益气补肾调脂方通过调控CDKN1A改善非酒精性脂肪性肝炎的研究

Study on improvement of non-alcoholic steatohepatitis by regulating CDKN1A by Yiqi-Bushen-Tiaozhi formula

目的网络药理学筛选益气补肾调脂方(Yiqi-Bushen-Tiaozhi formula,YBTF)治疗非酒精性脂肪性肝炎(non-alcoholic steatohepatitis,NASH)的机制;体内实验完成验证。方法TCMSP数据库、HPLC-MS明确YBTF成分、靶点;GSE89632数据集,筛选NASH与正常组差异基因;GeneCards、Disgenet数据库筛选疾病基因。交集为YBTF治疗NASH的靶基因。基因本体论(Gene Ontology,GO)、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)、疾病本体论(Disease Ontology,DO)富集分析,蛋白互作网络(protein-protein interaction,PPI)、拓扑分析,明确治疗NASH关键基因。分子对接(molecular docking)预测活性成分、靶点能否结合,发挥疗效;油红O、HE染色检测肝脏脂肪变、炎症;β-半乳糖苷酶(SA-β-Gal)检测肝细胞衰老;荧光定量PCR(Quantitative Real-time PCR,qRT-PCR),蛋白质免疫印迹法验证靶基因mRNA、蛋白表达。结果YBTF可调控周期依赖性激酶抑制剂1A(cyclin dependent kinase inhibitor 1A,CDKN1A)改善细胞衰老,治疗NASH。结论网络药理学结合实验可为研究YBTF治疗NASH的机制提供新的思路。

Aim To screen the mechanism of Yiqi-Bushen-Tiaozhi formula(YBTF)in treating non-alcoholic steatohepatitis(NASH)by network pharmacology analysis and to verify it by animal experiments.Methods TCMSP database and HPLC-MS analysis were used to mine the active ingredients and targets of YBTF;GSE89632 dataset was used to screen the differential expressed genes(DEGs)between the normal and the NASH groups;GeneCards and DisGeNET databases were used to screen NASH-related disease genes.The intersection genes of the three are the target genes of YBTF treatment of NASH.The intersection gene of the three sets of genes was the target gene of YBTF in treating NASH.GO,KEGG,DO enrichment analysis,protein-protein interaction network,and network topology analysis were used to identify the hub genes of YBTF in the treatment of NASH.Molecular docking was used to judge whether crucial target genes,active ingredients could be combined and exerted a curative effect;Oil red O and HE staining were used to determine whether YBTF could treat NASH mice;β-galactosidase(SA-β-Gal)test was used to determine whether NASH mice had hepatocyte senescence and whether YBTF improved senescence;Western blot,Quantitative Real-time PCR(qRT-PCR)combined with sequencing results were used to verify whether YBTF could regulate the expression of the essential target genes screened from the protein and RNA levels.Results YBTF could improve cellular aging and treat NASH through CDKN1A.Conclusion The rational application of Traditional Chinese medicine(TCM)network pharmacology and experiments can provide new ideas and directions for studying the mechanism of YBTF.