网络药理学方法联合体外实验初探金水六君煎辅助放化疗治疗恶性黑色素瘤的分子机制

Network pharmacological combined with in vitro experimental to explore the molecular mechanism of Jinshui Liujun Decoction in adjuvant radiotherapy for malignant melanoma

目的网络药理学方法联合体外实验初步探索金水六君煎辅助放化疗治疗恶性黑色素瘤(malignant melanoma,MM)的分子机制。方法在TCMSP数据库中获取金水六君煎的活性成分及靶点,以“malignant melanoma”为关键词在GeneCards、TTD、OMIM数据库中获得MM相关靶点,得到金水六君煎有效成分-MM交集靶点;通过STRING数据库获取蛋白质互作(PPI)网络,利用Metascape数据库进行基因本体论分析(GO)和基因组的京都基因与基因组百科全书富集分析(KEGG)并在AutoDockTools软件中进行分子对接;MTT法检测金水六君煎含药血清对小鼠B16黑色素瘤细胞增殖活性影响;蛋白免疫印迹法(Western blotting)检测金水六君煎含药血清对P38、p-P38、JNK、p-JNK、Beclin1、P62、LC3蛋白表达调控;透射电镜观察金水六君煎含药血清对B16细胞自噬影响。结果共获得金水六君煎相关靶点248个,MM相关靶点2147个,交集靶点149个。GO显示主要生物过程是DNA结合转录因子、激酶结合、细胞因子受体结合等;KEGG显示主要是癌症通路和MAPK信号通路,网络拓扑分析显示金水六君煎治疗MM的主要有效活性成分是槲皮素、山奈酚、柚皮素等,主要核心靶点为AKT1、TP53、JUN等;核心靶点与有效活性成分分子对接显示结合活性较好。MTT结果显示,与空白组比较,金水六君煎含药血清能有效抑制B16细胞的增殖且呈浓度依赖性。Western blotting实验表明,较空白组,中高剂量组能下调B16细胞中p-P38、p-JNK、P62蛋白表达,上调LC3Ⅱ、LC3Ⅱ/LC3Ⅰ、Beclin1蛋白表达。透射电镜下显示金水六君煎含药血清能促进B16细胞中自噬体生成,减少黑素体生成。结论金水六君煎的有效成分和药物靶点很多,其治疗MM的作用靶点与通路众多,分子机制可能是通过下调p-P38、p-JNK、P62的蛋白表达,上调Beclin1、LC3Ⅱ/LC3Ⅰ的蛋白表达,抑制P38通路和JNK通路的激活,从而抑制B16细胞增殖;同时促进细胞凋亡,激活细胞自噬,发挥抑癌作用。

Objective To explore the molecular mechanism of Jinshui Liujun Decoction in the treatment of malignant melanoma(MM)with adjuvant radiotherapy in combination with in vitro experiments.Methods The active ingredients and targets of Jinshui Liujun Decoction were obtained from TCMSP database,and the MM-related targets were obtained from GeneCards,TTD and OMIM databases with"malignant melanoma"as the keywords,so as to obtain the intersection targets of active ingredients and MM of Jinshui Liujun Decoction.The protein-protein interactions(PPI),network was obtained through STRING database,the gene ontology analysis(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were conducted using Metascape database,and molecular docking was performed in AutoDockTools software;MTT method was used to detect the effect of Jinshui Liujun Decoction-containing serum on the proliferative activity of mouse B16 melanoma cells;Western blotting was used to detect the effect of Jinshui Liujun Decoction-containing serum on the regulation of protein expression of P38,p-P38,JNK,p-JNK,Beclin1,P62,and LC3;and the effect of Jinshui Liujun Decoction-containing serum on autophagy of B16 cells was observed by transmission electron microscopy.Results A total of 248 targets related to Jinshui Liujun Decoction,2147 targets related to MM,and 149 intersecting targets were obtained;GO showed that the main biological processes were DNA binding to transcription factors,kinase binding,cytokine receptor binding,etc.;KEGG showed that it was mainly the cancer pathway and the MAPK signaling pathway;and the topology analysis of the network showed that quercetin was the main active ingredient in the treatment of MM by Jinshui Liujun Decoction,The network topology analysis showed that the main active ingredients of Jinshui Liujun Decoction for MM were quercetin,kaempferol,naringenin,etc.,and the main core targets were AKT1,TP53,JUN,etc.Docking between the core targets and the active ingredients showed that the binding activity was better.The MTT results showed that,compared with the blank group,the serum of the Jinshui Liujun Decoction containing the drug could inhibit the proliferation of B16 cells in a concentration-dependent manner.Western blotting showed that compared with the blank group,the medium and high dose groups could down-regulate the expressions of p-P38,p-JNK and P62 proteins,and up-regulate the expressions of LC3II,LC3II/LC3Ⅰand Beclin1 proteins in B16 cells.Transmission electron microscopy showed that the serum containing Jinshui Liujun Decoction promoted autophagy and reduced melanosome production in B16 cells.Conclusion Jinshui Liujun Decoction has many active ingredients and drug targets,and its targets and pathways for the treatment of MM are numerous.The molecular mechanism may be to inhibit the activation of the P38 pathway and JNK pathway by down-regulating the protein expression of p-P38,p-JNK,and P62,and up-regulating the protein expression of Beclin1,and LC3Ⅱ/LC3Ⅰ,thus inhibiting the proliferation of B16 cells;at the same time,it promotes apoptosis,activates cell autophagy,and plays a role in inhibiting the growth of melanosomes.