| Background:Ankylosing spondylitis(Ankylosing Spondylitis,AS)is a chronic progressive disease,which mainly involves the axial joint.Sacroiliac arthritis and tendon inflammation are the main pathological changes.Repeated attacks of chronic inflammation led to inflammatory bone destruction and new bone reconstruction,leading to progressive fusion of spinal joints,late spinal ankylosis deformity,and eventually disability,seriously affecting the quality of life of patients.Inflammatory low back pain is a typical clinical manifestation of AS,but the onset of the disease is hidden.Early diagnosis and treatment can effectively reduce the disability caused by AS.Judging disease activity is very important for the treatment of AS.At present,the evaluation criteria of AS disease activity include C-reactive protein(CRP),erythrocyte sedimentation rate(ESR),imaging index and Bath ankylosing spondylitis disease activity index(BASDAI),but they all have some limitations.In 2009,the Assessment in Ankylosing Spondylitis international Society(AS AS)introduced the concept of standard treatment(T2T)into AS treatment,using the ankylosing spondylitis disease activity score(ASDAS)as the evaluation criteria,including subjective indicators such as low back pain,morning stiffness,peripheral joint pain and fatigue,as well as ESR and CRP two objective laboratory indicators.Previous studies have shown that ASDAS is significantly better than BASDAI in evaluating the disease activity of AS.Inflammation of sacroiliac joint and tendon runs through the course of AS.Inflammation,bone destruction and new bone formation are the key links of AS disability.Once inflammation starts,it indicates that the disease has entered an irreversible period.Control of AS inflammation is the key to reduce disease activity.The occurrence of AS inflammation is highly related to Helper T cell 17(Th17)and I Interleukin 17(IL-17).Th17 cells specifically secrete IL-17,promote the recruitment and activation of neutrophils,mediate inflammation,and promote the secretion of a variety of cytokines to magnify systemic inflammation.Th17 differentiation and IL-17 secretion are regulated by the upstream specific transcription factor Retinoic acid receptor-related orphan receptors γt(RORγt).RORγt is encoded by RORc gene,which turns on JAK2/STAT3 signaling pathway under the stimulation of many inflammatory cytokines.STAT3 forms a dimer structure into the nucleus,activates the transcription and expression of RORγt gene,and promotes AS inflammation.The transcriptional activation of the gene is closely related to the acetylation of the specific site of nucleosome histone.P300 and HDAC1 can specifically acetylate the H3K56 site and activate gene transcription.The activation of RORyt gene is affected by the acetylation modification of its promoter region H3K56.P300 and HDAC1 are acetylation modification enzymes at H3K56 site,and acetylation modification at H3K56 site,transfer the negatively charged acetyl group from acetyl coenzyme A to histone H3N-terminal lysine residue,neutralize the positive charge of histone,reduce the interaction between histone and DNA,loose chromatin structure,expose gene binding site.Promote gene transcriptional expression.In the JAK2/STAT3 signaling pathway,there are also H3K56 acetylation binding sites in the promoter region of the signal molecule STAT3 gene,which can turn on or block the expression of STAT3 signal molecules through acetylation modification,regulate the expression of downstream transcription factor RORyt,and affect the differentiation of Th 17.At the same time,acetylation of H3K56 site in the promoter region of IL-17 gene can promote IL-17 secretion and mediate inflammation.By regulating the expression of p300 and HDAC1 and reducing the acetylation state of H3K56 site,it can inhibit the transcriptional activation of RORγt,block the expression of JAK2/STAT3 signal pathway,reduce IL-17 secretion,regulate Th17 differentiation and function in AS patients,and control AS inflammation.In 2015,the American Rheumatic Society put forward guidelines for the treatment of AS,with non-steroidal anti-inflammatory drugs,biological agents,sulfasalazine and glucocorticoids and other drugs as the main drugs to control AS inflammation and delay disability.Traditional Chinese medicine has its unique advantages in the treatment of AS based on syndrome differentiation,emphasizing the deficiency of kidney and governor,the obstruction of dampness and heat as the standard,and blood stasis throughout the pathogenesis of the disease.In the earlier stage,our research group conducted a clinical study on the treatment of AS with Qingre Qiangji decoction,which took Sophora flavescens as the king medicine and the legislative method of clearing heat and dampness,which had a significant effect in reducing disease activity,controlling inflammation and improving joint function.Matrine,an effective monomer of Sophora flavescens,interfered with AS-PBMC,which could inhibit Th17 differentiation and control AS inflammation in patients with AS.On the basis of previous studies,this study observed the Thl7 differentiation of as patients treated with p300/HDAC1,and explored the molecular mechanism of Qingre Qiangji Decoction in the treatment of AS inflammation;At the same