(*)AG490 could suppress bone marrow mesenchymal stem cells migration, mineralization and bone defect healing via inhibiting Jak2-STAT3 pathway

Abstract

Objective: To study the effect of Jak2-STAT3 pathway on cell proliferation, migration, mineralization and bone defect healing via simulating Jak2-STAT3 pathway inhibitor AG490 to bone marrow mesenchymal stem cells (BMSC) and bone defect mice models. Methods: The effect of AG490 on BMSC proliferation was measured by MTT (methyl thiazolyl tetrazolium) assay. Regulation of AG490 on BMSC migration was tested by scratch assay and transwell assay. The BMSC migration related gene, matrix metalloproteinase (MMP)-7, MMP-9 and CXC subfamily receptor 4 (CXCR4), regulated by AG490 was studied by real-time PCR. Western blotting was adopted to analyze the regulation of Jak2-STAT3 phosphorylation through the simulation of AG490. The alizarin red staining and alkaline phosphatase (ALP) activity assay were performed to measure the effect of AG490 on BMSC mineralization and osteogenic differentiation. Mice femur bone defect models were built to analyzed the effect of AG490 on bone remodeling. Results: AG490 significantly suppressed the migration rate of BMSC at 1 d and 2 d in the experiment group [(12.42±7.50) %, (41.8±2.6)%] compared with the control group [(55.5±9.9)%, (86.9±8.7)%] in scratch assay (P=0.000, P=0.000), the number of migrated BMSC in the experiment group (22.8± 5.9) was significantly suppressed compared with the control group (58.3±6.6) in Transwell assay (P=0.000). The expression of MMP-7, MMP-9 and CXCR4 were significantly downregulated in experiment group [(0.5± 0.1), (0.1±0.1) and (0.35±0.07)] compared with the control group [(1.1±0.1), (1.06±0.33), (1.08±0.13)] (P= 0.0003, P=0.000 and P=0.000). Also, the phosphorylation of Jak2-STAT3 was downregulated by AG490 in western blotting. After BMSCs were osteogenic induced for 14 days, the formation of mineralized nodule and the ALP activity of BMSC is significantly suppressed in experiment group (8.0±2.1) compared with the control group (35.7 ± 1.8) (P=0.0005). AG490 suppressed the bone defects healing,the expression level of phosphorylated Jak2 and phosphorylated STAT3, and the number of alkaline phosphatase positive cell at defect area in vivo is lower in experiment group than the control group. AG490 suppressed the relatively bone density at the defect area significantly (P=0.0004) at 5(th) week after the surgery. Conclusions: AG490 could suppress proliferation, migration and mineralization to of BMSCs regulate bone defect healing via inhibiting Jak2-STAT3 pathway.