage repair of intestinal mucosa. This study was to observe influence of KD medicated serum on MUC-2and TGF-α in CECs treated with TNF-α and to explore another effect target of KD in promoting damage repair of intestinal mucosa.
Method:The CECs intervened with50ng/ml TNF-α for6h, and cell damage model was made by this method;The effects of different concentration of KD serum on the MUC-2and TGF-α protein expression was detected by using Elisa kit.
Results:①Compared with blank group, the MUC-2content in the cell damage model induced by TNF-α was obviously increased (p<0.01);②The contents of MUC-2and TGF-α in high-dosage group(10%KD medicated serum)was significantly higher than that in model group (p<0.05,p<0.01).
8、Effect of KD medicated serums on the protein expression of pMEK1/2、pERK1/2in damaged rat colonic epithelial cell induced by TNF-α
Ras/MEK/ERK pathway is an important message pathway, which exists widely in eukaryotic cells. It is the key message pathway of many factors to perform function and participate in regulating the process of cell differentiation, proliferation, cleavage and apoptosis, and so on. The extracellular signal regulated kinase(ERK1/2) is the key mitogen kinase to transmite message in cells. The activated ERK could induct cells to take place some nuclear reactions, such as differentiation and proliferation. This study was to observe the effect of KD medicated serum on changes of pERKl/2and pMEK1/2protein expression in in CECs treated with TNF-α and to explore the mechanism of them.
Method:Whole-cell protein was extracted from colonic epithelial cell,Western blot technique was used to detect the effects of KD medicated serum on the protein expression of pERKl/2and pMEKl/2of CECs damage model which induced by intervening with50ng/mlTNF-αfor6h.
Results:①The protein expression of pERK1/2in the CECs induced by TNF-α for6h was increased;②The protein expression of pMEK1/2was obviously higher than that in blank group (p<0.05);③The protein expression of pMEK1/2in the medium (5%KD medicated serum)and high (10%KD medicated serum) groups were obviously higher than that in model group (p<0.05);④5%KD medicated serum could enhance the protein expression of pERK1/2significantly (p<0.05). The relative protein expression of pERK1/2and pMEK1/2in CECs induced by TNF-α were enhanced after using KD medicated serum and ERK message pathway may be one of pathways to protect and repair intestinal mucosal injury in UC rats bv KD treatment.
Conclusions
1、The results of this study showed that CECs expressed ITFmRNA, EGFmRNA, protein of MUC-2and TGF-α;ITF and MUC-2are both cell protecting factors secreted by goblet cells. So the CECs can be used as a cell model in the reaserch of intestinal mucosa damage repairation.
2、KD medicated serum could promote CECs proliferation, improve ultrstructual changes of CECs intervened with serum of UC rats, which suggested promote CECs proliferation is one mechanism of KD to treat UC.
3、KD medicated serum could increasse gene expression of ITF and protein expression of MUC-2、TGF-α, up-regulated the protein expression of pMEK1/2、 pERK1/2in the CEC damage model induced by TNF-α, indicating that activate ERK signal pathway to stimulate the migration and proliferation of CECs is also one of the molecular mechanism of KD to treat UC.
