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Title: [(*)AG490 could suppress bone marrow mesenchymal stem cells migration, mineralization and bone defect healing via inhibiting Jak2-STAT3 pathway].
Author: Yu X, Wan QL, Li Z, Li ZB.
Journal: Zhonghua Kou Qiang Yi Xue Za Zhi; 2018 May 09; 53(5):293-300. PubMed ID: 29972985.
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. 目的： 探讨Jak2-STAT3信号通路抑制剂AG490对骨髓间充质干细胞（bone marrow mesenchymal stem cell，BMSC）迁移、矿化及骨缺损愈合的调控机制。 方法： 应用甲基噻唑基四唑（methyl-thiazolyl tetrazolium，MTT）实验、细胞划痕实验及Transwell实验检测AG490对小鼠来源的原代BMSC增殖和迁移能力的调控。荧光定量PCR和蛋白质印迹法研究AG490调控BMSC增殖和迁移的相关基因及通路机制。通过茜素红染色及碱性磷酸酶活性实验探究AG490对BMSC矿化及成骨向分化能力的影响。构建小鼠股骨缺损模型，研究AG490对骨缺损愈合的影响及机制。 结果： 细胞划痕实验中第1、2天实验组[（12.42±7.50）%，（41.8±2.6）%］的细胞迁移率均显著小于对照组[（55.5±9.9）%，（86.9±8.7）%]（P=0.006，P=0.005），Transwell实验中实验组[（22.8±5.9）个]中单位视野细胞迁移数显著少于对照组[（58.3±6.6）个]（P=0.000）。实验组MMP-7 （0.5±0.1）、MMP-9(0.1±0.1）及CXCR4 （0.35± 0.07）的相对基因表达量均显著少于对照组[（1.1±0.1），（1.06±0.33）, （1.08±0.13）]（P=0.0000，P= 0.0003，P=0.0000 ）。蛋白质印迹法结果中实验组磷酸化Jak2、磷酸化STAT3的蛋白表达在AG490的作用下与对照组相比受到明显抑制。BMSC矿化诱导14 d后，实验组的碱性磷酸酶相对活性（8.0± 2.1）较对照组（35.7±1.8）受到显著抑制（P=0.0005），实验组较对照组矿化结节小且数量较少。体内实验中，术后第5周时，实验组的相对骨密度显著低于对照组（P=0.0004）。HE染色及免疫组化染色可见实验组中骨缺损断端处磷酸化Jak2、磷酸化STAT3和碱性磷酸酶阳性细胞较对照组稍多。 结论： AG490可通过抑制Jak2-STAT3信号通路抑制BMSC的增殖、迁移和矿化，从而调控骨缺损愈合。.

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