高迁移率族蛋白B1调控Janus激酶2/信号转导与转录激活子3信号促进骨髓间充质干细胞成骨分化

High mobility group protein 1 promotes bone marrow mesenchymal stem cells osteogenesis differentiation and mineralization by activating the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway

目的:探讨高迁移率族蛋白B1(HMBG1)对骨髓间充质干细胞(BMSCs)成骨分化的影响,分析可能的基础机制。方法:根据成骨培养基含HMGB1浓度的不同,分组处理BMSCs,使用蛋白质印迹法(Western blot)、QPCR和碱性磷酸酶(ALP)染色、茜素红染色等方法检测其信号激活、成骨标志蛋白表达和成骨分化矿化情况。使用含Janus激酶2/信号转导与转录激活子3(JAK2/STAT3)通路抑制剂和含HMGB1的成骨培养基处理BMSCs,通过Western blot验证JAK2/STAT3通路与HMGB1成骨分化能力的相关性。选用C57BL/6小鼠构建股骨骨折模型,使用HMGB1腹腔注射骨折模型小鼠,μCT扫描统计两组BV/TV值,以上实验使用t检验用于两组间比较。结果:在BMSCs中HMGB1能刺激关键转录因子JAK2和STAT3蛋白磷酸化,对照组成骨标志蛋白与基因的表达水平低于50 ng/ml HMGB1组(1.035±0.280比11.560±0.317,t=49.420,P<0.001;1.000±0.078比12.910±2.115,t=11.280,P<0.001),ALP染色及茜素红染色结果证明,HMGB1促进了BMSCs的成骨分化与矿化。使用JAK2/STAT3通路抑制剂后,HMGB1组成骨标志物的表达高于Stattic组和AG490组(0.670±0.036比0.171±0.031,t=18.140,P<0.001;0.670±0.036比0.138±0.019,t=22.630,P<0.001。1.025±0.038比0.482±0.046,t=15.770,P<0.001,1.025±0.038比0.539±0.038,t=6.124,P<0.01),骨折模型小鼠对照组的BV/TV值低于HMGB1组(0.442±0.044比0.605±0.015,P<0.05)也验证了其促骨折愈合的作用。结论:HMGB1在体外可以通过JAK2/STAT3通路促进BMSCs的成骨分化,由此推测HMGB1可能是在PMOP的骨循环中起复杂作用的重要靶点。

Objective Investigating the effects of high mobility group box 1 protein(HMGB1)on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and analyzing the potential underlying mechanisms.Methods According to the different concentrations of HMGB1 in osteogenic culture medium,BMSCs were grouped and treated.Western blotting,QPCR,alkaline phosphatase(ALP)staining,and Alizarin Red staining were used to detect the activation of key signaling proteins,expression of osteogenic marker proteins,and osteogenic mineralization.BMSCs were treated with osteogenic media containing janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)pathway inhibitors and HMGB1,and the correlation between JAK2/STAT3 pathway and HMGB1 osteogenic differentiation ability was verified by Western blotting.C57BL/6 mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co.,Ltd.to establish femoral fracture models.HMGB1 was intraperitoneally injected into the fracture models,and BV/TV values of the two groups were calculated usingμCT scanning,the above experiment used the t-test for comparison between the two groups.Results HMGB1 in BMSCs can stimulate the phosphorylation of key transcription factors JAK2 and STAT3.The expression levels of osteogenic marker proteins and genes in the control group were lower than those in the group treated with 50 ng/ml HMGB1(1.035±0.280 vs.11.560±0.317,t=49.420,P<0.001;1.000±0.078 vs.12.910±2.115,t=11.280,P<0.001).ALP staining and Alizarin Red staining results confirmed that HMGB1 promoted osteogenic differentiation and mineralization of BMSCs.After the use of JAK2/STAT3 pathway inhibitors,the expression of osteogenic markers in the HMGB1 group was higher than that in the Stattic group and the AG490 group(0.670±0.036 vs.0.171±0.031,t=18.140,P<0.001;0.670±0.036 vs.0.138±0.019,t=22.630,P<0.001,1.025±0.038 vs.0.482±0.046,t=15.770,P<0.001,1.025±0.038 vs.0.539±0.038,t=6.124,P<0.01).The BV/TV value in the control group of the femoral fracture mouse model was lower than that in the HMGB1 group(0.442±0.044 vs.0.605±0.015,P<0.05),which also confirmed its role in promoting fracture healing.Conclusion HMGB1 can promote osteogenic differentiation of BMSCs through the JAK2/STAT3 pathway in vitro,suggesting that it may be an important target playing a complex role in bone metabolism in postmenopausal osteoporosis(PMOP).