高浓度葡萄糖通过p38MAPK-TXNIP/TRX-ROS通路抑制MC3T3-E1细胞成骨分化

目的：研究高浓度葡萄糖抑制MC3T3-E1细胞成骨分化的机理。方法：建立MC3T3-E1细胞成骨分化诱导体系,观察不同浓度葡萄糖（5.5mM和22mM）对MC3T3-E1细胞成骨分化的影响;用不同浓度的p38 MAPK抑制剂Fr167653（0.1μM、1.0μM和10μM）进行药物干预,观察MC3T3-E1细胞在22mM葡萄糖浓度下成骨分化的变化情况。通过钙含量检测、Real time PCR检测相关分化的变化;用Western Blot方法检测MC3T3-E1细胞分化过程中p38 MAPK磷酸化状态、TXNIP表达水平的变化;使用胰岛素二硫键还原法检测细胞内TRX活性水平;使用活性氧检测试剂盒检测细胞内自由氧生成水平。结果：体外诱导条件下,高浓度（22mM）葡萄糖通过升高p38 MAPK磷酸化水平,上调TXNIP表达水平,同时降低TRX活性,使细胞内自由氧生成增加,抑制MC3T3-E1细胞的成骨分化;Fr167653通过抑制p38 MAPK磷酸化,下调TXNIP表达同时升高TRX活性,抑制细胞内自由氧生成,解除高浓度葡萄糖对细胞成骨分化的抑制作用。结论：高浓度葡萄糖通过p38 MAPK-TXNIP/TRX-ROS信号通路抑制MC3T3-E1细胞成骨分化。

High Glucose Suppresses Osteoblastic Differentiation of MC3T3-E1 Cells Via p38 MAPK-TXNIP/TRX-ROS Signal Pathway

Objective：Our study focus on the role of p38 MAPK and ROS in osteoblast differentiation of MC3T3-E1 cell under high concentration of glucose in vitro.Methods：MC3T3-E1 cells were divided into four groups：negtive control group（NC,MC3T3-E1 cells cultured in α-MEM medium）,normal glucose and calcification induced control group（NIC,MC3T3-E1 cells cultured in calcification medium with 5.5mM glucose）,high glucose group（HG,MC3T3-E1 cells cultured in calcification medium with 22mM glucose） and Fr167653 intervention group（Fr～,MC3T3-E1 cells cultured in calcification medium with 22mM glucose,in the presence of p38 MAPK inhibitor,Fr167653）.The calcium deposition,alkaline phosphatase activity and the mRNA levels of osteoblast markers were tested to evaluate the osteoblast differentiation potency of MC3T3-E1 cells.P38 MAPK phosphorylation level and TXNIP protein expression level were detected by Western blot.Intracellular TRX activity was detected using the insulin disulfide reduction assay method.Intracellular ROS generation level was detected by Reactive Oxygen Species Assay Kit.Results：We showed in this report that 22mM glucose suppressed the osteoblast differentiation process of MC3T3-E1 cells,as indicated by the reduced calcium deposition,ALP activity and osteoblast markers.In the presence of Fr167653,the specific inhibitor of p38 MAPK,could significantly reverse the suppression effect of high glucose and the decrease of above-mentioned osteoblast differentiation markers.Furthermore,high glucose increased p38 MAPK phosphorylation level.The TXNIP expression level was up-regulated and the TRX activity was decreased,which further increased the intracellular ROS generation.Fr167653,which blocked the p38 MAPK phosphorylation,down-regulated the TXNIP expression,enhanced the TRX activity and reduced the ROS generation.Conclusions：These data suggest that high concentration of glucose had a deleterious effect on osteoblast differentiation of MC3T3-E1 cells probably via p38 MAPK-TXNIP/TRX-ROS signal path