基于UHPLC-MS/MS、网络药理学、分子对接及体内外实验探究藏药“解吉那保”抗肝癌机制

Mechanism of Tibetan Medicine “Jiejinabao” against Hepatocellular Carcinoma Based on UHPLC?MS/MS, Network Pharmacology, Molecular Docking, and Experimental Validation in vivo and in vitro

关键词:  解吉那保  肝癌  PI3K/AKT通路  UHPLC-MS/MS  网络药理学  分子对接

Key words:  Jiejinabao  hepatocellular carcinoma  PI3K/AKT pathway  UHPLC-MS/MS  network pharmacology  molecular docking

摘要:

目的 应用UHPLC-MS/MS、网络药理学、分子对接及实验掲示藏药解吉那保(Gentiana crassicaulis Duthie ex Burk,GC)抗肝癌的活性及作用机制。方法 采用UHPLC-MS/MS联用技术,鉴定GC的化学物质及种类；通过TCMSP、TCM-ID、HERB、HIT 2.0及SymMap数据库筛选GC活性成分,应用Swiss Target prediction获取其潜在靶点；利用GeneCards、OMIM数据库筛选HCC靶点基因信息。使用Cytoscape 3.9.1软件构建“药物-成分-作用靶点”网络并筛选核心成分；应用STRING数据库构建交集靶点PPI网络,筛选核心作用靶点；利用DAVID数据库进行GO及KEGG富集分析。采用Chembio3D、AutodockTools-1.5.6及PyMOL 3.1.0软件进行分子对接,确定GC与枢纽靶点的对接稳定性。通过CCK-8、Transwell侵袭、细胞划痕愈合和流式细胞术检测GC对肝癌细胞(Hep-G2)活力、侵袭、迁移和凋亡、周期的影响；建立H22-LUC皮下移植瘤小鼠模型,通过动物活体成像技术、HE染色、免疫组化评估GC体内抗HCC小鼠作用；最后qPCR及免疫印迹法验证GC的抗肿瘤作用及分子机制。结果 鉴定及筛选出GC主要以Hydroxyevodiamine、Rutaecarpine、Diosgenin等5个活性成分为主,STAT3、PI3KCA和AKT1等10个核心作用靶点,PI3K/AKT为关键通路；分子对接表明PIK3CA、AKT1、BAX、Bcl-2和CDK4与Rutaecarpine、Diosgenin、Oleanolic Acid、Evodiamine具有较强的结合活性。体外实验表明GC抑制了肝癌细胞的增殖、侵袭、迁移作用,并通过诱导细胞凋亡、阻滞细胞周期来发挥抗肝癌作用。体内实验显示,GC可有效降低肿瘤荧光强度、重量和体积,抑制肿瘤细胞Ki-67增殖,且表现出显著的体内安全性；瘤组织内BAX和Cleaved caspase3免疫染色阳性面积显著升高(P< 0.001),伴随BCL-2、Cyclin D1及P-AKT显著降低(P< 0.001)。同时,在体内外检测均显示GC上调Caspase-3和Bax基因、蛋白,下调BCL-2、CDK4、Cyclin D1表达,降低P-PI3K/PI3K和P-AKT/AKT蛋白比值。结论 GC可通过抑制PI3K/AKT信号通路,诱导细胞凋亡和周期阻滞,从而发挥抗肝癌作用。

Abstract:

ABSTRACT: OBJECTIVE To reveal the active components and mechanism of the Tibetan medicine Jiejinabao (Gentiana crassicaulis Duthie ex Burk, GC) against hepatocellular carcinoma using UHPLC-MS/MS, network pharmacology, molecular docking, and experimental validation. METHODS UHPLC-MS/MS was employed to identify the chemical substances and types of GC. Active components of GC were screened through TCMSP, TCM-ID, HERB, HIT 2.0, and SymMap databases, and potential targets were obtained using Swiss Target Prediction. HCC target gene information was screened via GeneCards and OMIM databases. A "drug-component-target" network was constructed using Cytoscape 3.9.1 software to identify core components. The STRING database was used to construct a PPI network of intersecting targets and screen core targets. GO and KEGG enrichment analyses were performed using the DAVID database. Molecular docking was performed using AutoDock and PyMOL to determine the binding stability between GC and hub targets. The effects of GC on the viability, proliferation, invasion, migration, apoptosis, and cell cycle of hepatocellular carcinoma cells (Hep-G2) were assessed via CCK-8, colony formation, Transwell invasion, wound healing, and flow cytometry. A subcutaneous H22-LUC transplanted tumor mouse model was established, and the in vivo anti-HCC effects of GC were evaluated using in vivo animal imaging, HE staining, and immunohistochemistry. Finally, qPCR and Western blotting were used to confirm the anti-tumor effects and molecular mechanisms of GC. RESULT Five active components, primarily Hydroxyevodiamine, Rutaecarpine, and Diosgenin, were identified and screened from GC, along with ten core targets including STAT3, PIK3CA, and AKT1, with PI3K/AKT identified as the key pathway. Molecular docking indicated strong binding activity of PIK3CA, AKT1, BAX, Bcl-2, and CDK4 with Rutaecarpine, Diosgenin, Oleanolic Acid, and Evodiamine. In vitro experiments showed that GC reduced the proliferation, invasion, and migration of hepatocellular carcinoma cells and exerted anti-hepatocellular carcinoma effects by inducing apoptosis and cell cycle blockade. In vivo experiments demonstrated that GC effectively reduced tumor fluorescence intensity, weight, and volume, inhibited Ki-67 proliferation in tumor cells, and was well tolerated in vivo. The positive staining areas of BAX and Cleaved caspase3 were significantly increased (P < 0.0001), accompanied by significant decreases in BCL-2, Cyclin D1, and P-AKT (P < 0.0001). Additionally, both in vivo and in vitro assays showed that GC upregulated Caspase-3 and Bax genes and proteins, downregulated BCL-2, CDK4, and Cyclin D1 expression, and reduced the p-PI3K/PI3K and p-AKT/AKT protein ratios. CONCLUSION GC can exert anti-hepatocellular carcinoma effects by inhibiting the PI3K/AKT signaling pathway, inducing apoptosis and cell cycle blockade.