中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (28): 4498-4504.doi: 10.12307/2024.459

• 骨组织构建 bone tissue construction • 上一篇    下一篇

圣草素促进成骨细胞自噬减轻糖皮质激素诱导的细胞凋亡

马运锋1,韩小飞2   

  1. 河南中医药大学第二附属医院(河南省中医院),1骨病一科,2风湿病科,河南省郑州市  450002
  • 收稿日期:2023-05-21 接受日期:2023-08-16 出版日期:2024-10-08 发布日期:2023-11-27
  • 通讯作者: 马运锋,硕士,医师,河南中医药大学第二附属医院(河南省中医院)骨病一科,河南省郑州市 450002
  • 作者简介:马运锋,男,1983年生,河南省长葛市人,汉族,硕士,医师,主要从事中医药治疗骨关节疾病方面的研究。
  • 基金资助:
    2019年河南省卫健委中医药科学研究专项课题(2019ZY2149),项目负责人:韩小飞;2020年度河南省卫健委中医药科学研究专项课题(20-21ZY3013),项目负责人:韩小飞

Eriodictyol accelerates glucocorticoid-induced apoptosis by promoting osteoblast autophagy

Ma Yunfeng1, Han Xiaofei2   

  1. 1Department of Osteopathy, 2Department of Rheumatology, Second Affiliated Hospital of Henan University of Chinese Medicine (Henan Province Hospital of Traditional Chinese Medicine), Zhengzhou 450002, Henan Province, China
  • Received:2023-05-21 Accepted:2023-08-16 Online:2024-10-08 Published:2023-11-27
  • Contact: Ma Yunfeng, Department of Osteopathy, Second Affiliated Hospital of Henan University of Chinese Medicine (Henan Province Hospital of Traditional Chinese Medicine), Zhengzhou 450002, Henan Province, China
  • About author:Ma Yunfeng, Master, Physician, Department of Osteopathy, Second Affiliated Hospital of Henan University of Chinese Medicine (Henan Province Hospital of Traditional Chinese Medicine), Zhengzhou 450002, Henan Province, China
  • Supported by:
    2019 Special Project for Scientific Research on Traditional Chinese Medicine of Henan Provincial Health Commission, No. 2019ZY2149 (to HXF); 2020 Special Project for Scientific Research on Traditional Chinese Medicine of Henan Provincial Health Commission, No. 20-21ZY3013 (to HXF)

摘要:


文题释义:

糖皮质激素诱导的骨质疏松症:是继发性骨质疏松症最常见的形式,主要特征是快速刺激骨吸收,持久抑制成骨细胞。
自噬:是真核生物中的一个基本细胞过程,涉及胞质成分在双膜结合自噬体内的螯合,随后与内溶酶体囊泡融合,导致螯合底物的降解和回收。自噬是一种高度保守的细胞内降解过程,用于消除真核生物中受损的细胞器、蛋白质聚集体和入侵病原体。


背景:糖皮质激素诱导的骨质疏松症是系统性糖皮质激素治疗的常见并发症,主要表现为对成骨细胞的抑制作用。圣草素可抑制破骨细胞分化和卵巢切除术引起的骨质疏松,然而,圣草素是否调节糖皮质激素诱导的成骨细胞凋亡尚不清楚。

目的:探究圣草素是否在地塞米松诱导的成骨细胞凋亡中发挥作用,及其潜在的作用机制。
方法:采用不同浓度(0,0.5,1,2.5,5,10 μmol/L)的圣草素或自噬抑制剂3-甲基腺嘌呤(5 μmol/L)处理地塞米松预处理的成骨细胞MC3T3‐E1,然后用血红素加氧酶1的过表达载体(pcDNA-HMOX1)和空载体(pcDNA vector)转染MC3T3‐E1细胞。分别采用CCK-8和流式细胞术检测细胞活力和凋亡;采用ELISA检测半胱氨酸天冬氨酸蛋白酶3活性;采用Western blotting检测自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ、p62、Atg5和Atg12表达,凋亡相关蛋白Bax和Bcl-2的表达,以及AMP蛋白激活激酶(AMPK)和p-AMPK蛋白表达。

