Establishment Of ACPA-positive Rheumatoid Arthritis Animal Model And Intervention Of Tripterygium Wilfordii Polyglycoside Tablets

Objective:Rheumatoid arthritis(RA)is a kind of autoimmune disease with a high incidence rate and disability rate.Its clinical characteristics are mainly chronic inflammation and irreversible bone erosion.Pathogenic anti-citrullinated Protein antibodies(ACPA)are considered a key factor in inducing the onset and immune maintenance of RA.However,animal models of RA characterized by ACPA have not yet been reported,which greatly limits the exploration of the pathogenesis of ACPA and the development of related therapeutic drugs.In view of this,this study intends to establish an ACPApositive RA animal model based on the mechanism of ACPA Production,using two methods:citrullinated Proteins(CPs)immunization and Porphyromonas gingivalis(Pg)with unique peptide arginine deiminase infection,and compare and evaluate the clinical characteristics(including inflammation,bone destruction,and pain)and pathological characteristics.It is known that follicular helper T(Tfh)cells and their associated follicular regulatory T(Tfr)and germinal center(GC)B cells are the main immune cell subpopulations that regulate the Production of high-affinity autoantibody ACPA and also play a crucial role in the occurrence and development of RA.Based on the established and optimized ACPA-positive RA animal model,this study intends to further observe the effect of Tripterygium wilfordii polyglycoside tablets,a listed Chinese patent medicine with good efficacy on RA,on the intervention of this model,and start from the characteristics of Tfh,Tfr,and GCB cell subsets,to reveal the potential mechanism of Tripterygium wilfordii polyglycoside tablets on the regulation of Tfh cells based on energy metabolomics,so as to Provide a reference for the clinical rational use of Tripterygium wilfordii preparations,It also Provides evidence for the establishment of ACPA positive RA animal models.Method:1.Preliminary exploration on the construction of ACPA-positive RA animal modelIn vitro preparation of CPs,RA susceptible animal DBA/1 mice were divided into normal control(Control)group,type Ⅱ collagen(CⅡ)group,citrullinated type Ⅱcollagen(Cit-CⅡ)group,fibrinogen(Fib)group,and citrullinated fibrinogen(Cit-Fib)group.Antigen combined with complete Freund’s adjuvant immunization was used to investigate the clinical symptoms,and serum biochemistry to compare the pathological characteristics of RA mouse models induced by four different antigens such as CⅡ,Fib,and citrullinated Protein in terms of histopathology and imaging.For establishing an ACPA-positive animal model for Pg infection,we selected DBA/1 mice and C3H/He mice(a class of IEβ-K chain and human beings β1 chain HLADRB1*04:01 highly homologous mice)were initially explored.The animals were divided into the Control group,Pg infection alone(Pg)group,collagen-induced arthritis(CIA)group,and Pg infection combined with collagen-induced(Pg+CIA)group.The characteristics of the models of two different species of mice were studied from the aspects of clinical symptoms,serum antibody detection,and pathological changes.Finally,using ACPA as an evaluation index,two different strains of mouse models were further evaluated through the AUC curve.2.Pathological characteristics of arthritis in ACPA-positive RA animal modelsC3H/He mice were divided into the Control group,Pg group,CIA group,and Pg+CIA group.Observe and record the symptoms,incidence rate,and clinical scores of the mice in each group,such as joint redness,swelling,deformity,etc.The changes in mechanical pain threshold and cold pain sensitivity of mice in each group at different time points were detected by up down method and chemical stimulation method.The dynamic changes of anti-CCP antibodies at different time points were detected using a serum antibody kit,and the expression of CPs in different organs and tissues of mice was detected by Western blot(WB)assay.