| Background:Ankylosing spondylitis(AS)is a common chronic inflammatory spondy-loarthropathy Which is characterized by high disability rate,spine and ankle arthritis and tendon ligament attachment inflammation are common in clinical.Inflammation is a key factor in bone destruction and pathological ossification in patients with AS.Bone erosion and heterotopic ossification with bone and joint fusion and stiffness are caused by repeated inflammation,which restricts patient activity and seriously affects daily life and work.Therefore,controlling inflammation is the key to delaying the course of AS and pre-venting disability.T helper cell 17,Also called Th17,is the main effector cell of AS inflammation,and induces proinflammatory mediators and chemokines through the characteristic secretory cytokine interleukin 17(IL-17).For controlling AS inflammation,the important targets are mediating AS inflammation,inhibiting Th17 differentiation,and reducing IL-17 secretion.Th17 cells are differentiated from naive CD4+T cells(Naive CD4+T cell)and belong to one of the CD4+T cell subsets.When naive CD4+T cells are activated by stimulation of external inflammatory factors,the characteristic transcri-ption of Thl7 cell differentiation is triggered.The expression of Retinoid-acid receptor-related orphan receptor yt(RORyt)turned on the process of Th17 differentiation,and the differentiation into other subpopulations was inhibited.Therefore,inhibition of RORyt transcriptional activation is a key step in inhibiting the shift of Th 17 differen-tiation.The transcription factor RORyt is regulated by the upstream Janus kinase/signal transducer and transcriptional activator(JAK/STAT)pathways.JAK2/STAT3 is an important pathway involved in the differentiation of Th17 cells.After activation by external IL-23,JAK/STAT signaling pathway,phosphorylation of STAT3 is induced.Its dimer enters the nucleus,which binds to the transcri-ption factor RORc.The increased transcription activity of RORc turns on Th17 cell differentiation,and bind to a specific site of the IL-17 gene promoter sequence to promote IL-17 transcription and secretion.Therefore,Inhibiting of the transcriptional activation of RORyt by JAK/STAT signaling pathway,is important for controlling AS inflammation.In recent years,a large number of studies have found thatextensive histone H3 lysine 27-trimethylation exists in the encode regions of promoter of transcription factor RORyt,the upstream signal molecule STAT3 and the downstream effector IL-17.Modification of histone methylation is a common modification in epigenetics that can depolymerize or fold cellular chro-mosome structures,thereby the process of activating or inhibiting transcription is dynamic and reversible,and the regulatory genes are turned to silent activation just likethe "switch".Among them,the trimethylation of histone H3K27 mainly maintains gene silencing.It was found that H3K27me3 can inhibit the transcri-ptional activation of RORyt,STAT3 and IL-17.When the H3K27me3 marker was removed from the promoter regions of RORyt,STAT3 and IL-17 genes,the transcriptional repression of genes is weakened,the silence is lifted and turned to activation,and Thl7 cell differentiation is turned on.Therefore,histone H3K27me3 level may be an important regulatory factor that affects the differentiation of Th17 cells in AS patients..The level of histone H3K27me3 is regulated by histone demethylase JMJD3 and histone methyltransferase EZH2.The JmjC domain-containing protein 3(JMJD3)and the Zener homolog 2(EZH2)plays an antagonistic role on the histone H3K27 locus.The histone methylation-modifying enzyme,JMJD3 as a histone demethylase can specifically degrade H3K27me3,and EZH2 inhibits gene expression by specifically catalyzing the trimethylation of H3K27 site.After knocking out JMJD3 or applying its inhibitor,Naive CD4+T cells hardly differentiated into Th17 cells;in contrast,after EZH2 knockdown,Naive CD4+T cells turned to Th17 differentiation,indicating thatthe histone methylation-modification of enzyme JMJD3/EZH2 may play an upstream regulatory role in Th17 differentiation by regulating H3K27me3 levels.Clearing away heat and dampness and promoting blood circulation is one of the common treatments for clinical treatment of AS in traditional Chinese medicine.In the previous study of the major difficult diseases of the 11th Five-Year Science and Technology Support Program,the Qingre Qiangtang Decoction for the treatment of active period AS.The international effective efficacy AS AS 20,50,and 70 was 86.75%,60.68%,and 41.45%,respectively,and