海南医科大学学报2025，Vol.31Issue(22)：1705-1717,13.DOI:10.13210/j.cnki.jhmu.20250116.001
复方苗药九仙罗汉接骨汤通过提高自噬促进MC3T3-E1Subclone14成骨细胞分化防治骨质疏松
Compound Miao medicine Jiuxian Luohan Jiegu Decoction prevents osteoporosis by enhancing autophagy to promote MC3T3-E1Subclone14 osteoblast differentiation
摘要
Abstract
Objective:To investigate the effects of the compound Miao medicine Jiuxian Luohan Jiegu Decoction on the prolif-eration and differentiation of MC3T3-E1Subclone14 osteoblasts as well as the roles of PI3K/AKT/mTOR signaling pathway and cellular autophagy in it.Methods:The precursor osteoblast cell line MC3T3-E1Subclone14 was selected from logarithmic growth stage rats,and intervened with different concentrations of compound Miao medicine Jiuxian Luohan Jiegu Decoction(0%-20%),and the optimal concentration of the compound Miao medicine Jiuxian Luohan Jiegu Decoction was determined by the observation of cell viability through CCK8;the optimal concentration of compound Miao medicine Jiuxian Luohan Jiegu Decoction was used for treating MC3T3-E1Subclone14 cells.E1Subclone14 cells,the osteoblasts were divided into Group A(osteogenic differentia-tion induction group),Group B(group A+compound Miao medicine Jiuxian Luohan Jiegu Decoction containing serum),Group C(group A+5 mmol/L autophagy inhibitor 3-methylpurine(3-MA)),Group D[group B+5 mmol/L autophagy inhibitor 3-methylpurine(3-MA)]and the blank control group.Alizarin red staining was used to detect the relative mineralized nodule con-tent of each group;CCK8 assay was used to observe cellular value-added rate in each group;Western Blot was used to detect the expression levels of osteogenic differentiation proteins Runx2 and Osterix,and autophagy-related proteins LC3 and p62;q-PCR was used to detect the expression levels of osteogenic differentiation genes Runx2 and Osterix mRNA,autophagy-related genes LC3,p62 mRNA,and signaling pathway-related genes PI3K,AKT,mTOR mRNA expression;immunofluorescence staining was used to detect the expression of LC3,p62 in the cells of each group;transmission electron microscopy to detect the formation of autophagic vesicles in the cells of each group.Results:Compound Miao medicine Jiuxian Luohan Jiegu Decoction could pro-mote the proliferation and differentiation of osteoblasts,and the effect was most obvious when the concentration was 10%(P<0.01);the mineralized nodule formation in the osteogenic differentiation induction group was significantly more than that in the blank control group;the mineralised nodule formation in the compound Miao medicine Jiuxian Luohan Jiegu Decoction group was significantly more than that in the osteogenic differentiation induction group(P<0.01),and the cellular viability and activity signif-icantly increased(P<0.01);compared to the osteogenic differentiation induction group,the expression of osteogenic differentia-tion markers Runx2 and Osterix and autophagy-related markers LC3 gene and protein were up-regulated by the compound Miao medicine Jiuxian Luohan Jiegu Decoction(P<0.01),while the expression of p62 gene and protein was down-regulated(P<0.01),and the addition of 3-MA to inhibit autophagy was able to antagonise these effects(P<0.01).The immunofluorescence showed that osteogenesis differentiation inducing group was able to promote the expression of LC3 in the cells while inhibiting the expression of p62 in the cells compared to the compound Miao medicine Jiuxian Luohan Jiegu Decoction(P<0.05);the addition of 3-MA suppressed the expression of LC3 in the cells,while promoting the expression of p62 in the cells and the cellular viability and activity significantly reduced(P<0.01);transmission electron microscopy showed that the compound Miao medicine Jiuxian Luohan Jiegu Decoction promoted the increase of autophagic vesicles in osteoblasts,and 3-MA inhibited the increase of autophagic vesicles;compared to the osteogenic differentiation induction group,the expression of PI3K,AKT,and mTOR mRNA in the group of the compound Miao medicine Jiuxian Luohan Jiegu Decoction was significantly increased(P<0.01),the expression of PI3K,AKT and mTOR mRNA was significantly reduced after the addition of 3-MA to inhibit autophagy(P<0.01).Conclusion:Compound Miao medicine Jiuxian Luohan Jiegu Decoction may promote osteogenic differentiation of MC3T3-E1Subclone14 cells through activation of PI3K/AKT/mTOR signaling pathway and enhancement of autophagy,and its mechanism of action may be related to the regulation of Runx2,Osterix,LC3,p62 proteins and gene expression.关键词
复方苗药九仙罗汉接骨汤/自噬/成骨细胞/细胞分化/PI3K/AKT/mTOR信号通路Key words
Compound Miao medicine Jiuxian Luohan Jiegu Decoction/Autophagy/Osteoblasts/Cell differentiation/PI3K/AKT/mTOR signalling pathway分类
医药卫生引用本文复制引用
宁众,余梁,唐良华,殷天坪,李义,杨彦君,王洪发..复方苗药九仙罗汉接骨汤通过提高自噬促进MC3T3-E1Subclone14成骨细胞分化防治骨质疏松[J].海南医科大学学报,2025,31(22):1705-1717,13.基金项目
贵州省科学技术科技计划项目[黔科合基础-ZK(2024)一般414]This study was supported by the Guizhou Provincial Science and Technology Program Project[Qiankehe Basic Foundation-ZK(2024)General 414] （2024）