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of various immunoglobulins and inflammatory factors in mice in each group.Bone and cartilage damage in mice in each group were evaluated by HE staining,TRAP staining,safranine O-fast green staining,inflammatory ankle and knee X-ray,and inflammatory knee Micro-CT scanning.Then,the expression of osteoclast-related factors in bone marrow macrophages(BMMs)in vivo and in vitro was detected by q-PCR and WB experiments,and the co-labeling of CPs with osteoclast precursors and osteoclast marker CD68 was detected by immunofluorescence double labeling method.Finally,TRAP staining,toluidine blue staining,and actin ring staining were used to evaluate the effects of different groups of mouse BMMs on osteoclast formation,differentiation,and bone resorption in vitro.3.Immunocyte characteristics of ACPA-positive RA animal modelC3H/He mice were divided into the Control group,Pg,CIA group,and Pg+CIA group.The spleen and lymph nodes of mice in each group were isolated for macroscopic and pathological observation.Lymphocytes from the spleen and lymph nodes of mice in each group were isolated by density gradient centrifugation,and the Proportions of CD4+T,B220+B,Tfh,Tfr,and GCB cells were measured by flow cytometry.Through magnetic bead sorting,CD4+T cells in the Control and Pg+CIA groups were further obtained,and mRNA transcriptional genomic expression Profiling was performed.Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis was performed on differential genes.Gene Set Enrichment Analysis(GSEA)analysis was performed on upregulated genes in differential genes and Tfh cell upregulated gene sets.4.Intervention effect of TGT on ACPA-positive RA model animalsC3H/He mice were divided into the Control group,Pg+CIA group,TGT high-dose group(TGT-H,27.3 mg/kg,twice the clinical equivalent dose),and TGT low-dose group(TGT-L,13.65 mg/kg,one time the clinical equivalent dose).The intervention effect of TGT on the ACPA-positive RA model induced by Pg+CIA was evaluated from macroscopic,autoantibody detection,pathology and imaging,bone destruction factor detection,and Tfh-GCB flow cytometry.In addition,based on energy metabolomics,we explored the regulatory characteristics of energy metabolism of spleen CD4+T cells in ACPA-positive animal models through glycolysis and pentose phosphate pathways.Result:1.Comparative study on the establishment of animal models of ACPA-positive rheumatoid arthritisThe incidence of RA in DBA/1 mice induced by different antigens was significantly different,among which CⅡ(100%)>Cit Fib(37.5%)>Cit CⅡ(25%)>Fib(0%).In terms of clinical symptoms and manifestations of arthritis,mice in the CⅡ group had an onset of inflammation and significant redness and deformity in their limbs.The onset of arthritis in the Cit-Fib and Cit-CⅡ groups was mild and generally limited to the hind legs,while the FIB group did not have symptoms of arthritis.Further histopathological and imaging examination results showed that the CⅡ group had the most severe synovitis infiltration,pannus formation,cartilage,and bone destruction in the knee joint,followed by the Cit-Fib group,followed by the Cit-CⅡ group,and there were almost no significant pathological and imaging changes in the Fib group.CⅡ,Cit-CⅡ,and Cit-Fib groups all Produced a certain level of anti-CCP antibodies,with Cit-Fib group>Cit-CⅡ group>CⅡ group.Further testing of the levels of two citrullinated epitope-specific antibodies showed that Cit-CⅡ and Cit-Fib-induced mice expressed significantly higher levels of anti-Cit-CⅡ and anti-Cit-Fib antibodies,respectively,and both were slightly higher than those in the CⅡ group.Among the mice induced by four antigens,the expression levels of serum Proinflammatory factors and joint bone destruction factors in the CⅡ group were the highest,followed by the Cit-Fib group,and the Cit-CⅡ group was the third.There was almost no difference between the Fix group and the Control group.In both DBA/1 and C3H/He mice,Pg alone infection did not induce RA symptoms.In DBA/1 mice,Pg did not significantly aggravate the clinical symptoms and pathological scores of CIA-induced RA,and there was no statistical difference between the Pg+CIA group and the CIA group in terms of the expression of two types of autoantibodies,ACPA and IgG.Unlike DBA/1 mice,it has been demonstrated in the C3H/He mouse model that oral infection with Pg aggravates CIA-induced RA and mice in the Pg+CIA group exhibit more significant redness,swelling,and deformity,as well as more significant bone destruction and synovial inflammation in pathology compared to those in the CIA group.Autoantibody detection results showed that the expression levels of ACPA and IgG in the C3H/He mice Pg+CIA group were significantly higher than those in the other three groups.The AUC curve results using ACPA as an evaluation index indicate that the C3H/He mouse model induced by Pg+CIA is closer to the ideal ACPA-positive RA animal model.2.Pathological characteristics of arthritis in ACPA-positive RA animal modelsIn this experiment,we determined that in C3H/He mice,gingival Pg oral infection exacerbates collagen adjuvant-induced arthritis and