Effects And Mechanisms Of Total Glucosides Of Paeony On Collagen-induced Arthritis In Mice

The etiology and pathogenesis of rheumatoid arthritis (RA) is still poorly unknown. RA is a chronic progressive inflammatory disease involving multiple joints. It is characterized by T and B lymphocytes infiltration and the overgrowth of synovial fibroblasts. This invasive growth of synovial tissues corresponds with the progressive destruction of articular cartilage and bone, sometimes causing functional status decline and premature mortality in RA patients. Although it is accepted that RA is correlated with the immune system, and moreover, many studies showed that T cells, B cells, macrophages, synoviocytes and many proinflammatory cytokines, such as TNFa, IL1, IL-6and CD40, played an important role in mediating tissue damage and maintaining the chronicity of RA, but the initiation, perpetuation of RA and relationships between the above factors are still unclear. For this reason, it is difficult to treat RA effectively. Animal models of RA including rats and mice have provided the possibility of substantial insights into basic pathogenic mechanisms of RA and remedies. Among these, CIA mice has been the most popular.Total glucosides of paeony (TGP) is a kind of traditional Chinese medicine used for the treatment of rheumatoid immune diseases. It has bidirectional immunomodulatory effects and larger doses of which can suppress immune recationtion. It also has the function of protecting the liver, thus the drug toxicity can be minimized. However, there are few deep reports on the mechanism of TGP for the treatment of RA. There was no document was found on the effects of TGP to cell cycle.microRNA(miRNA) is a kind of non-coding endogenous RNA, of which molecular weight are about 22 nucleotides. It is a adjustment factor of T-cell growth, apoptosis, metabolic and other biological functions which is necessary to survive. So far there was no report having been found on the impact of TGP to miRNA in RA patients.Therefore, CIA model was established in our study. Effects and mechanisms of TGP on collagen-induced arthritis in mice was studied comprehensively and systematically from the following aspects:the effects of TGP on apoptosis of CIA immune cells, synovial cell, the regulatory protein of these processes, the influence on the inflammatory cytokines such as TNF-α, IL-1, IL-6, the effects on bone marrow cell cycle and the impact on expression of miR-15a in synovial cells.Objective:To establish a collagen-induced arthritis (CIA) mice model for RA by using DBA/1 mice and examine the clinical and pathological features as well as anti-collagen II antibodies, to evaluate the significance of CIA mice in the study of RA; to observation changes of localized swelling and histopathological of CIA rats and to identify the curative effect of TGP on CIA mice; to observe synovial cell morphologic changes of apoptosis, to clarify induction of TGP to synovial cells'apoptosis in CIA mice; to detect immune cell apoptosis, proliferation and cell cycle, to explore the Immune mechanisms of TGP to CIA mice; to detect serum TNF-α, IL-1, IL-6 concentration, to explore the mechanisms of inhibition of inflammatory response of TGP to CIA mice; to detect apoptosis protein, apoptosis regulatory proteins and the expression of miR-15a and to explore the mechanism of TGP-induced apoptosis of synoviocytes in CIA rats.Methods:CIA mice models were established by twice intradermal injection of bovine collagen II (CⅡ) in eight DBA/1 male mice. In Vivo, different dose of TGP was injected by intraperitoneal injection,250 mg·kg-1·d-1,50,100 mg·kg-1·d-1, respectively. In vitro, different concentrations of TGP were 62.5mg/L,125mg/L,250mg/L, respectively. Histopathological changes were examined under light microscope after H-E staining. Serum IgG antibodies to CII of CIA mice were also determined by enzyme-linked immuno-sorbent assay (ELISA) at 3 weeks and 6 weeks after second immunization. We also examined the apoptosis of synovial cells of CIA rats and related cytokines and protein expression after the treatment of TGP. Apoptosis rate of bone marrow cells and lymphocytes and changes in cell cycle of bone marrow cells were detected by flow cytometry. Morphological changes in apoptosis of synovial cells were observed by confocal microscopy. Cell proliferation assay was performed by MTT. The concentrations of TNF-α, IL-land IL-6 in serum were detected by radioactive immunoassay. Expression of miRNA-15a was detected by real-time quantitative RT-PCR. Expression of Bcl-2 protein was detected by Western blot analysis. Expression of Bcl-2 protein and caspase 3 in cells were detected by irnmunofluorescence.Results:1, clinical and pathological features and the levels of anti-C11 antibodies in CIASymptoms of arthritis, including