基于网络药理学和体外实验探讨蒲公英治疗卵巢早衰的作用机制

Discussion on the mechanism of action of dandelion in the treatment of premature ovarian failure based on network pharmacology and in vitro experiments

目的 通过网络药理学技术和体外实验探讨蒲公英治疗卵巢早衰的作用机制。方法 从中药系统药理学数据库与分析平台、STITCH、BATMAN-TCM、GEO、中药研究数据库、Bindingdb、DAVID数据库获取蒲公英的活性成分及卵巢早衰的潜在靶点,构建靶蛋白质-蛋白质相互作用网络、成分-靶点-疾病网络,并进行KEGG富集分析。通过卵巢颗粒细胞在不同浓度H2O2条件下培养不同时间,选择最佳造模条件构建卵巢细胞早衰模型(模型组),另采用不同浓度的蒲公英甾醇预处理细胞,选择后续干预的最佳浓度。将细胞设置空白组、模型组及蒲公英甾醇高、低剂量组。蒲公英甾醇高、低剂量组分别给予蒲公英甾醇预处理24 h后和模型组一同加入H2O2,空白组不做处理。干预结束后,采用CCK-8法测定细胞活力,DCFH-DA荧光探针检测细胞内活性氧(ROS)水平,蛋白质印迹法分别检测细胞质、细胞核中的低氧诱导因子-1α(HIF-1α)。结果 数据库共筛选出蒲公英活性成分23个,相应的作用靶点345个,卵巢早衰相关靶点基因497个,二者共同靶点基因61个,涉及的通路有HIF-1信号通路、蛋白聚糖作用等。当用600μmol/L H2O2处理细胞4 h,细胞活力最接近IC50。蒲公英甾醇浓度为7.5、15.0μmol/L时,细胞活力高于模型组(P<0.05)。模型组ROS相对荧光强度高于空白组,蒲公英甾醇高、低剂量组低于模型组(P<0.05)。四组细胞质HIF-1α蛋白表达比较,差异无统计学意义(P>0.05)。模型组细胞核HIF-1α蛋白表达高于空白组,蒲公英甾醇高、低剂量组低于模型组(P<0.05)。结论 蒲公英可通过多成分、多靶点、多通路治疗卵巢早衰,其代表性成分蒲公英甾醇可能通过抗氧化作用发挥治疗卵巢早衰的功能。

Objective To explore the mechanism of dandelion in the treatment of premature ovarian failure by network pharmacology technology and in vitro experiments.Methods The active ingredients of dandelion and potential targets for premature ovarian failure were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,STITCH,BATMANIC-TCM,GEO,Traditional Chinese Medicine Information Database,Bindingdb,and DAVID databases.Target protein-protein interaction network and component-target-disease network were constructed,and KEGG enrichment analysis was performed.Ovarian granulosa cells were cultured in different concentrations of H2O2 for different times,and the optimal model conditions were selected to construct a premature ovarian cell senescence model(model group).Then the cells were pretreated with different concentrations of dandelion sterol,and the optimal concentration of subsequent intervention was selected.The cells were divided into blank group,model group,dandelion sterol-high dose,and low dose groups.The cells in the dandelion sterol-high dose,and low dose groups were pretreated with dandelion sterol for 24 h and H2O2 was added toghter with model group.After the intervention,the cell viability was measured by CCK-8 method,the level of reactive oxygen species(ROS)in the cells was detected by DCFH-DA fluorescent probe,and the hypoxia-inducible factor-1α(HIF-1α)in the cytoplasm and nucleus of the cells was detected by Western blotting.Results A total of 23 active ingredients of dandelion were screened out from the database,345 corresponding target genes,497 target genes related to premature ovarian failure,and 61 common target genes.The pathways involved HIF-1 signaling pathway,proteoglycan interaction,and so on.When the cells were treated with 60μmol/L H2O2 for 4 h,the cell viability was closest to the IC50.When the concentration of dandelion sterol was 7.5 and 15.0μmol/L,the cell viability was higher than that of the model group(P<0.05).The relative fluorescence intensity of ROS in model group was higher than that in blank group,and those in dandelion sterol high-dose and low-dose groups were lower than that in model group(P<0.05).There was no significant difference in the expression of HIF-1αprotein in cytoplasm among the four groups(P>0.05).The expression of nuclear HIF-1αprotein in the model group was higher than that in the blank group,and those in dandelion sterol high-dose and low-dose groups were lower than that in model group(P<0.05).Conclusion Dandelion can treat premature ovarian failure through multi-component,multi-target,and multi-pathway,and its representative component dandelion sterol may play a role in the treatment of premature ovarian failure through antioxidant effects.