溃疡性结肠炎是一种病变部位主要位于结肠及直肠的慢性非特异性炎症反应。临床治疗多以抗炎、调节免疫为主。近年研究发现,肠道损伤后,除炎症损伤因素外,修复因素在病程的发展中同样具有非常重要的作用,两者的相互作用决定了UC病情的转归,许多生长因子参与肠黏膜修复过程。随着研究的发展,一些生长因子新制剂已逐渐应用于炎症损伤的治疗,并且取得不错的效果。肠三叶因子(ITF)是近年发现的一种由肠道杯状细胞分泌新型细胞生长因子,与其它肠道保护修复因子(MUC-2、EGF、TGF-α等)可一起共同作用,具有很强的细胞保护作用,可明显减轻多种损伤因子介导的肠黏膜损伤。因此,研究中药复方对这些特异保护因子的调节作用,有望为UC治疗提供新的思路。
经验方溃结灵以清热健脾活血为治则,临床研究表明其对UC疗效较好。前期研究采用三硝基苯磺酸灌肠复制UC大鼠模型,发现溃结灵可明显减轻UC大鼠肠黏膜炎症反应,可下调炎症因子如Toll样受体、IL-1β、TNF-α、NF-κB等的表达,改善肠黏膜超微结构,上调造模3、7、14天模型大鼠肠黏膜ITFmRNA、MUC-2蛋白的表达及14天TGF-α和pERK1/2的蛋白表达；对UC血清致Caco-2细胞损伤模型的研究发现,溃结灵含药血清可促进Caco-2细胞损伤模型细胞增殖,增加ITF基因、MUC-2蛋白的表达以及上调pERK、pMEK蛋白的表达。本研究拟继续从促黏膜修复角度入手,观察溃结灵含药血清对大鼠结肠上皮细胞ITF表达及其信号转导通路相关蛋白ERK、MEK磷酸化变化的影响,进一步探讨溃结灵治疗UC的作用机理,和前期的实验互为佐证。
二.研究方法与结果
1.原代大鼠结肠上皮细胞分离培养方法的建立
原代大鼠结肠上皮细胞来源于正常大鼠结肠组织,能真实反映机体生理和病理变化,然而分离培养技术是一个难题,限制了其应用。在参考国内外文献的基础上,本研究建立了大鼠原代结肠上皮细胞分离培养方法。可为结肠上皮形态及功能研究提供理想的细胞模型。
方法：采用6-15日龄乳鼠,取结肠剪碎、反复清洗后,弃上清,加10ml、0.1%Ⅰ型胶原酶和透明质酸酶联合配制的消化液,37℃消化25min,分离上皮细胞团；吹打消化液,取上清,加入培养基重悬反复离心3次后,细胞沉淀接种于含10%胎牛血清的完全培养基中,37℃、5%CO2细胞培养箱中培养。采用差速贴壁法及相差消化法去除成纤维细胞,细胞贴壁长满瓶80%-90%后用2.5g/L的胰酶消化液消化并传代。结果：成功分离结肠上皮细胞团且活力较高。24h后可见部分细胞团贴壁,贴壁细胞为多角形,4-8d逐渐融合成片,呈现明显的“铺路石”样。细胞经传代处理后成纤维细胞明显减少。第2代细胞在生长过程中逐渐生长成性似IEC-6上皮细胞,状态较好,增殖较快。细胞经免疫荧光染色及透射电镜观察鉴定为上皮细胞。冻存处理复苏后细胞状态较佳。
2.大鼠结肠上皮细胞ITF/EGF、MUC-2/TGF-α及pERK/pMEK表达的研究
胃肠黏膜保护及促修复因子ITF、MUC-2、EGF、TGF-α在肠黏膜损伤修复及肠黏膜屏障重建过程中具有重要的作用。本研究对结肠上皮细胞是否有以上生长因子的表达进行了检测。为后续开展药物对肠上皮细胞作用的指标选择提供基础及参考。
方法：采用荧光定量PCR (Real-Time PCR)检测结肠上皮细胞ITF、EGFmRNA的表达；采用Elisa的方法检测MUC-2/TGF-α蛋白表达；采用Western bolt方法检测pERK/pMEK蛋白表达。
结果：结肠上皮细胞表达ITF/EGFmRNA、MUC-2/TGF-α蛋白及pERK/pMEK蛋白。可用于肠黏膜损伤修复的相关实验研究。
3.溃结灵含药血清对UC模型血清致损的结肠上皮细胞超微结构的影响
本课题整体实验研究发现,溃结灵可明显减轻TNBS灌肠复制的UC大鼠的炎症反应,可促进肠道上皮杯状细胞的分泌,减轻结肠组织超微结构损伤,进而促进肠黏膜修复及重建。本实验在此基础上,观察溃结灵含药血清对UC大鼠血清致结肠上皮细胞损伤模型细胞超微结构的影响。进一步从细胞形态学方面探讨溃结灵的作用。
方法：三硝基苯磺酸灌肠法复制溃疡性结肠炎大鼠模型,3天后腹主动脉采血,分离获取血清,作为损伤剂作用结肠上皮细胞24h模拟细胞损伤模型,透射电镜法观察结肠上皮细胞超微结构的改变。
结果：正常对照组及空白对照组细胞可见丰富的微绒毛,有的呈小片状。细胞内可见丰富的线粒体,还可见核糖体、内质网及高、中、低等电子密度分泌颗粒；UC模型血清造模组可见较少微绒毛,胞质内可见粗面内质网扩张,少量线粒体及线粒体肿胀,空泡样变甚至溶解,有大量的胞质内空泡形成,微绒毛较少或消失,分泌颗粒较少,为中低等电子密度。给予溃结灵含药血清组细胞形态较好,细胞微绒毛及线粒体等细胞器丰富,可见较多高、中、低等电子密度分泌颗粒。
4.溃结灵含药血清对正常大鼠结肠上皮细胞增殖的影响
结肠上皮细胞在肠黏膜损伤修复中具有重要的作用,UC发病时,促进结肠上皮细胞的增殖、移行、分化,可加速受损黏膜的修复以及肠黏膜屏障功能的恢复。本实验采用溃结灵含药血清干预结肠上皮细胞,探讨溃结灵含药血清对结肠上皮细胞增殖的影响。
方法：采用血清药理学方法制备溃结灵含药血清；MTT法测定溃结灵含药血清干预大鼠结肠上皮细胞12、24、48h后细胞增殖情况。
结果：空白大鼠血清对大鼠结肠上皮细胞(CEC)生长无影响,溃结灵中剂量(5%含药血清)作用24h、48h均明显促进CEC增殖(p<0.05)
5.溃结灵含药血清对TNF-α致结肠上皮细胞损伤模型细胞增殖的影响
TNF-α为公认的UC致病因子,是肠道上皮细胞增殖和凋亡的主要促炎因子,UC发病时,TNF-α大量分泌,可使结肠上皮细胞凋亡,肠黏膜屏障的通透性增加,NF-κB被激活,进一步扩发炎症反应,损伤肠黏膜。本实验观察不同浓度TNF-α对大鼠上皮细胞增殖的影响,以确定TNF-α合适的作用浓度及作用时间建立结肠上皮细胞损伤模型,并测定溃结灵含药血清对TNF-α干预下结肠上皮细胞增殖的情况,从细胞增殖的方面,进一步探讨溃结灵对受损结肠上皮细胞的保护作用。
