基于网络药理学和体内实验探究七福饮治疗2型糖尿病认知障碍的分子机制

Molecular Mechanism of Qifu Decoction(七福饮)in the Treatment of Cognitive Impairment in Type 2 Diabetes:Based on Network Pharmacology and In Vivo Experiment

目的:通过网络药理学探讨七福饮改善2型糖尿病(Type 2 Diabetes,T2DM)认知障碍的作用机制,并通过动物体内实验验证分析。方法:通过TCMSP平台和文献研究的方法筛选七福饮活性成分,在Swisstarget平台和TCMSP平台预测出药物活性成分对应靶点,在GeneCard、OMIM等数据库检索T2DM认知障碍靶点。利用韦恩图得到药物-疾病交集靶点,用Cytoscape3.7.2软件分别构建“中药-活性成分-靶点”作用网络及PPI网络。用R语言对交集靶点进行GO和KEGG分析。结合网络药理学分析结果,采用链脲佐菌素STZ 30 mg/kg联合高脂高糖诱导建立T2DM认知障碍大鼠模型,验证七福饮改善T2DM认知障碍可能的分子机制。结果:确定七福饮治疗T2DM认知障碍的活性成分221个,治疗靶点561个。PPI网络拓扑分析筛选得到30个核心靶点,其中包括蛋白激酶(AKT1)、肿瘤坏死因子(TNF)、白介素6(IL-6)、半胱氨酸天冬氨酸蛋白酶3(Caspase3)等。GO分析表明靶点主要涉及营养水平反应、细胞钙离子稳态等多种生物过程;KEGG分析提示AGE-RAGE糖尿病并发症信号通路在七福饮治疗T2DM认知障碍过程中起着关键作用。动物实验表明,与正常对照组相比,模型对照组血糖含量、逃避潜伏期和海马DG区细胞凋亡数明显升高、p-P38、p-JNK、BAX、Caspase3蛋白表达明显上调,穿越平台次数及Bcl-2蛋白表达明显降低(P<0.05或P<0.01);与模型对照组相比,七福饮4.3、8.6 g/kg组能明显降低血糖含量、降低逃避潜伏期和海马DG区细胞凋亡数、明显下调p-P38、p-JNK、BAX、Caspase3蛋白表达,明显上调穿越平台次数及Bcl-2蛋白表达(P<0.05或P<0.01)。结论:提示七福饮可能通过调节AGE-RAGE信号通路上相关蛋白表达改善T2DM认知障碍,这些结果可为七福饮的临床应用提供理论基础和依据。

Objective:To investigate the mechanism of Qifu Decoction(七福饮)(QFD)in alleviating cognitive impairment in type 2 diabetes(T2 D)through network pharmacology and to verify the mechanism through in vivo animal experiment.Methods:The active components of QFD were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and previous research,and the corresponding targets were predicted by SwissTargetPrediction and TCMSP.Targets related to the cognitive impairment in T2 D were searched from GeneCard,Online Mendelian Inheritance in Man(OMIM),and other databases.The common targets of the disease and the decoction were yielded according to Venn diagram.The“Chinese medicinal-active component-target”network and protein-protein interaction(PPI)network were constructed by Cytoscape 3.7.2,respectively,followed by the Gene Ontology(GO)term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment of the common targets by R language.As for the in vivo animal experiment,streptozotocin(STZ,30 mg/kg)was combined with high-fat and high-sugar diet to induce T2 D with cognitive impairment in rats.Results:A total of 221 active components and 561 therapeutic targets of QFD for the treatment of cognitive impairment in T2 D were retrieved,and 30 core targets were screened out based on PPI network,including protein kinase(AKT1),tumor necrosis factor(TNF),interleukin-6(IL-6),and cysteine-aspartate protease 3(CASP3).The targets were mainly involved in the GO terms of response to nutrient level and cellular calcium homeostasis.KEGG analysis suggested that AGE-RAGE signaling pathway played a key role in treatment of cognitive impairment in T2 D.As to the animal experiment,the blood glucose level,escape latency,apoptosis level in hippocampal DG region,and expression of p-P38,p-JNK,Bax,CASP3 were improved,and the times of crossing the platform and Bcl-2 expression were reduced in the model group compared with those in the blank control group(P<0.05 or P<0.01).Compared with the model group,QFD reduced blood glucose level,escape latency,apoptosis level in hippocampal DG area,and the expression of p-P38,p-JNK,Bax,and CASP3,and increased the times of crossing the platform and Bcl-2 expression(P<0.05 or P<0.01).Conclusion:QFD relieves cognitive impairment in T2 D by regulating the expression of related proteins in the AGE-RAGE signaling pathway.These results can lay a theoretical basis for the clinical application of QFD.