| ObjectiveT helper cells play an significant role in the process of occurrence and development of Immune-mediated disease,such as Inflammatory Bowel Diseases including Ulcerative Colitis and Crohn Disease.The researches have found that large numbers of T helper cells invaded into the inflamed bowel in UC patients,which noted the cytokines secreted by T helper cells and then triggered and aggravated the inflammation.T helper 17 cells and T regulatory cells rival to each other in function and the process of differentiation so as to maintain the stability of immunization in human body. If some cytokines are in abnormal expression and differentiation, they might break the balance of Treg/Th17.The previous researches have proved that Kuijieling(KD) have an effect on TNBS method UC rat model,through up-regulating expression of Foxp3 and TGF-β1,down-regulating IL-6 expression that strengthening Treg function and differentiation and suppress Th17 differentiation to recover the balance of Treg/Th17. Therefore,the research on Th17 cells and related cytokines, such as TGF-β1, IL-6, IL-21, RORyt, STAT3,IL-17 might be helpful to discover the mechanism of KD treating UC.Contents1.The effect of KD on the mRNA expression of TGF-β1, IL-6, IL-21, RORyt, STAT3,IL-17 in colonic mucosa of Ulcerative Colitis Model RatsThe cytokines,such as TGF-β1,IL-6,IL-21,RORyt,STAT3, could promote Th17 differentiation.TGF-β1 in low concentration and IL-6 together could induce RORyt expression to advance initiative T cells to differentiation toward Thl7,while TGF-β1 in higher concentration might improve Foxp3 expression to advance initiative T cells to differentiation toward Treg that might suppress Thl7 differentiation. IL-6 and IL-21 can activate JNK and then STAT3 to promote Thl7 differentiation and IL-17 expression.The low expression of STAT3 might down-regulate IL-17,otherwise up-regulate RORyt that might suppress Foxp3 and promote initiative T cells to differentiation toward Thl7 instead of Treg. RORyt that is the specific transcription regulation factor of Th17 cells can promote IL-17 expression and induce Th17 cells differentiation and be effective on inflammation Th17 cells mediated in vitro.RORyt is necessary for IL-17 variable expression.In the process of T helper cells differentiation toward Th17 cells stimulated by antigen,IL-6,IL-21 and TGF-(31 could induce RORyt expression,while it and IL-17 might be ignored without IL-6.The research on the effect of KD on the mRNA expression of TGF-(31, IL-6, IL-21, RORyt,STAT3,IL-17 in colonic mucosa of UC model rats is to discover its mechanism.Method:UC model SD male rats are induced by TNBS.The rats were randomly divided into model control group,a blank control group, SASP group, high-does KD, medium-does KD, low-does KD. After the drug treatment with KD in three various dosage and SASP, the rats were sacrificed and scraped their colonic mucosa. Real-time PCR was used to assay the expression of TGF-β1, IL-6, IL-21, STAT3, RORyt,IL-17mRNA.Result:The expression of TGF-(31mRNA in UC model rats colonic mucosa membrane tissue of colon in high-dosage groups of KD and SASP group was significantly lower than model group (P<0.05).The expression of IL-6 mRNA in UC model rats colonic mucosa membrane tissue of colon in model group was significantly higher than that in normal group (P < 0.01),which expression in high and middle dosage groups of KD and SASP group were obviously lower than that in model group (P<0.01).The expression of IL-21 mRNA in UC model rats colonic mucosa membrane tissue of colon in model group was significantly higher than that in normal group (P < 0.01),which expression in high and middle dosage groups of KD and SASP group were obviously lower than that in model group (P<0.01 or P<0.05).The expression of STAT3 mRNA in UC model rats colonic mucosa membrane tissue of colon in model group was significantly higher than that in normal group (P < 0.01),which expression in high and middle dosage groups of KD and SASP group were obviously lower than that in model group (P<0.01 or P<0.05).The expression of RORyt mRNA in UC model rats colonic mucosa membrane tissue of colon in model group was significantly higher than that in normal group (P < 0.01),which expression in high and middle dosage groups of KD and SASP group were obviously lower than that in model group (P<0.01).The expression of IL-17 mRNA in UC model rats colonic mucosa membrane tissue of colon in model group was significantly higher than that in normal group (P < 0.01),which expression in high and middle dosage groups of KD and SASP group were obviously lower than that in model group (P<0.01 or P<0.05).2.The effect of KD on the protein expression of RORyt,STAT3 in colonic mucosa of Ulcerative Colitis Model RatsSTAT3 can regualte RORyt expression to suppress Thl7 cells differentiation and then lead to RORyt down-regulated.STAT3 is expressed in the cytoplasm that might be also in the mononuclear cells and neutrophile granulocyte and epithelial cell of lamina propria in bowel mucosa.RORyt can be seen in the inflammatory cells nuclear and cytoplasm. The research is to locate and quantitate the STAT3 and RORyt protein expression by Immunohistochemical technology.Method:Molding、grouping、dosing and drawing materials were the same as the first experiment.Neutral buffered formalin is used to fix colon tissue and then make pathological section.Immunohistochemical technology is used to detect the protein expression of STAT3 and RORyt.Result:The protein expression of RORyt in UC model rats colonic mucosa membrane tissue of colon in model group was higher than that in normal group (P< 0.01),which expression in high,middle and low dosage groups of KD and SASP group were obviously lower than that in model group (P<0.01 or P<0.05).The protein expression of STAT3 in UC model rats colonic mucosa membrane tissue of colon in model group was higher than that in normal group (P< 0.01),which expression in high,middle and low dosage groups of KD and SASP group were obviously lower than that in model group (P<0.01 or P<0.05)3.The effect of KuiJieling decoction(KD) on the protein expression of IL-21 in colonic mucosa of Ulcerative Colitis Model Rats.IL-21 can be largely expressed in the Th17 cells.Some related research have found that IL-21 can obviously promote Th17 cells differentiation, besides,IL-21 replace IL-6 to come together with TGF-β1 to induce Th17 cells differentiation.The research is to discuss the effect on the protein expression of IL-21 in colonic mucosa of Ulcerative Colitis Model Rats and the mechanism of KD.Method:Molding、grouping、dosing and drawing materials were the same as the first experiment.the protein expression of IL-21 in colonic mucosa will be examined by ElISA.ResuIt:The protein expression of IL-21 in UC model rats mucous membrane tissue of colon in model group was significantly higher than that in normal group (P < 0.05),which expression in high and middle dosage groups of KD and SASP group were obviously lower than that in model group (P<0.05).Conclusion:The results can be got by comprehensive analysis as follows:The large expression of such inflammation cytokines as IL-6 and IL-21 could activate significant cytokines STAT3 that promote Th17 differentiation and secrete IL-17 through RORyt. After treatment with KD,up-regulating TGF-β1 and IL-6 and IL-21 were suppress could down-regulate RORyt and then suppress STAT3 expression and finally suppress Th17 differentiation and secrete IL-17.The conclusion have indicated that KD had an effect on Th17 differentiation by regulating the above cytokines to recover the balance of Treg/Th17 and then to kill UC. |
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