二肽激肽酶4抑制剂对帕金森病模型细胞形态和增殖的干预作用及其机制

Intervention effects of DPP-4 inhibitors on morphology and proliferation of model cells with Parkinson's disease and the mechanism

目的基于体外实验及网络药理学,初步探讨二肽激肽酶4(DPP-4)抑制剂对鱼藤酮(ROT)诱导神经细胞帕金森病(PD)的干预作用及其相关机制。方法神经细胞株PC-12分为对照组、模型组、模型+西格列汀组、模型+利格列汀组、模型+维格列汀组,除对照组外各组均使用ROT建立PD体外模型,模型+西格列汀组、模型+利格列汀组、模型+维格列汀组在使用ROT建模的同时在培养基中分别加入DPP-4抑制剂西格列汀、利格列汀、维格列汀。倒置显微镜观察各组细胞形态学变化,CCK-8法观察各组细胞增殖能力。从Swiss Target Prediction、SEA等数据库分别获取DPP-4抑制剂西格列汀、利格列汀、维格列汀、沙格列汀及阿格列汀的药物靶点,通过DisGeNet、OMIM及GeneCards等数据库获取PD相关的疾病靶点,利用韦恩图将药物与疾病靶点取交集得到DPP-4抑制剂作用于PD的相关靶点。通过String数据库构建蛋白—蛋白相互作用(PPI)网络,选取基因满足度(Degree)值>平均值的基因作为DPP-4抑制剂对PD产生作用的关键靶点,将DPP-4抑制剂作用于PD的关键靶点基因输入到DAVID数据库进行GO基因功能和KEGG作用通路分析。将DPP-4抑制剂治疗PD的关键靶点以及KEGG信号通路导入到Cytoscape 3.7.2软件构建药物—疾病—靶点—通路网络,筛选与PD相关信号通路关系最为密切的靶点作为DPP-4抑制剂治疗PD的核心靶点,采用CB-Dock 2对接平台进行核心靶点的分子对接验证。结果对照组细胞形态完整,增长状态良好,细胞呈多角形,细胞之间类似突触样连接,且突触较长;模型组细胞密度减少,细胞皱缩,细胞与细胞间的突触样连接断裂,且观察到较多圆形的损伤细胞;模型+西格列汀组、模型+利格列汀组、模型+维格列汀组较模型组细胞形态得到改善,恢复到正常状态下的细长、多角形态。模型组细胞存活率低于对照组、模型+西格列汀组、模型+利格列汀组、模型+维格列汀组(P均<0.05)。共收集西格列汀药物靶点486个、利格列汀药物靶点665个、维格列汀药物靶点524个、沙格列汀药物靶点507个、阿格列汀药物靶点408个,PD疾病相关靶点1121个;将DPP-4抑制剂靶点与PD疾病相关靶点取交集共得到DPP-4抑制剂作用于PD的36个相关靶点。PPI网络显示,DPP-4抑制剂作用于PD相关靶点中Degree值>平均值的关键靶点共有16个,分别为ALB、IGF1、STAT3、CASP3、EGFR、ESR1、MAPK14、PPARG、MAPK1、HMOX1、NOS3、REN、MMP3、IL2、AR、IGF1R。GO分析结果显示,DPP-4抑制剂作用于PD的关键靶点共同涉及的生物过程主要包括信号转导、细胞迁移的正向调控和细胞凋亡的负向调控等,细胞组分主要包括细胞质、细胞质膜、细胞核等,分子功能主要包括酶结合、蛋白质结合、蛋白丝氨酸/苏氨酸/酪氨酸激酶活性等。KEGG通路富集分析共得到20条信号通路,与PD较为相关的信号通路为PI3K-AKT信号通路、FoxO信号通路、MAPK信号通路。药物—疾病—靶点—通路网络图显示,在PI3K-AKT、FoxO及MAPK信号通路中均有富集的靶点为IGF1、EGFR、IGF1R及MAPK1。分子对接结果显示,5种DPP-4抑制剂均与PD相关核心靶点蛋白有较好的结合,其结合能均小于-5.0 kcal/mol。结论DPP-4抑制剂对PD体外模型细胞具有积极地干预作用,其可能通过IGF1、EGFR、IGF1R及MAPK1等核心靶点作用于PI3K-AKT、FoxO、MAPK等信号通路来发挥作用。