方法：以25,50,100,150,200ng/ml的TNF-α干预结肠上皮细胞,MTT法测定不同干预时间(3h,6h,24h)细胞的增殖情况,同法测定预先给予溃结灵含药血清对50ng/mlTNF-α干预结肠上皮细胞6h细胞增殖的影响。
结果：①50ng/ml TNF-α干预结肠上皮细胞6h后可明显抑制细胞增殖(p<0.05)；②24h时,各浓度TNF-α均明显抑制结肠上皮细胞增殖(p<0.01)③与模型组比较,溃结灵含药血清各剂量组均有一定的促进TNF-α干预下结肠上皮细胞增殖的作用,但无统计学意义(p>0.05)。
6.溃结灵含药血清对TNF-α干预下大鼠结肠上皮细胞ITF、EGFmRNA表达的影响
ITF、EGF在胃肠道上皮保护和促进黏膜愈合方面发挥着重要的作用,与UC的修复愈合关系密切。本研究观察溃结灵含药血清对TNF-α干预下结肠上皮细胞EGF及ITFmRNA表达的影响,以进一步在离体细胞水平探讨溃结灵促进肠黏膜损伤修复的作用靶标。
方法：以50ng/mlTNF-α干预结肠上皮细胞6h,造成细胞损伤模型,采用荧光定量PCR (Real-Time PCR)法检测不同浓度溃结灵含药血清对此细胞损伤模型ITF及EGFmRNA的表达的影响。
结果：①模型组EGF、ITFmRNA表达较空白对照组减低；②溃结灵各剂量组(?)GFmRNA相对表达量与模型组相比,无统计学意义(p>0.05)；③溃结灵高(10%含药血清)、中剂量组(5%含药血清)ITFmRNA表达量明显高于模型组(p<0.05,p<0.01)。
7.溃结灵含药血清对TNF-α干预下大鼠结肠上皮细胞MUC-2、TGF-α蛋白表达的影响
MUC-2、TGF-α在黏膜保护和肠道疾病修复机制中占有重有地位,且在肠道黏膜修复中MUC-2、TGF-α、EGF、与ITF具有协同作用,保护和促进黏膜修复。本研究进一步观察了溃结灵含药血清对TNF-α干预下结肠上皮细胞MUC-2及TGF-α蛋白表达的影响,探讨溃结灵促进肠黏膜损伤修复的又一可能作用靶标,以进一步揭示溃结灵治疗UC的分子作用机理。
方法：TNF-α(50ng/ml)干预结肠上皮细胞生长6h造成结肠上皮细胞损伤模型,采用Elisa方法检测不同浓度溃结灵含药血清对此细胞损伤模型MUC-2及TGF-α蛋白含量的影响。
结果：①TNF-α作用于细胞后,与空白对照组比较,模型对照组MUC-2含量增加(p<0.01), TGF-α含量降低,但无统计学差异；②与模型对照组比较,溃结灵含药血清高剂量组MUC-2含量增加(p<0.05),溃结灵含药血清高剂量组TGF-α含量显著升高(p<0.01)。
8.溃结灵含药血清对TNF-α干预下大鼠结肠上皮细胞pERKl/2、pMEK1/2蛋白表达的影响
ERK信号转导通路参与调节细胞的增生、分化、凋亡等过程。其在真核细胞内广泛存在,许多因子通过ERK通路发挥作用。而细胞信号中传递丝裂原信号的关键激酶是胞外信号调节蛋白激酶(ERK1/2), ERK被激活后,可诱导细胞产生分裂、增殖等效应。本研究观察溃结灵含药血清对结肠上皮细胞损伤模型pERK1/2、pMEK1/2蛋白表达的影响。以期进一步揭示溃结灵治疗UC的作用机理。
方法：TNF-α(50ng/ml)干预结肠上皮细胞生长6h造成结肠上皮细胞损伤模型,提取细胞全蛋白,采用WesternBlot法检测溃结灵含药血清对此上皮细胞损伤模型pMEK1/2, pERK1/2蛋白表达水平的影响,内参蛋白为β-actin,目的蛋白的相对含量为目的蛋白与β-actin密度的比值,结果进行组间比较分析。
结果：①50ng/ml TNF-α作用于结肠上皮细胞6h后,细胞内pMEK1/2、pERK1/2蛋白表达均有增加；②溃结灵高剂量(10%含药血清)、中剂量(5%含药血清)明显促进pMEK1/2蛋白表达(p<0.05),溃结灵中剂量(5%含药血清)明显促进pERK1/2蛋白表达(p<0.05)。提示溃结灵含药血清对TNF-α法干预下结肠大鼠上皮细胞pERK1/2、 pMEK1/2蛋白表达有上调作用,ERK信号通路可能是溃结灵保护及修复肠黏膜损伤的转导途径之一
三、研究结论
1.本研究建立了大鼠结肠上皮细胞原代培养体系,为结肠上皮生理、病理及药物作用机制提供了体外研究平台。对研究肠上皮细胞与炎症性肠病病因学的相关性及其中药对炎症性肠病的防治具有重要的意义。
2、本研究结果显示,结肠上皮细胞表达ITFmRNA, EGFmRNA, MUC-2、TGF-α蛋白,ITF,MUC-2由肠道杯状细胞分泌,证明,此细胞可用于肠黏膜损伤修复的相关实验研究。
3.溃结灵含药血清能够促进正常大鼠结肠上皮细胞的增殖,改善UC大鼠血清致结肠上皮细胞损伤模型的超微结构,对TNF-α干预下的结肠上皮细胞具有一定的促增殖作用。提示,促进结肠上皮细胞增殖是该方的疗效机理之一
4.溃结灵含药血清能上调TNF-α干预下的结肠上皮细胞ITF基因、MUC-2、TGF-α蛋白表达以及pERK1/2、pMEK1/2蛋白表达。提示,溃结灵可通过激活ERK通路,促进上皮细胞的增殖移行,进而加速肠黏膜损伤修复。也可能是其作用的分子机制之一
Backgrouds
