Therapeutic Effects Of Matrine Derivate MASM In Rheumatoid Arthritis And Its Underling Mechanism

Bachgrounds:Rheumatoid arthritis(RA)is a chronic inflammatory autoimmune disorder,featured by synovial hyperplasia and inflammatory cell filtration,leading to chronic inflammation of the joints and subsequent erosion of the cartilage and the bone.rheumatoid arthritis fibroblast-like synoviocytes(RA-FLSs)are important compositions of the inflamed synovial,key effectors of RA and plays a pivotal role in the pathogenesis of RA.Conventional disease-modifying anti-rheumatic drugs(DMARDs),such as methotrexate,may gradually loss of efficacy in parts of RA patients,probably due to development of drug resistance during long-term usage.The development and clinical application of biologics greatly revolutionized the treatment of RA,improved the therapy in patients who were not sensitive to conventional DMARDs.However,the high expenses,parenteral administration required,increase the risk of infection and cancer are major drawbacks of biologics DMARDs,besides,some patients are refractory to the biologics.Thus,develop new drugs for the treatment of RA carry substantial meaning.Matrine is an active alkaloid isolated from traditional Chinese herbs such as Sophora flavescens,it carries a wide spectrum of pharmacological properties.But,until recently,matrine has not developed into a clinical drug due to its low drug efficacy and large dosage needed.The matrine derivate MASM [(6aS,10 S,11aR,11 bR,11cS)210-Methylamino-dodecahydro-3a,7a-diaza-benzo(de)anthracene-8-thione] exhibited better pharmacological activities.Previous reports have shown that matrine derivate MASM comprises anti-inflammatory,immune modulation,anti-hepatic fibrosis and radio protective properties.However,no study has ever applied it in the therapy of RA,thus in this study,we cultured original RA-FLSs from synovial tissues from RA patients and established collegen-induced arthritis(CIA),we aim to evaluate whether matrine derivate MASM has potential treatment effects on RA through in vitro and in vivo experiment.Methods:1.Primary human derivate RA-FLSs were isolated and cultured in vitro through collagenase digestive method,after the third passage,cells were subjected to flow cytometry analysis;2.The effect of matrine derivative MASM on proliferation of RA-FLSs were analyzed using CCK8 assay;3.The effect of matrine derivative MASM on the expression of proinflammatory cytokines(TNF-?,IL-6,IL-8)and MMPs(MMP-1,MMP-3,MMP-13)in RA-FLSs were analyzed using real-time PCR;4.The IL-1? activated RA-FLSs were treated with MASM and cell apoptosis were detected using Annexin V-FITC and PI double staining,then analyzed by fow cytometry;5.After pretreatment of matrine derivate MASM,RA-FLSs were then activated and treated by IL-1? for 48 hours.After JC-1 dye,the decrease in mitochondrial membrane potential were analysed by flow cytometry;the proteins were extracted and then subjected to analysis by western blot,the expression level of internal mitochondrial apoptotic pathway related proteins Bcl-2,Bax and cytochrome c cleaved caspase 9,pro-caspase 3,cleaved caspase 3,cleaved PARP were detected by western blot;The activity of capsase 3 in RA-FLSs after correspongding treatment was detected by caspase 3 activity assay;6.After the corresponding treatment,detect expression changes in autophagic markers p62,Beclin-1,LC3 in RA-FLSs;7.Investigate the apoptosis inducing effects of MASM in RA-FLSs after blockade of autophagy by CQ;8.RA-FLSs were pretreated with MASM,and then activated by IL-1? co-treatment.The expression level of phosphorylate Akt and total Akt in the Akt/PKB signaling pathway were detected by western blot;9.The MAPKs and NF-?B signaling pathway are closely related to the pathogenesis of RA.RA-FLSs were pretreated with matrine derivative MASM,and then co-treated by IL-1? to activate the MAPKs and NF-?B signaling.After extraction of the total protein,the phosphorylation of Erk1/2,JNK,p38 and corresponding total protein in MAPKs signal pathway were detected by Western blot;Nuclear protein extraction kit was adopted to separate the cytoplasmic protein and nuclear protein.The expression level of NF-?B/65 in the nuclear protein and the expression of I?B? in cytoplasmic protein were also detected by western blot;10.DBA / 1 mice were divided into 4 groups: normal group,positive CIA control group,low dosage group(matrine derivative MASM 10mg/kg group)and high dosage group(matrine derivative MASM 30 mg/kg group).The widely used animal model of RA,namely collagen-induced arthritis,was induced by type ? collagen.The administration of drugs in the experimental group and saline in the control group was given by oral garage once a day.The clinical manifestations of joint swelling and redness was scored every 2 days since the 28 th day after induction;11.The tissue samples of mice were collected and fixed,decalcified and stained with hematoxylin and eosin(HE)to observe the pathological changes of the joints of the foot and ankle in mice.The synovial inflammation was also scored;12.The samples from the hind foot of the mice were collected and fixed,and then subjected to Micro-CT scan.After three-dimensional reconstruction,the severity of bone erosion was scored;13.The blood was collected through orbit,coagulatied at room temperature and centrifuged to collect serum.The serum levels of TNF-?,IL-1? and IL-6 were detected by ELISA;14.Immunohistochemical staining was adopted to detect the local TNF-? expression level in ankle joints.Results:1.Primary RA-FLSs can be cultured from synovial tissues through collagenase digestion method,after culture medium replacemtent and cell passage,a rather singular cell type resembling fibroblasts can be obtained.After labled by CD90,cells were subjected to flow cytometry,CD90 positive cells were more than 90%,indicating