| Background:Ankylosing Spondylitis is a chronic inflammatory diseasemainly involving the attachment point of the middle axis and tendon,and the spinal ankylosis can occur at the end of the disease,which seriously affects the quality of life of the patients.Inflammation of the sacroiliac joint and spinal attachment point is the main feature of AS.Inflammation is closely related to the differentiation and effect of helper T cell 17(T helper cell 17,Th17).Th17 is derived from the differentiation of initial CD4+T cells(Naive CD4+T cell),which secretes interleukin 17(Interleukin 17,IL-17)with specific secretion of cytokines,which promotes the occurrence of inflammation.Th17 differentiation and IL-17 secretion are regulated by the transcriptional factor retinoic acid related orphan nuclear receptor gamma t(Retinoic acid receptor related orphan receptor,ROR gamma T).Histone methylation plays an important role in regulating the differentiation of Th17.The Zeste gene enhancer homologue 2(enhancer of zeste homolog 2,EZH2)is one of the histone methylation modifier,and its main function is the expression of the target gene in the downstream of silencing.It is found that EZH2 can significantly inhibit the differentiation of Na ve CD4+T cells into Th17.EZH2 silencing gene expression is achieved by specific methylation of histone H3K27 locus,which inhibits transcription of downstream target genes.There are a large number of histone H3K27 loci in the promoter region of the encoding gene RORc of ROR gamma t.EZH2 can catalyze H3K27 methylation modification,inhibit the expression of RORc gene and seal the differentiation of Th17.Through up regulation of EZH2 expression,maintenance of RORc gene silencing and inhibition of Th 17 differentiation,AS inflammation can be effectively controlled.Matrine is an effective monomer for Sophora flavescens.Earlier experimental studies showed that the application of different concentration of matrine monomers in AS patients PBMC in vitro could inhibit the differentiation of Th17 and inhibit the AS inflammation.Triptolide,an effective monomer of Tripterygium wilfordii,can regulate the differentiation of Th17 by regulating the expression of histone H3K27 methylation and interfere with AS inflammation.On the basis of previous research,we continue to explore the expression of EZH2 in AS patients and the relationship with Th17 differentiation,and explore the histone modification mechanism of the intervention of matrine and triptolide to control the Thl7 differentiation of AS patients and control the AS inflammation.Objective:To explore the mechanism of Th17 differentiation in AS patients with histone methylation enzyme EZH2,to explore the expression of PBMC EZH2 and RORc in AS patients with single matrine and triptolide at active stage,to influence the level of methylation of H3K27 loci and to regulate the histone modification mechanism of Th17 differentiation and control of AS inflammation.Methods:1.30 active AS patients,15 stable AS patients and 10 normal people were selected in this study.We use gradient density centrifugation to get serum and peripheral blood mononuclear cells(Peripheral Blood Mononuclear Cells,PBMC).We use enzyme linked immunosorbent assay(ELISA)to detect the IL-17 of Normal,AS-Active and AS-Steady.We use Western Blotting to detect the protein EZH2,H3K27me3,RORc expression in Normal,AS-Active and AS-Steady.We use PCR to detect the EZH2,RORc mRNA expression in Normal,AS-Active and AS-Steady.Also,we analyse the relativity of EZH2.RORc.2.In vitro,we isolate and culture active AS patients' PBMC.We use ELISA and CCK8 method to determine the optimal concentration of matrine and triptolide.The protein and mRNA expression levels of EZH2,H3K27me3 and RORc were detected.In the same period,the effects of triptolide and matrine on H3K27me3,EZH2,RORc and Th17 differentiation in active AS patients were observed in the same period.Results:1.Compared with the normal control,AS-Active IL-17 expression in PBMC were significant higher(p<0.05),while AS-Steady had no significant different.AS-Active EZH2 protein and mRNA expression was significant higher than the normal control(protein:p<0.05,mRNA:p<0.05),while AS-Steady had no significant different.AS-Active H3K27me3 protein was significant higher than the normal control(p<0.05),while AS-Steady had no significant different.AS-Active RORc mRNA expression was significant higher than the normal control(mRNA:p<0.05),while protein had no significant different.AS-Steady RORc protein was significant higher than the normal control,while mRNA expression had no significant different.2.After intervened by matrine,EZH2 protein and mRNA expression in AS-Active PBMC were significant higher than before(protein:p<0.05,mRNA:p<0.05).H3K27me3 protein in AS-Active PBMC were significant higher than before(protein:p<0.05).RORc protein and mRNA expression in AS-Active PBMC were significant lower than before(protein:p<0.05,mRNA:p<0.05).3.After intervened by triptolid,EZH2 protein and mRNA expression in AS-ActivePBMC were significant higher than before(protein:p<0.05,mRNA:p<0.05).H3K27me3 protein in AS-Active PBMC were significant higher than before(protein:p<0.05).RORc protein and mRNA expression in AS-Active PBMC were significant lower than before(protein:p<0.05,mRNA:p<0.05).Conclusions:1.In AS-Active PBMC,EZH2 and H3K27me3 were significant lower than the normal controls.EZH2 is related to RORc.Promoter region of RORc gene H3K271oci may be regulated by EZH2,which means important to Th 17 differentiation and AS inflammation and treatment.2.Matrine is an effective monomer of Kuh-seng.Matrine could increase EZH2 expression in AS-Active PBMC.It can control the H3K37me3 expression,which plays an important role in RORc expression and Thl7differentiation in AS inflammation.3.Triptolide is an effective monomer of tripterygium.Matrine could increase EZH2 expression in AS-Active PBMC.It can control the H3K37me3 expression,which plays an important role in RORc expression and Thl7differentiation in AS inflammation. |
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