通络糖泰方治疗糖尿病周围神经病变作用机制的网络药理学和分子对接分析

Network pharmacology and molecular docking analysis on mechanism of Tongluo Tangtai Power in treatment of diabetic peripheral neuropathy

目的:利用网络药理学筛选通络糖泰方(TLTT)治疗糖尿病周围神经病变(DPN)的活性成分,预测作用靶点及信号通路,探讨其作用机制。方法:采用中药系统药理学分析平台(TCMSP)收集TLTT的主要化学成分及作用靶点;利用基因卡片(Genecards)数据库检索与DPN相关的蛋白;利用STRING数据库和网络可视化软件Cytoscape3.9. 1构建蛋白-蛋白互作(PPI)网络;通过京都基因与基因组百科全书(KEGG)和分子对接技术对靶点进行通路富集分析及成分-靶点对接。将30只SD大鼠随机分为对照组、弥可保组和TLTT组,每组10只,给予弥可保和TLTT灌胃制备含药血清;复苏1株RSC96雪旺细胞,设置对照组、高糖模型组、弥可保组和TLTT组,采用细胞计数检测法观察各组细胞存活率,Western blotting法检测各组细胞中丝氨酸/苏氨酸激酶1(AKT1)和抑癌基因TP53蛋白表达水平。结果:TLTT治疗DPN的有效化合物成分95个,463个靶点,DPN相关蛋白1 187个,137个药物-疾病共同靶点;基因本体论(GO)分析中生物过程、细胞组成和分子功能分别得到5 505个、466个和791个结果;KEGG分析得到253个结果,提示TLTT可能通过调节磷脂酰肌醇3激酶/蛋白激酶B (PI3K-Akt)信号通路、液体剪切应力和动脉粥样硬化、晚期糖基化终末产物及其受体(AGE-RAGE)和丝裂原活化蛋白激酶(MAPK)等信号通路发挥治疗作用;分子对接分析,TLTT通过槲皮素、豆甾醇、β-谷甾醇、山柰酚和常春藤皂苷元等核心成分,作用于AKT1、TP53、肿瘤坏死因子(TNF)和表皮生长因子受体(EGFR)等核心靶点。CCK-8法检测,与对照组比较,高糖模型组细胞存活率明显降低(P<0.05);与高糖模型组比较,TLTT组细胞存活率明显升高(P<0.05)。Western blotting法,与对照组比较,高糖模型组细胞中AKT1蛋白表达水平明显降低(P<0.05), TP53蛋白表达水平明显升高(P<0.05);与高糖模型组比较,TLTT组细胞中AKT1蛋白表达水平明显升高(P<0.05), TP53蛋白表达水平明显降低(P<0.05)。结论:TLTT的主要活性成分槲皮素等可能通过靶向PI3K-Akt等信号通路,促进雪旺细胞存活,升高AKT1蛋白和降低TP53蛋白表达水平,进而发挥治疗DPN的作用。

Objective:To screen the active components of Tongluo Tangtai Power(TLTT)in the treatment of diabetic peripheral neuropathy(DPN),predict the action targets and signaling pathways by network pharmacology,and to explore its mechanism.Methods:The Traditional Chinese Medicine System Pharmacological Analysis Platform(TCMSP)was used to collect the main chemical components and action targets of TLTT;Genecard Database was used to retrieve the proteins associated with DPN;the protein-protein interaction(PPI)network was constructed by STRING Database and network visualization software Cytoscape3.9.1;pathway enrichment analysis and composition-target docking were analyzed by the Kyoto Encyclopedia of Genes and Genomes(KEGG)and molecular docking technique.Thirty SD rats were divided into control group,mecobalamin group,and TLTT group,and there were 10 rats in each group.The rats were given mecobalamin and TLTT by gavage to make the drug-containing serum;one stain of RSC96 Schwann cells was resuscitated;control group,high glucose model group,mecobalamin group,and TLTT group were set up;then cell counting method was used to detect the survival rates of Schwann cells in various groups;Western blotting method was used to detect the expression levels of serine/threonine kinase 1(AKT1)and tumor suppressor gene TP53 proteins in the cells in various groups.Results:There were 95 active compound components and 463 targets in TLTT for the treatment of DPN,1187 DPN-related proteins,and 137 drug-disease common targets.The Geno Ontology(GO)analysis,including biological processes,cell composition,and molecular function results obtained 5505,466,and 791 results;the KEGG analysis obtained 253 results,suggesting that TLTT may played a therapeutic role by regulating the phosphatidyl inositol 3 kinase/protein kinase B(PI3K-Akt)signaling pathway,liquid shear stress and atherosclerosis,advanced glycosylation end products and their receptors(AGE-RAGE),mitosolysis-activated protein kinase(MAPK)singling pathways and so on;the molecular docking results showed that TLTT acted on AKT1,TP53,tumor necrosis factor(TNF),epidermal growth factor receptor(EGFR),and other core targets by some core components such as quercetin,sterol,betasitosterol,kaempferol,ivy saponin,and so on.The CCK-8 results showed that compared with control group,the survival rate of the cells in high glucose model group was significantly decreased(P<0.05);compared with high glucose model group,the viability of the cells in TLTT group was significantly increased(P<0.05).The Western blotting results showed that compared with control group,the expression level of AKT1 the cells in high glucose model group was significantly decreased(P<0.05),and the expression level of TP53 protein was significantly increased(P<0.05);compared with high glucose model group,the expression level of AKT1 protein in the cells in TLTT group was significantly increased(P<0.05),and the expression level of TP53 protein was significantly decreased(P<0.05).Conclusion:The main active components of TLTT,such as quercetin,may promote the survival of the Schwann cells,increase the expression level of AKT1 protein and decrease the expression level of TP53 protein by targeting PI3K-Akt and other signaling pathways, so as to play a role in the treatment of DPN.