基于糖尿病骨质疏松细胞模型探究ELAVL1对破骨细胞铁死亡的调控作用

Investigating roles of ELAVL1 in regulating ferroptosis of osteoclasts based on cell models of diabetic osteoporosis

摘要:

目的 探究胚胎致死性异常视觉样蛋白1（ELAVL1）是否通过破骨细胞死亡调控糖尿病骨质疏松症（DOP）。方法 选取2021年1月—2023年2月长沙市中心医院就诊的50例DOP患者和50例糖尿病但不伴随骨质疏松的患者作为研究对象，分别作为DOP组和对照组。比较两组患者血清ELAVL1、GPX4、Nrf2、ACSL4水平差异，分析ELAVL1与铁死亡相关基因（GPX4、Nrf2、ACSL4）的相关性。采用50 ng/mL RANKL孵育小鼠骨髓源性巨噬细胞RAW264.7以诱导破骨分化。对DOP组进行亚分组，以患者血清ELAVL1含量的平均值（14.94 ng/mL）为标准，分为ELAVL1低表达组（血清ELAVL1<14.94 ng/mL，28例）及ELAVL1高表达组（血清ELAVL1> 14.94 ng/mL，22例）。将破骨细胞分为对照组、高糖组、高糖+sh-NC组、高糖+sh-ELAVL1组，比较各组ELAVL1、GPX4、核因子红细胞系2相关因子2（Nrf2）、酰基辅酶A合成酶长链家族成员4（ACSL4）、活性氧、丙二醛（MDA）、谷胱甘肽（GSH）、总铁含量、铁离子（Fe²⁺）表达量、线粒体膜电位。结果 ELAVL1高表达组与ELAVL1低表达组性别构成、年龄、糖尿病病程、BMI比较，差异均无统计学意义（P >0.05）。ELAVL1高表达组T-score值低于ELAVL1低表达组（P <0.05），血清β-CTX、血清PINP高于ELAVL1低表达组（P <0.05）。DOP组血清GPX4、Nrf2比对照组低（P <0.05），ACSL4比对照组高（P <0.05）。经Pearson相关性分析结果表明，DOP患者血清ELAVL1表达与ACSL4呈正相关（r =0.689，P <0.05），与GPX4、Nrf2呈负相关（r =-0.312和-0.447，均P <0.05），血清ELAVL1与ACSL4呈正相关（r =0.689，P <0.05），血清ELAVL1与GPX4呈负相关（r =-0.559，P <0.05），血清ELAVL1与Nrf2呈负相关（r =-0.669，P <0.05）。高糖组GPX4、Nrf2 mRNA较对照组降低（P <0.05），ELAVL1、ACSL3 mRNA较对照组升高（P <0.05），高糖+sh-ELAVL1组GPX4、Nrf2 mRNA较高糖+sh-NC组升高（P <0.05），ELAVL1、ACSL3 mRNA较高糖+sh-NC组降低（P <0.05）。高糖组GPX4、Nrf2蛋白较对照组降低，ELAVL1、ACSL4蛋白较对照组升高（P <0.05），高糖+sh-ELAVL1组GPX4、Nrf2蛋白较高糖+sh-NC组升高（P <0.05），ELAVL1、ACSL4蛋白较高糖+sh-NC组降低（P <0.05）。高糖组总铁含量及Fe²⁺表达量较对照组升高（P <0.05），高糖+sh-ELAVL1组较高糖+sh-NC组降低（P <0.05）。高糖组MDA较对照组高（P <0.05），GSH较对照组低（P <0.05），高糖+sh-ELAVL1组MDA较高糖+sh-NC组低，GSH较高糖+sh-NC组高（P <0.05）。结论 高糖条件诱导破骨细胞铁死亡，ELAVL1与DOP铁死亡有关，敲低ELAVL1可抑制高糖诱导的破骨细胞铁死亡。

Abstract:

