基于PI3K/Akt/HIF-1α信号通路探讨补肾活血汤干预乳腺癌内分泌治疗相关骨质疏松的作用机制

Intervention Mechanism of Bushen Huoxuetang on Osteoporosis Related to Endocrine Therapy in Breast Cancer Based on PI3K/Akt/HIF-1α Signaling Pathway

目的:利用网络药理学预测补肾活血汤干预乳腺癌内分泌治疗相关骨质疏松的潜在作用机制,并通过体外细胞模型进行实验验证。方法:首先通过网络药理学筛选补肾活血汤的主要有效化学成分和作用靶点,进一步获取乳腺癌内分泌治疗相关骨质疏松相关的疾病靶点。将二者的共同靶点信息进行基因本体(GO)功能注释和京都基因与基因组百科全书(KEGG)通路富集分析。其次利用生存分析网站(Kaplan-Meier Plotter)对关键靶点进行生存分析。最后通过体外实验噻唑蓝(MTT)比色法评估细胞增殖抑制活性,蛋白免疫印迹法(Western blot)验证关键靶点及通路,并使用实时荧光定量聚合酶链式反应(Real-time PCR)评估关键靶点mRNA表达。结果:网络药理学研究共获得补肾活血汤活性成分716个,关键靶点249个,补肾活血汤和乳腺癌内分泌治疗相关骨质疏松的共同靶点135个,其中蛋白激酶B(Akt)1、低氧诱导因子-1α(HIF-1α)是两个关键靶点,靶点共涉及生物过程531种,细胞组成62种,分子功能162种,参与乳腺癌、内分泌抵抗等细胞信号通路145条;靶点有效地富集在磷脂酰肌醇3-激酶(PI3K)/Akt和低氧诱导因子(HIF)-1两条信号通路。体外实验中,MTT比色法显示,与空白组比较,22.5、45、90 g·L-1及45、90、180 g·L-1质量浓度的补肾活血汤作用48 h后分别能不同程度的降低人Luminal A型乳腺癌细胞系MCF-7和T47D细胞增殖率和细胞运动能力。将0、15、60 g·L-1的补肾活血汤干预MCF-7细胞48 h,Western blot实验表明,与空白组比较,不同质量浓度的补肾活血汤作用于MCF-7细胞中磷酸化(p)-PI3K、PI3K、p-Akt、Akt和HIF-1α蛋白相对表达水平均降低(P<0.05,P<0.01),且呈浓度依赖性。采用Real-time PCR检测关键靶点HIF-1α的mRNA表达,结果显示,与空白组比较随浓度升高MCF-7细胞中HIF-1α的mRNA表达显著降低(P<0.01),且呈浓度依赖性。结论:补肾活血汤通过PI3K/Akt/HIF-1信号通路作用于关键靶点Akt1、HIF1A,进而干预乳腺癌内分泌治疗相关的骨质疏松。

Objective:To predict the underlying mechanism of Bushen Huoxuetang in treating osteoporosis related to endocrine therapy in breast cancer by network pharmacology and to verify the results through in vitro cell model.Method:The main effective components and targets of Bushen Huoxuetang were screened out through network pharmacology,and the targets of osteoporosis related to endocrine therapy in breast cancer were further obtained.The intersected targets were analyzed by Gene Ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment.Kaplan Meier plotter was used to analyze the survival of crucial targets.Finally,the inhibitory activity against cell proliferation was evaluated by in vitro methye thiazolye telrazlium(MTT)assay.The key targets and pathways were verified by Western blot,and the mRNA expression of the key targets was evaluated by real-time polymerase chain reaction(Real-time PCR).Result:A total of 716 active components and 249 key targets of Bushen Huoxuetang were obtained from network pharmacology.There were 135 common targets,among which protein kinase B(Akt)1and hypoxia-inducible factor-1α(HIF-1α) were two key targets.Additionally,531 biological processes,62 cellular components,162 molecular functions,and 145 signaling pathways including breast cancer and endocrine resistance were involved.The key targets were effectively enriched in phosphatidylinositol 3-kinases(PI3K)/Akt and HIF-1 signaling pathways.According to the MTT assay,the cell proliferation rate and cell motility of MCF-7 and T47D cells in the luminal A cell line were reduced by Bushen Huoxuetang treatment(22.5,45,90 g·L-1,and 45,90,180 g·L-1)for 48 h as compared with the blank group.As revealed by Western blot,MCF-7 cells were treated with Bushen Huoxuetang(0,15,60 g·L-1)for 48 h,and the relative expression of p-PI3K,PI3K,p-Akt,Akt,and HIF-1α was decreased in a dose-dependent manner as compared with the blank group(P<0.05,P<0.01).Real-time PCR was used to detect the mRNA expression of the key target HIF-1α.The results showed that the mRNA expression of HIF-1α in MCF-7 cells was decreased with the increase in the dose(P<0.01),and the change was in a concentration-dependent manner.Conclusion:The mechanism of Bushen Huoxuetang in the treatment of osteoporosis related to endocrine therapy in breast cancer may be related to the key targets including Akt1 and HIF-1α through the PI3K/Akt/HIF-1α signaling pathway.