Ulcerative colitis (UC) is a chronic and non-specific inflammatory disease of the colorectal mucosa. Anti-inflammatory and regulating immunity are the main clinical treatment of UC. Recent studies have found that after the damage of intestinal tract, in addition to inflammatory and injury factors, the repair factors also play an equally important role in the development of disease, the prognosis of disease is determined by the interaction between them, and many growth factors participate in the whole repair process. With the development of research, some growth factors have been gradually applied to cure inflammatory injury and obtained good results. Intestinal Trefoil Factor (ITF) is a new cell growth factor found recently,which is secreted by the goblet cells and mainly distributes in the intestinal tract. It has a strong cell protecting effect, synergizing with other intestinal repair factors (MUC-2, EGF, TGF-a,etc.) to reduce the multiple factor-mediated injury of intestinal mucosa significantly. Therefore, the study of formula on the regulation of specific protective factors may help to develop a new way to treat UC. Kui jie ling decoction (KD)was frequently used to treat UC patients. The therapeutic principle of KD is clearing away heat, invigorating the spleen and activating blood.It showed affirmative therapeutic effect on patients. Some experiments about effect of KD on UC model rats induced by TNBS were observed before. The observation showed that the number and area of ulcer in model rats were significantly reduced with the administration of KD. Pathological changes such as inflammatory cell infiltration and edema were improved. The expression of imflammatory factors such as Toll-like receptor, IL-1β, TNF-α, NF-κB were reduced. At the same time KD formula could improve ultrstructual changes, increasse gene expression of ITF and protein expression of MUC-2on the3th,7th and14th, raise gene expression of EGF, protein expression of TGF-α and pERK1/2on the14th in colonic mucosa of UC model rats. And in the reaserch of Caco-2cells injuried model induced by Serum of UC Model Rats have showed that KD medicated serum could promote Caco-2cells prolifiration, enhance mRNA expression of ITF and protein expression of MUC-2, pER、pMEK. In short, KD may regulate and enhance the expression of ITF and MUC-2, The interaction of ITF and MUC-2may reinforce the effect of protecting colonic mucosa and Caco2cells injuried model induced by Serum of UC Model Rats. In this article, the gene and the key elements of MEK/ERK pathway such as the activation of MEK and ERK in rat colonic epithelial cells were observed to evaluate the interventional effect of KD medicated serum, which would help to further explore mechanism of KD in treatment of UC and provide evidence for the previous reaserch.
