Effects Of HDND-11 On The Proliferation Of Fibroblast-like Synoviocytes In Adjuvant Arthritis Rats And Its Mechanism

Rheumatoid arthritis(RA)is characterized by synovial inflammation and synovial dysplasia,which erodes adjacent cartilage and bone tissue,leading to irreversible joint damage.It is one of the most common autoimmune diseases in humans.Most researches demonstrate that abnormal proliferation and activation of fibroblast-like synoviocytes(FLS)are important events in the development and progression of rheumatoid arthritis(RA),which can serect many inflammatory cytokines.Therefore,the inhibition of FLS cell activation and proliferation may be a good way to treat RA.According to the research,Wnt signaling pathway regulates many cellular biological processes,including cell growth,proliferation and apoptosis.Among them,the classic Wnt signaling pathway plays an important role in the development of a variety of tumors,such as liver cancer,gastric cancer,and melanoma.Secreted frizzled-related proteins 2(SFRP2)is an antagonist of the Wnt signaling pathway,and it can be catalyzed by DNA methyltransferase 1(DNMT1)in a variety of cancer.SFRP2 methylation can activate Wnt signaling pathway due to the decreasing expression of SFRP2.In view of the similar characteristics of FLS with tumor cells,it is suggested that Wnt pathway might play an inhibitive role in inhibiting FLS proliferation through SFRP2-dependent molecular mechanism.More and more literature shows that traditional Chinese medicine plays a vital role in rheumatoid arthritis prevention and treatment.Hesperidin(HDN)is a kind of flavonoids which mainly exist in citrus fruits,and it is also the main component of traditional Chinese medicine Chenpi.Flavonoids,including HDN,are known to have a variety of biological functions,such as antioxidant,anti-inflammatory,antiviral and antitumor activities.Therefore,our group synthesized and improved a number of hesperetin derivatives,and selected a number of derivatives which can treat rheumatoid arthritis by the MTT experiment.HDND-11 is one of the derivatives we screened,and HDND-11 was found to inhibit the proliferation and activation of FLS cells by MTT assay,and can promote the expression of SFPR2.However,it was not reported whether it could treat rheumatoid arthritis and the mechanism was related to the promotion of the expression of SFRP2.This study was to investigate the effect of HDND-11 on the treatment of rheumatoid arthritis and the possible mechanism.The experiment was divided into the following six parts.1.Therapeutic effect of HDND-11 on AA rat model.We prepared AA model rats with Complete Freund’s Adjuvant(CFA)(0.1ml)by paw injection to male Sprague Dawley(SD)rats.The inflammation of the joints was measured by HE staining,arthritis and paw swelling scores and ELISA.The experimental results showed that HDND-11 has obvious therapeutic effect on CFA-induced adjuvant arthritis rats.2.Effect of HDND-11 on the proliferation of AA-FLS.FLS were treated with different HDND-11(0,12.5,25,50,100,200μM)in 48 h and HDND-11(100μM)in 6,12,24,48 and 72 h in order to detect the effect of HDND-11 on the proliferation of FLS cell by MTT assay.In addition,the effect of HDND-11 on FLS proliferation and cell cycle distribution was examined by flow cytometry.The results showed that HDND-11 could significantly inhibit the proliferation of FLS cells.3.Effect of HDND-11 on the expression of SFRP2 in synovial tissue and FLS cells of AA rats.The expression of SFRP2 in synovial tissue was detected by immunohistochemistry,Western blot and RT-q PCR.The expression of SFRP2 in FLS was detected by Western blot,RT-q PCR and immunofluorescence.The results showed that the expression of SFRP2 in AA synovial tissue and AA-FLS was significantly lower than that in normal group,and HDND-11 could significantly promote the expression of SFRP2 in the synovial tissue and FLS cells of model group.4.The role of HDND-11 inhibited the proliferation of FLS by promoting SFRP2.After detecting the effect of HDND-11 on SFRP2 in vivo and in vitro,we studied the effects of over-expression of SFRP2 on FLS proliferation by using MTT assay and Flow cytometry.The results showed that overexpression of SFRP2 significantly inhibited the proliferation of AA-FLS.5.The mechanism of HDND-11 on the expression of SFRP2 in synovial tissue and FLS of AA rats.The expression of DNMT1 in synovial tissue was detected by Western blot and RT-q PCR.The expression of DNMT1 in FLS was detected by Western blot,RT-q PCR and immunofluorescence.The results showed that the expression of DNMT1 in AA synovium and AA-FLS was significantly higher than that in normal group,and HDND-11 significantly inhibited the expression of DNMT1 in the synovial tissue and FLS cells of the model group.The effect of DNMT1 on SFRP2 expression was detected by Western blot and RT-q PCR.The results showed that inhibition of DNMT1 significantly promoted the expression of SFRP2.6.Effect of HDND-11,SFRP2 and DNMT1 on the Wnt signaling pathway.The effects of HDND-11 on the expression of β-catenin,C-myc and cyclin D1 in synovial tissue and FLS were detected by Western blot.The results showed that HDND-11 could significantly inhibit the expression of β-catenin,C-myc and cyclin D1 in synovial tissue and FLS.The results also showed HDND-11 could inhibit Wnt signaling pathway.The effects of overexpressed SFRP2 and silenced DNMT1 on the expression of β-catenin,C-myc and cyclin D1 in FLS cells were detected by Western blot.The results showed that overexpressed SFRP2 and silenced DNMT1 could significantly inhibit the expression of β-catenin,C-myc and cyclin D1 in FLS cells,so the results showed overexpression of SFRP2 and silenced DNMT1 could inhibit the Wnt signaling pathway.