【摘要】 背景：骨髓间充质干细胞成骨分化功能减弱是骨质疏松发生发展的关键病理环节。姜黄素可通过调控磷脂酰肌醇3激酶/蛋白激酶B信号通路促进成骨分化治疗骨质疏松，但具体作用机制尚未明确。目的：研究姜黄素调控磷脂酰肌醇3激酶/蛋白激酶B信号通路促进成骨分化治疗骨质疏松的机制。方法：通过网络药理学筛选姜黄素药物作用靶点及成骨分化、骨质疏松的相关靶点基因，三者取交集后，绘制蛋白质相互作用网络图并进行基因本体论、京都基因与基因组百科全书富集分析及分子对接处理。通过生物信息学筛选蛋白质相互作用网络中潜在的标志基因，构建临床预测模型；运用单细胞转录组学技术分析标志基因在成骨细胞谱系分群中的表达模式；最后进行细胞及动物实验验证。结果与结论：(1)筛选出姜黄素的药物作用靶点366个，姜黄素与成骨分化及骨质疏松的交集靶点有134个；(2)京都基因与基因组百科全书富集分析显示，交集靶点涉及磷脂酰肌醇3激酶/蛋白激酶B信号通路；(3)分子对接显示姜黄素主要活性成分与磷脂酰肌醇3激酶/蛋白激酶B信号通路相关靶点均可良好对接；(4)由基因表达综合数据库下载骨质疏松相关基因集，将蛋白质相互作用网络图中的134个靶点基因通过113种机器学习的算法组合筛选得到14个潜在的标志基因，其中包括磷脂酰肌醇3激酶/蛋白激酶B信号通路的靶点基因PIK3R1、PIK3CG、GSK3β、AKT1，这14个基因可能在姜黄素促进成骨分化治疗骨质疏松方面发挥着重要作用；(5)单细胞转录组学分析显示骨质疏松患者的成骨细胞谱系分群中存在一群GSK3β高表达的细胞群，且该细胞分群可能与骨质疏松的发生发展密切相关；(6)拟时序分析发现在骨质疏松发展过程中GSK3β表达量逐渐升高，PIK3R1、PIK3CG、AKT1表达量逐渐降低；(7)细胞实验发现，敲除GSK3β后，促进了小鼠颅顶成骨前体细胞亚克隆14(MC3T3-E1 subclone 14)的成骨分化，具体表现在碱性磷酸酶染色、茜素红染色着色加深；碱性磷酸酶活性以及钙盐沉积显著增加(P <0.05);β-catenin mRNA以及蛋白表达量显著升高(P <0.05);CCK-8与EdU结果显示，5,10μmol/L姜黄素无明显毒性且可以明显促进MC3T3-E1 subclone 14细胞增殖；此外，5,10μmol/L姜黄素干预后明显促进了MC3T3-E1 subclone 14细胞成骨分化；(8)动物实验显示，姜黄素通过增加骨小梁数量并改善骨小梁排列紊乱程度来改善去势卵巢大鼠股骨组织的骨质疏松情况；姜黄素干预后骨质疏松大鼠股骨远端骨组织中磷脂酰肌醇3激酶、蛋白激酶B1表达及磷酸化修饰水平显著升高(P <0.05),GSK3β磷酸化修饰水平显著下降(P <0.05)，此外，c-MYC、Cyclin d1以及骨形态发生蛋白2、骨钙素、骨桥蛋白及Osterix的表达显著升高(P <0.05)。综上所述，磷脂酰肌醇3激酶/蛋白激酶B信号通路中的GSK3β表达升高可能是导致成骨分化功能下降及骨质疏松发生发展的关键因素，姜黄素通过激活磷脂酰肌醇3激酶/蛋白激酶B信号通路促进成骨分化来治疗骨质疏松。更多还原

【Abstract】 BACKGROUND:The diseased osteogenic differentiation function of bone marrow mesenchymal stem cells is a key pathological link in the occurrence and development of osteoporosis.Curcumin can promote osteogenic differentiation and treat osteoporosis by regulating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway,but the specific mechanism of action remains unclear.OBJECTIVE:To investigate the mechanism by which curcumin regulates the phosphatidylinositol 3-kinase/protein kinase B signaling pathway to promote osteogenic differentiation for the treatment of osteoporosis.METHODS:Network pharmacology was used to screen the drug action targets of curcumin and target genes related to osteogenic differentiation and osteoporosis.The intersection targets of the three were used to construct a protein-protein interaction network diagram and perform Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses and molecular docking.Bioinfo rmatics was used to screen potential signature genes in the proteinprotein interaction network and construct a clinical prediction model.Single-cell transcriptomics technology was used to analyze the expression patterns of signature genes in osteoblast lineage subpopulations.Finally,cell and animal expe riments were conducted for ve rification.RESULTS AND CONCLUSION:(1) A total of 366 drug targets of curcumin were screened,and 134 inte rsection targets were identified among curcumin,osteogenic differentiation,and osteopo rosis.(2) Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the intersection targets were involved in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.(3) Molecular docking showed that the main active components of curcumin could be well docked with the targets related to the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.(4) Osteoporosis-related gene sets were downloaded from the Gene Expression Omnibus database.The 134 target genes in the protein-protein interaction network were screened by 113 combinations of machine learning algo rithms to obtain 14 potential signature genes,including target genes in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway:PIK3R1,PIK3CG,GSK3β,and AKT1.These 14 genes may play important roles in curcumin promoting osteogenic differentiation for the treatment of osteoporosis.(5) Single-cell transcriptomics analysis showed that there was a cell subpopulation with high GSK3β expression in the osteoblast lineage of osteoporosis patients,and this cell subpopulation might be closely related to the occurrence and development of osteoporosis.(6) Pseudotime analysis found that during the development of osteopo rosis,the expression level of GSK3β gradually increased,while the expression levels of PIK3R1,PIK3CG,and AKT1gradually decreased.(7) Cell experiments showed that after GSK3β knocko ut,the osteogenic differentiation of mouse MC3T3-E1 subclone 14 was promoted,as evidenced by deeper staining in alkaline phosphatase staining and Alizarin Red S staining,and significantly increased alkaline phosphatase activity and calcium deposition(P <0.05).The mRNA and protein expression levels of β-catenin were significantly increased(P <0.05).Cell counti ng kit-8 and EdU results showed that 5 and 10 μmol/L curcumin had no obvious toxicity and could significantly promote the prolife ration of MC3T3-E1 subclone 14 cells.In addition,intervention with 5 and 10 μmol/L curcumin significantly promoted the osteogenic differentiation of MC3T3-E1 subclone 14.(8) Animal expe riments showed that curcumin improved osteoporosis in the femoral tissue of ovariectomized rats by increasing the number of trabecular bones and ameliorating the disorder of trabecular bone arrangement.After curcumin intervention,the expression and phosphorylation levels of phosphatidylinositol 3-kinase and protein kinase B1in the distal femoral bone tissue of osteoporotic rats were significantly increased(P <0.05),and the phosphorylation level of GSK3β was significantly decreased(P<0.05).In addition,the expression levels of c-MYC,Cyclin D1,bone morphogenetic protein 2,osteocalcin,osteopontin,and Oste rix were significantly increased(P<0.05).In conclusion,the increased expression of GSK3β in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway may be a key factor leading to the decline of osteogenic differentiation function and the occurrence and development of osteoporosis.Curcumin treats osteoporosis by activating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway to promote osteogenic differentiation.更多还原