Methods and Results
1. Establishment of the method for primary culture of colonic epithelial cell
The primary Colonic epithelial cell originated from normal colon tissue in rats, which can really reflect physiological and pathological changes of colon. But isolation and culture technology of CEC is a problem, which limited its application. After researched on domestic and foreign literatures, the method for primary culture of colonic epithelial cell was established in this study, which could provide a research platform in vitro for the study on physiological, physiological and medicine function mechanism of colonic epithelial cell.
Methods:Colons obtained from suckling rats (6-15d) were cut into small pieces and washed. Colonic epithelial cell cluster were separated by10ml0.1%collagenase I and hyaluronidase digestion for25min at37℃. After pipetting digestion, the supernant was transfered into another new tube and DMEM solution was added in to centrifuge for3times. Then cells were cultured in DMEM solution containing10%fetal bovine serum in a CO2incubator with a saturated humidity at37℃. Fibroblasts were removed by using the phase difference digestion and adherence. When80%-90%of the cells were adherent to the culture plat passaged with2.5g/L pancreatin digestion.
Results:The colonic epithelial cell clusters were successfully obtained which showed high viability and became adherent after24hours culture, the cells represented polygonal shape, and grew into pavestone-like monola yer gradually in4-8days,which showed an excellent proliferate ability. Fibroblasts were significantly decreased after passage. During cell growth, passage2cells grew into polygonal shape just like the shape of IEC-6. The colonic epithelial cells were identified by the ways of immunofluores cence staining and TEM observation. The cells were in good condition after being frozen and thawed.
2. Study on expression of ITF/EGF、MUC-2/TGF-αand pERK/pMEK in colonic epithelial cells
Protective and repair factors in gastrointestinal mucosa such as ITF、MUC-2、 EGF and TGF-α play an important role in the process of repair and reconstruction of intestinal mucosal barrier. In this research, the expression of ITF and other repaire factors were detected to provide basic and reference for index selection in the preceeding research on colonic epithelial cells.
