探究肉苁蓉水提物(the aqueous extract of Cistanche tubulosa,AECT)化学成分和AECT治疗糖尿病肾病(dia-betic nephropathy,DN)的作用机制.利用UPLC-MS/MS对化合物进行分析鉴定;借助PubChem、GeneCards、OMIM、DAVID等数据库预测有效成分防治DN的作用靶点及通路,对核心基因进行GO、KEGG富集分析,并使用Cytoscape软件构建"成分-靶点-通路"可视化网络图.MTT法测定AECT的抗DN活性;生化试剂盒检测过氧化指标超氧化物歧化酶(superoxide dismutase,SOD)活性和谷胱甘肽过氧化物酶(glutathione peroxide,GSH-Px)含量水平;ELISA法检测炎症因子白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子(tumor necrosis factor-a,TNF-a)和转化生长因子(transforming growth factor-β,TGF-β)水平;Annexin V-FITC/PI 法检测细胞凋亡率;Western blot 检测相关蛋白的表达.从AECT中共鉴定出84个化学成分,并推测出15个化合物可能的裂解途径;筛选出AECT治疗DN的70个潜在靶点和多个信号通路.体外实验发现AECT能够增加高糖高脂环境中的HK-2细胞存活率抑制细胞凋亡;并上调SOD活性和GSH-Px含量,抑制炎症因子IL-1β、TNF-a和TGF-β表达水平;Western blot结果证明AECT影响PI3K-AKT信号通路.本研究初步探明了 AECT中的主要有效成分及其治疗DN的潜在靶点与作用机制,为AECT治疗DN后续的深入研究提供思路.
Mechanism of Cistanche tubulosa water extract in the treatment of diabetic nephropathy based on UPLC-QE-Orbitrap-MS/MS and network pharmacology
To investigate the chemical composition of the aqueous extract of Cistanche tubulosa(AECT)and the mechanism of action of AECT in the treatment of diabetic nephropathy(DN),UPLC-MS/MS was used to analyse and identify the com-pounds,Using PubChem,GeneCards,OMIM,DAVID and other databases,we predicted the targets and pathways of the com-pounds to prevent and control DN,performed GO and KEGG enrichment analysis on the core genes,and constructed a visual'compound-target-pathway'network diagram using Cytoscape software.Determination of Anti-DN Activity of AECT by MTT Method,Biochemical kits were used to detect the activity of superoxide dismutase(SOD)and the levels of glutathione peroxidase(GSH-Px).ELISA was used to measure the levels of inflammatory factors interleukin-1β(IL-1β),tumour necrosis factor(TNF-α),and transforming growth factor(TGF-β).Detection of apoptosis was performed using the Annexin V-FITC/PI assay.Western blot was performed to detect the expression of relevant proteins.AECT contained 84 chemical components,with 15 compounds identified as possible cleavage pathways.Screening 70 potential targets and multiple signaling pathways of AECT for the treatment of DN.The results of the in vitro experiments showed that AECT increased the survival of HK-2 cells and inhibited apoptosis in a high-sugar and high-fat environment,And SOD activity and GSH-Px content were up-regulated,while the expression levels of inflammatory factors IL-1β,TNF-α and TGF-β were inhibited.The Western blot results indicate that AECT has an impact on the PI3K-AKT signaling pathway.This study investigates the potential targets and mechanisms of action of the main active ingredients in AECT for the treatment of DN.The findings suggest avenues for further research into the use of AECT for DN treatment.
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