| BackgroundIntestinal mucoasal barrier is recognized as an important protective defense against the hacterial translocation and endotoxin penetrating from intestinal lumen. The integrity of of intestinal mucosal barrier largely depends on the proliferation, differentiation, migration and adhesion of the stem cells reside in small intestinal crypt. The rapid proliferative characteristic of intestinal crypt cells greatly contributes to the healing of intestinal mucosal injuries in pathological conditions. Licorice belongs to the category of herbs that tonifies the spleen and strengthens the Qi, in which the herbs are reported to possess treatment properties for the intestinal mucosal lesion due to Spleen Qi Deficiency through promoting the growth of intestinal epithelium cells. Moreover, licorice has a potent healing effect on the mucosal wounds in varied diseases especially in peptic ulcer. However, the cellular and molecular mechanism about the protective action of licorice on small intestinal epithelium cells is still elusive. The rat small intestinal epithelial cell line, IEC-6 is a commonly-used physiological cellular model for studying the proliferation, migration, adhesion and differentiation of intestinal epithelia cells. Correspondingly, IEC-6 cells treated with DFMO, an agent inhibits the growth of cells, are used as a pathological model. Based on the researches into the protective mechanism of herbs for invigorating spleen, small intestinal crypt cells are considered as a pharmacological target of spleen-strengthening and qi-invigorating herbal medicine. A MTT assay preliminarilly revealed that licorice extracts have a pro-proliferative effect on normal IEC-6 cells, but either the underlying molecular mechanism or the action of licorice on the cells under pathological conditions still remain to be uncovered. Based upon the researches on DFMO-treated cells, it was revealed that the expression of some genes that related with the growth of intestinal epithelial cells were regulated at post-transcriptional level. The mRNA stability of these genes are influenced by the interaction between their AU-rich elements within 3'-UTR (3'untranslated region) and HuR, a typical RNA binding protein, consequently, the quantiy of mRNA available for translation decides the production of proteins and the biological function they exert. A preliminary research conducted in our group discovered that the cytoplasmic localization of HuR in IEC-6 cells could be detected in the presence of glycyrrhizic acid, one of active principles of licorice. Therefore, we hypothesize that the post-transcriptional regulation might be involved in the proliferation of IEC-6 triggered by licorice, and HuR could be one of the potential molecular targets during the process.ObjectiveIn the present research, we aim at:1. evaluating the impact of licorice on the proliferation of normal IEC-6 cells and DFMO-induced growth inhibitory IEC-6 cells.2. examining the gene expression that correlated with the pro-proliferative action of licorice on IEC-6 cells.3. investigating the role of HuR within the process of licorice regulating gene expression and the possible post-transcriptional regulatory mechanism mediated by HuR.Methods1. Cellular models and drugsNormal IEC-6 cells and DFMO-treated IEC-6 cells were separately used as physiological and pathological cellular models under corresponding conditions. Licorice granules (a commercial product of locorice aqueous extract), glycyrrhizic acid monoammonium salt and liquiritin (two major marker compounds defined by Pharmacopoeia of the People's Republic of China) were used as the drug to determine the effect of licorice on the proliferation of IEC-6 cells.2. Measuring the pro-proliferation action of licorice granules, glycyrrhizic acid monoammonium salt and liquiritin on IEC-6 cells and determining corresponding effective dosageDirect cell number measurement, cell distribution detection and CFSE-labeled cell division tracking were employed as three major flow cytometry-based techniques in our research to evaluate the proliferative activity of normal IEC-6 cells and DFMO-treated IEC-6 cells after the administration of licorice. MTT assay and flow cytometry-based cell counting were respectively applied to determine the action of glycyrrhizic acid monoammonium salt and liquiritin on the proliferation of IEC-6 cells.3. Analyzing the effect of licorice on the expression of target genes Quantitative real-time PCR (qRT-PCR) and western blot were performed to analysize transcriptional and translational expression of genes that associated with the pro-proliferative effect of licorice.4. Investigating the post-transcriptional mechanism that licorice regulates mRNA stabitlity through HuRFollowing transcriptional repression induced by actinomycin D, the changes of mRNA stability after licorice treatment were assessed by qRT-PCR. Then the cytoplasmic and nuclear HuR levels were detected by western blot analysis to identify whether the changes of mRNA stability were accompanied with the alterning intracellular localization of HuR. Furthermore, it is necessary to verify whether the synchronous changes of mRNA stability and nucleo-cytoplasmic distribution of HuR after licorice treatment were dependent on the interaction between HuR and its target mRNAs. mRNP immunoprecipitation was used to facilitate analysis of the level of target mRNAs in endogenously formed HuR-mRNA complexes from cellular extracts. In addition, biotin pull-down assay was employed to identify whether AREs within untranslated region contributes to the binding of HuR and its mRNAs. The biological effect of ARE containing-fragment from HuR targeted mRNAs was validated throuth gene recombination and luciferase reporter gene assay.Results1. Licorice can stimulate the proliferation of DFMO-treated IEC-6 cells by reversing the growth-inhibitory effect of DFMO.Within the dosage studied in our research, licorice was found to significantly promote the growth of IEC-6 cells that had been treated with DFMO, while it did not cause similar effect on the normal IEC-6 cells. The pro-proliferative action of licorice on IEC-6 cells was accompanied with the changes in cell cycle progression. Although glycyrrhizic acid and liquiritin are two major mark components of licorice, they failed to stimulate the proliferation of IEC-6 cells as licorice.2. The pharmacological action of licorice on DFMO-treated IEC-6 cells was associated with the expression of p21 and p53.The raised levels of p21 and p53 mRNA and protein in DFMO-treated cells were significantly down-regulated by licorice in a dose-dependant manner within certain concentration range. It suggested that p21 and p53 might be target genes of licorice in our experiments.3. Licorice exerted its pharmacological action through HuR-mediated post-transcriptional regulatory mechanism.The levels of p21 and p53 mRNAs in DFMO-treated IEC-6 cells could be down-regulated by licorice via stabilizing p21 and p53 mRNAs, meanwhile, the cytoplasmic translocation of HuR induced by HuR could be reversed by licorice. Based on mRNP immunoprecipitation, the decreasing p21 and p53 mRNAs within endogenously formed HuR-mRNP complexes were detected in the IEC-6 cells treated with DFMO plus licorice, compared with DFMO-treated cells. The result indicated that licorice abolished the binding of endogenous HuR to p21 and p53 mRNA, and hence destabilized these two mRNAs and consequently decreased their expression in cytoplasm. It was further supported by biotin pull down assay that the binding of HuR and p21mRNA was due to the specific affinity of HuR to the AREs resided in 3'-UTR of p21 mRNA. Luciferase reporter gene assay proved that licorice modulated gene expression through HuR-targeted fragment from p21mRNA.ConclusionsTaken together, our conclusion from this work is that licorice post-transcriptioinally down-regulates the levels of p21 and p53 mRNAs via HuR and therefore stimulates the growth of DFMO-treated IEC-6 cells. This study presents an interpretation that licorice exerts its healing effect on intestinal mucosa through the modulationg of cell kinetics. More importantly, HuR was identified as a molecular drug target of licorice at post-transcriptional level.
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