significantly increases the levels of CPs(joints,lungs,intestines,and spleen),ACPA,and immunoglobulin(IgG,IgM,and IgA).The trend of changes in mechanical pain and cold sensitivity pain showed that the pain sensation of mice in the Pg+CIA group was higher than that of the other three groups.The levels of inflammatory factors in the Pg+CIA group were significantly higher than those in the other three groups,and there was a significant positive correlation between them and the expression of serum ACPA.In terms of pathology and imaging,the Pg+CIA group showed more significant cartilage and bone destruction.WB and PCR results showed that the expression of osteoclastrelated factors in the Pg+CIA group was significantly higher than that in the other three groups,and there was a significant positive correlation between the expression of serum ACPA.Further observation of the co-labeling of CPs and CD68 in bone marrow sections and BMMs showed that mice in the Pg+CIA group exhibited more significant osteoclasts,and high levels of CPs were expressed in the cytoplasm.After 5-7 days of induction of BMMs in each group,the osteoclast formation,differentiation,and bone resorption in the Pg+CIA group were significantly higher than those in the other groups.3.Immunocyte characteristics of ACPA-positive RA animal modelThe macroscopic and pathological results showed that the spleen and peripheral lymph nodes in the Pg+CIA group were significantly larger than those in the other three groups,indicating that the Pg+CIA group mice showed more significant spontaneous lymphoid hyperplasia.Compared with the Control group,the Proliferation of white pulp and marginal areas in the spleen tissue of mice in the Pg+CIA group was significant,with inflammatory cells infiltrating,and a large number of germinal centers forming.The results of flow cytometry showed that the Proportion of Tfh cells in mice in the Pg+CIA group was significantly higher than that in the other three groups,indicating that Pg infection and CIA induction may play a synergistic role in the differentiation of CD4+T cells into Tfh cells.GCB cells can be activated with the assistance of Tfh cells in peripheral immune organs,thereby Producing high-affinity antibodies.In this study,the Proportion of GCB cells to Tfh cells in mice in the Pg+CIA group was consistent,and significantly increased compared to the other three groups.In addition,Tfh cells and GCB cells were significantly positively correlated with ACPA.However,in this study,we found that the Proportion of Tfr cells in mice in the Pg+CIA group increased compared to other groups,but there was no statistical difference,and there was no significant correlation between the Proportion of Tfr cells and ACPA.Finally,GSEA analysis confirmed that CD4+T cells from mice in the Pg+CIA group had a significant tendency to differentiate into Tfh cells compared to CD4+T cells from the Control group.4.Intervention effect of TGT on ACPA-positive RA model animalsThe experimental results show that TGT can reduce the incidence rate of animal models induced by Pg+CIA,and imProve pathological damage and bone destruction.In addition,TGT has a good inhibitory effect on the Production of autoantibody ACPA.Through flow cytometry,it was further found that TGT can reduce the Proportion of Tfh cells and GCB cells in mice spleen induced by Pg+CIA.The results of energy metabolomics showed that in the Pg+CIA group,the key metabolites of the glycolytic pathway,such as fructose 6-phosphate,glucose 6-phosphate,D-glucose-1phosphate,fructose 1,6-diphosphate,and D-ribose 5-phosphate,were significantly increased in spleen CD4+T cells.After oral TGT treatment;the metabolic disorder of the glycolytic pathway was imProved.Conclusion:1.Pg infection combined with type Ⅱ collagen and complete Freund’s adjuvant immunization in C3H/He mice can successfully establish an ACPA-positive RA animal model with similar clinical characteristics,manifested by high levels of citrullinated Protein expression and serum ACPA content,severe arthritis symptoms,and degree of bone destruction.The pathogenesis may be related to the activation of immune organ Tfh GCB cells induced by modeling,which Promotes the Production of ACPA.2.Using the above mouse model,oral TGT intervention can imProve the symptoms of arthritis,reduce the incidence rate,and inhibit the Production of ACPA and bone destruction,to effectively treat ACPA-positive RA mice.The mechanism may be related to regulating the glycolysis pathway and inhibiting the differentiation of Tfh cells,thereby inhibiting the activation of GCB.