that the majority of cells were RA-FLSs,and the cells can be used for subsequent experiments;2.CCK8 assay showed that matrine derivative MASM could inhibit the proliferation of RA-FLSs,in a dose-and time-dependent manner;3.The results of real-time PCR showed that IL-1? could activate RA-FLSs,and up-regulate the expression of proinflammatory cytokines(TNF-?,IL-6 and IL-8)and MMPs(MMP-1,MMP-3,MMPs-13),the matrine derivative MASM can inhibit the expression of the aformentioned proinflammatory cytokines and MMPs in RA-FLSs,and the inhibitory effects were dose-dependent;4.After MASM pretreatment for an hour,IL-1? continued to stimulate RA-FLSs for 48 hours.Annexin V-FITC and PI double staining was used to detect cell apoptosis.The results indicated that 5-20 ?M matrine derivative MASM could induce the apoptosis of RA-FLSs in a dose-dependent manner5.After Jc-1 staining,the decrease of mitochondrial membrane potential in RA-FLSs was detected by flow cytometr,the decrease of mitochondrial membrane potential showed a dose-dependent manner;Western blot analysis of mitochondrial pathway-related apoptotic proteins showed that,with the increased treatment dosage of matrine derivate MASM,the expression of anti-apoptotic protein Bcl-2 decreased,the expression of pro-apoptotic protein Bax increased,the level of cytochrome c protein in the cytoplasm increased,and the expression of cleaved caspase 9 increased,the expression of pro-caspase 3 decreased and cleaved caspase 3 increased,the expression of apoptotic marker Cleaved PARP was also increased;The results of Caspase 3 activity assay showed that MASM treatment of matrine could obviously increase the activity of caspase 3 in a dose-dependent manner,which was consistent with the result of protein expression;6.After the corresponding treatment,the immunoflorenscence of the cells revealed that the treatment of matrine derivate MASM could induce the formation of LC3 puncta in RA-FLSs;Western blot was adopted to detect the expression of autophagic related protein p62,Beclin-1,LC3,and the results showed that treatment of RA-FLSs with 5~20?M MASM could inhibit the expression of p62 while upregulate the expression of Beclin-1 and LC3B-II;7.After block the autophagy in RA-FLSs by CQ,the apoptosis induction effect of MASM was further enhanced;8.The results of Western blot showed that matrine derivate MASM inhibited the phosphorylation of Akt,but showed little effect on the expression of Akt;9.Western blot analysis of proteins in MAPKs and NF-?B signaling pathway showed that 2ng/ml IL-1? treatment for 30 minutes could activate RA-FLSs,significantly induced the phosphorylation of Erk1/2,JNK and p38,5~20?M matrine derivatives MASM can inhibit the phosphorylation of Erk1/2,JNK and p38,with the most obvious inhibition at 20?M,but matrine derivate MASM showed little effect on the expression of total Erk1/2,JNK and p38;Treatment of RA-FLSs with 2ng/ml IL-1? 30 mins can significantly reduced the expression of I?B? in the cytoplasm and induced NF-?B/p65 nuclear translocation,but the matrine derived MASM(5~20?M)treatment can reverse this trend in a dose-dependent manner;10.Arthritis was successfully induced by type ?collagen in DBA / 1 mice,the RA animal model,namely the CIA,was established.The clinical arthritis score showed that there was no arthritis in the normal control group,the score in CIA positive control group was significantly increased,While the matrine derivative MASM administration group can alleviate the severity of arthritis,and the larger the dosage,the more obvious the degree of remission;11.Hematoxylin and eosin staining showed that obviouos synovial proliferation,synovial inflammation,erosion of cartilage and bone,and joint destruction were observed in CIA-positive control mice.The score of synovial inflammation showed that MASM administration could relief the corresponding pathologic impairment,and the greater the dosage,the more obvious the relief;12.Three-dimensional reconstruction after Micro-CT scan showed that the bone of the negative control group was smooth,with no obvious bone destruction,and the joint space was clearly visible.The CIA positive control group had multiple bone injuries,some of the joints were completely destroyed,In the low dosage group(10 mg/kg MASM group),roughness,mild and moderate bone injuries was observed in multiple spots,but much better than those in the CIA positive control group.In the high dosage group(MASM 30 mg / kg group),the bone injury was less severe than the low dosage group(10 mg/kg MASM group);13.ELISA was used to detect the serum levels of TNF-?,IL-1 and IL-6 in mice,the results showed that the expression levels of TNF-?,IL-1 and IL-6 in CIA positive control group were markedly increased,the administration of matrine derivate MASM can dose-dependently inhibit their expression;14.The results of immunohistochemistry showed that the TNF-? expression levels of in local tissues of CIA-positive control group were significantly higher than those in normal control group,while those of MASM-treatment(30mg/kg or 10mg/kg)Significantly lower than the CIA positive control group.Conclusions:Matrine derivate MASM could inhibit the expression of inflammatory mediators and MMPs in RA-FLSs,inhibit the proliferation of RA-FLSs,promote the apoptosis of RA-FLSs through mitochondrial pathway,and upregulate the autophagy level in RA-FLSs.Inhibition of autophagy could further enhance the apoptosis inducing effects of MASM.Besides,MASM could inhibit signal transduction of the MAPKs and NF-?B signaling pathway,which were pivatol to the pathogenesis of RA,in RA-FLSs.In vivo experiments showed that matrine derivative MASM can attenuate the severity of arthritis and tissue destruction in CIA.In summary,MASM may be a potential therapeutic drug for RA,combinate treatment with CQ or HCQ may further enhance its treatment effects.