time,matrine was used to intervene AS-PBMC in vitro to observe the expression of histone acetyltransferase p300/HDAC1 before and after matrine intervention in PBMC of active as patients,and to explore the molecular mechanism of matrine regulating Th17 differentiation in PBMC of AS patients.Objective:To explore the expression of histone acetylating enzyme p300 and HDAC1 and the acetylation level of histone H3K56 site,and the molecular mechanism of controlling AS inflammation;to explore the molecular mechanism of Qingre Qiangji decoction regulating the expression of histone acetylation modifying enzyme p300 and HDAC1 and regulating Thl7 differentiation in AS patients;and to explore the molecular mechanism of Matrine regulating the expression of histone acetylation modifying enzyme p300 and HDAC1 in AS-PBMC and regulating Th17 differentiation.Methods:1.AS patients treated in the rheumatology Department of Guang’anmen Hospital,China Academy of Traditional Chinese Medicine from January 2020 to December 2020 were selected.ASDAS-CRP>1.3 was considered AS active group(AS-A group),ASDAS-CRP≤1.3 was considered AS stable group(AS-S group),and healthy control group(N group),with 30 cases in each group.Western Blot was used to detect the protein expression and phosphorylation of histone acetylation modification enzyme p300,HDAC1,H3K56ac,transcription factor RORyt and JAK2/STAT3 signaling pathway.The histone acetylation modification enzymes p300 and HDAC1,transcription factor RORyt,JAK2/STAT3 signaling pathway signal molecules and IL-17 mRNA expression were detected by RT-PCR method.Serum IL-17 expression level was detected by ELISA method,and correlation analysis was conducted.2.The AS-A group was treated with Qingre Qiangji Decoction for 3 months,and the protein expression and phosphorylation of histone acetylation modification enzyme p300,HDAC1,H3K56ac,transcription factor RORyt,JAK2/STAT3 signaling pathway and IL-17 expression were observed before and after treatment.3.From January 2020 to December 2020,30 AS patients and 30 healthy people in the Rheumatology Department of Guang’anmen Hospital,China Academy of Chinese Medical Sciences were selected.PBMC of active AS patients were isolated in vitro,and Matrine was used for 24h in vitro intervention.4.The protein expression and phosphorylation of histone acetylation modification enzyme p300,HDAC1,H3K56ac,transcription factor RORγt,JAK2/STAT3 signal pathway and IL-17 expression in AS-PBMC were observed before and after Matrine intervention.Results:1.Study on expression of histone acetylation modification enzyme P300/HDAC1 and Differentiation of Th17 in AS patients1.1 Expression of histone acetylation modification enzymes p300 and HDAC1Compared with the N group,the expression of p300 in the AS-A group was significantly increased(p<0.05),and the difference was statistically significant.There was no difference between the AS-S group and the N group(p<0.05),but it was significantly decreased compared with the AS-A group(p<0.05).Compared with the N group,the expression of HDAC1 in the AS-A group was significantly decreased(p<0.05),while the expression of HDAC1 in the AS-S group was significantly decreased(p<0.05)and significantly increased(p<0.05)compared with the N group.Compared with the N group,the expression of H3K56ac in the AS-A group was significantly increased(p<0.05),while the expression of H3K56ac in the AS-S group was not different from that in the N group(p<0.05),but significantly decreased compared with the AS-A group(p<0.05).P300 mRNA expression in AS-A group was higher than that in N group(p<0.05),p300 mRNA expression in AS-S group was significantly higher than that in N group(p<0.05),and p300 mRNA expression in AS-A group was significantly higher than that in AS-S group(p<0.05).The expression of HDAC1 mRNA in AS-A group and AS-S group was significantly higher than that in N group(p<0.05),and the expression of HDAC1 mRNA in AS-A group was significantly lower than that in AS-S group(p<0.05).1.2 Study on serum IL-17 levelIL-17 mRNA expression in AS-A group was significantly higher than that in N group(p<0.05),IL-17 mRNA expression in AS-A group was significantly higher than that in AS-S group(p<0.05),and il-17 mRNA expression in AS-S group was not significantly different from that in N group(p>0.05).Serum IL-17 in AS-A group was significantly higher than that in N group(p<0.05),serum IL-17 in AS-S group was significantly higher than that in N group(p<0.05),and serum IL-17 level in AS-A group was significantly higher than that in AS-S group(p<0.05).1.3 Study on the expression of transcription factor RORytThere was no significant difference in RORyt protein expression among the three groups included in this study(p>0.05).The expression of RORc mRNA in The AS-A group was significantly higher than that in the N group(p<0.05),the expression of RORc mRNA in the AS-A group was significantly higher than that in the AS-S group(p<0.05),and there was no significant difference between the AS-S group and the N group(p>0.05).1.4 Studies on the expression of JAK2/STAT3 signal molecules and activation of signal pathwaysThere was no significant difference in JAK2 and STAT3 protein phosphorylation among the three groups(p>0.05).In JAK2 gene expression detection,the