Effect Of Kuijieling Decoction On The Imbalance Of Treg And Th17 Cells In Vitro And Its Mechanism

Object:Ulcerative colitis(UC)is a chronic idiopathic inflammation involving colorectum,its main clinical symptoms include bloody diarrhea with mucus,abdominal pain and so on.In Chinese medicine theory,deficiency of spleen is the intrinsic basis of UC,heat induced by blood stasis presents the manifestation of UC.Because of the basic pathogenesis of UC,our institute designed a Chinese medicine named Kuijieling Decoction(KD),which is following the method of clearing heat,reinforcing spleen and activating blood.KD works well in clinic,but its mechanism isn't fully clear.In previous study,we found that KD ameliorates trinitrobenzene sulfonic acid induced colitis in rats via regulating the balance between regulatory T cells(Treg cells)and helper T cells 17(Thl7 cells).Treg cells come from CD4+ naive T cells induced by transforming growth factor-?1(TGF-?1).Treg cells maintain immune tolerance by secreting interleukin(IL)-10.Th17 cells come from CD4+naive T cells induced by the co-action of TGF-?1 and IL-6.Thl7 cells secret IL-17 and IL-21,promote development of many autoimmunity disease.Anti-inflammatory Treg cells decreasd,pro-inflammatory Thl7 cells increasd,and the balance between Treg and Thl7 cells destroyed is a crucial link in the pathogenesis of UC.Forkhead box p3(Foxp3)and retinoid acid-related orphan receptor ?t(ROR?t)are the specific transcription factors of Treg cells and Thl7 cells,respectively.Signal transducers and activators of transcription(STAT)3 and STAT5 are the most important intracellar signaling pathway in CD4+ naive T cells for Treg and Th17 cell differentiation.The present study aimed to established a model of imbalance between Treg and Thl7 cells in vitro,detect the effect of serum containing KD on the proportion,function and differentiation of Treg and Thl7 cells,testify that KD regulates the balance between Treg and Th17 cells at cell level,and explore the effect of serum containing KD on STAT3 and STAT5 sinaling pathway in lymphocyte,further reveal the potential mechanism.Methods:1.Rat's peripheral blood lymphocytes were isolated by density gradient centrifugation method.Anti-CD3 antibody and anti-CD28 antibody were administered to it for inducing the activation and proliferation of T lymphocytes.10%,5%,2.5%serum containing KD were administered for 12h,24h or 48h,then the effect of serum containing KD on the activation and proliferation of T lymphocytes in vitro were detected by MTT cell proliferation activity assay.2.Rat's peripheral blood lymphocytes were isolated by density gradient centrifugation method.Stimulated the T lymphocytes in it by Anti-CD3 antibody,anti-CD28 antibody,transforming growth factor-?1(TGF-?1),interleukin(IL)-6 and IL-23 to induce the polarization to Th17,and establish model of imbalance of Treg and Thl7 cells in vitro.The proportion of Treg or Thl7 cells in CD4+T cells were measured with flow cytometry technology.The model was stimulated by 10%,5%or 2.5%serum containing KD for 48h,the effect of serum containing KD on the proportion of Treg an Th17 cells in CD4+ T cells was detected by flow cytometry technology3.The imbalance between Treg and Th17 cells was induced by TGF-?1,IL-6 and IL-23,and stimulated by 10%,5%or 2.5%serum containing KD for 48h.Enzyme-linked immuno sorbent assay was performed to detected the effect of serum containing KD on IL-10,IL-17 and IL-21 concentration in the cell culture supernatant.Real time fluorescence quantified reverse-transcription PCR were performed to detected the effect of serum containing KD on IL-10,IL-17 and IL-21 mRNA.4.The imbalance between Treg and Th17 cells was induced by TGF-?1,IL-6 and IL-23,and stimulated by 10%,5%or 2.5%serum containing KD for 48h.Flow cytometry technology