Methods:real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of ITF、EGFmRNA;The protein expression of MUC-2/TGF-α were determined by the method of Elisa kit;And western blot technique was used to detect protein levels of pERK1/2and pMEK1/2.
Results:the Gene expression of ITF/EGF and the protein expression of MUC-2/TGF-α、pERK/pMEK in CECs were dected.
3. Effect of KD medicated serum on the rat colonic epithelial cell ultramicrostructure in cell damage model induced by serum of UC model rats
The previous reaserch found that KD could improve the morph and function of globlet cells and alleviate the ultrastructure damage in colonic mucosa of UC Model Rats. Based on that, the effect of KD medicated serum on epithelial cell ultrastructures damaged by serum of UC model rats was further studied in this study to explore effect of KD from the aspect of cytomorphology
Methods:Rats received enteroclysis of trinitro-benzene-sulfonic acid (TNBS) to induce ulcerative colitis models for3days. Then, blood samples were collected from abdominal aorta to get serum, rat colonic epithelial cell damage model was made by intervened with the serum for24h. Then, rat colonic epithelial cell ultramicrostructure were observed by transmission electron microscopy.
Results:Ultrastructures of colonic epithelial cell in normal and blank control group were abundant microvilli、organelles and secretroy granules with high、moderate and low electrondensity;In model group with the appearance of a few microvilli, expand of rough, cytoplasmic vacuolization, a small number of mitochondria, a few secretory granule, many vacuoles in the cytoplasm;While in the group of10%KD medicated serum showed that abundant microvilli exist, mitochondrions and other organelles were abundant, secretroy granules with high、moderate and low electrondensity could be seen.
4. Effect of KD medicated serum on the proliferation of normal rat colonic epithelial cell
colonic epithelial cells play an important role in intestinal mucosal microstructural damage and reparation. Stimulation of colon epithelial cell proliferation, migration and differentiation could promote restoration of damaged intestinal mucosa and intestine mucosal barrier function with UC. In this study, KD medicated serum was used to intervene CECs growth to explore the effects of KD medicated serum on the proliferation of CECs.
Methods:Prepared KD medicated serum with Serum Pharmacological. MTT assay was used to determine cell growth of CECs under the effects of Kuijieling Decoction medicated serum for12、24、48h.
Results:There was no effect on the CECs proliferation with rat serum without medicine. And the CECs proliferation was promoted significantly after intervened with5%KD medicated serum for24h and48h (p<0.05)
5. Effect of KD medicated serum on the proliferation of damaged rat colonic epithelial cell induced by TNF-α
TNF-α is a generally accepted pathogenic factor in UC, and its also an inflammatory factors in the process of hyperplasia and apoptosis of intestinal epithelial cell In UC, the increased secretion of TNF-α can induce CEC apoptosis, increase Permeability of intestinal mucosal barrier, activate NF-κB pathway to further enlarge response of inflammation and damage intestinal mucosa. In this reaserch the effect of different concentrations of TNF-α on the proliferation of rat CECs was dected to determine the appropriate concentrations and action time of TNF-α to establish cell damage model, and then effect of KD medicated serum on the proliferation of damaged rat CECs induced by TNF-α was measured to explore cell protection effect of KD on CECs.
Method:CECs were treated with different concentration of TNF-α (25,50,100,150,200ng/mL)for3h,6h and24h. And then the proliferous activity in CECs was measured by MTT assays, and the same method was used to detect cell prolifiration treated with50ng/mlTNF-α for6h under the protect effect of KD medicated serum.
Results:①The CECs proliferation was inhibited significantly after intervened with50ng/ml TNF-α for6h (p<0.05);②All concentrations of TNF-a could inhibit CECs proliferation obviously after intervened with TNF-α for24h(p<0.01);③Different concentration of KD medicated serum could stimulate colonic epithelial cells to reproduce in a certain degree after damaged by TNF-α, but there was no significantly difference compaired to model group(p>0.05).
6. Effect of KD medicated serums on the mRNA expression of ITF and EGF in damaged rat colonic epithelial cell induced by TNF-α.
ITF and EGF are both cell protecting factors.They play an important role in epithelial protection of gastrointestinal tract and promoting mucosal healing which are closely related to UC repair. In this article the changes of ITF、EGFmRNA in CECs intervened with TNF-α under the effect of KD medicated serum were studied to evaluate the treatment mechanism of KD.
