不同冲击性负荷运动对小鼠Wnt信号通路及骨合成代谢的影响

Effects of different impact loading on Wnt signaling pathway and anabolic metabolism of bone in mice

目的:寻求有利于骨量积累和提高峰值骨量的运动方式,并探讨不同冲击性负荷对骨合成代谢的影响及其分子生物学机制。方法:C57BL/6小鼠48只,分成下坡跑台(DT)组、平坡跑台(T)组、游泳(S)组和对照(C)组,每组12只。DT组、T组和S组小鼠每组8只,分别进行下坡跑台训练、平坡跑台训练和游泳训练,C组小鼠不训练。训练8周后,取左后肢用原子吸收分光光度法测胫骨骨矿含量,取右后肢用荧光定量PCR法测Wnt信号通路相关蛋白的mRNA表达;每组4只小鼠,取间充质干细胞培养后诱导分化,用于成骨细胞基因表达、ALP染色和Von Kossa染色。结果:(1)8周运动后,与C组相比,DT组(P<0.01)和T组(P<0.05)小鼠胫骨无机盐中钙含量均显著提高,DT组和T组胫骨无机盐中磷的含量均显著提高(P<0.05),而S组与C组相比,钙和磷含量均无差异(P>0.05)。在3个运动组比较中,DT组钙和磷含量提高最多。(2)8周运动后,不同方式运动对Wnt信号通路中Fz、DVL、β-catenin、Rock、JNK的mRNA表达产生显著影响(P<0.05);(3)4组小鼠原代间充质干细胞分化后ALP和OCN基因表达差异显著,ALP活性及矿化结节也明显不同。结论:高冲击性负荷运动显著提高生长期小鼠胫骨骨盐中钙和磷元素含量,提高骨强度。不同冲击性负荷运动调节骨合成代谢的途径不同,高冲击性负荷运动激活Wnt/PCP信号通路,下调通路中效应蛋白ROCK和JNK的基因表达,促进骨合成代谢;低冲击性负荷运动运动激活经典Wnt信号通路,调节骨合成代谢。高冲击性负荷运动对ALP和OCN基因表达影响更显著,通过ALP活性和骨矿化调节骨的理化特性。

Objective:The purpose of this study is to find out the movement mode which is beneficial to bone mass accumulation and increase the peak bone mass,and to explore the influence of different impact loading on bone synthesis and metabolism and its molecular biological mechanism.Methods:48 C57BL/6 mice were divided into DT group,T group,S group and C group,12 in each group.In DT group,T group and S group,8 mice in each group were trained in downhill running platform,treadmill exercise and swimming,respectively.After 8 weeks of training,bone mineral content of tibial in mice was determined by atomic absorption method.The mRNA expression of wnt signaling pathway-related proteins was measured by fluorescence quantitative PCR,and the material was taken from the right hind limb.Four mice in each group were sacrificed,take mesenchymal stem cell culture and then induce differentiation,and which can be used to detect gene expression.It can also be used for ALP staining and Von kossa staining.Results:(1)The calcium content in DT group(P<0.01)and T group(P<0.05)was significantly increased after 8 weeks of exercise compared with the C group.The contents of phosphorus in tibial inorganic salt in DT and T groups were significantly increased(P<0.05).There was no difference in calcium and phosphorus content between the S group and the C group(P>0.05).Among the three exercise groups,the calcium and phosphorus contents of DT group increased most.(2)After 8 weeks of exercise,different modes of exercise had a significant effect on the mRNA expression of Fz,DVL、β-Catenin、Rock、JNK genes in Wnt signaling pathway(P<0.05);(3)It was significantly different that the expression of ALP and OCN genes after differentiation of primary mesenchymal stem cells in four groups of mice.Moreover,the activity of ALP and the mineralized nodules were also significantly different.Conclusion:High impact loading exercise significantly increased the content of calcium and phosphorus in tibial bone and increased bone strength.The pathways of regulating bone synthesis metabolism by different impact loading exercise are different.High impact loading activates Wnt/PCP signaling pathway and then down-regulates gene expression of effector protein ROCK and JNK,and promotes bone synthesis metabolism.Low impact loading exercise activates classical Wnt signaling pathway and regulates bone synthesis metabolism.The effect of high impact loading exercise on ALP and OCN gene expression was more significant,and the physicochemical properties of bone were regulated by ALP activity and bone mineralization.