中国中西医结合杂志2023，Vol.43Issue(12)：1478-1485,8.DOI:10.7661/j.cjim.20231107.307
基于转录组学筛选左归丸促进MC3T3-E1细胞成骨细胞增殖分化的关键基因
Transcriptomics-based Screening of Key Genes for Promoting Osteoblast Proliferation And Differentiation in MC3T3-E1 Treated by Zuogui Pill
摘要
Abstract
Objective To investigate the molecular mechanism of osteoporosis(OP)in MC3T3-E1 regulated by Zuogui Pill solution.Methods MC3T3-E1 cells were treated with Zuogui Pill solution at concentrations of 0,100,200,400,and 1 000 μg/mL.Western Blot was performed to assess the protein expressions of total β-catenin,active β-catenin,Runt-related transcription factor 2(Runx2),and osxterix(Osx).qPCR was used to detect the mRNA expressions of Runx2,Osx,type Ⅱ alpha 1 collagen gene(Col2a1),osteocalcin(Ocn),and β-catenin.The proliferation activity of MC3T3-E1 cells was measured using CCK-8 assay,while alkaline phosphatase(ALP)staining and alizarin red staining(ARS)were used to evaluate the osteoblast differentiation ability.Differential gene expression related to MC3T3-E1 cell proliferation and differentiation induced by Zuogui Pill solution was confirmed through qPCR analysis of RNA-Seq results.Results Different concentrations of Zuogui Pill solutions(0,100,200,400,1 000 μg/mL)were used to treat MC3T3-E1 cells for 48 hours,the expression levels of osteogenic marker genes(Runx2,Osx,Col2A1,Ocn,β-catenin)were significantly increased(P＜0.05).Additionally,the proliferation rate of MC3T3-E1 cells was significantly increased(P＜0.05),and ALP and ARS showed significant enhancement(P＜0.05).Notably,the intervention effect of the 400 μ g/mL Zuogui Pill solution was the most prominent among the tested concentrations.The osteogenic differentiation ability of MC3T3-E1 cells was significantly reduced(P＜0.05)when the intervention concentration of Zuogui Pill solution reached 1 000 μg/mL.After being intervened with 400 μg/mL Zuogui Pill solution for 48 h,RNA-seq analysis identified 10 genes with the most differential expression,including 6 up-regulated genes and 4 down-regulated genes.qPCR verification confirmed a significant increase in the mRNA expression levels of EF-hand calcium-binding domain-containing protein 9(Efcab9),forkhead box J1(Foxj1)(P＜0.05),while the mRNA expression levels of Ras protein specific guanine nucleotide releasing factor 1(RasGRF1),growth differentiation factor 9(GDF9),and achain of type Ⅳ collagen(Col4a3)were significantly reduced(P＜0.05).Conclusion Zuogui Pill solution(400 μg/mL)can regulate the mRNA expression levels of Efcab9,FOXj1,RasGRF1,GDF-9,and Col4a3,and promote osteoblast proliferation and differentiation of MC3T3-E1 cells.关键词
左归丸/MC3T3-E1细胞/转录组学/成骨分化/中药复方/骨质疏松Key words
Zuogui Pill/MC3T3-E1 cells/transcriptomics/osteogenic differentiation/Chinese herbal compound/osteoporosis引用本文复制引用
霍少川,韩霞,熊仪,俞建萍,郭海,廖昔威,程文昌,钟嘉漫,温刘莹,黄婷婷..基于转录组学筛选左归丸促进MC3T3-E1细胞成骨细胞增殖分化的关键基因[J].中国中西医结合杂志,2023,43(12):1478-1485,8.基金项目
国家自然科学基金青年项目(No.82004395) （No.82004395）
中国博士后科学基金及出站博士后科研资助(No.2018M633088) （No.2018M633088）
广东省中医药局科研项目(No.20212203) （No.20212203）
深圳市福田区卫生公益性科研项目(No.FTWS2020058,No.FTWS2020037) （No.FTWS2020058,No.FTWS2020037）
