Exploring The Mechanism Of Th17 Differentiation And Treatment Of Traditional Chinese Medicine In Ankylosing Spondylitis Through TAZ Regulated By Hippo Signaling Pathway

Background:Ankylosing spondylitis(AS)is a common chronic inflammatory disease which can involve the sacroiliac joints,central axis joints,peripheral joints,and bone attachment points of tendon ligament.It is characterized by inflammatory pain of lumbago,back,hip and other joints.Inflammation is one of the key factors leading to bone destruction in AS.Repeated episodes of inflammation can lead to new bone formation and granulation tissue hyperplasia.Eventually,the involved ligaments and joints gradually develop into the fibrous and bony rigidity,leading to sacroiliac joint fusion,joint ankylosis and other consequences,which restricts patients activity and seriously affects the their daily life and work.Therefore,it is necessary to control AS inflammation to delay the progression of disease,protect joint function,prevent disability and improve the quality of life of patients.AS an important component of effector T cells,T helper 17 cell(Th17)play an important role in a variety of inflammatory diseases and are the core effector cells that lead to AS inflammation.Th17 cells can secrete interleukin 17(IL-17),interleukin 21(IL-21),interleukin 22(IL-22),tumor necrosis factor?(TNF?)and many other cytokines.However,IL-17 is the marker cytokine of Th17 cells and the main pathway by which Th17 cells exert its inflammatory effect.IL-17 can stimulate multiple cells to produce multiple inflammatory factors by binding to the IL-17 receptor(IL-17R)on different cells,and then opening a variety of inflammatory pathways.Therefore,the important targets are inhibiting the differentiation and function of Th17 cell and reducing the secretion of IL-17 for controlling AS inflammation.Naive CD4+T cells are stimulated by different cytokines to express different specific transcription factors,and then differentiate into different subgroups such as T helper cell 1(Th1),T helper cell 2(Th2),T regulatory cells(Treg)and Th17 respectively.Retinoid-acid receptor-related orphan receptor ?t(ROR?t),as a specific transcription factor of Th17 cell,is a key transcription factor necessary for the differentiation of Th17 cell.ROR?t is encoded by the gene of RORc,accordingly,inhibiting the translation of RORyt protein and the expression of the RORc gene is a key step in regulating the differentiation of Th17 cell.translation.Recent studies have found that TAZ(transcriptional co-Activator with PDZ-binding motif)is the most important transcriptional co-activator of ROR?t.After binding to TAZ through ligand binding region,RORyt can enhance its own transcriptional activity,thereby promoting the differentiation of Th17 cell and playing the role in inflammation.TAZ is the main effector in the downstream of Hippo signaling pathway,and its activity is regulated by Hippo signaling pathway.Hippo signaling pathway is a highly conserved growth-inhibiting pathway,which has been a research hotspot in the field of tumor since its discovery because of its functions of regulating the proliferation,differentiation and apoptosis of cells and controlling organ size.As the research progressed,the regulatory role of Hippo signaling pathway in the immune system has been gradually revealed.Mammalian sterile 20-like kinases 1/2(MST1/2)and nuclear Dbf2-related kinases 1/2(NDR1/2)constitute the core kinase of the Hippo signaling pathway,and negatively regulates TAZ levels through upstream and downstream cascade phosphorylation reactions.At present,TAZ,as a key transcription co-activator of the characteristic transcription factor ROR?t of Th17 cells,has an exact effect on Th17 differentiation shift.While the Hippo signaling pathway as an upstream regulatory pathway of TAZ,may play an important role in AS inflammation.QingreLishiHuoxue therapy is one of the basic treatment methods in the clinical treatment of active period AS pateints in traditional Chinese medicine.Preliminary clinical studies have found that QingreLishiHuoxue therapy can effectively control AS inflammation and disease activity.In the previous research on major and difficult diseases in the Eleventh Five-Year National Science and Technology Support Program,QingreQiangji