The Effect Of Kuijieling On Treg Differentiation-related Factors In Ulcerative Colitis Rats

ObjectiveRegulatory T cells (Treg) is a subset of CD4+ cell with unique function, whose main function is immunosuppression in autoimmune diseases, including Inflammatory Bowel Disease(IBD). Treg cells can effectively inhibit response caused by harmful pathogens excessive stimulating T cell, control inflammation, and play an effective anti-inflammatory effect in experimental colitis, which is closely related to the relapse and treatment of Ulcerative Colitis. Studies have shown that, the balance with Treg and Th17 may play a key role in the pathogenesis of chronic inflammation and autoimmune diseases. The two maintain the immune balance of physiological state, mutually antagonistic on differentiation pathways and functions. The increase of Th17 cells or the decrease of Treg cells can break the balance between Treg/Th17, resulting in a series of inflammatory reactions. Our early research has shown that experimental ulcerative colitis in rats induced by trinitrobenzene sulfonic acid (TNBS) can be suppressed effectively by Kuijieling(KD), the proportion of CD4+Foxp3+ Treg cells can be improved and the proportion of CD4+IL-17A+Th17 cell can be reduced in peripheral blood. Furthermore, through the down regulation of IL-6, IL-21, IL-17, RORγ t and STAT3 expression, Kuijieling can inhibit Th17 cell differentiation and function, regulate the balance of Treg/Th17 and maintain the body’s steady state. Therefore, this study further explore factors related to Treg differentiation and function, such as IL-2, IL-10, IL-6, TGF-β1, Foxp3, STAT5, to reveal the mechanism of action and targets deeply in the treatment of UC by Kuijieling.Contents1. The effect of Kuijieling on the content of IL-2, IL-10 in plasma and TGF-β1, IL-6, IL-2, IL-10 in colonic mucosa of Ulcerative Colitis rats.Treg cells’generation mainly affected by TGF-β1, IL-2, IL-10 and so on. The differentiation of Treg can be induced by high concentrations of TGF-β1, while IL-6 and TGF-β1 jointly promote T lymphocyte differentiate into Thl7 cells. The IL-2 signaling pathway contributes the growth of the Treg in thymus, to maintain and amplification of Treg in peripheral and the immunosuppressive function of Treg cells. The immunosuppressive function of Treg can work through the secretion of IL-10. The experiment observed the effect of Kuijieling on content of IL-2, IL-10 in UC rats plasma and TGF-β1, IL-6, IL-2, IL-10 in UC rat colonic mucosa, and the related mechanism was discussed.Method:SD male rats were induced into UC model by trinitrobenzene sulfonic acid(TNBS) except normal group rats, then model rats were random divided into model group, kuijieling high dose group, kuijieling medium dose group, kuijieling low dose group and SASP group. experimental groups’intervention according to the corresponding drugs, dosage, normal group and model group water supply. After the end, plasma and colonic mucosa collected, using ELISA kits operate according to instructions, to detect content of IL-2, IL-10 in plasma and TGF-β1, IL-6, IL-2, IL-10 in colonic mucosaResults:In plasma of rats, the UC model group’content of IL-2 was significantly lower than the normal group (P<0.01), Kuijieling high dose group and SASP group’content of IL-2 was significantly higher than the model group (P<0.05 or P<0.01).In plasma of rats, the UC model group’content of IL-10-was significantly lower than the normal group (P<0.05), Kuijieling high dose group and Kuijieling low dose group’content of IL-10 was significantly higher than the model group (P<0.05).In colonic mucosa of rats, the UC model group’content of IL-2 was significantly lower than the normal group (P<0.05), Kuijieling high dose group and SASP group’content of IL-2 was significantly higher than the model group (P<0.05).In colonic mucosa of rats, the UC model group’content of IL-10 was significantly lower than the normal group (P<0.01), Kuijieling high, medium, low dose group and SASP group’content of IL-10 was significantly higher than the model group (P<0.05 or P<0.01).In colonic mucosa of rats, the UC model group’content of IL-6 was significantly higher than the normal group (P<0.05), Kuijieling high dose group and SASP group’content of IL-6 was significantly lower than the model group (P<0.05).In colonic mucosa of rats, the UC model group’content of TGF-β1 was significantly lower than the normal group (P<0.05), Kuijieling high, medium, low dose group and SASP group’content of TGF-β1 was significantly higher than the model group (P<0.05).2. The Effect of Kuijieling on mRNA of STAT5 and IL-2 in