The Action Of Mechanism By Which DOCA Inhibits The TNF-α-TNFR Interaction To Protect Against Rheumatoid Arthritis

Rheumatoid arthritis（RA）is an autoimmune disease with chronic arthritis as the main symptom and humoral immunity plays a key role in the development of RA.TNF-αis one of the important pro-inflammatory factors in humoral immunity.The content of TNF-αin synovial fluid of RA patients was significantly higher than that in healthy people.TNF-αbinds to its receptor TNFR to activate the downstream NF-κB signaling pathway,then to promote inflammatory gene transcription and cause the continuous development of joint inflammation.Blocking the interaction between TNF-αand TNFR is an important strategy for the treatment of RA.Monoclonal antibody can block TNF-α-TNFR binding and has significant efficacy in clinical applications.But biological agents are characterized with large molecular weight and expensive price,so the development of TNF-α/TNFR small molecule inhibitors is of great significance.Virtual database or combinatorial chemistry methods are used for the development of TNF-α/TNFR small molecule inhibitors.Based on the previous research,TNF-α/TNFR were regarded as the target to screen some inhibitors among 186 natural triterpenoids.We found that1-decarboxy-3-oxotheochic acid（DOCA）could inhibit the binding of TNF-αto TNFR.Based on this,this paper aims to explore the effect and mechanism of DOCA in improving RA by blocking TNF-αbinding to TNFR.Some methods were performed in this paper,including surface plasmon resonance（SPR）,solid-binding experiment,luciferase reporter gene detection,MTT,flow cytometry,cellular immunofluorescence,collagen-induced rheumatoid arthritis mouse model,articular cartilage staining and immunohistochemical analysis.The main findings are as follows:1.DOCA directly binds to TNF-α,TNFR1 and TNFR2,and DOCA could block the interaction between TNF-αand TNFR.We examine the interaction between DOCA and TNF-α,TNFR1 and TNFR2 through affinity testing,competitive experiments,and solid combination experiments.（1）The affinity detection results showed that the binding affinity（K_D）between DOCA and TNF-αwas 6.548×10^-4 M,the K_D value between DOCA and TNFR1 was 8.405×10^-6 M,and the K_D value between DOCA and TNFR2 was 4.835×10^-6 M,indicating that DOCA could bind to TNF-α,TNFR1 and TNFR2 directly.（2）The competitive binding results showed that DOCA（4μM,8μM,16μM）could inhibit the interaction between TNF-αand TNFR1 or TNFR2 with the decreased binding signal value（R1:49%、68%and 69%;R2:28%、39%and 46%）.（3）The results of solid-binding experiments showed that DOCA（5μM,10μM,20μM）could block the binding of TNF-αto TNFR1 and TNFR2（R1:4%-78%;R2:3%-79%）.These results showed that DOCA could directly bind to TNF-α,TNFR1and TNFR2 and block the interaction between TNF-αand TNFR.2.DOCA could inhibit TNF-α-induced biological effects.Since DOCA blocks the interaction between TNF-αand TNFR,we used four cell models to validate the biological effects induced by TNF-αwith or without DOCA.（1）L929 cells are mouse fibroblasts,and their cell membrane surface expresses TNFR.L929 cells are induced apoptosis by TNF-α,so L929 cells are commonly used to determine TNF-αactivity.Our experimental results showed that TNF-α（10 ng/m L）could significantly induce apoptosis of L929 cells within 10 hours,and DOCA（6.5μM）could significantly inhibit apoptosis and morphological changes of L929 cells caused by TNF-α.（2）293（NF-κB）reporter cells are stably transferred Luciferase gene,so this cell line is sensitive to TNF-αstimulation.293（NF-κB）reporter cells could respond to NF-κB transcription factor activity by detecting relative Luciferase activity.The results suggested that TNF-αsignificantly induced NF-κB activation（5ng/m L and 10 ng/m L）within 4 hours,and DOCA（4.5μM,5.5μM,and 6.5μM）significantly inhibited the transcriptional activity of NF-κB in 293（NF-κB）reporter cells after TNF-αstimulation.（3）Synovial cells（human normal fibroblast-like synovial cells,HFLS;Rheumatoid arthritis fibroblast-like synovial cells,MH7A）are important components of the synovial membrane,and TNF-αcould cause increased aggressiveness and proliferative capacity of HFLS and MH7A,which could result in synovial hyperplasia and joint damage.Experimental results showed that TNF-αcould induce NF-κB signaling activation in HFLS and MH7A cells within 1 hour,manifested by increased phosphorylation levels of NF-κB signaling pathway marker proteins（IKKα/β,IκBαand p65）,and NF-κB（p65）subunits transferred from cell plasma to nucleus.DOCA treatment significantly reduced phosphorylation of marker proteins after TNF-αstimulation,and significantly inhibited TNF-α-induced p65 nuclear translocation.Our results suggested that DOCA could inhibit the activation of the effector molecule NF-κB by blocking TNF-αbinding to TNFR.3.DOCA improvs RA in animal models.We took the RA model established by collagen-induced DBA mice as the research object（Collagen-induced arthritis,CIA）.CIA mice were injected intraperitoneally at a dose of 6 mg/kg body weight of DOCA for 23 days,and adalimumab acts as a positive control.The main organs and joint tissues of mice were used to evaluate the efficacy of DOCA in improving RA.