| BackgroudsUlcerative 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 Results1. Establishment of the method for primary culture of colonic epithelial cellThe 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 cellsProtective 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 ratsThe 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 cytomorphologyMethods: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 cellcolonic 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-αTNF-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-αTGF-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-αRas/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. Conclusions1、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. |
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