Astragaloside IV alleviates oxidative stress injury and promotes osteogenesis in MC3T3-E1 cells

doi:10.12307/2025.706

Abstract

Abstract: BACKGROUND: Oxidative stress is one of the main causes of osteoporosis, and reducing the level of oxidative stress with increasing antioxidant defense is an important research direction for the treatment of osteoporosis. Studies have confirmed that astragaloside IV has anti-osteoporosis effects, but its mechanism of action is not clear.
OBJECTIVE: To investigate the osteogenic effect of astragaloside IV in MC3T3-E1 cells under oxidative stress conditions.
METHODS: MC3T3-E1 cells were randomly divided into four groups: the control group was cultured in a complete medium; the model group was cultured in the complete medium containing hydrogen peroxide which was replaced with another complete medium after 24 hours of intervention; the astragaloside IV group was cultured with the complete medium containing hydrogen peroxide and astragaloside IV which was replaced with another complete medium containing astragaloside IV after 24 hours of intervention; and the inhibitor group was cultured in the complete medium containing hydrogen peroxide, astragaloside IV, and extracellular signal-regulated kinases (ERK) inhibitor which was replaced with complete medium containing hydrogen peroxide, astragaloside IV, and ERK inhibitor after 24 hours of intervention. After 48 hours of intervention with hydrogen peroxide, malondialdehyde content was detected to evaluate the mitigating effect of astragaloside IV on the oxidative stress in MC3T3-E1 cells. Osteogenic induction was performed after 48 hours of intervention with hydrogen peroxide, and the osteogenic and mineralizing ability of MC3T3-E1 cells was verified by alkaline phosphatase staining and alizarin red staining; the expression of osteogenesis-related genes was detected by RT-qPCR; and the expression of osteogenesis-related proteins and ERK/AMP-activated protein kinase (AMPK) signaling pathway proteins was detected by western blot.
RESULTS AND CONCLUSION: The intracellular alkaline phosphatase content and mineralized nodule formation were less in the model group than in the control group (P < 0.05), and were more in the astragaloside IV group than in the model group (P < 0.05). Compared with the control group, intracellular malondialdehyde content increased in the model group (P < 0.05), mRNA and protein expression of osteocalcin, RUNX2, and type I collagen decreased (P < 0.05), and AMPK mRNA and p-AMPK protein expressions were elevated (P < 0.05); compared with the model group, intracellular malondialdehyde content in the astragaloside IV group decreased (P < 0.05), the mRNA and protein expressions of osteocalcin, RUNX2, and type I collagen were elevated (P < 0.05), the mRNA expressions of ERK1/2 and AMPK were elevated (P < 0.05), and the protein expressions of p-AMPK and p-ERK1/2 were elevated (P < 0.05). Additionally, ERK inhibitors partially inhibited the above effects of astragaloside IV. To conclude, astragaloside IV can promote osteogenic differentiation of MC3T3-E1 cells by activating the ERK/AMPK pathway.

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

Key words: astragaloside IV, osteoporosis, MC3T3-E1 cells, oxidative stress, ERK/AMPK signaling pathway, osteogenesis, engineered bone materials

CLC Number:

Zhang Jiahao, Li Jiacheng, Wen Mingtao, Guo Yanbo, Luo Di, Li Gang. Astragaloside IV alleviates oxidative stress injury and promotes osteogenesis in MC3T3-E1 cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(17): 3529-3536.