decoction,which is made up of by QingreHuoxue therapy,was used to treat active stage AS.The effective rates of international ASAS 20/50/70 were 86.75%,60.68%and 41.45%respectively,with satisfactory clinical efficacy.Moreover,many previous studies have shown that QingreLishiHuoxue therapy can effectively inhibit Th17 differentiation shift through multiple targets and reduce the secretion of IL-17,thus exert anti-inflammatory effects.Based on the above discussion,we speculate that QingreLishiHuoxue therapy may inhibit the transcriptional co-activator TAZ by activating the Hippo signaling pathway,or directly inhibit the transcriptional co-activator TAZ,finally reduce the expression of the transcription factor RORyt,and thereby inhibit the Naive CD4+T cells differentiate to Th17 cell,play an anti-AS inflammation effect.Objective:To explore the molecular mechanism of transcription co-activator TAZ and its upstream Hippo signaling pathway regulating the differentiation of Th17 cells in AS patients;To explore whether the anti-inflammatory effects of QingreLishiHuoxue therapy on active AS patients was influenced by the regulation of TAZ by Hippo signaling pathway on Th17 differentiation shift.Methods:Research one and two included 30 active AS patients(AS-A),20 stable AS patients(AS-S)and 20 healthy volunteers(N).Record patients' general conditions such as gender and age,CRP and ESR levels and BSDAI score.Peripheral blood mononuclear cells(PBMC)and serum were collected by ficoll-hypaque density gradient centrifugation from venous blood.Enzyme linked immunosorbent assay(ELISA)was used to detect the serum IL-17 expression level in each group;western blot(WB)was used to detect the protein expression of MST1/2,NDR1/2,TAZ and RORyt in PBMC of each group;quantitative real-time PCR(qPCR)was used to detect the mRNA expressions of MST1/2,NDR1/2,TAZ,and RORc in PBMC of each group.Research three included 30 active AS patients.Before and after the treatment with QingreLishiHuoxue therapy,the changes of CRP level,ESR level and BASDAI score were observed.Serum IL-17 expression levels were measured by ELISA before and after treatment;WB was used to detect the protein expressions of MST1/2,TAZ and RORyt in patients' PBMC before and after treatment.Results:1.Serum IL-17 level in active AS patients was significantly higher than that in stable AS patients and normal control group(p<0.01),although the IL-17 level in stable AS patients was somewhat higher than those in normal control group,there was no statistically significant difference between them(p>0.05).Spearman correlation coefficient was used for correlation test,and it was found that IL-17 level was significantly positively correlated with inflammatory activity indicators ESR,CRP and BASDAI score(IL-17-ESR:r=0.586,p<0.01;IL-17-CRP:r=0.422,p=0.020<0.05);IL-17-BASDAI:r=0.450,p=0.013<0.05).Compared with the normal control group,the expression of RORc mRNA and protein in active AS patients was significantly increased(p<0.01);compared with the stable AS patients,the expression of RORc mRNA and protein in active AS patients was also significantly increased(p<0.01);the mRNA expression of RORc was significantly increased in stable AS patients compared with the normal control group(p<0.05),but the RORyt protein expression was not significantly different(p>0.05).The expression level of TAZ protein in active AS patients was significantly higher than that in normal control group and stable AS patients(p<0.01);compared with normal control group,the expression level of TAZ protein in stable AS patients was also significantly increased(p<0.01).At the gene level,the expression of TAZ mRNA in the active AS patients was significantly higher than that in the normal control group and the stable AS patients(p<0.01),and there was no significant difference in the expression of TAZ mRNA in the stable AS patients compared with the normal control group.The Spearman correlation coefficient was used to perform the correlation test,and it was found that there was a significant positive correlation between TAZ expression level and inflammatory activity indicators ESR and CRP(TAZ-ESR:r=0.409,p=0.031<0.05;TAZ-CRP:r=0.362,p=0.039<0.05);there was no significant correlation between TAZ expression level and BASDAI(r=0.272,p=0.161>0.05).The