| ObjectiveThe present study was to explore the therapeutic mechanism of tripterginum wlfordii on collagen induced arthritis (CIA) from the mucosal immune, immunological and pathological aspects.Materials and methodsMale DBA/1 mice and DA rats were randomly allocated to normal control group , collagen induced arthritis group(CIA) and Triptolide-treated group (TT) respectively according to the experiment design. All the model DA rats were injected intradermally at the base of the tail of 150 ug type II collagen in 75 ul 0. 1 M acetic acid emulsified in an equal volume of complete Freund's adjuvant to induce arthritis. For DBA/1 mice, every mouse was injected intradermally at the base of the tail of 100 ug type II collagen in 50 ul 0. 1 M acetic acid emulsified in an equal volume of complete Freund's adjuvant, and mices were challenged again with the same antigen preparation 21 days later. The rats in the model group and normal group were fed with physiological saline, and TT groups were fed with Triptolide, 11. 62 ug /kg.d for DA rats and 16. 36 ug /kg.d for DBA/1 mice. The arthritis indexes, the weight of thymus gland and spleen indexes and the amounts of Peyer's patch in the small intestine of both DA rats and DBA/1 mice were accounted. The changes of T cell subsets in the serum and Payer's patch of the small intestine of both DA rats and DBA/1 mice in different time point were assayed by FACA Calibur flow cytometer.Anti-CII antibody, TGF-β , IFN-γ levels in serum of both DA rats and DBA/1 mice were detected with ELISA method. Proliferatios of T and B lymphocytes in spleen were tested with MTT method. In addition the changes of IEL and LPL T cell subset in the small intestine, apoptosis of Peyer's patch, the expression of IFN- γ injoints by RT-PCR were observed in DBA/1 mice. The expression of CD4\ CD8+ T cells, slgA, TGF- P , IFN- y in the small intestine and the expression of IFN- y in joints of DA rats were assessed by Immunohistochemical staining and immuonfluorescence methods. The pathological changes of the knee and ankle joints of DA rats and DBA/1 mice were detected by telescope.Results1. The changes of joints indicated that the arthritis indexes and the expression of IFN- y of both kinds of animals in CIA model group increased significantly. There were no pathological changes in the knees and ankle joints in normal group mice and rats. In the mice and rats of CIA model group, erosion of cartilage and bone, proliferation of synoviocytes and capillary vessels, the hyperemia and edema in synovium and the formation of pannus, the inflammatory cells could be observed. The CIA mice and rats joints of TT group showed that decreased arthritis indexes and the IFN- Y levels and slight pathological change in synovial tissue and cartilage.2. The amounts of Peyer's patch in the animals of CIA model group were significantly greater than that of normal animals, and the amounts of Peyer's patch in the animals of TT group was less than that of CIA group animals.3. There were no differences of thymus gland indexes in the DA rats and DBA/1 mice among in three groups. The spleen indexes of CIA model group animals were higher than normal group animals, and the spleen indexes of TT group animals were lower significantly than CIA model group animals and had no differences from the normal group animals. In this experiment, proliferation of splenocytes induced by Con-A stimulation in the CIA model group was more active than that in normal group and it could be derease by Triptolide. As for the lymphocytes proliferation reaction by LPS, there were no discrepancy among animals of three groups in DBA/1 mice and increase in CIAgroup of DA rats.4. TGF- P levels in serum of animals in CIA model group decreased significantly which indicated that TGF- P , which belongs to Th2 cytokines, had some negative effect on the pathogenesis of CIA; and TGF- P levels in serum of animals in TT group increased significantly indicated that Triptolide could inhibit the processof CIA by improving the TGF- (3 levels in serum. The levels of IFN- v in serum of animals in CIA model group increased significantly and it indicated that CIA was the kind of diseases which Thl cytokines dominated as RA, Triptolide could inhibit the progress of the disease by decreasing the IFN-y levels in serum. From above, we can see that Triptolide could up-regulate the TGF- (3 levels and down-regulate the IFN- y levels in serum to treat CIA by adjusting the balance of Thl/Th2.5. The amount of anti-CII antibody in both kinds of animals of CIA model group was higher than that of normal group and the content of them in CIA model group was higher compared with TT group. All that indicated that anti-CII antibody had some effect on the pathogenesis of CIA, and Triptolide could lessen the higher level of anti-CII antibody in serum, and have some inhibition effect on immunity.6. In the 28th day of the DBA/1 mice were immunized, the CD4 + lymphocytes of DBA/1 mice in the CIA model group increased and CD4/CD8 lymphocytes increased significantly compared with whose in the normal control group; in the mice of TT group the CD4+ lymphocytes increased and the CD8+ lymphocytes decreased which led to the improvement of the ratio of CD4/CD8 lymphocytes. In the 42nd day of the DBA/1 mice were immunized , the CD4+ lymphocytes, the CD3+ lymphocytes and the ratio of CD4/CD8 lymphocytes of CIA model group increased and these indexes of TT group decreased significantly compared to that of CIA model group, but there were no differences between TT group and normal control group. In the 60th day, the expression of CD4+ lymphocytesdecreased compared with other time points, but the expression of CD4+ lymphocytes and the ratio of CD4/CD8 lymphocytes were still higher than that of normal control group.On the 21st day of immunization, comparing to the negative control group, the CD8+ lymphocytes of DA rats in both CIA model group and TT group decreased and the ratio of CD4/CD8 lymphocytes increased accordingly. On the 30th day, CD4+ lymphocytes in CIA model increased, thus CD3+ and the ratio of CD4/CD8 increased consequently. These indexes in TT group showed significant decrease when compared with CIA model group, but did not show much difference from negative control. On the 60th day, the CD4+ lymphocytes of CIA model mice decreased in certain level. Compared to the negative control group, the expression of CD4+ lymphocytes and the ratio of CD4/CD8 had significant change.7. The levels of CD4+ and CD8+ lymphocytes in the Peyer's patch of the small intestine increased significantly in the CIA model group when compared with negative control group, but the ratio of CD4/CD8 decreased in the CIA model group. In apoptosis detection with PI assay, the lymphocytes in Peyer' patch of CIA model did not undergo apoptosis.LPL: The levels of CD4+ and CD8+ lymphocyte in CIA model were higher than those in negative control, but the ratio of CD4/CD8 in CIA model group was lower. Comparing to CIA model group, the CD8+ lymphocytes in TT group decreased, but the expression of CD4+ lymphocytes and the ratio of CD4/CD8 increased. There was no significant change between TT group and negative control group. IEL: The level of CD4+ lymphocyte in CIA model was higher than that in negative control, but y 5 TCR and CD8+ lymphocyte in CIA model showed significant decrease. The level of CD8+ lymphocyte in TT group was higher than that in CIA group.8. The results of immunohistochemistry for CD4, CD8, SigA, TGF-(3 and IFN- Y showed that the expression of CD4, slgA and IFN- y in...
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