Figures/Tables 6

2.1 不同浓度过氧化氢对MC3T3-E1细胞增殖活力的影响干预12 h后，0.4，0.6 mmol/L过氧化氢组细胞增殖率低于对照组；干预24 h后，0.1，0.2，0.4，0.6 mmol/L过氧化氢组细胞增殖率均低于对照组；干预48 h后，0.2，0.4，0.6 mmol/L过氧化氢组细胞增殖率均低于对照组，并且各组干预24 h的细胞增殖率较低，见图1A。后续实验选择0.2 mmol/L过氧化氢干预24 h作为氧化应激造模条件。 2.2 不同浓度黄芪甲苷组对MC3T3-E1细胞增殖活力的影响干预24 h后，25，50，100 μmol/L黄芪甲苷组细胞增殖率均高于对照组，200 μmol/L黄芪甲苷组细胞增殖率低于对照组；干预48 h后，25，50，100 μmol/L黄芪甲苷组细胞增殖率较干预24 h后升高，并且均高于对照组，200 μmol/L黄芪甲苷组细胞增殖率低于对照组；干预72 h后，25，50，100 μmol/L黄芪甲苷组细胞增殖率较干预48 h后降低，但仍高于对照组，200 μmol/L黄芪甲苷组细胞增殖率低于对照组，见图1B。说明100 μmol/L以内的黄芪甲苷对MC3T3-E1细胞无毒性作用。 2.3 黄芪甲苷对氧化应激条件下MC3T3-E1细胞增殖活力的影响在加入过氧化氢基础上，干预24 h后，25，50，100，200 μmol/L黄芪甲苷提高了MC3T3-E1细胞增殖率；干预48 h后，25，50，100 μmol/L黄芪甲苷提高了MC3T3-E1细胞增殖率，而200 μmol/L黄芪甲苷降低了MC3T3-E1细胞增殖率；干预72 h后，25，50，100 μmol/L黄芪甲苷提高了MC3T3-E1细胞增殖率，而200 μmol/L黄芪甲苷降低了MC3T3-E1细胞增殖率，见图1C。并且可见过氧化氢+100 μmol/L黄芪甲苷组干预48 h的细胞增殖率最高。综合2.2、2.3实验结果，在保证MC3T3-E1细胞足够活性的条件下，选择100 μmol/L黄芪甲苷干预48 h进行实验。 2.4 黄芪甲苷可增强氧化应激条件下MC3T3-E1细胞的成骨与矿化能力 2.4.1 丙二醛含量检测结果模型组细胞内丙二醛含量高于对照组、黄芪甲苷组，抑制剂组细胞内丙二醛含量高于黄芪甲苷组，见图2。 2.4.2 碱性磷酸酶染色与茜素红染色结果模型组碱性磷酸酶染色较对照组浅，黄芪甲苷组碱性磷酸酶染色较模型组加深，表明黄芪甲苷可促进氧化应激条件下MC3T3-E1细胞的成骨分化；定量分析结果显示，模型组碱性磷酸酶含量低于对照组、黄芪甲苷组(P < 0.001，P < 0.05)，见图3。茜素红染色及定量分析结果显示，模型组成骨矿化结节形成少于对照组、黄芪甲苷组(P < 0.001，P < 0.05)，见图3。表明黄芪甲苷能够促进过氧化氢诱导MC3T3-E1细胞的成骨分化与矿化能力。 2.4.3 RT-qPCR检测结果模型组骨钙素、RUNX2、Ⅰ型胶原蛋白mRNA表达均低于对照组(P < 0.01，P < 0.001)，黄芪甲苷组骨钙素、RUNX2、Ⅰ型胶原蛋白mRNA表达均高于模型组(P < 0.01，P < 0.001)，抑制剂组骨钙素、RUNX2、Ⅰ型胶原蛋白mRNA表达均低于黄芪甲苷组(P < 0.01)，见图4。表明黄芪甲苷可以明显提高氧化应激条件下MC3T3-E1细胞成骨相关基因mRNA的表达，并且ERK抑制剂可部分拮抗黄芪甲苷的作用。模型组AMPK mRNA表达高于对照组(P < 0.01)，黄芪甲苷组ERK1/2、AMPK mRNA表达均高于模型组(P < 0.001)，抑制剂组ERK1/2、AMPK mRNA表达均低于黄芪甲苷组(P < 0.001，P < 0.01)，见图5。 2.4.4 Western blot检测结果模型组骨钙素、RUNX2、Ⅰ型胶原蛋白的蛋白表达均低于对照组(P < 0.01，P < 0.001)，黄芪甲苷组骨钙素、RUNX2、Ⅰ型胶原蛋白的蛋白表达均高于模型组(P < 0.05，P < 0.01)，抑制剂组骨钙素、RUNX2、Ⅰ型胶原蛋白的蛋白表达均低于黄芪甲苷组(P < 0.05)，见图6。各组ERK1/2蛋白表达无差异，模型组、黄芪甲苷组、抑制剂组AMPK蛋白表达高于对照组(P < 0.05)，黄芪甲苷组p-AMPK、p-ERK1/2蛋白表达高于模型组(P < 0.05，P < 0.001)，抑制剂组p-AMPK、p-ERK1/2蛋白表达低于黄芪甲苷组(P < 0.01)，见图7。"

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