| Background:Ankylosing Spondylitis(AS)a chronic autoimmune disease,is characterized as the central axis joint pain and stiffness.It often happens more commonly in young men with high rate of disability.Inflammation,bone destruction and new bone formation are three major pathological changes.Inflammation is the primary pathological change of AS,which T helper cell 17(Th17)is the most important effector cell that triggers it.Thl7 differentiate from the naive CD4+T cell,and regulated by the epigenetic regulation.Histone demethylase JMJD3 catalyzes demethylation of H3K27,which may be a key regulator of Th17 differentiation.In the pre-clinical study,we found that QingreHuoxue therapy to alleviate the clinical efficacy of AS inflammation satisfaction.The expression level of IL-17 in AS patients was significantly decreased,the expression of Th17 and the expression of RORc decreased significantly,and the activation level of JAK/STAT pathway was significantly decreased.The results showed that the QingreHuoxue therapy could intervening the differentiation of Th17 to remission the AS inflammation.Based on the preliminary study,this study further explored the epigenetic mechanism of JMJD3 regulation of AS Th17 differentiation and the intervention effect of QingreHuoxue therapy.Objective:To investigate the expression of JMJD3 in AS patients and the effect of demethylated H3K27 on AS Th 17 differentiation.We also want to explore the effect of traditional Chinese medicine QingreHuoxue therapy and triad of triptolide on the expression of JMJD3 and Th17 differentiation in patients with AS in vitro.Methods:Researche one,two and three took the serum and PBMC of active AS patients as the research object.Western blot was used to measure the expression of protein.RT-PCR was used to measure the expression of the mRNA.ELISA was used to measure the expression of inflammatory cytokines in serum.All of the experimental method was used to detected the relationship between JMJD3 expression,H3K27 methylation and Thl7 differentiation status in AS patients was detected in activation stage,stable stage and 3 months of treatment of disease.Researches fore was took the PBMC of active AS patients as the research object.Western blot was used to measure the expression of protein.RT-PCR was used to measure the expression of the mRNA.The study aimed to detected the effect of triptolide on the expression of JMJD3 in AS patients,the demethylation of H3K27 catalyzed by JMJD3 and the differentiation of Th17.Results:1.The expression of cytokine IL-17 secreted by Th17 in the serum of AS patients was significantly higher than that of the normal control group(p<0.001).IL-17,ESR and CRP were highly correlated with each other(IL-17-ESR:p<0.001;IL-17-CRP:p<0.01).And IL-17 expression level and patients with BASDAI no significant correlation.JMJD3 was significantly correlated with ESR and CRP(ESR:r= 0.631,p= 0.003<0.01)(CRP:r= 0.567,p= 0.009<0.01),and the expression of JMJD3 mRNA in PBMC was significantly higher than that in normal control group.There was no significant correlation between the expression of JMJD3 and the BASDAI index(r = 0.140,p = 0.634>0.05).JMJD3 was significantly associated with inflammatory cytokine IL-17(IL-17:p<0.01).2.The effect of JMJD3 demethylation of H3K27 on Th17 differentiation in patients with active AS was studied.Three groups of Active AS patients(AS-Active),stable AS patients(AS-Stable)and normal control group(N)were detected JMJD3,H3K27me3,and so on in the interventional study of JMJD3 demethylated H3K27 and Th17 differentiation in AS patients.JAK/STAT signal pathway and characteristic transcription factor RORc protein and mRNA expression.The expression level of JMJD3 protein in patients with active AS was significantly higher than that in normal controls and inpatients with AS(p<0.001),and the expression level of JMJD3 protein in patients with stable AS was not significantly different from that in normal control(p>0.05).At the gene level,the expression of J.MJD3 mRNA in patients with active AS and non-active AS was significantly higher than that normal control group(AS-Active:P<0.001;AS-Stable:P>0.05).There was also a significant difference in the mRNA expression level of JMJD3 between active and inactive AS patients(p<0.001).The level of H3K27me3 methylation in patients with active AS and non-active AS was significantly lower(P<0.01).The level of H3K27 methylation in patients with active AS and non-active AS was also significant differences(p<0.001).The phosphorylation level of STAT3(pSTAT3/STAT3)in JAK/STAT signaling pathway was significantly higher