was performed to detected the effect of serum containing KD on retinoid acid-related orphan receptor ?t(ROR?t)and Forkhead box p3(Foxp3)protein expression in lymphocytes.Real time fluorescence quantified reverse-transcription PCR were performed to detected the effect of serum containing KD on ROR?t and Foxp3 mRNA.5.The imbalance between Treg and Th17 cells was induced by TGF-?1,IL-6 and IL-23,and stimulated by 10%,5%or 2.5%serum containing KD for 48h.Flow cytometry technology was performed to detected the effect of serum containing KD on signal transducers and activators of transcription 3(STAT3),p-STAT3,signal transducers and activators of transcription 5(STAT5),p-STAT5 protein expression in lymphocytes.Real time fluorescence quantified reverse-transcription PCR were performed to detected the effect of serum containing KD on STAT3 and STAT5 mRNA.6.The imbalance between Treg and Thl7 cells was induced by TGF-?1,IL-6 and IL-23,and stimulated by 10%,5%or 2.5%serum containing KD for 48h.Real time fluorescence quantified reverse-transcription PCR were performed to detected the effect of serum containing KD on IL-6,IL-23 and TGF-?1 mRNA.Results:1.10%,5%and 2.5%serum containing KD didn't have any significant effect on anti-CD3 antibody and anti-CD28 antibody induced T lymphocyte proliferation in vitro at 12h,24h and 48h.2.After the co-stimulation of TGF-?1,IL-6 and IL-23,the proportion of Treg cells in CD4+ T cells decreased,the proportion of Thl7 cells in CD4+T cells increased,the ratio of Th17 to Treg cells was enhanced,and the model of imbalance of Treg and Thl7 cells was established.10%,5%and 2.5%serum containing KD increased the proportion of Treg cells in CD4+T cells,decreased the proportion of Thl7 cells in CD4+T cells,reduced the ratio of Th17 cells to Treg cells.3.After the co-stimulation of TGF-?1,IL-6 and IL-23,IL-10,IL-17 and IL-21 concentration in cell culture supernatant didn' t change remarkly,IL-10 mRNA expression in cells decreased,IL-17 and IL-21 mRNA expression in cells increased.Serum containing KD didn' t have any significant effect on IL-10,IL-17 and IL-21 concentration in cell culture supernatant,Serum containing KD increased IL-10 mRNA in cells,decreased 11-17 and IL-21 mRNA in cells.4.After the co-stimulation of TGF-?1,IL-6 and IL-23,ROR?t protein expression in lymphocytes was increased,Foxp3 protein expression in lymphocytes was decreased,ROR? t mRNA expression in cells increased,Foxp3 mRNA expression in cells decreased.Serum containing KD decreased ROR?t protein expression in lymphocytes,increased Foxp3 protein expression in lymphocytes,decreased ROR?t mRNA in cells.5.After the co-stimulation of TGF-?1,IL-6 and IL-23,p-STAT3 protein expression and STAT3 mRNA expression in lymphocytes was increased,STAT3,STAT5,p-STAT5 protein expression and STAT5 mRNA expression in lymphocytes didn' t change remarkly.Serum containing KD decreased STAT3 and p-STAT3 protein expression in lymphocytes,increased STAT5 and p-STAT5 protein expression in lymphocytes,decreased STAT3 mRNA in cells,increased STAT5 mRNA in cells.6.After the co-stimulation of TGF-?1?IL-6 and IL-23,IL-6 mRNA expression in cells increased,TGF-?1 and IL-23 expression in cells didn't change remarkly.Serum containing KD decreased IL-6 mRNA expression,increased TGF-?1 mRNA expression in cells.Conclusion:Serum containing KD regulates the imbalance of Treg and Thl7 cells and their abnormal function induce by TGF-?1?IL-6 and IL-23.The potential mechanism may be this:serum containing KD inhibits STAT3 signaling in lymphocytes,promote STATS signaling in lymphocytes,and regulates lymphocytes secreting TGF-?1?IL-6,thereby promotes Treg cell differentiation,inhibits Th17 cell differentiation.The effect on differentiation and function of Treg and Thl7 cells may be parts of the mechanism of KD cure UC.