Qingreqiangji Decoction significantly improved AS disease activity,inflammation index and clinical symptoms.In the previous Beijing Natural Science Foundation project,we found that Qingre Huoxue method can inhibit Th17 differentiation and exert anti-inflammatory effects.In addition,in the previous study,we found that there were high expression of JMJD3 and low expression of EZH2 in PBMC of AS patients,and Qingrehuoxue method can significantly reduce the expression level of JMJD3 in PBMC of patients with AS.Based on the above findings,we hypothesize that the inhibitory effect of traditional Chinese medicine on Th17 differentiation may be influence ofhistone methylation modifying enzyme JMJD3/EZH2 through interfering with transcription factor RORc,JAK/STAT pathway signaling molecule and effector IL-17 gene promoter by realizing the level of trimethylation of the region H3K27.Tripterygium wilfordii is a rheumatoid drug with anti-inflammatory,immun-osuppressive and analgesic functions.Triptolide is one of the active constituents of the traditional Chinese medicine Tripterygium wilfordii.In the previous study,we found that the traditional Chinese medicine triptolide can inhibit the expression of histone demethylase JMJD3 in peripheral blood mononuclear cells,or up-regulation of histone methyltransferase EZH2 expression,affects the differentiation and function of Th17 cells,in AS patients.Based on this,our study is going to further explore the interven-tional effect of the traditional Chinese medicine triptolide on the level of histone H3K27me3 and the epigenetic mechanism of Th17 cell differentiation in AS patients.Study One:Th17 differentiation status and histone H3K27me3 expression in AS patientsObjective:To investigate the expression level of H3K27me3 in AS patients and its epigenetic mechanism of regulating Th17 cell differentiation during AS inflammatory process;Methods:1.30 patients with active AS(AS-Active)and 15 patients with stable AS(AS-Stable)were enrolled in the outpatient or inpatient hospitals of Guang’anmen Hospital of China Academy of Chinese Medical Sciences.10 healthy volunteers were included as normal control group(N).Serum was collected from the blood of the elbow and venous blood.PBMC was isolated by Ficoll-hypaque density gradient centrifugation.Serum IL-17 expression levels were detected by enzyme-linked immunosorbent assay(ELISA).IL-17 levels and inflammatory markers ESR and CRP were studied.Correlation of BASDAI with disease activity was also observed.2.Western blot(WB)was used to observe the expression of H3K27me3 in PBMC of each groupincluding AS patients,AS-Stable and healthy control.The proteinexpression and phosphorylation of JAK/STAT signaling pathway,and the expression of characteristic transcription factor RORc was assessed.Real-time quantitative PCR(Quantitative Real-time PCR)was used to detect mRNA expression.Spearman correlation coefficient was used to correlation analysis to study the correlation between H3K27me3 and inflammatory activity and Thl7 differentiation in AS patients.Results:1.Serum levels of IL-17 were significantly lower in active and inactive AS patients than the normal control group(p<0.05);active IL patients had significantly lower serum IL-17 levels than non-active patients(p<0.01).There was a significant positive correlation between serum IL-17 levels and inflammatory markers CRP and ESR in patients with AS(IL-17-CRP:p<0.05;IL-17-ESR:p<0.05);2.Compared with the normal control group,JAK2 protein expression level was increased in active AS patients,and there was no significant difference protein expression of STAT3(JAK2:p<0.05;STAT3:p>0.05);compared with inactive period,active period there was no significant difference protein expressionof JAK2 and STAT3 in AS patients(JAK2:p>0.05;STAT3:p>0.05),but the expressionof pJAK2 and pSTAT3 were significantly increased(pJAK2:p<0.05;pSTAT3:p<0.01).However,compared with the normal control group,the expression of JAK2 and STAT3 protein in inactive AS patients was not significantly different(JAK2:p>0.05;STAT3:p>0.05),there was no significant difference in pJAK2 level,while pSTAT3 level was significantly increased(pJAK2:p>0.05;pSTAT3:p<0.05).The levels of RORc mRNA and protein in patients with active and inactive AS were significantly higher than those in the normal control group(p<0.05).The expression of RORc mRNA and protein in active patients was significantly higher than that in the inactive phase(p<0.01)..3.Compared with the normal control groupand the inactive phase,the level of H3K27me3 in active AS