基于网络药理学和实验验证探讨金水相生方逆转前列腺癌去势抵抗的分子机制研究

Molecular Mechanism of Jinshui Xiangsheng Formula(金水相生方)in Reversing Castration Resistance of Prostatic Cancer Based on Network Pharmacology and Experimental Verification

目的:基于网络药理学和实验验证探讨金水相生方逆转前列腺癌去势抵抗的有效成分、作用靶点及相关机制。方法:利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)和高通量基因表达数据库(Gene Expression Omnibus,GEO)筛选前列腺癌与前列腺增生差异基因;利用中药系统药理分析平台并结合文献报道筛选金水相生方的有效活性成分和作用靶点,获得活性成分-靶点互作交集基因,利用Cytoscape3.7.2软件对交集基因进行蛋白互作分析、基因本体论(GO)功能和京都基因与基因组百科全书(KEGG)通路进行基因富集分析,根据分析结果进行实验验证。金水相生方含药血清12.5、25、50 g/kg组处理人前列腺癌细胞PC-3细胞24、48和72 h后,检测细胞增殖、细胞凋亡、周期的变化和磷脂酰肌3-羟激酶(PI3K)蛋白、丝氨酸-苏氨酸蛋白激酶(AKT)及磷酸化(p)-AKT和κB激酶抑制剂(IKKβ)蛋白的表达情况。结果:共得到金水相生方潜在活性成分101个,药物-疾病共同靶点187个,其中上调基因59个,下调基因128个,核心靶点20个及关联的有效成分58个,主要包括黄芩新素、薯蓣皂苷、二甲氧基黄酮、圣草素、黄芩黄酮II、等;KEGG富集分析主要在代谢途径、癌症的途径、钙信号通路、神经活性配体受体相互作用、PI3K/AKT信号通路等,GO富集分析在BP中主要包括信号转导、蛋白质磷酸化等;CC中主要包括质膜、胞浆等;MF中主要包括蛋白质结合、三磷酸腺苷结合等通路与金水相生方逆转前列腺癌去势抵抗作用机制相关。试验结果表明,与空白对照组比较,金水相生方含药血清12.5、25、50 g/kg组PC-3细胞增殖受抑制程度增加,细胞G2期比例增加,S期比例降低(P<0.01),PI3K、AKT、IKKβ及p-AKT蛋白表达显著下调(P<0.01)。结论:本研究证实金水相生方逆转前列腺癌去势抵抗的作用机制较为复杂,其过程中涉及多种信号通路和潜在靶点及有效活性成分,但其主要的机制可能与其调控PI3K/AKT信号通路活性有关。

Objective:To explore the effective components,targets and related mechanism of Jinshui Xiangsheng Formula(金水相生方)in reversing castration resistance of prostatic cancer based on network pharmacology and experimental validation.Methods:The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)were used to screen out the differential genes between prostatic cancer and prostatic hyperplasia.The pharmacological analysis platform of traditional Chinese medicine system was used combining with the literature reports,to screen out the effective active components and action targets of Jinshui Xiangsheng Formula.The active component-target interaction intersection genes were obtained.The protein-protein interaction analysis,gene ontology(GO)functional enrichment analysis and Kyoto Gene and Genome Encyclopedia(KEGG)pathway enrichment analysis of the intersection genes were carried out by using cytoscape 3.7.2,and the experimental verification was carried out according to the analysis results.After human prostate cancer cell PC-3 cells were treated with serum containing 12.5,25,and 50 g/kg of Jinshui Xiangsheng Formula for 24,48,and 72 hours,the cell proliferation,apoptosis,and cycle changes were observed,while the protein expression levels of phosphatidyl muscle 3-hydroxykinase(PI3K),serine-threonine protein kinase(AKT),phosphorylation(p)-AKT,andκB kinase inhibitor(IKKβ)were determined.Results:A total of 101 potential active components,187 common drug-disease targets of Jinshui Xiangsheng Formula were obtained,including 59 up-regulated genes,128 down-regulated genes,20 core targets,and 58 associated active ingredients,mainly including baicalin,diosgenin,dimethoxyflavone,Shengcao,and baicalein II.KEGG pathway enrichment analysis mainly included metabolic pathway,cancer pathway,calcium signaling pathway,neuroactive ligand receptor interaction,and PI3K/AKT signaling pathway.GO functional enrichment analysis mainly included signal transduction and protein phosphorylation in the biological process(BP),plasma membrane and cytoplasm in the cellular component(CC),and protein binding and adenosine triphosphate binding in the molecular function(MF),which were related to the mechanism of Jinshui Xiangsheng Formula in reversing the castration resistance of prostatic cancer.The experimental results showed that as compared with the blank control group,the inhibition of PC-3 cell proliferation was increased in groups of serum containing 12.5,25,and 50 g/kg of Jinshui Xiangsheng Formula,the proportion of G2 phase was increased,and the proportion of S phase was decreased in a time-concentration dependent manner(P<0.01).The protein expression levels of PI3K,AKT and IKKβand p-AKT were significantly reduced in a concentration-dependent manner(P<0.01).Conclusion:This study confirmed that the mechanism of Jinshui Xiangsheng Formula in reversing the castration resistance of prostatic cancer is relatively complex,involving a variety of signaling pathways,potential targets,and active components,but its main mechanism may be related to its regulation of the activity of PI3K/AKT signaling pathway.