erythema, swelling, vague outlines of multi-articulus in anterior limb and posterior limb, appeared after second immunization in all DBA/1 mice with collagen II injection. The mean time of onset of arthritis was 12.25±9.498. The highest mean score is 10.875±5.167, which manifested the different severe state of CIA. The formation of pannus, pannus combined with the articular surface and the erosion of articular cartilage and subchondral bone was observed by histopathology method in mice with a high score. Local damage of bone cells and cartilage cells, many new blood vessels, capillaries and mononuclear cells were observed in the joint cavity of the CIA mouse with moderate score. Persistent high levels of IgG were observed in all CIA mice stimulated by anti-C11 antibodies in ELISA. Positive correlation between the severity of arthritis and the titers of IgG antibodies was observed.2, Impacts of TGP on arthritis and joint pathology in CIA mice14 days later after intraperitoneal injection of TGP, it was observed that joint swelling of anterior and posterior limb in CIA mice significantly reduced and some changes took place in the joint pathological sections that the infiltration of neutrophils and lymphocytes decreased, the proliferation of synovial tissue reduced, the formation of capillary pannus was inhibited.3, Effects of TGP on the immune function in CIA ratsAfter administration of TGP (50,100mg.kg-1.d-1), the apoptosis of bone marrow cell increased significantly, reaching more than 38.8%, the apoptosis of spleen cells increased significantly, reaching more than 41.3%, the apoptosis of thymic B lymphocyte also increased significantly, reaching more than 36%, the apoptosis of apoptosis thymic T lymphocyte increased significantly, reaching more than 40.6%, these difference have Statistically significance compared with blank controlgroup and CIA group (P<0. 01). The proliferation of T lymphocytes and B lymphocytes in the spleen were inhibited after the administration of different concentrations of TGP (62.5mg/L,125mg/L,250mg/L) detected by MTT. The proportion of G0/G1 phase bone marrow cells significantly increased while S phase cells decreased significantly after the administration of medium and high dose TGP (50,100 mg.kg-1.d-1) detected by flow cytometry (P<0.05).4, Effects of TGP on the inflammatory response in CIA ratsAfter administration of TGP (25,50,100mg.kg-1.d-1), the expression of Serum TNF-α, IL-1, IL-6 was lower than CIA mice (P<0.01).It indicated that TGP can inhibite the expression of TNF-α. IL-1 and IL-6. There was no significant difference of the expression of Serum TNF-α, and IL-1 between the high dose group and the medium dose group (P> 0.05);,the expression of IL-6 in high dose group was significantly lower than that in the medium dose group (P<0.05).5, Effects of TGP on the apoptosis of synovial cell apoptosis in CIA ratsResults of real-time quantitative RT-PCR showed that the expression of miR-15a in synovial cells is lower in CIA rat compared with normal control group,(P<0.05). After administration of TGP (50mg.kg-1.d-1), the expression of miR-15a in synovial cells increased significantly than that of miR-15a in CIA mice (P<0.05). Expression reduction of Bcl-2 protein in TGP group compared with CIA rats detected by Western blot and immunohistochemistry was found. The expression of caspase 3 increased in TGP group compared with control CIA group detected by immunofluorescence.Conclusion:(1) Collagen-induced murine model of rheumatoid arthritis were established successfully.. Our results support the views that antibodies to CII may take part in the progression of arthritis, active phase of inflammation can be choiced as a sensitive period of observating arthritis indicators. CIA model is a essential method in researching the pathogenesis and new of remedy assessment of rheumatoid arthritis.(2)TGP can relieve the symptoms CIA mice and reduce the pathological changes of synovial tissue.(3) TGP can induce lymphocytes apoptosis, suppress cell proliferation, suppress conversion of bone marrow cells from a relatively quiescent period to active proliferation and inhibit autoimmune response in immune organs of CIA rats,.(4) TGP can induce apoptosis of synovial cells and inhibit the inflammatory response in CIA mice by inhibiting serum levels of TNF-α, IL-1 and IL-6.(5) The expression of miR-15a in synovial cells in CIA mice group is lower campared with the normal control group.(6) The expression of Bcl-2 protein significantly reduced, while the expression of miR-15a and caspase 3 increased in synovial cells in CIA rats after the administration of TGP. It suggests that TGP induced apoptosis of synovial cells possibly by increasing the expression of miR-15a, which provides a new idea for the research on the mechanism of TGP to the treatment of rheumatoid arthritis.