patients was significantly increased(p<0.05),and(p<0.01),respectively,in active AS.levels of H3K27me3 in inactive AS were some increased than thoseinthe normal group.But there wasno significant difference.There was a negative correlation between H3K27me3 level and CRP and ESR(H3K27me3-CRP:p<0.05;H3K27me3-ESR:p<0.05),which was negatively correlated with RORc protein expression(p<0.05).2.There is a low level of H3K27me3 expression in AS patients,and its expression level is opposite to JAK2,STAT3 phosphorylation level,RORc and IL-17 expression level,and negatively correlated with inflammation index CRP and ESR,indicating low level of H3K27me3.It may be one of the important regulatory factors that induce JAK2/STAT3 pathway activation and RORc transcriptional activation,promote Th17 differentiation,and mediate AS inflammation.Study Two:The effect of histone methylation modification enzyme JMJD3/EZH2 on H3K27me3 levels and Th17 differentiationObjective:To investigate the expression of histone methylation modifying enzymes JMJD3 and EZH2 in AS patients and their intervention effects on Th 17 differentiation.Methods:Western Blot(WB)was used to evaluate the expression of JMJD3 and EZH2 in PBMC of each group,and compared with healthy volunteers.Real-time quantitative PCR(Quantitative Real-time PCR)was used to detect mRNA expression.Spearman correlation coefficient was used for correlation analysis.The correlation between JMJD3,and EZH2 in AS patients with inflammatory activity and Th17 differentiation was studied.Results:Compared with the groups ofnormal control and the inactive phase,the expression of JMJD3 mRNA and protein was significantly increased in active AS patients(protein:p<0.01;mRNA:p<0.05).There were no significant differences between the inactive AS patients and the normal ones.Compared with the normal control group,the expression of EZH2 mRNA and protein in patients with active and inactive AS decreased significantly(p<0.05).The expression of EZH2 mRNA and protein in active AS patients was significantly lower than that in that non-active phase(p<0.05).After correlation analysis,it was found that the expression level of JMJD3 was negatively correlated with H3K27me3 level(p<0.05);the expression level of EZH2 was positively correlated with H3K27me3 level(p<0.05).The expression level of JMJD3 was positively correlated with IL-17 and RORc levels(p<0.05).The expression level of EZH2 was negatively correlated with IL-17 and RORc levels(p<0.05).Study Three:Effect of QingreLishiHuoxue therapy on H3K27me3 expression and Th17 DifferentiationObjective:To investigate the effect of QingreLishiHuoxue therapyrecipe on the H3K27me3expression of PBMC in AS patients and its regulation on Thl7 differentiation.Methods:Before and after treatment with QingreLishiHuoxue therapy,the changes of inflammation index and disease activity of AS patients were observed.The expression of serum IL-17 was detected by ELISA before and after treatment with traditional Chinese medicine.The expression of H3K27me3 in PBMC of patients was detected by Western Blot before and after treatment with clearing heat,dampness and promoting blood circulation.And the expression level of the characteristic transcription factor RORc of Th17 differentiationwas also assessed.Results:1.After 3 months of treatment with QingreLishiHuoxue therapy,the ESR and CRP of AS patients were significantly decreased than those before treatment(ESR:p<0.01;CRP:p<0.05).The disease activity index of BASDAI was improved than that before treatment.The decrease was significantly(p<0.01);the serum IL-17 level was significantly declined than that before the Chinese medicine treatment(p<0.01).2.After 3 months of treatment with QingreLishiHuoxue therapy,the expression level of H3K27me3 and RORc in AS patients was significantly decreased than that before Chinese medicine treatment(p<0.01)respectively.Study Four:Effect of QingreLishiHuoxue therapy on Expression of Histone Methylation Modification Enzyme JMJD3/EZH2Objective:To investigate the effect of QingreLishiHuoxue therapy on the expression of histone methylation-modifying enzyme JMJD3/EZH2 in PBMC of patients with AS.Methods:Western Blot was used to detect the expression of JMJD3/EZH2 in PBMC of patients before and after treatment with clearing away heat and dampness and promoting blood circulation.Results:After 3 months of treatment with QingreLishiHuoxue therapy,the expression level of JMJD3 in AS patients was significantly downregulated than that of before Chinese medicine treatment(p<0.01),and the expression level of EZH2 was