| Objectives:To investigat the effects and mechanisms of combination therapy with triptolide (TP) and glycyrrhizin (GL) in rats of collagen-induced arthritis (CIA), the peripheral blood, synovial membrane and gut mucosal immune system are observed from the immunogical and pathological aspects. And the combination therapy is compared with TP for finding the appropriate compatibility program, providing the new therapeutic regimen for rheumatoid arthritis.Methods:For the induction of CIA, CII was dissolved in sterilized 0.1 mol/L acetic acid to a final concentration of 2 mg/ml. CII (2 mg/ml) and complete Freund's adjuvant were homogenized at a 1:1 ratio. Each rat was injected intradermally at the right hind paws (day 0). 70 successful model rats were randomly divided into 7 groups: CIA group, TP22.33μg group (TPH), TP17.86μg group (TPM), TP13.40μg group (TPL), TP17.86μg/GL26.78mg group (TPM/GL), TP13.40μg/GL26.78mg group (IPL/GL), GL26.78mg group (GL). 10 normal rats were set in normal group. From the day 28 after immunization, all treated rats were administered with different dosages of TP and/or GL. The rats in normal group and CIA group were administered with same volume of saline. The treatment lasted for 4 weeks with a frequency of once a day.The edema rate of ankle joints and the arthritic indexes were examined; the pathological injuries of ankle were observed by light microscope and analyzed by a semi-quantitative joint disruption analysis.The levels of Anti-CII IgG, TNF-a, IL-10 in serum were measured by enzyme linked immunosorbent assay (ELISA).The expression of vascular endothelial growth factor (VEGF) and Endostatin in synovial membrane of knee joint were assessed by immunohistochemical staining method.The indexes of spleen and thymus and the amounts of Peyer's patch were accounted. The T lymphocytes of peripheral blood, Peyer's patch and intraepithelial and lamina propria lymphocytes were obtained by graduated digestion and grads centrifugation, and the subsets were assayed by FACS- calibur flow cytometer.Results:The edema rate of ankle joints and the arthritic indexes in CIA group were increased significantly, and these were significantly decreased after the treatment of TP and/or GL. The effects of TPH, TPM, TPM-GL and TPL-GL were superior to TPL and GL, and effects of TPM and TPL-GL were similar.In CIA group, the proliferation of synoviocytes and capillary vessels, the inflammatory cells and the erosion of cartilage and bone could be observed by light microscope. Those pathological changes were decreased after the treatment. The effects of TPH, TPM, TPM-GL and TPL-GL were similar and superior to TPL and GL.Higher level of anti-CII IgG in the serum was observed in the rats of CIA group. The antibody level was significantly decreased in the rats of all treated groups. In TPH, TPM and TPL groups, the antibody levels were negative related to the dosage of TP in certain degree. There were no significant differences in anti-CII IgG between the rats in TPM and TPL-GL groups.The level of TNF-a in the serum in the rats of CIA group was significantly higher than that in normal group. In the rats of all treated groups, the level of TNF-a in the serum was significantly lower than that in CIA group. In TPH, TPM and TPL groups, the levels of TNF-a had a negative relation to the dosage of TP in certain degree. There were no significant difference among the rats in TPM, TPM-GL and TPL-GL groups.The level of IL-10 in the serum in the rats of the CIA group was significantly higher than that in the normal group. In the rats of all treated groups, the level of IL-10 in the serum was significantly higher than that of the CIA group. In TPH, TPM and TPL groups, the levels of IL-10 had a positive relation to the dosage of TP. Compared to TPM group, the level of IL-10 in the rats of GL group was significantly lower, while the levels in the rats of TPM-GL and TPL-GL groups were significantly higher. There were few vascular endothelial growth factor (VEGF) and Endostatin positive cells in synovium of normal group. The expression of VEGF increased in synovium of CIA group, while the expression of Endostatin was no change.Compared to CIA group, the expressions of VEGF were decreased in synovium