Objective To explore whether embryonic lethal abnormal visual-like protein 1 (ELAVL1) regulates osteocyte death to affect diabetic osteoporosis (DOP).Methods DOP patients (n = 50) and diabetes mellitus patients without osteoporosis (n = 50) hospitalized in Changsha Central Hospital from January 2021 to February 2023 were included as the DOP group and the control group, respectively. The serum levels of ELAVL1, GPX4, Nrf2 and ACSL4 were compared between the two groups of patients, and the correlations between ELAVL1 and ferroptosis-related genes (GPX4, Nrf2, ACSL4) were analyzed. The macrophage cell line RAW264.7 derived from bone marrow of mice were incubated with 50 ng/mL of RANKL to induce their differentiation into osteoclasts. The DOP group was subcategorized based on the average serum ELAVL1 level (14.94 ng/mL) of patients, dividing them into the low ELAVL1 expression group (serum ELAVL1 < 14.94 ng/mL, 28 cases) and the high ELAVL1 expression group (serum ELAVL1 > 14.94 ng/mL, 22 cases). Osteoclasts were divided into the control group, the high glucose group, the high glucose + sh-NC group, and the high glucose + sh-ELAVL1 group, and levels of ELAVL1, GPX4, Nrf2, ACSL4, ROS, MDA, GSH, total iron, and Fe²⁺ as well as the mitochondrial membrane potential were compared among the groups.Results There was no difference in the sex composition, the age, the course of diabetes mellitus, or BMI between the low ELAVL1 expression group and the high ELAVL1 expression group (P > 0.05). Compared with the low ELAVL1 expression group, the T-score was lower (P < 0.05) and the serum levels of β-CTX and PINP were higher in the high ELAVL1 expression group (P < 0.05). Compared with the control group, the serum levels of GPX4 and Nrf2 were lower (P < 0.05) and the serum level of ACSL4 was higher in the DOP group (P < 0.05). As demonstrated by Pearson correlation analysis, the serum level of ELAVL1 was positively correlated with that of ACSL4 (r = 0.689, P < 0.05) but negatively correlated with the serum levels of GPX4 and Nrf2 (r = -0.312 and -0.447, both P < 0.05) in DOP patients. The serum level of ELAVL1 was positively correlated with that of ACSL4 (r = 0.689, P < 0.05) but negatively correlated with that of GPX4 (r = -0.559, P < 0.05) and that of Nrf2 (r = -0.669, P < 0.05). The mRNA expressions of GPX4 and Nrf2 in the high glucose group were lower than those in the control group (P < 0.05), while the mRNA expressions of ELAVL1 and ACSL4 in the high glucose group were higher than those in the control group (P < 0.05). The mRNA expressions of GPX4 and Nrf2 in the high glucose + sh-ELAVL1 group were higher than those in the high glucose + sh-NC group (P < 0.05), while the mRNA expressions of ELAVL1 and ACSL4 in the high glucose + sh-ELAVL1 group were lower than those in the high glucose + sh-NC group (P < 0.05). The protein expressions of GPX4 and Nrf2 in the high glucose group were lower than those in the control group (P < 0.05), while the protein expressions of ELAVL1 and ACSL4 in the high glucose group were higher than those in the control group (P < 0.05). The protein expressions of GPX4 and Nrf2 in the high glucose + sh-ELAVL1 group were higher than those in the high glucose + sh-NC group (P < 0.05), while the protein expressions of ELAVL1 and ACSL4 in the high glucose + sh-ELAVL1 group were lower than those in the high glucose + sh-NC group (P < 0.05). The levels of total iron and Fe²⁺ in the high glucose group were higher than those in the control group (P < 0.05), whereas those in the high glucose + sh-ELAVL1 group were lower than those in the high glucose + sh-NC group (P < 0.05). Compared with the control group, the level of MDA was higher (P < 0.05) but the level of GSH was lower (P < 0.05) in the high glucose group. Compared with the high glucose + sh-NC group, the level of MDA was lower (P < 0.05) but the level of GSH was higher (P < 0.05) in the high glucose + sh-ELAVL1 group (P < 0.05).Conclusions High glucose condition triggers ferroptosis of osteoclasts. ELAVL1 is significantly associated with the ferroptosis in DOP, and knockdown of ELAVL1 inhibits the ferroptosis of osteoclasts induced by high glucose.