significantly upregulated than that of before Chinese medicine treatment(p<0.01).Study Five:Effect of Chinese medicine monomer triptolide on H3K27me3 level and Th 17 differentiationObjective:To observe the effect of in vitro intervention of Chinese herbal medicine triptolide on the expression of PBMC H3K27me3 in active AS patients and its regulation on Th 17 differentiation.Methods:PBMCs from active AS patients were isolated and cultured in vitro.The treatment withChinese medicine monomer triptolide was usedin vitro,and the small molecule inhibitor GSK-J4 of JMJD3 was used as the positive control group.The optimal intervention concentration of the traditional Chinese medicine triptolide and GSK-J4 was determined by Cell Counting Kit-8(CCK-8)method and ELISA.The effects of triptolide on the expression of H3K27me3 phosphorylation of transcription factor RORc in JAK/STAT signaling pathway were observed under the optimal drug concentration.Results:1.The level of H3K27me3 in the triptolide monomer intervention group(AS-TP)and the small molecule inhibitor GSK-J4 intervention group(positive control group)was significantly upregulated than that in the blank control group(p<0.05);There was no significant difference between the AS-TP group and the positive control group.2.Triptolide monomer intervention group(AS-TP)and small molecule inhibitor GSK-J4 intervention group(positive control group)can significantly inhibit phosphorylation level ofJAK2/STAT3 signaling factor(p<0.05);The difference between the AS-TP group and the positive control group was not statistically significant.There was no significant difference in the expression of JAK2 and STAT3 mRNA between the triptolide monomer intervention group(AS-TP)and the small molecule inhibitor GSK-J4 intervention group(positive control group)compared with the blank control group.3.mRNA and protein levels of RORc was significantly inhibited by triptolide monomer intervention group(AS-TP)and small molecule inhibitor GSK-J4 intervention group(positive control group)(p<0.05);There was no significant difference in the positive control group and AS-TP group.Study Six:Effect of Chinese medicine monomer triptolide on expression of histone methylation modifying enzyme JMJD3/EZH2Objective:To observe the effect of in vitro intervention of traditional Chinese medicine triptolide on the expression of PBMC JMJD3 and EZH2 in active AS patients.Methods:The effects of triptolide on the expression of JMJD3 and EZH2 and the expression of phosphorylation and phosphorylation of transcription factor RORc in JAK/STAT signaling pathway were observed under the optimal drug concentration.See "Experiment l" for details.Results:1.The mRNA and protein levels of JMJD3 in the triptolide monomer intervention group(AS-TP)and small molecule inhibitor GSK-J4 intervention group(positive control group)were significantly decreased than those in the blank control group(p<0.05).There was no significant difference between the AS-TP group and the positive control group.2.EZH2 mRNA and protein levels in the triptolide monomer intervention group(AS-TP)and small molecule inhibitor GSK-J4 intervention group(positive control group)were significantly increased than the blank control group(p<0.05);positive The level of EZH2 in the control group was significantly increased than that in the AS-TP group(p<0.05).Conclusion:1.The normal human H3K27 exists in a trimethylated state,and the Th17 gene remains silent;the level of H3K27 trimethylation in AS patients decreases,thereby activating the Th17 characteristic transcription factor RORc and the upstream JAK/STAT signaling pathway,possibly AS One of the important mechanisms of inflammation.2.The high expression of histone demethylase JMJD3 in AS patients and the low expression of methyltransferase EZH2 may demethylate H3K27,which may be one of the factors inducing AS inflammation.3.QingreLishiHuoxue therapy inhibits the activity of demethylase JMJD3,up-regulates the expression of methyltransferase EZH2,promotes trimethylation of H3K27 locus,and inhibits Th17 differentiation,which may be one of the mechanisms to alleviate AS inflammation.4.In vitro intervention of Chinese herbal medicine triptolide in PBMC can significantly inhibit the expression of Th 17-characteristic transcription factor RORc and the activation of upstream JAK2/STAT3 signaling pathway.The mechanism may be through inhibition of demethylase JMJD3 activity and up-regulation the expression ofEZH2,which promotes the trimethylation of histone H3K27. |
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