Method:The CECs were cultured in vitro and treated with50ng/ml TNF-α for6h, and cell damage model was made by this method, Real Time PCR was used to detect the expression of ITF、EGFmRNA under the effects of differrent concentration of Kuijieling Decoction medicated serum.
Results:①The mRNA expression of EGF and ITF in model group induced by TNF-α was lower than that in the blank control group;②The mRNA expression EGF in all KD groups had no significantly difference compaired to model group(p>0.05);③The expression of ITFmRNA in high-dosage group(10%KD medicated serum) and medium-dosage group(5%KD medicated serum) were significantly higher than that in the model group(p<0.05, p<0.01).
7. Effect of KD medicated serums on the protein expression of MUC-2and TGF-a in damaged rat colonic epithelial cell induced by TNF-α
TGF-α and MUC-2play an important role in mucosal protection and repair mechanisms of intestinal disease. And they have a good synergism effect with ITF and EGF to promte damage repair of intestinal mucosa. This study was to observe influence of KD medicated serum on MUC-2and TGF-α in CECs treated with TNF-α and to explore another effect target of KD in promoting damage repair of intestinal mucosa.
Method:The CECs intervened with50ng/ml TNF-α for6h, and cell damage model was made by this method;The effects of different concentration of KD serum on the MUC-2and TGF-α protein expression was detected by using Elisa kit.
Results:①Compared with blank group, the MUC-2content in the cell damage model induced by TNF-α was obviously increased (p<0.01);②The contents of MUC-2and TGF-α in high-dosage group(10%KD medicated serum)was significantly higher than that in model group (p<0.05,p<0.01).
8、Effect of KD medicated serums on the protein expression of pMEK1/2、pERK1/2in damaged rat colonic epithelial cell induced by TNF-α
Ras/MEK/ERK pathway is an important message pathway, which exists widely in eukaryotic cells. It is the key message pathway of many factors to perform function and participate in regulating the process of cell differentiation, proliferation, cleavage and apoptosis, and so on. The extracellular signal regulated kinase(ERK1/2) is the key mitogen kinase to transmite message in cells. The activated ERK could induct cells to take place some nuclear reactions, such as differentiation and proliferation. This study was to observe the effect of KD medicated serum on changes of pERKl/2and pMEK1/2protein expression in in CECs treated with TNF-α and to explore the mechanism of them.
Method:Whole-cell protein was extracted from colonic epithelial cell,Western blot technique was used to detect the effects of KD medicated serum on the protein expression of pERKl/2and pMEKl/2of CECs damage model which induced by intervening with50ng/mlTNF-αfor6h.
Results:①The protein expression of pERK1/2in the CECs induced by TNF-α for6h was increased;②The protein expression of pMEK1/2was obviously higher than that in blank group (p<0.05);③The protein expression of pMEK1/2in the medium (5%KD medicated serum)and high (10%KD medicated serum) groups were obviously higher than that in model group (p<0.05);④5%KD medicated serum could enhance the protein expression of pERK1/2significantly (p<0.05). The relative protein expression of pERK1/2and pMEK1/2in CECs induced by TNF-α were enhanced after using KD medicated serum and ERK message pathway may be one of pathways to protect and repair intestinal mucosal injury in UC rats bv KD treatment.
Conclusions
1、The results of this study showed that CECs expressed ITFmRNA, EGFmRNA, protein of MUC-2and TGF-α;ITF and MUC-2are both cell protecting factors secreted by goblet cells. So the CECs can be used as a cell model in the reaserch of intestinal mucosa damage repairation.
2、KD medicated serum could promote CECs proliferation, improve ultrstructual changes of CECs intervened with serum of UC rats, which suggested promote CECs proliferation is one mechanism of KD to treat UC.
3、KD medicated serum could increasse gene expression of ITF and protein expression of MUC-2、TGF-α, up-regulated the protein expression of pMEK1/2、 pERK1/2in the CEC damage model induced by TNF-α, indicating that activate ERK signal pathway to stimulate the migration and proliferation of CECs is also one of the molecular mechanism of KD to treat UC.
引文
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