than that in normal control(AS-Active:p<0.001;AS-Stable:p<0.01).Compared with inactive patients,the expression level of JAK2 in patients with active AS was not significantly different,but the phosphorylated JAK2 expression was significantly increased(p = 0.013<0.05);STAT3 protein expression and phosphorylation were significantly higher patients with active AS were significantly higher(STAT3:p<0.01;pSTAT3:p<0.001).The expression levels of JAK2 and STAT3 mRNA in AS patients were significantly higher than those in normal controls(AS-Active:p<0.001;AS-Stable:p<0.01).There was also a significant difference in the mRNA expression of the above-mentioned signal molecules in patients with active AS compared with non-active AS patients(p<0.001).The expression of RORc in patients with active AS and non-active AS was significantly higher than that in normal control group(p<0.001).The expression of RORc in AS patients was significantly higher than that in non-active patients(p<0.001).At the level of gene expression,the mRNA levels of RORc were significantly increased in patients with active AS with the normal control group(p<0.001).There was also a significant difference between AS patients and non-active AS patients(P<0.01).3.In the study of the regulation of JMJD3 expression and Th17 differentiation in patients with active AS by QingreHuoxue therapy,we selected the effective treatment of active AS patients with wet heat stasis syndrome,and 3 months after taking the detection of its Th17 cell activity and function.After 3 months of treatment with QingreHuoxue therapy,the levels of active AS patients’ ESR,CRP and BASDAI were significantly decreased before treatment(p<0.001).The expression of JMJD3 mRNA and relative gray value were significantly different from those before treatment(p<0.05).The expression of IL-17 in the serum was significantly lower than that before treatment(p<0.01).The level of methylation of H3K27me3 in patients was significantly lower than that before treatment(p<0.001).The gene and protein expression levels of RORc were significantly lower than those before treatment(p<0.001).4.Patients with active AS were treated with triptolide,the level of JMJD3 protein in patients with AS was significantly lower than that in the control group(p<0.001),detection of gene levels results in the same result(p<0.001).After treatment with triptolide,the level of H3K27 methylation was significantly higher than that of the control group(p<0.001).There was no significant difference in the level of H3K27me3 between the TP group and the normal control(p=0.08>0.05).After activation with triptolide,the activation of JAK2/STAT3 signal pathway was significantly inhibited,and the phosphorylation level of each signal molecule was significantly decreased(p<0.001).At the gene level,the mRNA expression of JAK2 and STAT3 was significantly decreased(p<0.001).After treatment with triptolide,RORc protein and mRNA expression decreased significantly(p<0.001).Conclusion:1.JMJD3 is an important regulatory factor for Th17 differentiation and function,and is involved in the onset of AS in the event of inflammation.The expression level of JMJD3 is highly correlated with the differentiation and function of Thl7.It is also closely related to the inflammation index of AS,which may be one of the therapeutic targets for controlling AS inflammation.2.There was a significant difference between the expression of JMJD3 and the decrease of H3K27me3 level in AS patients,and there was significant difference in the expression level of JMJD3.At the same time,the expression of RORc,JAK/STAT signaling pathway and IL-17 secretion were different in patients with different disease activity levels.JMJD3 may be one of the mechanisms that regulate the differentiation,activation and function of Th17,thereby affecting AS inflammation and disease activity.3.The method of QingreHuoxue therapy has a good control effect on the inflammation index and disease activity of the patients.It may be through the intervention of JMJD3 expression and its catalytic H3K27 methylation level,which affects the epigenetic regulation of Th17 differentiation Th17 activation and IL-17 expression.4.The expression of JMJD3 in PBMC of AS patients was inhibited by triptolide in vitro.The effects of JMJD3 on the regulation of H3K27me3 demethylation,affecting Th17 transcription factor RORc,signal pathway JAK/STAT and IL-17 secretion,this may be one of the significant therapeutic agents that modifies the differentiation and function of Th17,thereby relieving AS inflammation. |
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