| Ulcerative colitis (UC), generally also called non-specific ulcerative colitis or idiopathic ulcerative colitis, is regarded as inflammatory bowel disease (IBD) combined with Crohn's disease. Although the pathogenesis is still uncertain clearly, the rate is increasing among adult patients. As a disease of digestive tract, it has a profound impact on health, and is the subject of intense global research focusing on elucidating its mechanism and treatments all the time.Although the mechanism of UC is poorly understood, it has been reported to associate with many factors, such as heredity, environment, infection and immunity. Recently, Toll-like receptors (TLRs) and many cellular signaling pathways implicated to play a role in IBD. In the study of UC, we discovered that Chinese herb drugs may also affect these pathways. We studied the effect of Chinese herbal monomer on MAPKs signaling pathways and TLR4 expression in HT-29 cells, with the aim of exploring the pathologic mechanism of IBD combined with the effect sites of Chinese herbs.Initially, TLRs were discovered in Drosophila, where they act as transmembrane signaling receptors. In a subsequent study according to homology, we found that there is a TLR family in mammal which consists of about 12 kinds of TLRs. It is a host defense mechanism of all living beings, which has been conserved throughout evolution. The pathway has the ability to activate anti-infection mechanism directly through recognizing pathogen microbes. The most extensively studied are TLR2 and TLR4, which can recognize lipopolysaccharide and peptidoglycan respectively, activate transcription factors NF-kB, induce the expression of cytokines and defensin and mediate the release of inflammatory mediator.Intestinal epithelial cells (EC) express TLRs widely, however, because of low levels of TLR proteins and high levels of some Toll signaling negative-regulators, normally TLRsare hyporesponsive to microbe and their products in intestinal epithelial cells. However, the expression of TLR4 is enhanced in IBD, which can induce NF-kB activation and initiate a defense mechanism. Over-activation of NF-kB can lead to extension of inflammation.The mitogen-activated protein kinase (MAPK) pathway is an important signaling mechanism for mediating cellular responses, which includes several subgroups such as extracellular-signal regulated (ERK), c-Jun amino-terminal kinase (JNK)/Stress-activated protein kinase (SAPK), p38. They signal through three highly conserved kinase cascades: extracellular stimuli activate MAP kinase kinase kinase (MKKK), which then activate MKK kinase kinase (MKK), which in turn activate MAPK via dual Tyr and Thr phosphorylation. EPK1/ EPK2, JNK/SAPK, p38MAPK, included in MAPKs signaling pathways, take part in intestinal mucosal injury in IBD.The NF-kB family in mammals is composed of five members: NF-kB 1, NF-kB2, RelA, RelB and c-Rel, which function as homo- or hetrodimers. NF-kB is most commonly a hetrodimer of p50/p65. After activation, NF-kB translocates to the nucleus and activates transcription of multiple KB-dependent genes. Over-activation of NF-kB can lead to many diseases, for example, Patients with IBD activation of NF-kB results in intestinal epithelial cells producing elevated levels of pro-inflammatory cytokines, causing the impairment of epithelial cells.In recent years, the curative effect of Chinese traditional medicine and Chinese herbs in the treatment of UC gave promising results with minimal side effects. In the present study, we also found that Chinese herbs and their components such as Dahuang Mudan decoction of herbal medicine, emodin, berberine, synephrine, and astragloside can affect the expression and production of defensin. As defensin expression is related to TLRs, we studied the effect of emodin and other Chinese herbal monomer on signal transduction.In this study, we established inflammatory cellular model with HT-29 cells, and observed the changes of TLRs expression, MAPKs signaling pathways, IkB/NF-kB expression, IL-8 secretion, and the interference Chinese herbs have on inflammatory cellular model. 1. Experimental Research 1.1. Establishment of cell culture methodsIn this study, a HT-29 cell line was cultured in vitro. We optimized the conditions of cellular passage, cryopreservation and resuscitation, observed their shape and state by microscope, and used WST-8 and MTT assay to measure growth curve.Results showed that HT-29 cells could passage at ratio 1/2 when they covered 80% of the bottom of the flask, and optimal cryopreservation concentration is 5xl06/ml. When resuscitated, cells were removed from liquid nitrogen to a waterbath at 38 degree centigradeand defrosted within 1~2 min. Cells were inoculated at a density of 1x10 /ml, and adhered well after 24 hours. The growth curve showed that cells reached the exponential phase growth after 3 or 4 days, and were fit for research.1.2. Biological