Regulation Of MeCP2 On PTCH1 In Adjuvant Arthritis Rats And The Research Of Anti-inflammatory Mechanism Of Hesperidin Derivative-XIV

Rheumatoid arthritis（RA）is an inflammatory autoimmune disease characterized by thickening of synovial tissue,destruction of cartilage and bone tissue.Fibroblast-like synoviocytes（FLS）proliferate abnormally and produce a variety of inflammatory cytokines.Some inflammatory cytokines,such as IL^-1,IL-6,IL-8,TNF-α,matrix metalloproteinases and collagenase form a complex network that causes joint damage.RA has a high morbidity,and the pathogenesis is still unknown,not only affected people’s daily life seriously and causing discomfort,but also lead to the loss of labor capacity.Therefore,it is an urgent problem to be solved that search for an effective therapy for RA treatment.Methylation is a form of epigenetic modification that regulates protein expression by inhibiting transcription.The study found that the pathogenesis of RA accompanied by a specific gene methylation process.Methylated Cp G binding protein 2（MeCP2）,a member of the MBD family,can recognize and bind to methylated DNA sequences specifically,thereby inhibiting gene expression.MeCP2 was found to be highly expressed in RA and involved in the regulation of methylation of specific genes.It has been reported that the Hedgehog signaling pathway plays an important role in tissue repair and persistent inflammation.Patch1（PTCH1）is a negative regulatory gene in this pathway.In many cancers,the hypermethylation of its promoter leads to a decrease in protein expression.It is not clear how PTCH1 changes in RA.At present,steroidal anti-inflammatory drugs,non-steroidal anti-inflammatory drugs,immunosuppressive agents and glucocorticoids were used for the treatment of RA,but have toxicity and side effects.Hesperidin（HDN）belongs to dihydroflavonoid glycosides.Previous studies have shown that HDN has strong anti-inflammatory and antioxidant activity.It has a therapeutic effect on adjuvant arthritis（AA）rats.But it short half-life,poor water solubility and oral failure,limiting its clinical application.At present,most of the domestic and foreign focus on the structural transformation of HDN,through the transformation and modification of its structure,its half-life,water solubility and stability were improved significantly.A series of melanin derivatives（HDND）were obtained and the anti-inflammatory activity was screened for the structural modification of HDN.It was found that 5-（4-chlorophenylethyl）imino-7-O-acetyl-4-chlorophenethylaminopyelin（HDND-XIV）had good anti-inflammatory activity.In this study,AA rats were selected as animal model,because its similar pathological features and immunological with RA.FLS were used for cell research.The effect of MeCP2 on the expression of PTCH1 protein was studied by using methylation inhibitor 5-hybrid-4-deoxycytidine（5-Azad C）and gene silencing.At the same time,the effect of HDND-XIV on FLS inflammation and the possible mechanism were explored.The main research contents include two parts:1.Effect of MeCP2 on expression of PTCH1 in rheumatoid arthritis and its mechanism.To study how to MeCP2 regulate PTCH1.AA rats were induced by Freund’s complete adjuvant（FCA）and normal rats were given an equal volume of PBS.After 24 days of injection,the primary FLS was extracted.Using 5-azadc in FLS and silencing MeCP2 by gene silencing techniques to observe the regulation of PTCH1 in vitro and the above effects on inflammation.HE staining was used to observe the pathological changes of rat joint,and the degree of secondary lateral swelling was also detected.The expression of MeCP2 and PTCH1 protein was detected by immunofluorescence and immunohistochemistry.The expression of MeCP2,PTCH1,Gli1 and Shh protein was detected by Western blot.Enzyme linked immunosorbent assay（ELISA）was used for detection of inflammatory cytokines.Methylation-Specific PCR（MSP）and pyrophosphate sequencing were used to detect the degree of methylation.Pathological results indicate that the AA rat model was established and the knee was significantly diseased and swollen.Immunohistochemistry,immunofluorescence and western blot showed that the expression of PTCH1 protein was decreased and the expression of MeCP2 protein was increased in both AA rat tissue and cells.At the same time,the expression of Gli1 and Shh were increased in AA FLS,indicating that the Hedgehog signaling pathway of AA group was activated.Compared with AA group,the levels of MeCP2,Gli1 and Shh protein were decreased and the expression of PTCH1 protein was increased in AA FLS treated 5-azadc group.MSP and pyrophosphate sequencing showed that the methylation level of PTCH1 gene decreased after AA FLS was stimulated by 5-azadc.The expression of PTCH1 was elevated after silencing MeCP2,accompanied by a decrease in inflammatory cytokines.It is suggested that MeCP2 regulates the expression of PTCH1 through DNA methylation in rheumatoid arthritis,which provides a new target for the prevention and research of RA.2.The inhibitory effect of hesperidin derivatives XIV on FLS inflammation and its mechanismResearch the effect of Hesperidin derivative XIV（HDND-XIV）on FLS inflammation and its mechanism.AA rats were induced by FCA.The Half maximal inhibitory concentration（IC50）of HDND-XIV was detected by MTT assay.ELISA was used to detect the inflammatory cytokines in each group.The expression of MeCP2,PTCH1,Gli1 and Shh protein in FLS was detected by Western blot.Pyrosequencing was used to detect the degree of methylation.The IC50 of HDND-XIV was 161 μmol·L^-1.In view of the toxicity of the drug,five drug concentrations(80 μmol·L^-1,40 μmol·L^-1,20 μmol·L^-1,10 μmol·L^-1,5 μmol·L^-1)were selected in the range of less than 0.5 times IC50.It was found that when the drug concentration was 10 μmol·L^-1,the inhibition of inflammation was most significant.And then,the concentration of 10 μmol·L^-1 was used for treating AA group.The expression of MeCP2,Gli1 and Shh protein were decreased and the expression of PTCH1 protein was increased after AA FLS treated with HDND-XIV,and the results of pyrosequencing showed that the degree of methylation of PTCH1 gene was significantly lower than AA FLS.MeCP2 was overexpressed on AA FLS,followed by HDND-XIV stimulation.The results showed that after successful overexpression,PTCH1 protein expression was decreased and inflammatory cytokines were increased.It is suggested that HDND-XIV inhibits the inflammation of FLS,the mechanism of which may be associated with inhibiting the expression of MeCP2 to reduce the methylation level of PTCH1 gene.Eventually,downregulate the secretion of inflammatory cytokines.