of TPH, TPM, TPM-GL and TPL-GL groups. Compared to TPM group, the expreesions of VEGF in GL group were increased, and there were no significant differences between TPM and TPL-GL groups.. Compared to CIA group, the expressions of Endostatin in synovium of all treated groups were increased. Compared to TPM group, the expreesions of Endostatin in GL group was decreased, and there were no significant differences between TPM and TPL-GL groups.The spleen indexes of CIA group were significantly higher than normal group. Compared to CIA group, the spleen indexes of all treated groups are lower, and the difference is notable in TPM and TPL-GL groups.There was no difference of thymus gland indexes between CIA and normal groups, while the thymus gland indexes of TPH, TPM and TPM-GL groups were significantly lower than that of CIA group. And there were no significant differences between TPM and TPL-GL groups.The numbers of peyer's patch of CIA group were significantly more than normal group. Compared to CIA group, the numbers of all treated groups were significantly decreased, and there were no significant differences between TPM and TPL-GL groups.In periphery, the CD3~+cells of CIA groups were increased, primarily were CD4~+ cells. The ratios of CD4~+ to CD8~+ were increased accordingly. Compared to CIA group, the CD4~+ cells of TPH, TPM, TPL, TPM-GL and TPL-GL groups were decreased and the ratios of CD4~+ to CD8~+ were decreased accordingly. There were no significant differences between TPM and TPL-GL groups.Although the levels of CD3~+, CD4~+ and CD8~+ lymphocytes in peyer's patch were no significant difference between CIA and normal groups, the ratio of CD4~+ to CD8~+ were increased in CIA group. Compared to CIA group, the CD8~+ cells of all treated groups were increased and the ratios of CD4~+ to CD8~+ were decreased accordingly. There were no significant differences between TPM and TPL-GL groups.Compared to normal group, the CD4~+ cells in intraepithelial of CIA group were increased while the CD8~+ cells decreased and the ratio of CD4~+ to CD8~+ were increased accordingly. Compared to CIA group, the CD4~+ cells in IEL of TPH, TPM, TPL, TPM-GL and TPL-GL groups were decreased and the CD8~+ cell increased, the ratio of CD4~+ to CD8~+ were decreased. There were no significant differences between TPM and TPL-GL groups.Compared to normal group, the CD4~+ cells in lamina propria of CIA group were increased and the ratios of CD4+ to CD8+ were increased. Compared to CIA group, Although the CD4~+ and CD8~+ cells of TPH, TPM, TPM-GL and TPL-GL groups were no significant difference, the ratio of CD4~+ to CD8~+ were decreased. There were no significant differences between TPM and TPL-GL groups.Conclusions:Triptolide could significantly decrease the edema rate of ankle joints and the arthritic indexes in CIA rats, and had positive relation to the dosage. Glycyrrhizin also had the effects but inferior to Triptolide. The combination of TP 13.40μg with GL 26.78mg could achieve the similar effects of TP 17.86μg, and these indicated that we can decreased the dosage of triptolide for reducing the side-effects by combined with glycyrrhizin.Triptolide and glycyrrhizin also can depressed the abnormal self-immunoreaction, it could balanced the network of T helper 1 and T helper 2 by degrading the level of TNF-a and upgrading the level of IL-10, and alleviated the inflammation of synovial membrane sequentially. Combined therapy showed synergistic effect.The balance of stimulator and inhibitor of angiogenesis in synovial membrane of CIA rats was upset, and had correlation with the inflammation and proliferation of synovial membrane. Triptolide could inhibit the proliferation and inflammation by regulating the level of VEGF and Endostatin. Although glycyrrhizin had few effects, it could enhanced the effect of triptolide. Triptolide can suppressed the immunoreaction by regulating the subsets of T lymphocytes in intestinal mucosa-associated lymphoid tissue, degrading the ratio of CD4~+ to CD8~+ T lymphocyte. Glycyrrhizm also had effect of immuno-suppressive, although it could not degraded the ratio of CD4~+ to CD8~+ T lymphocyte markedly, but it could synergized the effect of triptolide.
|
PDF Full Text Request