基于网络药理学及体外实验发现汉黄芩素介导膀胱癌细胞凋亡及细胞周期阻滞

Wogonin Mediating the Apoptosis and Cell Cycle Arrest in Bladder Cancer Cells Based on Network Pharmacology and in vitro Experiments

目的:探究汉黄芩素抑制膀胱癌细胞活性的作用机制。方法:利用TCMSP和Swiss Target Prediction数据库对汉黄芩素的潜在靶标进行筛选。从GeneCards、DisGeNET和OMIM数据库中获取膀胱癌疾病靶点。通过STRING在线分析平台和Cytoscape 3.8.2插件获取靶标相互作用的蛋白-蛋白相互作用关系(PPI)网络。然后在Metascape在线平台上进行基因本体(GO)富集分析和京都基因与基因组百科全书(KEGG)通路富集分析,并通过Cytoscape 3.8.2软件制作“药物-疾病-核心靶点-通路”网络图。通过CCK-8和克隆形成实验观察汉黄芩素抗膀胱癌的作用;Hoechst 33342染色检测汉黄芩素诱导膀胱癌细胞的凋亡情况;流式细胞术检测汉黄芩素诱导细胞凋亡及周期阻滞的请况;蛋白免疫印迹(Western blot)法检测汉黄芩素对凋亡相关蛋白B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2关联X蛋白(Bax)、活化的聚腺苷二磷酸核糖聚合酶(Cleaved-PARP)、活化的含半胱氨酸的天冬氨酸水解酶3(Cleaved-Caspase3)、细胞周期相关蛋白周期素B1(Cyclin-B1)以及核心基因P53蛋白表达情况的影响。结果:共获得汉黄芩素-膀胱癌相关靶点77个,并筛选出核心靶点26个,包括细胞肿瘤抗原P53(TP53)、细胞周期检查点激酶(CHEK1)、Bcl-2及Caspase3等。GO功能富集分析共获得43个条目(P<0.01),其中生物过程20条、细胞组成9条、分子功能13条。KEGG通路富集分析确定了24个具有代表性的信号通路(P<0.01),结果包括癌症通路、蛋白多糖与癌症、磷酸肌醇-3-激酶-蛋白激酶B(PI3K-AKT)信号通路、凋亡、P53信号通路、丝裂原活化蛋白激酶(MAPK)信号通路、细胞周期等。汉黄芩素对膀胱癌细胞活性具有抑制作用,呈时间及浓度依赖。通过流式细胞术及Hoechst 33342染色提示汉黄芩素可介导膀胱癌细胞凋亡。流式细胞术结果显示细胞周期阻滞在G2/M期。Western blot显示与对照组相比汉黄芩素组P53、Bax、Cleaved-Caspase3、Cleaved-PARP蛋白表达显著升高,Cyclin-B1、Bcl-2蛋白表达显著下降(P<0.05或P<0.01)。结论:汉黄芩素可通过促进凋亡和细胞周期阻滞抑制膀胱癌细胞活性。

Objective: To explore the mechanism of Wogonin in inhibiting the activity of bladder cancer cells. Methods: Potential targets of Wogonin were screened using TCMSP and Swiss Target Prediction databases. Bladder cancer disease targets were obtained from GeneCards, DisGeNET and OMIM databases. Protein-protein interaction(PPI) networks of target interactions were obtained through STRING online analysis platform and Cytoscape 3.8.2 plugin. Subsequently, gene ontology(GO) enrichment analysis and Kyoto Encyclopedia of genes and Genomes(KEGG) pathway enrichment analysis were performed on the Metascape online platform, and the network diagram of “drug-disease-core target-pathway” was constructed by Cytoscape 3.8.2 software. The effect of Wogonin against bladder cancer was observed by CCK-8 and clone formation experiment;the apoptosis of bladder cancer cells induced by Wogonin was detected by Hoechst 33342 staining;the apoptosis and cycle arrest induced by Wogonin were examined by flow cytometry;the effect of Wogonin on the expression of apoptosis-related proteins Bax, Bcl-2, Cleaved-PARP and Cleaved-caspase3, cell cycle-related protein Cyclin-B1 and P53 was examined by protein immunoblotting(Western blot). Results: A total of 77 Wogonin-bladder cancer-related targets were obtained, and 26 core targets were screened, including TP53, CHEK1, Bcl-2 and Caspase3, etc. GO functional enrichment analysis yielded a total of 43 entries(P<0.01), of which 20 were for biological processes, 9 for cellular composition and 13 for molecular functions. KEGG pathway enrichment analysis identified 24 representative signaling pathways(P<0.01), including intra-cancer pathway, intra-cancer proteoglycan, PI3 K-AKT signaling pathway, apoptosis, P53 signaling pathway, MAPK signaling pathway and cell cycle. Wogonin inhibited the activity of bladder cancer cells in a time-and concentration-dependent manner. Flow cytometry and Hoechst 33342 staining suggested that Wogonin could mediate the apoptosis in bladder cancer cells. Flow cytometry results showed that Wogonin mediated the cell cycle arrest at G2/M phase in bladder cancer cells. Western blot showed that P53, Bax, Cleaved-Caspase3 and Cleaved-PARP protein expression was up-regulated, and Cyclin-B1 and Bcl-2 protein expression was down-regulated in Wogonin group when compared with the control group(P<0.05 or P<0.01). Conclusion: Wogonin inhibits bladder cancer cell activity by promoting the apoptosis and cell cycle blockade.