The Research Of The Effect Of Physalis Calyx Seu Fructus Active Ingredient On The Acute Myeloid Leukemia Cells HL60 And Its Mechanism Based On Network Pharmacological

Acute myeloid leukemia（AML）is one of the most common types of leukaemia in adults today.It is caused by an abnormal clonal proliferation of primitive cells in the bone marrow,resulting in the failure of these primitive cells to develop normally into mature cells.Clinical treatment of AML mainly focus on radiotherapy,chemotherapy and the combination of multiple anti-cancer drugs,but relapse is common in patients with intermediate to high risk AML.It is well documented that a variety of herbal medicines have been effective in the treatment of leukaemia.Physalis Calyx seu Fructus（PCF）was first published in the Materia Medica,and has been found to be effective in relieving dampness and heat,relieving coughs and phlegm,etc.Modern pharmacology has also found that it has anti-infective and anti-inflammatory properties.Its active ingredient,β-sitosterol,is commonly used in the treatment of inflammatory disorders such as colitis and nephritis,etc.The pathogenesis of AML is closely related to inflammatory factors,but there are no studies on the effects of PCF and its active ingredients on AML,and the exact mechanism of action is unclear.Therefore,this project applied network pharmacology and molecular docking techniques to analyze the effects of the active ingredients of PCF in AML,and applied in vitro experiments to verify the effects of its active ingredientβ-sitosterol on AML cells HL60 and related mechanisms of action,providing a rational basis and experimental foundation for the treatment of AML with Chinese medicine.The bioactive components of PCF were screened based on pharmacokinetic parameters（oral bioavailability（OB）and drug-likeness（DL））from the TCMSP database and the potential targets were evaluated and retented using Swiss Target Prediction database;Online Mendelian Inheritance in Man（OMIM）database,Drug Bank database and Gene Cards database were used to search for AML-related genes,AML-related disease targets were removed duplicates and consolidated.the potential targets of PCF action on AML were obtained by Wayne analysis and they were imported into the STRING database to obtain the relevant file,Cytoscape3.7.2software was used to analyze the file for metric values,the PPI network of the target proteins were extracted,the PPI network were analyzed and the highest scoring targets were filtered out by using the Cyto Hubba plugin in the software;the specific mechanism of action was elucidated,the cross-targets were introduced into the DAVID database for GO function enrichment and KEGG signaling pathway enrichment analysis;the above active ingredients and target pathways were imported into Cytoscape3.7.2 software to map the"Drug-Core Active Ingredient-Core Target-Pathway"network of PCF and AML and the core active ingredients were screened.The final core components and core targets were connected and visualized by Py Mol and Autodock software.Afterwards,AML HL60 cells were used as the experimental model,the IC50 of the core component was determined by primary screening of drug concentration and cytotoxicity assay;the effect of the core component on the proliferation ability of HL60 cells was detected by CCK-8 assay;the cell cycle and apoptosis of HL60 cells were unpacked by flow cytometry;q PCR was used to detect the effect of the core component on the expression of HL60cell-related target genes.BSP assay was used to detect the effect of core components on the methylation of the promoter region of target genes in HL60 cells.Results:Six active ingredients of PCF were obtained after screening the results of the TCMSP database search,luteolin,sitosterol,β-sitosterol,gramisterol,obtusifoliol,cycloartenol,respectively;1206 genes related to AML were obtained from OMIM database,Drug Bank database and Gene Cards database,and 29 common targets of PCF and AML were collected by Wayne’s analysis.GO enrichment analysis focuses on biological processes such as development of haematopoietic and lymphoid organs,drug response and apoptosis;KEGG enrichment analysis focuses on signalling pathways such as tumour necrosis factor（TNF）and T-cell receptor（TCR）.Topological network analysis using Cytoscape3.7.2 software yielded 10 core targets,including protein kinase B1（AKT1）and cysteine-containing aspartate protein hydrolase 3（Caspase-3）,which were combined with the KEGG signalling pathway to identify the core active ingredient asβ-sitosterol.Molecular docking results showed that the active component of PCF,β-sitosterol,was best docked to the proto-oncogene（JUN）and prostaglandin endoperoxide synthase 2（PTGS2）,with energy savings of-8.8 and-7.9kcal-mol^-1respectively.The IC50 ofβ-sitosterol was determined to be508.78μM by primary screening and cytotoxicity assay;CCK-8 results showed thatβ-sitosterol inhibited the proliferation of HL60 cells in a dose-dependent manner（P<0.0001）;flow cytometry results showed that the G0/G1 phase of cells was elevated and the S and G2 phases were decreased along with the promotion of apoptosis（P<0.0001）;q PCR results showed that The expression of JUN and PTGS2genes decreased with increasing drug concentration（P<0.0001）,At the same time,the methylation levels of JUN and PTGS2 gene promoter regions decreased and increased to different degrees respectively（P<0.05）.In summary,the active component of PCF,β-sitosterol,and the core target genes,JUN and PTGS2,were screened using network pharmacology and molecular docking techniques.The effects ofβ-sitosterol on the growth and development of HL60 cells and its mechanism of action were verified by cellular experiments,gene expression levels and epigenetic aspects of gene methylation,which may be one of the potential mechanisms for PCF to inhibit AML cells.