Study Of Sijunzi Decoction On Treating UC And The Effect On Tight Junction Protein Expression

Background:Inflammatory bowel disease (IBD) is a group of unknown and nonspecific etiology inflammatory bowel disease, including ulcerative colitis (UC) and crohn’s disease (CD). The inflammation mainly happens in mucosal layer of colon with ulcer erosion. Mostly will extend to the distal colon and rectum. Controlling the inflammation of the intestinal mucosal barrier having great significance in the clinical treatment of ulcerative colitis disease aspect. In clinic, UC not only often performent for chronic and recurrent attacking, but also showing a sharp and fulminant onset. Due to change in the environment, personal habits, diet and so on, the UC disease is a rising trend by the statistics.Intestinal mucosal barrier is the most important intestinal barrier, constituting by intact intestinal epithelial cells (Intestinal epithelial cell, IEC) and the adjacent connection between intestinal epithelial cells. They regulate water and solute (such as monosaccharides, amino acids, nucleotides, vitamins and hormones, etc.) transepithelial transporting, Studies suggest that various connection structure between epithelial cells and other cells is the key of their nursing care for the harmful substances invasion from the external environment or or pathogen. Beyond that, it is the structural basis for the maintenance of the intestinal epithelium on selective permeability and other function. Connections between cells in different ways, such as tight junctions (Tight junction, TJ), gap junctions (Gap junction, GJ), adhesive connection (Adherence junction, AJ) and desmosomes (Desmosome), etc. TJ is the most important intercellular connection. Intestinal epithelial cells (IEC) TJ Once mutated, reduced or absent, IEC gap will increase the permeability of bacteria, endotoxin and macromolecules to enter the systemic circulation by TJ. One characterize of IBD is IEC bypass permeability increaseing, then allowing pathogens and toxins through the intestine and into the epithelial basement, leading to diseases. Therefore, to protect the integrity of the tight junctions of epithelial barrier function of defense plays a vital role in the treatment of inflammatory bowel disease.Sijunzi decoction (Sijunzi Decoction, SJZD) from the Song Dynasty, "Taiping Huimin Hejiju Prescription", the representative of prescription Spleen Qi, commonly used in clinical treatment spleen deficiency syndrome. A number of clinical studies using four SJZD unilaterally, subtraction prescription or with other drugs for treatment of ulcerative colitis, getting a significant effect. Previous studies of our team find that SJZD can promote intestinal epithelial cell proliferation, migration and inhibition of apoptosis, the repair of damaged gastrointestinal mucosa and immunomodulatory effects. Thus, we propose working hypothesis:SZJD treats UC may via affecting the intestinal epithelial tight junctions (TJ) protein.Objective:Establishment experimental model of UC, as well as the intestinal epithelial barrier damaged model of Caco-2 cells in vitro to study the SJZD impact intestinal tight junction protein expression, and to explore SJZD molecular mechanism of repairing intestinal mucosa barrier, and treatmenting UC of rats. And researching its impact on the signal protein expression of NF-κB pathway.Methods:In vivo exprimerits:1. Establishment TNBS induced SD rat UC modelAfter weighing, animals were randomly divided into 3 groups of normal group (n=6), TNBS 50mg/kg (n=6) group, TNBS 100mg/kg (n=6) group, TNBS 150mg/kg (n=6) group. After modeling 24h, oral administration of sterile water (lOmL/kg) once daily for 10 days. Rats were observed body weight, diet, mental state and feces, the DAI score. Collecting serum and colon of rats and gave CMDI score and took the colon to do HE staining, giving TDI score, selecting a comprehensive method of suitable modeling as criteria.2. Treatment observation of SJZD in TNBS-induced UC ratsThe experiment set up normal group, TNBS model group, SASP positive control group and SJZD compound group (including low, medium and high doses). Intragastric administration after TNBS-induced modeling 24h, continuing for 10 days. Next day the rats’ diet, mental status, body weight, stool and occult blood score, stool frequency and weight, etc were observed, and gave DAI score. Collecting rats’ serum, and animals were sacrificed, taking the part of the colon, visually colonic mucosa lesions and colon gross morphological damage score (CMDI), taking the lesion for HE staining. Evaluating the mucosal tissue damage index (TDI) by light microscope, and analysis the treatment of TNBS-induced UC SD rats cases by SJZD.3. SJZD affect on the expression of TNBS-induced UC SD rats’ colon tight junction mRNA and proteinBy RT-PCR method to detect lesions occludin, claudin-2 gene expression in rat colon tissue, and using immunohistochemistry to analysis the expression of tight junction proteins occludin and claudin-2, average optical density of occludin and claudin-2 protein was calculated.4. SJZD affect expression of TNBS-induced UC SD rats’ colon inflammatory cytokine mRNA and their secretion in serumAfter using Real-Time PCR to detect expression of inflammatory cytokines IL-6, TNF-α, IFN-γ,IL-1βmRNA in rat colon, we used Elisa assay to measure content of IL-6, TNF-α, IFN-γ, IL-1β, intestinal trefoil peptide TFF-3 in TNBS-induced UC SD rats serum.In