Objective To investigate the protective effects of DPP-4 inhibitors against rotenone-induced neuronal Parkinson's disease and its associated mechanism based on in vitro experiments and network pharmacology.Methods The neural cell line PC-12 was divided into the control group,model group,model+sitagliptin group,model+linagliptin group,and model+vildagliptin group,respectively.After 16-18 h,the PD in vitro models were established using ROT in all groups except the control group.In the model+sitagliptin,model+linagliptin,and model+vildagliptin groups,ROT was added along with sitagliptin,linagliptin,and vildagliptin for intervention.The morphological changes of the cells in each group were observed using the inverted microscope,and the proliferative ability of the cells in each group was ob⁃served by CCK-8.The drug targets of DPP-4 inhibitors,sitagliptin,linagliptin,vildagliptin,saxagliptin and alogliptin,were obtained from Swiss Target Prediction,SEA,Super-Pred,and PharmMapper databases,respectively.PD-related dis⁃ease targets were obtained from DisGeNet,OMIM,and GeneCards databases.A visualization of the common targets of DPP-4 inhibitors acting on PD was obtained from the Venn diagram database.The protein-protein interaction(PPI)net⁃work was constructed through the String database,and the genes with degree values>the average were selected as the key targets of DPP-4 inhibitors on PD,and the key target genes were input into the DAVID database for the analysis of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway.The key targets of DPP-4 inhibitors for PD treatment and KEGG signaling pathway were imported into Cytoscape 3.7.2 software to construct a drug-disease-targetpathway network,and the targets most closely related to PD-related signaling pathways were screened as the core targets of DPP-4 inhibitors for PD treatment.The CB-Dock 2 docking platform was used to validate the molecular docking of the core target.Results The cells in the control group were morphologically intact,with good growth status,and the cells were in a polygonal state,with synapse-like connections between the cells and long synapses.In the model group,cell density was reduced,cells became wrinkled,synapse-like connections between cells were broken,and more rounded damaged cells were observed.The cell morphology of the model+sitagliptin,model+linagliptin and model+vildagliptin groups was im⁃proved compared to the model group and returned to the elongated and polygonal morphology as in the normal state.The cell survival rate in the model group was lower than that in the control,model+sitagliptin,model+linagliptin,and model+vildagliptin groups(all P<0.05).We collected a total of 486 sitagliptin drug targets,665 linagliptin drug targets,524 vildagliptin drug targets,507 saxagliptin drug targets,408 alogliptin drug targets,and 1121 PD disease targets.We took the intersection of DPP-4 inhibitor targets with PD disease-related targets to obtain 36 DPP-4 inhibitor-associated targets acting in PD.The PPI network showed that DPP-4 inhibitors acted on a total of 16 key PD targets with greater degree val⁃ues than the average,namely ALB,IGF1,STAT3,CASP3,EGFR,ESR1,MAPK14,PPARG,MAPK1,HMOX1,NOS3,REN,MMP3,IL2,AR,and IGF1R.GO analysis showed that the biological processes involved in the key targets of DPP-4 inhibitors acting on PD mainly included signal transduction,positive regulation of cell migration and negative reg⁃ulation of apoptosis,etc.,the cellular components mainly included cytoplasm,cytoplasmic membrane and nucleus,etc.,and the molecular functions mainly included enzyme binding,protein binding,and protein serine/threonine/tyrosine ki⁃nase activity.KEGG pathway enrichment analysis resulted in a total of 20 signaling pathways,and the signaling pathways more relevant to PD were PI3K-Akt signaling pathway,the FoxO signaling pathway,and MAPK signaling pathway.The drug-disease-target-pathway network map showed that the targets enriched in the PI3K-Akt,FoxO,and MAPK signaling pathways were IGF1,EGFR,IGF1R,and MAPK1.The molecular docking results demonstrated that all five DPP-4 inhibi⁃tors bound well to the PD-associated core target proteins,and their binding energies were all less than-5.0 kcal/mol.Conclusion DPP-4 inhibitors were protective in the in vitro models of PD,possibly exerting their protective effects through core targets such as IGF1,EGFR,IGF1R,and MAPK1 acting on signaling pathways such as PI3K-Akt,FoxO,and MAPK,etc.