The Application Of Strategy Of Prevent DDR2 On Heterotopic Ossification And Rheumatoid Arthritis In Mice

Discoidin domain receptor 2(DDR2) is a kind of receptor tyrosine kinases(RTKs). DDR2 and its signal pathway play an important role in development and contribute to many diseases such as cancer, fibrosis, atherosclerosis, osteoarthritis(OA), rheumatoid arthritis(RA) and so on. Thus, DDR2 has become the potential target of the treatment of these diseases. In the present study, using the strategy of prevent DDR2, we respectively investaged the effect of GD856 and dasatinib which are DDR2 kinase small-molecule inhibitors on heterotopic ossification and rheumatoid arthritis in mice. Experiment 1 DDR2 deficiency on bone formation in mice in vivoDDR2 plays a key role in bone development and formation. DDR2 promotes proliferation and differentiation of chondrocytes, differentiation of osteoblasts. And DDR2 suppresses osteoclastogenesis. Many studies showed the role of DDR2 on bone formation cellular level. However, the evidence of role of DDR2 on bone formation in vivo is still not enough. In this part, we compared the bone development and bone formation after fraction and heterotopic ossification in DDR2 null mice and wild type mice.DDR2 null mice showed abnormal development on bone development. The body size and bone length of DDR2 null mice were significantly shorter than those of wild type mice. Bone formation of trabecula and cortical was markedly suppressed in DDR2 null mice than in wild type mice evaluated by microCT. Three point bending test showed the max force and stiffness of femurs from DDR2 null mice were markedly lower than those of wild type mice. Bone metabolism was significantly suppressed in DDR2 null mice than in wild type mice evaluated by double calcein labeling. We established open fraction model in mice by operation. The callus formation of DDR2 null mice were mardly surppressed than those of wild type mice, which meant fracture healing was impaired in DDR2 null mice. The heterotopic bone formation of calcaneus was markedly impaired in DDR2 null mice in 10 weeks after the establishment of mouse trauma-burn model of heterotopic ossification.These results indicated that bone generation, bone metabolism and bone formation after fraction and heterotopic ossification were suppressed in DDR2 null mice. Experiment 2 The effect of DDR2 kinase small-molecule inhibitor on heterotopic ossification in miceGD856 is a newly designed DDR2 kinase small-molecule inhibitor. It strongly inhibits DDR2 phosphorylation in low concentration. In this part we investaged the effect of DDR2 kinase small-molecule inhibitor on heterotopic ossification through MC-3T3-E1 osteoblast cell line and mouse heterotopic ossification model.We divided cells into 3 groups: control group with no GD856 stimulation, low concentration group with GD856 at 10 μM and high concentration group with GD856 at 50 μM. After osteogenic induction for 3 weeks, cells were performed alizarin red stain, alkaline phosphatase(ALP) stain and real-time PCR to assess the mRNA expression of osteogenesis-related genes. The positive degree of alizarin red stain was lower in low and high concentration groups than those in control group. The positive degree of ALP stain were higher in low concentration group than that in control group while the positive degree of ALP stain were lower in high concentration group than that in control group. The expression of OPN, OCN and BMP2 mRNA were significantly lower in in low and high concentration groups than those in control group. And the expression of ALP mRNA was significantly lower in high concentration group than that in control group. However, the expression of ALP and Col I A1 mRNA were markedly higher in low concentration group than those in control group. In order to study the effect of GD856 on heterotopic ossification, we established mouse trauma-burn model of heterotopic ossification. Mice were divided into control group and GD856 group with 0.08 mg/ml in drink water. In 10 weeks after the establishment of model, the bone metabolism and the heterotopic bone formation of calcaneus were markedly impaired in GD856 group compaired with control group.These results indicated that GD856 impaired osteogenesis and heterotopic ossification in mice. Experiment 3 The effect of dasatinib on mice with collagen-induced arthritisDasatinib is second generation of tyrosine kinase inhibitor and strongly inhibit DDR2 activity. And dasatinib inhibited many key genes durning RA pathology. The aim of this part was to investage the effect of dasatinib on mice with collagen-induced arthritis(CIA).DBA/1 mice were injected with chicken collagen II in the 1st and 21 th day to establish CIA model. Mice were divided into dasatinib group treated with 10 mg/kg dasatinib from the day 28 to the day 57 orally once a day and control group treated with placebo in the same time. Dasatinib treatment significantly decreased paw redness and swelling, the thickness of paws and the scores of arthritis. And the body weight of mice in dasatinib group is markedly higher than that in control group. The bone erosion and the volume of bone loss were much alleviated in mice of dasatanib group evaluated using micro-CT. H&E stain, toluidine blue stain, safranin O stain and CD31 immunohistochemistry stain showed less severity of synovium hyperplasia, inflammation, bone loss, cartilage destruction, cartilage damage and pannus formation in dasatinib group than those in control group.These results indicated that dasatinib decreased the severity of inflammation and joint destruction in CIA mice. Dasatinib has therapy effect in CIA mice. Experiment 4 The effect of dasatinb on rheumatoid arthritis fibroblast-like synoviocytesFibroblast-like synoviocyte(FLS) plays an key role durning RA pathology and FLS has been conserder as an important target cell in RA treatment. The aim of this part was to investage the effect of dasatinb on rheumatoid arthritis fibroblast-like synoviocytes to study the mechanism of dasatinb treatment on RA.FLS were separated from synovial tissue of RA patients. 5-bromodeoxyuridine(BrdU) was used to evaluate cell proliferation. After treated with dasatinib for 24 h, proliferation of cells was marked inhibited. Erasion trace test and transwell migration assay were used to assess the migration of cells. In erasion trace test, cells treated with dasatinib migrated much slow than the control cells treated with DMSO. In tramnswell migration assay, the number of cells on the lower surface of the inserts in dasatinib treated wells was much less than that in control wells. Flow cytometry was used to evaluate the apoptosis of FLS. The percentage of apoptotic cells of FLS treated with dasatinib for 3 d and 5 d were significantly higher than control cells. The mRNA expression of MMP 13, VEGF, FGF and DKK1 of FLS were measured using RT-PCR. The mRNA expression of MMP 13, VEGF, FGF and DKK1 were significantly lower in FLS treated with dasatinib for 24 h than that in FLS treated with DMSO.These results indicated that dasatinib suppressed proliferation and migration, and induced apoptosis in RA FLS. And the expression of MMP 13, VEGF, FGF and DKK1 were inhibited by dasatinb.