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Objective:To establish the methods of isolated culture and functional identification of mice bone marrow derived tolerogenic dendritic cells (CD11b+F4/80 +TDCs) in vitro.Methods: Mice bone marrow cells were isolated and cultured to obtain iDCs with the simulation of mouse rmGM-CSF and rmIL-4.CD11b+F4/80 +TDCs were purified by fluorescence-activated cell sorting on day 6.The morphological changes of TDCs were observed with the inverted microscope dynamically .The expression of CD11b+F4/80 +TDCs were analyzed by the flow cytometry .Tolerogenic function of CD11b+F4/80 +TDCs was evaluated by the expression of MHCⅡ, CD83, IDO, TLR-2, IL-10 and TGF-β1.The expression of MHCⅡ was analyzed by the flow cytometry , and the expression of CD83, IDO and TLR-2 were analyzed by immune-histochemistry.The levels of IL-10 and TGF-β1 in the supernatant of CD11b+F4/80 +TDC were analyzed by ELISA .Meanwhile mature DCs ( mDCs) induced by LPS were used as control .Results:The fresh isolated bone marrow cells look like round and small under microscope .After two days of culture , cells became big and formed into clusters . Five or six days later, cells clusters increased, and the morphology of cells became irregular .At the same time, more dendrite ap-peared on the surface of cells .The percentage of CD11b+F4/80 +TDCs induced by rmGM-CSF and rmIL-4 was about 23%, and the purity of the purified BM CD11b+F4/80 +iDC was about 99%.Compared with mDCs, CD11b+F4/80 +TDCs expressed low levels of MHCⅡand CD83 and high levels of IDO, TLR-2, IL-10 and TGF-β1.Conclusion:CD11b+F4/80 +TDCs derived from mouse bone marrow could be induced successfully by rmGM-CSF and rmIL-4 in vitro.CD11b+F4/80 +TDCs showed tolerogenic function by the expressions of IL-10, TGF-β1, IDO and TLR-2.
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Aim To investigate the regulatory effects of Paeoniflorin on G protein-coupled signaling by synoviocytes of collagen-induced arthritis (CIA) rats. Methods CIA was induced by chicken type Ⅱcollagen. Pae (25, 50, 100 mg?kg-1) was administered to CIA rats from d 16 to d 23 after immunization. Arthritis was evaluated by hind paw swelling and arthritis index. Expression of inhibitory G protein (Gi) was detected by Western blotting technique. The level of cAMP in synoviocytes was measured by radioimmunoassay. Protein kinase A (PKA) activity was assessed by Kinase-Glo Luminescent Kinase Assay. Results There was remarkably secondary inflammation in CIA rats. The expression of Gi in synoviocytes increased. While cAMP level and PKA activity of synoviocytes decreased. The inflammatory responses of CIA rats were inhibited after administration of Pae. At the dose of 100 mg?kg-1, Pae reduced Gi expression. cAMP level and PKA activity were enhanced by Pae at the doses of 50 and 100 mg?kg-1 respectively. In ivtro, Pae (2.5, 12.5, 62.5 mg?L-1) reduced Gi expression, but enhanced the level of cAMP and PKA activity. Conclusion G protein-coupled signaling was associated with the pathogenesis of synovitis in CIA rats. Pae has anti-inflammatory effects on CIA rats by modulating G protein -coupled signaling.
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Aim To investigate the expression of GRKs in rats of synovial tissue with collagen-induced arthritis(CIA) and the effect of total glucosides of paeony.Methods SD rats were divided into six groups including normal,model,TGP(25,50,100 mg?kg-1)groups,GTW(40 mg?kg-1)group.Chicken type Ⅱ collagen was used to induce CIA in rats.The expression of GRKs was detected by Westernblot.Results The expression of GRK2,5,6 increased in model group than that in normal group.Compared with the model group,the expression of GRK2,5,6 decreased in TGP groups.Conclusion In rats of synovial tissue with CIA,the expression of GRKs was abnormal,and TGP could change the variation of GRKs which may be one of the mechanisms of TGP improvement CIA.