mRNA expression of AS-A group was significantly higher than that of AS-S group and N group(p<0.05),and that of AS-S group was significantly higher than that of N group(p<0.05).The mRNA expression of STAT3 gene in AS-A group was significantly higher than that in AS-S group and N group(p<0.05),and that in AS-S group was significantly higher than that in N group(p<0.05).1.5 Correlation study1.5.1 Correlation between histone acetylation modification enzyme p300/HDAC1 and acetylation status of H3K56 site in AS patientsHistone acetylation enzyme p300 was positively correlated with H3K56ac(r=0.789,p<0.05),and histone deacetylation enzyme HDAC1 was negatively correlated with H3K56ac(r=-0.779,p<0.05),indicating a moderate correlation.1.5.2 Correlation between serum IL-17 and ASDAS-CRPIL-17 expression level was highly positively correlated with ASDAS-CRP(r=0.896,p<0.05).IL-17 expression level was positively correlated with ESR(r=0.489,p<0.05).IL-17 expression level was moderately positively correlated with CRP(r=0.732,p<0.05)1.5.3 Correlation between histone acetylation modification enzyme p300/HMAC1 and IL-17 secretion in patient with ASp300 was positively correlated with IL-17(r=0.905,p<0.05).HDAC1 was negatively correlated with IL-17(r=-0.646,p<0.05),and the correlation was moderate.Histone H3K56ac was positively correlated with IL-17(r=0.855,p<0.05).Histone acetylase p300 was positively correlated with ASDAS-CRP(r=0.997,p<0.05),and histone deacetylase HDAC1 was negatively correlated with ASDAS-CRP(r=-0.991,p<0.05).Histone H3K56ac was positively correlated with ASDAS-CRP(r=0.85,p<0.05).2.Regulation of Qinggre Qiangji Decoction on secretion of p300/HDAC1 and IL-172.1 Expression of p300/HDACl in AS patients treated with Qingre Qiangji DecoctionAfter Qinggre qiangji Decoction treatment,histone acetylation enzyme p300 was significantly decreased(p<0.05),histone deacetylation enzyme HDAC1 was significantly increased(p<0.05),and the acetylation level of Histone H3K56 site was significantly decreased(p<0.05).2.2 Study on serum IL-17 expression level of AS patients treated with Qingre Qiangji DecoctionAfter treatment with Qingre Qiangji Decoction,the expression of IL-17 in serum of AS patients was significantly decreased compared with that before treatment(p<0.05).2.3 Expression of RORyt transcription factor in AS patients treated with Qingre Qiangji DecoctionAfter Qingre Qiangji Decoction treatment,the specific transcription factor RORyt was significantly decreased(p<0.05).2.4 Qingre Qiangji Decoction interferes with activation of JAK2/STAT signaling pathway in AS patientsAfter treatment with Qinggre Qiangji Decoction,the phosphorylation levels of JAK2 and STAT3 in JAK2/STAT3 signaling pathway were significantly decreased(p<0.05).Effects of Matrine on expression of p300/HDAC1 and Th17 differentiation in AS-PBMC3.1 Matrine interferes with the expression of p300/HDAC1 in AS-PBMCThe expression level of p300 protein in AS-PBMC was significantly lower than before treatment(p<0.05),and the expression level of HDAC1 protein in AS-PBMC was significantly higher than before treatment(p<0.05).The expression level of H3K56ac in AS-PBMC was significantly lower than that before treatment(p<0.05).3.2 Study on IL-17 expression level in supernatant of AS-PBMC cells after Matrine interventionAfter Matrine intervention,the level of IL-17 in supernatant of AS-PBMC cells was significantly lower than before intervention(p<0.05).3.3 Matrine interferes with the expression of RORyt in AS-PBMCAfter Matrine intervention,the expression of RORyt protein in AS-PBMC was significantly decreased compared with before treatment(p<0.05).3.4 Matrine interferes with activation of JAK2/STAT signaling pathway in AS-PBMC After Matrine intervention,JAK2 phosphorylation level in AS-PBMC was significantly lower than that before treatment(p<0.05),and STAT3 phosphorylation level in AS-PBMC was significantly lower than that before treatment(p<0.05).Conclusion:1.AS patients have high expression of p300 and low expression of HDAC1,and H3K56 is in a high acetylation state,which may be one of the important mechanisms to activate the Th17 characteristic transcription factor RORyt and the upstream JAK2/STAT3 signaling pathway.And this mechanism regulates Th17 differentiation in AS inflammation.2.Qingre Qiangji Decoction interferes with PBMC of AS patients,reduces the expression level of p300,increases the expression level of HDAC1,up-regulates the acetylation level of H3K56 site,and reduces the transcription factor RORyt.The expression level of RORyt and the activity of JAK2/STAT3 signaling pathway inhibit the expression of IL-17,which may be an effective Chinese medicine prescription to inhibit Th17 in anti-inflammation in AS.3.Matrine,a traditional Chinese medicine monomer,down-regulates the expression level of histone acetylase p300 in vitro,up-regulates the expression level of histone deacetylase HDAC1.At the same time,it reduces the acetylation level of histone H3K56 site,and reduces the transcription factor RORyt and the activity of JAK2/STAT3 signaling pathway to inhibit the expression of IL-17.Matrine may be an effective Chinese medicine monomer to control the differentiation of Th17 in AS patients. |
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