高糖损伤对成骨细胞铁死亡及纤毛改变诱导骨质疏松的机制研究

Mechanism of osteoporosis induced by high glucose injury through osteoblast ferroptosis and ciliary changes

摘要

目的 探究高糖损伤诱导成骨细胞死亡的骨质疏松机制。方法 研究对象选取SD大鼠24只及成骨细胞（MC3T3-E1）。大鼠随机分为对照组及研究组各12只，对照组给予安慰剂，研究组建立糖尿病骨质疏松模型；成骨细胞随机分为正常组（12个样本）、高糖组（6个样本）、高糖+METTL3抑制组（6个样本）。检测两组大鼠骨矿物质密度（BMC），骨矿物质含量（BMD），血清碱性磷酸酶（ALP）、骨钙素（OCN）、铁蛋白水平；观察大鼠骨组织中ALP、Runx2、SLC7A11、GPX4蛋白及mRNA表达。观察各组细胞ALP、Runx2、SLC7A11、GPX4、METTL3、ASK1蛋白表达及细胞纤毛状态。结果 动物实验结果显示，研究组BMC、BMD、血清OCN、ALP水平低于对照组，血清铁蛋白水平高于对照组（P<0.05）；免疫组化结果显示，SLC7A11和GPX4蛋白在对照组中高表达，而在研究组中低表达；蛋白印迹分析和qRT-PCR结果显示，研究组ALP、Runx2、SLC7A11、GPX4蛋白及mRNA相对表达低于对照组（P<0.05）。细胞实验结果显示，高糖组ALP、Runx2、SLC7A11、GPX4蛋白相对表达显著低于正常组、高糖+METTL3抑制组（P<0.05），正常组和高糖+METTL3抑制组比较差异无统计学意义（P>0.05）；高糖组METTL3、ASK1蛋白相对表达显著高于正常组、高糖+METTL3抑制组（P<0.05），正常组和高糖+METTL3抑制组比较差异无统计学意义（P>0.05）。免疫荧光检测结果显示，正常组细胞中细胞纤毛旺盛，高糖组表现为纤毛数量减少及长度降低，高糖+METTL3抑制逆转了高糖引起的纤毛数量及长度变化。结论 高糖损伤诱导的成骨细胞存在铁死亡及细胞纤毛减少，高糖损伤引起的骨质疏松机制可能与METTL3/ASK1细胞通路引起的铁死亡及纤毛改变有关。

Abstract

Objective To explore the mechanism of high glucose-induced osteoblast cell death and osteoporosis. Methods Twenty-four SD rats and osteoblasts (MC3T3-E1) were included in the study. The rats were randomly divided into control group (n=12) and study group (n=12). The control group was given placebo, and a diabetic osteoporosis model was established in the study group. Osteoblasts were randomly divided into normal group (12 cell samples), high glucose group (6 cell samples), and high glucose + METTL3 inhibition group (6 cell samples). BMC, BMD, the levels of ALP, OCN, and ferritin in serum were measured; the relative protein and mRNA expressions of ALP, Runx2, SLC7A11, and GPX4 in bone tissue of rats were observed. The protein expressions of ALP, Runx2, SLC7A11, GPX4, METTL3, ASK1, and the condition of cell cilia were observed in the three groups of cells. Results The results of animal experiments showed that the study group had significantly lower BMC, BMD, and serum OCN and ALP levels and a significantly higher serum ferritin level than the control group (P<0.05). Immunohistochemical showed that SLC7A11 and GPX4 were highly expressed in the control groups but were significantly decreased in the study group. Western blotting and qRT-PCR showed that the relative expressions of ALP, Runx2, SLC7A11, and GPX4 protein and mRNA in the study group were lower than those in the control group (P<0.05). Cell experiments showed that the relative expressions of ALP, Runx2, SLC7A11, and GPX4 proteins in the high glucose group were significantly lower than those in the normal group and the high glucose + METTL3 inhibition group (P<0.05). There was no significant difference between normal group and high glucose + METTL3 inhibition group (P>0.05). The relative expressions of METTL3 and ASK1 proteins in the high glucose group were significantly higher than those in the normal group and the high glucose + METTL3 inhibition group (P<0.05). There was no significant difference between normal group and high glucose + METTL3 inhibition group (P>0.05). Immunofluorescence results showed that the cell cilia were abundant in the normal group, the number and length of cilia in the high glucose group were decreased, and high glucose + METTL3 inhibition reversed the changes in the number and length of cilia caused by high glucose. Conclusion High glucose induces ferroptosis and cilia reduction in osteoblasts, and the mechanism of osteoporosis induced by high glucose may be related to ferroptotic cell death and ciliary changes caused by the METTL3/ASK1 signaling pathway.

关键词

高糖损伤 / 成骨细胞 / 骨质疏松 / 作用机制 / METTL3 / ASK1

Key words

High glucose injury / Osteoblast / Osteoporosis / Mechanism of action / METTL3 / ASK1

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基金资助

云南省科技计划项目(202101BA070001-225)