（1）The results of liver and kidney pathological section staining showed that the administration matrix,DOCA and monoclonal antibody had no effect on mice;（2）The spleen is an important immune organ,and the development of RA is often accompanied by splenomegaly syndrome.The results of spleen wet weight weighing showed that the wet weight of spleen in CIA mice was significantly higher than that in control group,and DOCA or adalimumab could reduce the spleen wet weight of CIA mice to a certain extent（10%and 15%）.The results of spleen H&E staining and immunohistochemical analysis showed that the size and number of splenic lymph sheath germinal centers in CIA mice increased significantly.Both DOCA and adalimumab could reduce the number and density of lymphatic follicles,and significantly inhibit the expression of T cell markers CD3εand CD4 in spleen.（3）Joint swelling is an important clinical manifestation in the development of RA.The measurement results of hind paw thickness in mice showed that the hind paw thickness of CIA mice was significantly higher than that in control group.Both DOCA and adalimumab significantly reduced hind paw thickness（15%and 20%）and arthritis scores（24%and 36%）in CIA mice.（4）Synovial hyperplasia is an important pathological feature in the development of RA.The results of H&E staining of knee,ankle,and toe joints of the lower limbs in mice showed that obvious synovial hyperplasia,severe inflammatory cell infiltration and bone erosion symptoms appeared in the joints of mice,and the micro-joint structure was significantly changed.Both DOCA and adalimumab could significantly reduce synovial hyperplasia and cell infiltration in CIA mice and improve joint pathological structure.（5）Cartilage loss is one of the main causes of joint function loss in RA patients.The results of Safranin O/fast green cartilage staining,and toluidine blue cartilage staining showed that the knee,ankle,and toe joints of normal mice were covered by complete cartilage tissue,with obvious coloration and clear joint cavity gaps.The cartilage of CIA mice was severely damaged,without staining or very low degree of coloration were seen.Both DOCA and adalimumab reduced articular surface cartilage loss in CIA mice.（6）TNF-αcould cause osteoclast differentiation and cause irreversible bone damage during the development of RA.TRAP staining of knee,ankle and toe joints of mice showed that the number of osteoclasts in joint bone tissue in CIA mice increased significantly.Both DOCA and adalimumab effectively reduced the number of articular osteoclasts in CIA mice.（7）NF-κB（p65）,as a key effector molecule of TNF-α,is very important in the development of RA.The results of immunohistochemical staining and western blotting of bone tissue showed that the expression of p-p65 in the joint bone tissue of CIA mice was significantly higher than that in normal mice.Both DOCA and adalimumab significantly inhibited p-p65 expression in bone tissue of CIA mice.（8）Inflammatory response is important in the continuous development of RA.The serum inflammatory factor detection results showed that the levels of pro-inflammatory factors in serum（TNF-α,IL-1βand IL-6）of CIA mice were significantly higher than those in normal mice.Both DOCA and adalimumab significantly reduced the expression of relevant pro-inflammatory factors in the serum of CIA mice.Our results showed that DOCA could block the activation of NF-κB,a key effector molecule of TNF-αin joint tissues of CIA mice and improves RA in animal models.In summary,our study showed that DOCA,as a natural compound,could bind to TNF-αand TNFR directly,and block the interaction between TNF-αand TNFR to inhibit NF-κB signaling pathway activation induced by TNF-αand improve RA.These results provide a new strategy for obtaining RA therapeutics from natural compounds and provide a theoretical basis for the application development of edible and pharmaceutical homologous plants.