摘要
背景:PI3K/Akt通路作为一种胞内细胞转导通路,对于细胞增殖、生长与代谢的调控都有着密切的关系。菟丝子作为补肾中药,其总黄酮成分具有潜在骨保护作用,但具体分子机制尚未明确。目的:通过网络药理学结合体外实验的方式探讨菟丝子总黄酮对地塞米松致成骨细胞凋亡的保护作用及潜在的分子作用机制。方法:采用网络药理学的方法,对菟丝子治疗股骨头坏死的潜在靶点以及信号通路进行预测分析,并进行细胞学验证。体外培养hFOB1.19细胞,分别用不同质量浓度的菟丝子总黄酮(0,25,50,100,200,400和600μg/mL)处理24 h,CCK-8法检测细胞存活率,筛选后续菟丝子总黄酮的处理剂量。将hFOB1.19细胞分为对照组、地塞米松组、菟丝子总黄酮各剂量组(分别加入100,200,400μg/mL菟丝子总黄酮和2μmol/L地塞米松)及菟丝子总黄酮+LY294002组(加入菟丝子总黄酮400μg/mL和PI3K抑制剂LY29400210μmol/L)6组。处理结束后,采用CCK-8法测定细胞存活率,流式细胞术测定细胞凋亡率,免疫荧光测定凋亡相关蛋白表达,DCFH-DA探针测定活性氧水平,Western Blot测定PI3K/Akt通路相关蛋白表达。结果与结论:①网络药理学结果表明,菟丝子的作用靶点可能与调控肿瘤坏死因子、肿瘤蛋白p53、白细胞介素6、丝氨酸/苏氨酸蛋白激酶1、血管内皮生长因子A、半胱天冬酶3、白细胞介素1β、缺氧诱导因子1α表达以及PI3K/Akt通路有关;②CCK-8结果表明,菟丝子总黄酮在一定范围内能够促进hFOB1.19细胞的增殖,且菟丝子总黄酮能够缓解地塞米松对hFOB1.19细胞增殖的抑制作用;③菟丝子总黄酮能够抑制地塞米松诱导的细胞凋亡和活性氧水平升高,降低Cleaved Caspase-3和Bax蛋白表达以及活性氧水平,增加Bcl-2蛋白表达;④与地塞米松组相比,菟丝子总黄酮能增强p-PI3K和p-Akt蛋白表达;⑤PI3K抑制剂LY294002逆转了菟丝子总黄酮对地塞米松处理细胞凋亡和活性氧水平的影响。结果表明,菟丝子总黄酮能够减弱地塞米松介导的hFOB1.19细胞凋亡,其机制可能与激活PI3K/Akt通路有关。
BACKGROUND:The PI3K/Akt pathway,as an intracellular signal transduction pathway,plays a crucial role in regulating cell proliferation,growth,and metabolism.Semen Cuscutae,a traditional Chinese medicine used for kidney tonification,possesses potential bone-protective effects due to its total flavonoids;however,the specific molecular mechanisms remain unclear.OBJECTIVE:To investigate the protective effect of total flavonoids from Semen Cuscutae against dexamethasone-induced osteoblast apoptosis and its potential molecular mechanisms through a combination of network pharmacology and in vitro experiments.METHODS:Network pharmacology was used to predict potential targets and signaling pathways for Semen Cuscutae in the treatment of dexamethasoneinduced femoral head avascular necrosis.hFOB1.19 cells were cultured in vitro and treated with different concentrations of total flavonoids from Semen Cuscutae(0,25,50,100,200,400,and 600μg/mL)for 24 hours.Cell viability was assessed using the cell counting kit-8 assay to select the optimal treatment dose for subsequent experiments.hFOB1.19 cells were divided into six groups:control,dexamethasone,total flavonoids from Semen Cuscutae at 100,200,400μg/mL plus 2μmol/L dexamethasone,and total flavonoids from Semen Cuscutae plus LY294002 groups(400μg/mL total flavonoids from Semen Cuscutae and 10μmol/L LY294002,a PI3K inhibitor).Cell viability was determined using the cell counting kit-8 assay,apoptosis rate was measured by flow cytometry,apoptosisrelated protein expression was assessed by immunofluorescence,reactive oxygen species levels were quantified using the DCFH-DA probe,and expression of proteins related to the PI3K/Akt pathway was analyzed by western blot assay after treatment,.RESULTS AND CONCLUSION:(1)Network pharmacology results indicated that the potential targets of Semen Cuscutae may be related to the regulation of tumor necrosis factor,p53,interleukin-6,serine/threonine protein kinase 1,vascular endothelial growth factor A,caspase-3,interleukin-1β,hypoxia-inducible factor 1αexpression,and the PI3K/Akt pathway.(2)CCK-8 results showed that total flavonoids from Semen Cuscutae promoted the proliferation of hFOB1.19 cells within a certain range and alleviated the inhibitory effect of dexamethasone on the proliferation of hFOB1.19 cells.(3)Total flavonoids from Semen Cuscutae inhibited dexamethasone-induced apoptosis and increased reactive oxygen species levels,reduced the expression of cleaved caspase-3 and Bax,and decreased reactive oxygen species levels,while increased the protein expression of Bcl-2.(4)Compared with the dexamethasone group,total flavonoids from Semen Cuscutae enhanced the expression of p-PI3K and p-Akt proteins.(5)The PI3K inhibitor LY294002 reversed the effects of total flavonoids from Semen Cuscutae on apoptosis and reactive oxygen species levels in dexamethasone-treated cells.The results indicate that total flavonoids from Semen Cuscutae can attenuate dexamethasone-mediated apoptosis in hFOB1.19 cells,and the mechanism may be related to the activation of the PI3K/Akt pathway.
作者
赵宇
薛云
黄家俊
吴迪友
杨彬
黄俊卿
Zhao Yu;Xue Yun;Huang Jiajun;Wu Diyou;Yang Bin;Huang Junqing(College of Orthopedics and Traumatology,Henan University of Chinese Medicine,Zhengzhou 450002,Henan Province,China;Department of Pain,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450002,Henan Province,China)
出处
《中国组织工程研究》
北大核心
2026年第17期4289-4298,共10页
Chinese Journal of Tissue Engineering Research
基金
全国中医临床特色技术传承人才项目(中国中医药人教教育便函[2019]36号),项目负责人:杨彬
河南省中医药青苗人才培养项目(豫卫中医函[2021]16号),指导老师:黄俊卿
河南科技智库中医药强省战略研究基地2024年度专项课题(23104007-2024),项目负责人:黄俊卿
河南省中医药科学研究专项课题(2023ZY3013),项目负责人:黄俊卿
2024年度河南省中医药科学研究专项(2024ZY2066),项目负责人:杨彬
河南省中医药文化与管理研究项目(TCM2024027),项目负责人:杨彬
河南中医药大学2024年度研究生科研创新能力提升计划项目(2024KYCX075),项目负责人:赵宇
河南中医药大学2024年度研究生科研创新能力提升计划项目(2024SHDY012),项目负责人:薛云。