expression level of TAZ was positively correlated with the expression of serum IL-17 and RORyt(p<0.01).2.Compared with the normal control group,the expression of MST1 protein and mRNA in active AS patients was significantly decreased(p<0.01);compared with stable AS patients,the expression of MST1 protein expression in active AS patients was significantly decreased,but the difference of mRNA expression was not statistically significant(protein:p<0.01;mRNA:p>0.05);compared with the normal control group,stable AS patients had significantly reduced MST1 protein and mRNA expression(protein:p<0.05;mRNA:p<0.01).Compared with the normal control group,the expression of MST2 protein and mRNA in the active AS patients were significantly decreased(p<0.01);compared with stable AS patients,the expression of MST2 protein in active AS patients was significantly reduced,and there was no significant difference in mRNA expression(protein:p<0.01;mRNA:p>0.05);compared with the normal control group,the expression of MST2 mRNA and protein were significantly decreased in stable AS patients with(protein:p<0.05;mRNA:p<0.01).Compared with the normal control group,the expression of NDR1 protein and mRNA in active AS patients were significantly decreased(p<0.01);compared with stable AS patients,the expression of NDR1 protein in active AS patients was significantly decreased,but the difference of mRNA expression was not statistically significant(protein:p<0.01;mRNA:p>0.05);compared with the normal control group,there was no difference in NDR1 protein expression of stable AS patients,but the expression of mRNA was significantly decreased(protein:p>0.05;mRNA:p<0.05).Compared with the normal control group,the expression of NDR2 protein and mRNA in active AS patients were significantly decreased(p<0.01);compared in stable AS patients,the expression of NDR2 protein in active AS patients was significantly decreased,and the difference of mRNA expression was not statistically significant(protein:p<0.05;mRNA:p>0.05);compared with the normal control group,there was no statistically significant difference in the expression of NDR2 protein in stable AS patients,but the mRNA expression was significantly reduced(protein:p>0.05;mRNA:p<0.01).3.After 3 months of treatment with QingreLishiHuoxue therapy,the inflammatory indexes of AS patients,such as ESR and CRP were significantly lower than those before the treatment(p<0.01)).The disease activity index of AS patients was BASDAI,the score was also decreased significantly compared with that before the treatment(p<0.01).After 3 months of treatment with QingreLishiHuoxue therapy,the level of IL-17 in active AS patients decreased significantly compared with that before the treatment(p<0.01).After 3 months of treatment with QingreLishiHuoxue therapy,the expression of proteins of RORyt and TAZ in active AS patients were all decreased significantly compared with those before treatment(p<0.01),the expression of proteins of MST1 and MST2 were all increased significantly(p<0.01).Conclusions:1.The expression level of TAZ in PBMC of AS patients was significantly increased,and it was highly correlated with the inflammation indicators of AS and the differentiation and function of Th17 cell,indicating that the transcriptional coactivator TAZ binds to RORyt and enhances the expression of the transcription factor RORyt,thereby promoting the shift of Th17 differentiation,which may be one of the important mechanisms leading to the inflammation of AS.2.The expression levels of MST1/2 and NDR1/2 in PBMC of AS patients were significantly reduced,indicating that the Hippo signaling pathway was in a state of shutdown,which removed the inhibition of TAZ and activated the expression of transcriptional coactivator TAZ,thereby promoting the transcriptional activity of ROR?t and promoting Th17 differentiation shift,which may be one of the important mechanisms for regulating the inflammation of AS.3.QingreLishiHuoxue therapy activates the Hippo signaling pathway by enhancing the expression of MST1/2 kinase,inhibits the co-activation of TAZ,and further inhibits the expression of RORyt,a specific transcription factor of Th17 cells,and participates the Th17 differentiation shift,which may be one of the mechanisms to alleviate AS inflammation.