characteristics of HT-29 and effect of Chinese herbs on their proliferation No changes of HT-29 cells proliferation were detected after treatment with emodin,curcumin, astragloside, geniposide and niringin for 12 to 24 hours. There was also no change of HT-29 cells after co-incubation with emodin for one week.The results of reverse transcriptase PCR (RT-PCR) and flow cytometry assays showed that without any stimulants, HT-29 cells express TLR4, MD-2 and IL-8 at low levels, and these levels did not change post emodin, curcumin, astragloside and geniposide treatment. After HT-29 cells were stimulated with LPS, IL-8 secretion increased as expected according to models of inflammation. Thus, we stimulate HT-29 cells with LPS to establish a cellular model.1.3. Establishment of inflammatory cellular model and the influences of Chinese herbs RT-PCR results show that gamma-IFN can enhance the expression of TLR4 and MD2genes, which correlates to the character of inflammatory cells. gamma-IFN and LPS can coordinate to induce IL-8 secretion, so an inflammatory cellular model can be established with them respectively or together.In studying the influence of Chinese herbs on an inflammatory cellular model, we found that astragloside and emodin can inhibit the up-regulation of gamma-IFN-induced TLR4 gene expression, and furthermore, astragloside, geniposide and emodin can inhibit the up-regulation of IFN-gamma-induced MD-2 gene expression. IL-8 secretion data showed that astragaloside, geniposide, emodin and curcumin can obviously inhibit the increased secretion of IL-8 induced by LPS and gamma-IFN. We found all Chinese herbal monomers used in this study can resist inflammation of HT-29 cells to different extents. Specifically emodin has a relatively significant effect. Different concentrations of emodin (10umol/L~80umol/L) can significantly inhibit IL-8 secretion in HT-29 cells induced by gamma-IFN plus LPS in a dose-dependent manner. Hence, the remainder of this study focused on this particular monomer.1.4. The effect of Chinese herbs on MAPKs signaling pathwaysInhibition of the inflammatory cellular model by emodin was observed at protein and polypeptide levels in the above study. We investigated ERK/JNK/P38 phosphorylation in MAPKs pathways to find the mode of action of emodin. Results show that EPK/JNK/p38 phosphorylation was enhanced by different concentrations of LPS (10 ng/ml, 100 ng/ml, 1000 ng/ml), which peaked at 15 min, and started to weaken after 30 min. Moreover, this effect can be enhanced by gamma-IFN. Therefore, based on our cellular inflammatorymodel induced by LPS plus gamma-IFN, the interference of emodin on MAPKs signaling pathways was detected. And results showed that emodin can inhibit JNK and p38 phosphorylation of inflammatory cellular model, indicating emodin can affect MAPKs signaling pathways. 1.5. The interference of Chinese herbs on the activity of NF-kBIn order to explore the mechanism of MAPKs signaling pathways activation, we detected the activity and content of downstream molecule NF-kB. After activation, NF-kB was phosphorylated and translocated to the nucleus. Our results show that LPS or LPS plus gamma-IFN can induce NF-kB translocation, which can also be inhibited by emodin in a dose-dependent manner.2. ConclusionsHT-29 cells can be applied to studying TLR4, MD-2 and IL-8;LPS and gamma-IFN can be used to induce an inflammatory cellular model in HT-29 cells;emodin, geniposide, astragaloside, niringin and curcumin can interfere with this HT-29 inflammatory cellular model at several levels;emodin can inhibit both MAPKs signaling pathways and NF-kB activation in HT-29 cells;emodin may be an inhibitor of signaling pathways or signaling molecules.3. InnovationThis study initialed on a solid basis of scientific creativity. First, this new subject focused on studying signal transduction. Second, all methods in this study employed new techniques developed in recent years. Third, a technical platform for Chinese herbal research and HT-29 a model for cellular inflammatory were established successfully. Finally, levels of functional molecules, signaling pathways and transduction regulation system, anti-inflammation mechanism of emodin were clearly investigated which potentially provide the experimental basis for applying emodin to cure IBD.4. LimitationsProblems in this study are major in two aspects: one is that we only studied HT-29 cells;the other is lack of regularity in selection of Chinese herbal monomer.5. ProspectiveFurther study is required on the basis of this paper. Identification of targets for emodin will be helpful for subsequent study. Study can then combined in vivo with in vitro will be beneficial for the evaluation of Chinese herbal effect. Finally, investigation of more inflammatory molecules will be assistance for the study of functional mechanism of Chinese herbal monomer. |
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