colonic mucosa of Ulcerative Colitis rats.IL-2 play a critical role for establishing and maintaining of immune tolerance, it can promote the generation of Treg cells and maintain the function of Treg cells. IL-2 activates downstream STAT5 signaling pathways, as a nuclear transcription factor, STAT5 can regulates the proliferation and differentiation of Treg, etc. This experiment observed the effect of Kuijieling on mRNA of STAT5 and IL-2 in colonic mucosa of Ulcerative Colitis rats by fluorescence quantitative PCR.Method:Molding、grouping、dosing and drawing materials were the same as the first experiment. Homogenized colonic mucosa,add RNAios, chloroform, isopropyl alcohol to extract total RNA, reverse transcription into cDNA, reaction conditions on 20 ul reaction system for reverse transcription,37℃ for 15 min,85℃ for 5 s, terminates at 4 ℃. Rt-PCR set GAPDH for reference gene, followed reaction condition: 30 sec,95 ℃ and then at 95 ℃ for 5 sec,60 ℃ for 30 sec, repeatted 40 cycles.Results:In colonic mucosa of rats, the UC model group’mRNA expression of IL-2 and STAT5 was significantly lower than the normal group (P<0.05), Kuijieling high dose group and SASP group’mRNA expression of IL-2 and STAT5 was significantly higher than the model group (P<0.05 or P<0.01).3. The effect of Kuijieling on the situ protein expression of Foxp3、 STAT5 in colonic mucosa of Ulcerative Colitis rats.Transcription factors Foxp3 specific expression on CD4+CD25+Treg cells, related to growth, development, maturity, and inhibiting function of treg, and Foxp3 is the main regulatory factor of Treg cells. STAT5 and Foxp3 have binding sites at the gene level, STAT5 can bind directly to the conservated area of the Foxp3 gene, thus promotes the expression of Foxp3, regulate the function of Treg cells. Therefore, expression of STAT5 and Foxp3 are closely linked. This experiment observed the effect of Kuijieling on situ protein expression of Foxp3、STAT5 in colonic mucosa of Ulcerative Colitis rats by Immunohistochemical technique.Method:Molding、grouping、dosing and drawing materials were the same as the first experiment. Neutral buffered formalin fixed colon mucosa, making biopsy, immunohistochemical staining, to observe Foxp3 and STAT5 protein expression region, with an average optical density value as a semi-quantitative criterion.Results:In colonic mucosa of rats, the UC model group’protein expression of Foxp3 and STAT5 was significantly lower than the normal group (P<0.01), Kuijieling high, medium, low dose group and SASP group’ protein expression of Foxp3 and STAT5 was significantly higher than the model group (P<0.05 or P<0.01).4. The effect of Kuijieling on the protein expression of Foxp3、STAT5 in colonic mucosa of Ulcerative Colitis rats.Foxp3 and STAT5 play a decisive role on occurrence, development and function of Treg cells. Foxp3 protein is an important regulatory factor in Treg cell, and activation of STAT5 protein plays a key role for expression of Foxp3.Method:Molding, grouping, dosing and drawing materials were the same as the first experiment. Cytoplasmic protein extract from colonic mucosa, BCA method for protein assay. Protein (150 ug) was fractionated on 10% SDS-polyacrylamide gels and semi-dry transferred to nitrocellulose membranes, closed, incubated with polyclonal anti-rabbit antibody, then incubated with horseradish peroxidase-labeled goat anti-rabbit antibody. Density analysis of protein content was used by BIO-RAD scanner with ECL chemiluminescene light solution.Results:In colonic mucosa of rats, the UC model group’protein expression of Foxp3 and STAT5 was significantly lower than the normal group (P<0.05), Kuijieling high,medium, low dose group’protein expression of Foxp3 was significantly higher than the model group (P <0.05 or P<0.01); Kuijieling high, medium dose group and SASP group’ protein expression of STAT5 was significantly higher than the model group (P<0.05).Conclusion:Based on the results of the study, we thought that IL-6 as one of inflammatory factors expression in great quantities, the expression of TGF-β1, IL-2, IL-10, Foxp3, STAT5 decreased, which were related to Treg’ s differentiation;Kuijieling can inhibit the expression of IL-6, enhance the expression of TGF-β1, high concentrations of TGF-β1 can promote differentiation of Treg cells; Kuijieling can promote the expression of IL-2, again activate STAT5 signaling pathway of downstream through the IL-2 signal, promoting expression of Foxp3, then enhance Treg’s function, promote the secretion of IL-10 to inhibit the immune function, which may be one of mechanism of action on the treatment of UC with kuijieling.