结果与结论:①低浓度的圣草素对MC3T3-E1细胞无毒性,且促进细胞增殖,并以剂量依赖性的方式促进自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ、p62、Atg5和Atg12表达,降低半胱氨酸天冬氨酸蛋白酶3活性,抑制Bax蛋白表达,促进Bcl-2蛋白表达,减少地塞米松诱导的MC3T3-E1细胞凋亡;②此外圣草素可显著促进成骨细胞中血红素加氧酶1的表达;过表达血红素加氧酶1促进AMPK磷酸化,激活自噬,抑制地塞米松诱导的成骨细胞凋亡;③而3-甲基腺嘌呤处理抵消了血红素加氧酶1过表达对MC3T3-E1细胞的影响;④提示低浓度圣草素对成骨细胞无毒性,可以通过上调血红素加氧酶1的表达激活AMPK信号通路,从而促进自噬,抑制地塞米松诱导的成骨细胞凋亡;圣草素具有治疗糖皮质激素诱导骨质疏松症的巨大潜力。

https://orcid.org/0009-0006-6085-4931(马运锋)

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程

关键词: 圣草素, 地塞米松, 血红素加氧酶1, MC3T3‐E1细胞, AMPK通路

Abstract: BACKGROUND: Glucocorticoid-induced osteoporosis is a common complication of systemic glucocorticoid therapy, which is mainly characterized by its inhibitory effect on osteoblasts. Eriodictyol inhibits osteoclast differentiation and osteoporosis-induced by ovariectomy. However, it is unclear whether eriodictyol regulates glucocorticoid-induced osteoblasts. 
OBJECTIVE: To explore whether eriodictyol plays a role in glucocorticoid-induced osteoblast apoptosis and its potential regulatory mechanisms. 
METHODS: Dexamethasone-pretreated osteoblasts MC3T3‐E1 were treated with the different concentrations (0, 0.5, 1, 2.5, 5, 10 μmol/L) of eriodictyol or 5 μmol/L 3-methyladenine, an autophagy inhibitor, and then transfected with heme oxygenase 1 overexpression vector (pcDNA-HMOX1) and empty vector (pcDNA vector). Cell proliferation and apoptosis were assessed by using cell counting kit-8 assay and flow cytometry, respectively. The activity of caspase-3 was detected with ELISA. Western blot assay was used to detect the protein expression of autophagy-related proteins LC3-II/LC3-I, p62, Atg5 and Atg12, the expression of apoptotic related proteins Bax and Bcl-2, as well as the protein expression of AMPK and p-AMPK. 
RESULTS AND CONCLUSION: Low concentrations of eriodictyol were non-toxic to MC3T3-E1 cells and promoted cell proliferation, as well as increased the expression of autophagy related proteins LC3-II/LC3-I, p62, Atg5 and Atg12, decreased caspase-3 enzyme activity, inhibited Bax protein expression, promoted Bcl-2 protein expression and reduced dexamethasone-induced apoptosis in MC3T3-E1 cells in a dose-dependent manner. Moreover, eriodictyol significantly promoted heme oxygenase 1 expression in osteoblasts, whereas overexpression of heme oxygenase 1 promoted AMPK phosphorylation, activated autophagy, and inhibited dexamethasone-induced osteoblast apoptosis. While 3-methyladenine treatment counteracted the effects of heme oxygenase 1 overexpression on MC3T3-E1 cells. To conclude, low concentration of Eriodictyol is non-toxic to osteoblasts and activates AMPK signaling pathway by upregulating the expression of heme oxygenase 1, thereby promoting autophagy and inhibiting dexamethasone-induced osteoblast apoptosis. Eriodictyol has great potential for the treatment of glucocorticoid-induced osteoporosis. 

Key words: eriodictyol, dexamethasone, heme oxygenase 1, MC3T3-E1 cell, the AMPK pathway

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