The Rapeutic Effect Of Trichinella Spiralis P43 Recombinant Protein On Rheumatoid Arthritis Mice

Rheumatoid arthritis(RA)is a serious autoimmune disease,in which patients mainly present with swelling of limbs and certain degree of destruction of joints.In severe cases,patients even lose joint function,leading to their inability to live normally.It is mainly caused by the immune dysfunction of the body due to the excessive activation of CD4~+T cells in the body.Studies have shown that Trichinella spiralis(T.spiralis)and its derivatives play an important role in regulating the immunity of the host body,and even have a good intervention effect on some autoimmune diseases.After invading the host,T.spiralis secretes an excretory secretion(ES)antigen to promote the expression of indoleamine 2,3 dioxygenase(IDO)to regulate the host immune system and cause immunosuppression of the host organism.In this study,T.spiralis protein 43(Tsp43)with high content of T.spiralis ES antigen was selected to treat RA by its immunoregulatory ability,and the mechanism was further verified.The purpose of this study was to provide a new idea for the treatment of RA,and lay a foundation for the related research and development of parasitic biological preparations.In this experiment,Western blotting and Immunofluorescence were used to verify that Tsp43could promote the expression of IDO in Dendritic cells in vitro.In vivo experiments,200 BALB/c mice were randomly divided into four groups,Blank group,Model group(BCII),Protein group(BCII+Tsp43)and Inhibition group(BCII+Tsp43+1-MT).By detecting various characteristic indicators of RA,we verified whether Tsp43 can produce therapeutic effects on bovine type II collagen(BCII)-induced RA mice and whether such therapeutic effects have an IDO dependence.In this experiment,enzyme-linked immunosorbent assay(ELISA)was used to detect the serum IL-1βand TNF-αcontents of mice in each group.The pathological changes of ankle joints in the hind limbs of mice in each group were detected by Hematoxylin-eosin(H(5)E)staining.The ratio of CD4~+/CD8~+T cells in the spleen of mice in each group was detected by Flow cytometry(FCM).The results showed that the serum IL-1βand TNF-αcontents of RA mice were much higher than those of the blank group,and their hind limbs’ankle joints were seriously damaged,their bones were seriously damaged,and their cartilage was seriously damaged.The CD4~+/CD8~+T ratio of RA mice was also much higher than that of the blank group.After treatment with Tsp43,the contents of IL-1βand TNF-αin RA mice were significantly decreased,the damaged ankle joint was restored,and the ratio of CD4~+/CD8~+T in the spleen was also significantly reduced.1-MT(IDO inhibitor)could well inhibit the therapeutic effect of Tsp43 on RA mice.The results of this experiment showed that Tsp43 had certain therapeutic effect on RA mice,and the therapeutic effect was IDO dependent.In this experiment,the therapeutic mechanism of Tsp43 in RA mice was further explored.Western blotting was used to detect the expression of IDO in mice of each group and the subsequent changes in the concentrations of Tryptophan(Try)and Kynurenine(Kyn)in serum.In order to explore the effect of Tsp43 on the proliferation of CD4~+T cells,CD4~+T cells in the spleen of mice from each group were separated by magnetic beads,and the primary culture was conducted after the purity was verified.The expressions of amino acid sensing related proteins GCN2 and p-e IF2αin CD4~+T cells in the spleen of mice from each group were detected,and the proliferation changes of CD4~+T cells in each group at 0,6,12,24,48 and 72h were detected by CCK-8 method.In order to explore the effect of Tsp43 on the apoptosis of CD4+T cells,we detected the expression of CD4~+T cell-related apoptotic proteins(Bcl-2,Bax,cl-CASP9,cl-CASP3,and cl-PARP)in mice of each group and detected the apoptosis of CD4~+T cells in spleen by FCM.In addition,FCM was used to detect the ratio of Foxp3 in CD4~+CD25~+Treg cells in the spleen of mice in each group.The results showed that Tsp43 reduced the concentration of tryptophan through high expression of IDO in RA mice,and thus promoted the high expression of amino acid sensing related proteins GCN2and p-e IF2αin CD4~+T cells,and thus inhibited the proliferation of CD4+T cells.Moreover,after promoting the expression of IDO,Tsp43 increased the serum Kyn concentration in RA mice,resulting in increased expression of CD4~+T cell pro-apoptotic proteins(Bax,cl-CASP9,cl-CASP3,and cl-PARP)and decreased expression of anti-apoptotic protein(Bcl-2)in RA mice,in which CD4~+T cells were subjected to apoptosis.In addition,Tsp43 also significantly increased the ratio of Foxp3 in CD4~+CD25~+Treg cells in RA mice,thus exerting a negative regulatory effect on the immune function of RA mice.In summary,Tsp43 had a good therapeutic effect on RA mice,in that it could reduce the content of IL-1βand TNF-αin RA mice,repair the damaged ankle,and restore the ratio of CD4~+/CD8~+T cells.This therapeutic effect is in an IDO dependence.The specific mechanism is that Tsp43 causes high expression of IDO in RA mice and reduces Trp concentration,which leads to high expression of amino acid sensing related proteins GCN2 and p-e IF2α,and finally inhibits the proliferation of CD4~+T cells.At the same time,the decomposition of Trp by IDO produced high concentrations of Kyn,which led to the apoptosis of CD4+T cells in RA mice.Moreover,Tsp43was able to increase the ratio of Foxp3 in CD4~+CD25~+Treg cells though IDO,thereby negatively regulating the immunity of RA mice.This study has proved that Tsp43 works together through IDO in a variety of ways to regulate the immune function imbalance caused by the excessive activation of CD4+T cells in RA mice,which not only provides a new idea for the treatment of RA,but also lays a foundation for the development of parasite-derived drugs for the treatment of RA.