The Preparation Of Ginsenoside Rg3 Liposome And Its Anti-hepatoma Carcinoma Cells Activity And Molecular Mechanism

Object:(1)In order to improve the bioavailability of ginsenoside Rg3(Ginsenoside Rg3,Gs-Rg3)in vivo,the process of ginsenoside Rg3 liposome was optimized.(2)The target of ginsenoside Rg3 was combed by network pharmacology,and its regulatory effect on the life process and signal pathway of hepatocellular carcinoma cells was analyzed.(3)To reveal the anti-hepatoma carcinoma cells activity of ginsenoside Rg3 and its molecular mechanism.(4)To evaluate the anti-hepatoma carcinoma cells activity of ginsenoside Rg3 liposome.Method:(1)Preparation of ginsenoside Rg3 liposomes;firstly,the content of ginsenoside Rg3 in liposomes was determined by high performance liquid chromatography,and the methodology was investigated by precision,stability,sample recovery and other experiments.Then ginsenoside Rg3 liposomes were prepared by ethanol injection method,and the entrapment efficiency of the liposomes was determined by ultrafiltration centrifugation.The formulation and preparation process of ginsenoside Rg3 liposomes were optimized by single factor method with entrapment efficiency as the detection index,and then the appearance,particle size and Zeta potential of the liposomes were evaluated.(2)The mechanism of ginsenoside Rg3 inhibiting the proliferation of liver cancer was analyzed by network pharmacology;the relevant databases were searched to identify the overlapping protein targets of ginsenoside Rg3 and liver cancer.Cytoscape software and String database were used to generate(PPI),topology analysis of component-target and protein-protein interaction network to determine the highly connected nodes in the latter.The biological process of target protein gene ontology(GO)was annotated by David database,and the(KEGG)pathway of Kyoto encyclopedia and genome database was enriched and analyzed by Bioconductor platform.(3)Through MTT assay,it was confirmed that ginsenoside Rg3 inhibited the proliferation of hepatocellular carcinoma cells(Bel-7402 and HCCLM3)in a dose-dependent manner,and revealed that ginsenoside Rg3 induced cell cycle arrest in G1 phase and inhibited the protein expression of Cyclin D1 and CDK2/4.Immunofluorescence and immunoblotting experiments revealed that ginsenoside Rg3 inhibited the expression of SIRT2 protein,thus induced the modification level of whole cell histone H3K18 ac and H4K16 ac.(4)The cell uptake of ginsenoside Rg3 liposome was detected by cell uptake assay,and the effects of ginsenoside Rg3 liposome and monomer on the proliferation of hepatocellular carcinoma cells were compared by MTT method.Result:(1)First of all,there is a good linear relationship between peak area and concentration of ginsenoside Rg3 in the linear range.There are no other interference peaks in the retention time,and the precision,stability and recovery are good.Secondly,the optimum formulation and technological conditions of ginsenoside Rg3 liposome were determined by single factor experiment: the ratio of lipid to drug was 15:1,the ratio of membrane to material was 4:1,and the p H=6.8 of aqueous phase PBS.Finally,the liposome prepared by the optimum process has obvious blue opalescence phenomenon,and the particle size is 93.95 nm,The Zeta potential is-2.42 m V,and the entrapment efficiency is 94.57 ±1.15%.(2)Network pharmacology analysis of ginsenoside Rg3 through multi-target,multi-pathway synergistic effect to inhibit the proliferation of hepatoma carcinoma cells,sort out the related cell pathways and target proteins.(3)MTT assay confirmed that ginsenoside Rg3 inhibited the proliferation of hepatoma carcinoma cells(Bel-7402 and HCCLM3)in a dose-dependent manner,and revealed that ginsenoside Rg3 induced cell cycle arrest in G1 phase and inhibited the protein expression of Cyclin D1 and CDK2/4.Immunofluorescence and Western blotting experiments revealed that ginsenoside Rg3 inhibited the expression of SIRT2 protein,and then induced the modification level of histone H3K18 ac and H4K16 ac at the whole cell level.(4)Cell uptake experiment revealed that ginsenoside Rg3 in liposomes could enter the cells.Compared with ginsenoside Rg3,the inhibitory effect of ginsenoside Rg3 liposomes on Bel-7402 was significantly higher than that of ginsenoside Rg3 liposomes,which confirmed that ginsenoside Rg3 liposomes helped ginsenoside Rg3 transport across the membrane.Conclusion: The prescription of ginsenoside Rg3 liposome was determined,and ginsenoside Rg3 liposome with small particle size,uniform distribution,stable system and high entrapment efficiency was prepared.Network pharmacology analysis and summary of ginsenoside Rg3 action targets,and explore the synergistic effect of ginsenoside Rg3 on a variety of pathways related to the pathogenesis of HCC,including cell proliferation,apoptosis and so on.In vitro experiments showed that ginsenoside Rg3 inhibited the proliferation of hepatocellular carcinoma cells in a dose-dependent manner,induced G1 phase cell cycle arrest,decreased cyclin D1 and cyclin-dependent kinase 2/4,and ginsenoside Rg3 could inhibit the expression of SIRT2 protein and increase the modification level of H3K18 ac and H4K16 ac in whole cells.These findings provide a theoretical basis and support for further preclinical studies on the safety of ginsenoside Rg3 and the molecular mechanism of antihepatocellular carcinoma cells.It was confirmed that the anti-hepatoma carcinoma cells activity of ginsenoside Rg3 liposomes was improved.