基于加权网络药理学探究归芪白术方治疗葡萄膜黑色素瘤的药效物质基础及分子机制

Exploring the pharmacological basis and molecular mechanisms of GQBZP treatment for uveal melanoma based on weighted network pharmacology

关键词:  归芪白术方  加权网络药理学  葡萄膜黑色素瘤  MEK

Key words:  GQBZP  weighted network pharmacology  uveal melanoma  MEK

摘要:

摘要：目的 基于加权网络药理学、分子对接、体外实验探究归芪白术方（Guiqi Baizhu Formula，GQBZP）治疗葡萄膜黑色素瘤（Uveal melanoma，UM）的药效物质基础及分子机制。方法 通过裸鼠荷瘤实验考察归芪白术方对UM发展的影响，网络药理学筛选GQBZP作用靶点与UM的交集关键靶点，根据君臣佐使配伍对其赋予权重，构建“药味-潜在活性成分-关键靶点”网络，对网络中的关键靶点进行GO与KEGG通路富集分析，并利用kmeans clustering对关键靶点聚类分析。对GQBZP成分与MEK进行分子对接与聚类分析其潜在活性成分，运用CCK8筛选潜在活性成分槲皮素、山奈酚、芦荟大黄素、大黄素、毛蕊异黄酮对UM细胞的有效安全浓度。通过Hoechst染色检测代表性成分槲皮素、山奈酚对UM细胞凋亡的影响，通过流式细胞术检测代表性成分槲皮素、山奈酚对UM细胞周期的影响。运用Western Blot检测代表性成分对MEK、p-MEK、ERK、p-ERK、Bax、Bcl-2、CDK2、CDK4的蛋白表达影响，RT-qPCR检测Bax、Bcl-2、CDK2、CDK4的mRNA表达影响。结果 归芪白术方可通过促进肿瘤细胞凋亡、抑制肿瘤细胞增殖延缓UM的进展。网络药理学分析发现MEK可能是GQBZP治疗UM的关键靶点，分子对接结果发现其潜在活性成分可能是槲皮素、山奈酚、芦荟大黄素、大黄素、毛蕊异黄酮。体外实验发现GQBZP代表性成分槲皮素（40 μmol·L-1）、山奈酚（20 μmol·L-1）可以抑制UM细胞活力（P<0.05），且对HEK293T细胞具有保护作用（P<0.05）。Hoechst染色发现槲皮素、山奈酚可以促进UM细胞的凋亡，流式细胞术发现，代表性成分使UM细胞周期阻滞在G0/G1期，Western Blot结果表示，槲皮素和山奈酚可使p-MEK/MEK、p-ERK/ERK、Bcl-2、CDK2、CDK4蛋白表达下降，Bax蛋白表达上升，RT-qPCR检测显示，槲皮素和山奈酚可使Bcl-2、CDK2、CDK4的mRNA水平下降，Bax的mRNA水平上升。结论 体内实验表明GQBZP可有效延缓UM的发展，其中GQBZP代表性成分槲皮素、山奈酚可通过靶向MEK靶点，抑制MEK的磷酸化，不仅可促进UM细胞凋亡，还可使UM细胞周期阻滞在G0/G1期，从而抑制UM细胞的活力。

Abstract:

ABSTRACT: OBJECTIVE The present study investigates the pharmacological basis and molecular mechanisms underlying the therapeutic effects of Guiqi Baizhu Formula (GQBZP) on uveal melanoma (UM) through weighted network pharmacology, molecular docking, and in vitro experiments. Methods: The present study examined the effects of the GQBZP on UM progression through the use of a nude mouse tumour-bearing model. A network pharmacology screening was conducted to identify key intersections between GQBZP and UM. The weights were assigned based on the sovereign-minister-assistant-messenger formula. A "herbal ingredient-potential active component-key target" network was constructed. GO and KEGG pathway enrichment analysis was performed on key targets within the network. K-means clustering was applied for target clustering. Molecular docking and clustering analysis were performed between GQBZP components and MEK to identify potential active constituents. CCK8 assays were utilised to screen effective and safe concentrations of quercetin, kaempferol, aloe emodin, rhein and paeoniflorin against UM cells. The effects of representative components quercetin and kaempferol on UM cell apoptosis were assessed by Hoechst staining. The impact of quercetin and kaempferol on the UM cell cycle was evaluated by means of flow cytometry. The effects of representative components on the protein expression of MEK, p-MEK, ERK, p-ERK, Bax, Bcl-2, CDK2, and CDK4 were detected by Western Blot analysis. RT-qPCR was utilised to detect the effects on the mRNA expression of Bax, Bcl-2, CDK2, and CDK4. RESULTS The GQBZP has been demonstrated to retard the advancement of UM by inducing tumour cell apoptosis and impeding tumour cell proliferation. Network pharmacology analysis suggests that MEK may be a key therapeutic target for GQBZP in treating UM. The molecular docking results indicate the potential active components may include quercetin, kaempferol, aloe-emodin, emodin, and isoflavone. In vitro experiments demonstrated that representative GQBZP components, quercetin (40 μmol·L?1) and kaempferol (20 μmol·L?1), inhibited UM cell viability (P<0.05) while exhibiting protective effects on HEK293T cells (P<0.05). The Hoechst staining method revealed that quercetin and kaempferol promoted apoptosis in UM cells. The results of the flow cytometry analysis demonstrated that the components in question induced UM cell cycle arrest at the G0/G1 phase. The results of the Western blot analysis indicated that quercetin and kaempferol were able to reduce the levels of p-MEK/MEK, p-ERK/ERK, Bcl-2, CDK2, and CDK4 protein expression, whilst increasing the level of Bax protein expression. RT-qPCR analysis demonstrated that quercetin and kaempferol led to a decrease in the mRNA levels of Bcl-2, CDK2, and CDK4, whilst concurrently increasing Bax mRNA levels. CONCLUSION In vivo experiments demonstrate that GQBZP effectively delays the progression of UM. Its representative components, quercetin and kaempferol, target the MEK pathway by inhibiting MEK phosphorylation. This has been demonstrated to promote apoptosis in UM cells and to cause cell cycle arrest at the G0/G1 phase, thereby suppressing UM cell viability.