中国骨质疏松杂志

mTORC1抑制自噬调控Wnt/β-catenin信号通路在激素环境下对MC3T3-E1成骨分化的影响

Effect of inhibition of mTORC1 on the autophagy regulation of Wnt/β-catenin signaling pathway in the osteogenic differentiation by MC3T3-E1 under hormonal environment

DOI：10.3969/j.issn.1006-7108.2022.01.004

中文关键词: mTORC1 Wnt/β-catenin信号通路自噬激素成骨分化

英文关键词:mTORC1 Wnt/β-catenin signaling pathway autophagy hormone osteogenic differentiation

基金项目:新疆维吾尔自治区卫生计生委青年医学科技人才专项（WJWY-201831）；国家自然科学基金（81774338，81904225）；新疆维吾尔自治区自然科学基金（2018D01C011，2019D01C013）

作者	单位
招文华1 任辉2 沈耿杨2 尚奇1 张志达1 余翔2 陈弘林2 张鹏1 陈桂锋1 余富勇1 汤凯1 江晓兵2 梁德2 张玉新3 唐军伟3*	1.广州中医药大学第一临床医学院，广东广州 510405 2.广州中医药大学第一附属医院，广东广州510405 3.喀什地区第一人民医院，新疆喀什 844000

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中文摘要:

目的探讨激素环境下，mTORC1是否通过抑制自噬调控Wnt/β-catenin信号通路介导前成骨细胞MC3T3-E1向成骨细胞分化。方法对前成骨细胞MC3T3-E1进行以下分组及处理：对照组、地塞米松（Dexamethasone，DEX）组、DEX+mTORC1激活剂组，其中DEX为1 μmol/L，mTORC1激活剂为10 μmol/L的MHY1485。应用ALP及ARS染色检测MC3T3-E1的成骨能力。Real-time PCR检测mTORC1组成中的关键基因 Raptor，自噬相关基因Beclin-1、Atg5，Wnt/β-catenin信号通路相关基因Wnt3、β-catenin及成骨相关基因Runx2的表达。Western-Blot检测Raptor、Beclin-1、Atg5、Wnt3、β-catenin、Runx2的蛋白表达水平。结果与对照组相比，DEX组的ALP及ARS染色较浅，成骨相关基因Runx2 mRNA及蛋白质表达下降，说明DEX抑制MC3T3-E1的成骨分化，且Raptor的mRNA及蛋白质水平下降，自噬相关基因Beclin-1、Atg5 mRNA及蛋白表达水平上升，说明DEX诱导MC3T3-E1自噬，而Wnt3、β-catenin mRNA及蛋白质表达下降。与DEX组相比，DEX+mTORC1激活剂组ALP及ARS染色较深，Raptor、Wnt3、β-catenin、Runx2 mRNA及蛋白质表达上调，而Beclin-1、Atg5 mRNA及蛋白质表达下降，表明应用mTORC1激活剂后抑制自噬并上调Wnt/β-catenin信号通路，从而逆转DEX对MC3T3-E1向成骨细胞分化的抑制作用。结论在激素环境下，MC3T3-E1成骨分化显著被抑制，mTORC1下调，自噬激活且Wnt/β-catenin信号通路被抑制，而应用mTORC1激活剂则能逆转该过程，表明mTORC1抑制自噬并调控Wnt/β-catenin信号通路介导激素环境下的MC3T3-E1成骨分化。

英文摘要:

Objective To investigate whether mTORC1 mediates the differentiation of pre-osteoblast MC3T3-E1 into osteoblasts by inhibiting autophagy to regulate the Wnt/β-catenin signaling pathway under hormone environment. Methods Pre-osteoblasts MC3T3-E1 were grouped and processed as follows: Control group, Dexamethasone (Dexamethasone, DEX) group, DEX+mTORC1 activator group, in which DEX was 1 μM and mTORC1 activator MHY1485 was 10 μM, respectively. ALP and ARS staining were used to detect the osteogenic differentiation of MC3T3-E1. The expressions of key gene Raptor in the composition of mTORC1, autophagy-related genes Beclin-1 and Atg5, Wnt/β-catenin signaling pathway related genes Wnt3 and β-catenin, and osteogenic related gene Runx2 were detected with real-time PCR. The protein expression levels of Raptor, Beclin-1, Atg5, Wnt3, β-catenin, and Runx2 were detected with Western blotting. Results Compared to those in the control group, ALP and ARS staining in the DEX group were lighter, and the osteogenic related gene Runx2 mRNA and protein expression decreased, indicating that DEX inhibited the osteogenic differentiation of MC3T3-E1. The mRNA and protein levels of Raptor decreased, and mRNA and protein expression levels of autophagy-related genes Beclin-1 and Atg5 increased, and mRNA and protein expressions of Wnt3 and β-catenin decreased, indicating that DEX induced MC3T3-E1 autophagy. Compared to those in the DEX group, ALP and ARS staining in the DEX+mTORC1 activator group were darker, mRNA and protein expressions of Raptor, Wnt3, β-catenin, and Runx2 were up-regulated, and mRNA and protein expressions of Beclin-1 and Atg5 decreased, indicating that the autophagy was inhibited and Wnt/β-catenin signaling pathway was up-regulated after the application of mTORC1 activator, thereby reversing the inhibitory effect of DEX on the differentiation of MC3T3-E1 into osteoblasts. Conclusion Under hormone environment, MC3T3-E1 osteogenic differentiation is significantly inhibited, mTORC1 is down-regulated, autophagy is activated, and Wnt/β-catenin signaling pathway is inhibited. The application of mTORC1 activator reverses this process, indicating that mTORC1 inhibits autophagy and regulates the Wnt/β-catenin signaling pathway to mediate the osteogenic differentiation of MC3T3-E1 under hormone environment.

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