vitro experiments:1. Estabish TNBS induced human colon cancer Caco-2 cell damaged modelUsing different concentrations of TNBS solution (200μg/mL,300μg/mL, 400μg/mL,500μg/mL) injury Caco-2 cells at different times (4h,6h,12h, 24h,48h) produced Caco-2 cell model of inflammation, MTT method to measure cell viability, determining the appropriate concentration of TNBS modeling with injury time.2. Determination of Polysaccharide in SJZD total polysaccharide and simple quality controlDetermination of polysaccharide in SJZD total polysaccharides using sulfuric acid phenol method, and the stability SJZD total polysaccharides were investigated. In addition, the detection of the protein content of total polysaccharide using BCA method.3. Growth protective effect of SJZD polysaccharide on TNBS-induced Caco-2 damage modelAfter TNBS damage modeling, cells were treatde by different concentrations SJZD total polysaccharides (70μg/mL-200μig/mL), at different time (24h,36h,48h), MTT assay of cell viability, evaluate best treatment concentration of SJZD total polysaccharides.4. The SJZD total polysaccharide affect on tight junction protein in TNBS-damaged Caco-2 cellsExperiment was set up normal group, model group, SJZD total polysaccharide administered group, while setting different time of administration (including 24h,36h,48h), RTH-PCR was used to detect expression of claudin-1,2,4, ZO-1,2,3, occludin E-cad, JAM mRNA, and using Western-blot to measure expression of occludin, claudin-land ZO-3 protein.5. The impact of the SJZD total polysaccharide on NF-κB pathway of TNBS-damaged Caco-2 cellsExperiment was set up normal group, model group, total polysaccharide administered group, time of administration set 6,9,12h. After treating, extracting nuclear proteins of Caco-2 cells, then using NF-κB p50/p65 Tanscription Factor Assay Kit and Western-blot assay to measure expression of nuclear protein NF-κB.Results:In vivo experiment:1.50mg/kg-150mg/kg TNBS can induce SD rats colon mucosa appeared damage, preparing ulcerative colitis pathological model, model sustainable over 10 days, which TNBS 100mg/kg,150mg/kg TNBS can make more significant role damage on colon. From the stability of model and modeling cost considerations, we choose TNBS 100mg/kg TNBS dose modeling as in vivo studies.2. SJZD can reduce TNBS induced UC rats’DAI, CDMI, TDI scores. HE staining showed that SJZD can repair injuried mucosa, in which the high-dose group (crude drug 5.6g/kg) in the treatment can reach the most obvious result.3. The tight junction protein occludin and claudin-2 ranged from epithelial cells in rats’colon. TNBS induced rat colon tissue can reduce occludin, claudin-2 mRNA expression, while allowing occludin and claudin-2 protein expression was reduced. SJZD high-dose group (crude drug 2.8g/kg, 5.6g/kg) could significantly restore rat colon tight junction protein expression.4. Colon tissue of rats The model group TNF-α, IFN-γ, IL-6 mRNA express significantly upward trend than the control group. SJZD high dose group (cude drug 2.8g/kg,5.6g/kg) can reduce TNF-α, IFN-γ mRNA expression in rats’ colon, low, middle, and high-dose (crude drug 1.4g/kg,2.8g/kg,5.6g/kg) can downregulate IL-6 mRNA expression. Content of TNF-α, IFN-γ, IL-6, IL-1βin serum of model group were higher than the control group, IL-1β increased the value of having a significant difference (P<0.05), Intestinal Trefoil Factor TFF-3 expression decreased. SJZD can reduce content of inflammatory cytokines TNF-α, IFN-γ, IL-6, IL-1β in serum, increasing intestinal trefoil factor TFF-3 content.In vitro experiment:1. Different TNBS concentration has a certain vitality inhibition of Caco-2 cells, and in a dose and time dependent. We chose the concentration of TNBS 200μ/mL as modeling conditions, damaging cells for 24h, the results showed that it can cause stabilitied cell damaged model.2. SJZD total polysaccharide content of about 70% polysaccharides, polysaccharide content stabilize within one year and a half. Protein content of about 20%.3. SJZD total polysaccharide concentration between 100-150μg/mL treating can repair TNBS-induced Caco-2 cells damage, which 120μg/mL had most significant effect.4. SJZD total polysaccharides administration within 24,36,48h upregulate expression of TNBS-induced damaged Caco-2 cells tight junction protein claudin-1,2,4, ZO-1,2,3, occludin, E-cad mRNA, which ZO-3, occludin, claudin-1 are obvious, but JAM mRNA had little effect. Meanwhile, SJZD total polysaccharides can increase the TNBS-induced damaged cells ZO-3, occludin claudin-1 protein expression.5. After modeling by TNBS, Caco-2 cells nucleus protein NF-κB levels increased. SJZD total polysaccharide administration 9h can reduced content of NF-κB in TNBS-induced injuried Caco-2 cell nucleus protein, the results have a statistical significance comparing with the model group(P<0.05).Conclusion:SJZD can repair TJ protein of colonic mucosal epithelial cells of TNBS-induce damaged intestinal mucosal barrier, treating UC rats induced by TNBS. the active ingredient of Sijunzi is SJZD total polysaccharides, it can affect NF-κB related pathway, increased expression of TNBS-induced damaged Caco-2 cells tight junctions genes and proteins. The results of this study will be the basis for the treatment of UC disease in modern clinical pharmacology theoretical prescription, while laying a good foundation for the further elaboration of SJZD relating preliminary pharmacological mechanism of gastrointestinal tract.