Preliminary Study On The Effects Of Dobinea Delavayi Polysaccharides On Proliferation And Apoptosis Of Hep G2 And MFC Cells

Cancer is a serious threat to human life and health.At present,chemical drugs are mainly used in cancer treatment,while plant polysaccharides have highlighted some advantages in the field of cancer treatment in recent years.Dobinea delavayi is a perennial subshrubby herb belonging to Dobinea of Anacardiaceae.It has a long history of folk medicine and has the effects of anti-inflammatory,analgesic,relaxation of tendons and activating collaterals.During the preliminary screening of the biological activities of medicinal plant polysaccharides,the research group found that the polysaccharides of Dobinea delavayi has obvious anti-tumor activities.Therefore,in this study,D.delavayi polysaccharides were used as the research object,the effects of different alcohol precipitation parts of D.delavayi polysaccharides on the proliferation,cell cycle and apoptosis of human hepatoma Hep G2cells and mouse gastric cancer MFC cells were studied in vitro.Combined with transcriptomics,the molecular mechanism of its effect on Hep G2 cell proliferation and apoptosis was explored,so as to provide theoretical data for the mining of medicinal potential of D.delavayi.The main results of this study are as follows:（1）30%,60%and 95%alcohol precipitated polysaccharides of Dobinea delavayi were extracted by step-by-step alcohol precipitation method,which were named DDP-1,DDP-2and DDP-3,respectively.The total sugar contents of the three polysaccharides were 59.94%,35.40%and 41.21%by phenol-sulfuric acid method,respectively.Fourier transform infrared spectroscopy determined that the three polysaccharides had common characteristic absorption peaks of polysaccharides.The monosaccharide compositions of three polysaccharides were determined by PMP-HPLC.It was found that DDP-2 and DDP-3 both contained mannose,rhamnose,glucose,galactose and arabinose with molar ratios of 1.00:0.70:2.70:2.73:0.31and 1.00:0.83:1.93:2.93:0.47,respectively.DDP-1 contained mannose,glucose,galactose and arabinose,and the mole ratio was 1.00:8.73:0.43:0.09.（2）CCK-8 method was used to determine the effects of 30%,60%and 95%alcohol precipitated polysaccharides of Dobinea delavayi on the viability of Hep G2 cells and MFC cells.Within the concentration range of 25～1600μg/m L,the three polysaccharides can reduce the cell viability of the two cells.Among them,DDP-3 has the best effect,and the IC₅₀was1188.16μg/m L and 1188.16μg/m L,respectively.When 5-FU concentration of positive control was 100μg/m L,the survival rate of Hep G2 cells and MFC cells was 43.87%and44.73%,respectively.It can be seen that the antitumor activity of Polysaccharides from D.delavayi was weaker than that of 5-FU.The effects of DDP-3 on cell cycle and apoptosis of Hep G2 and MFC were measured by flow cytometry.It was found that when the concentration was 400～1600μg/m L,DDP-3 could significantly arrest the cycle of Hep G2cells in S phase;the concentration was between 800 and 1600μg/m L,DDP-3 could significantly arrest the cycle of MFC cells in G₂/M phase.In addition,flow cytometry was used to determine the effect of DDP-3 on the apoptosis of two kinds of cells.It was found that DDP-3 could significantly promote the apoptosis of Hep G2 cells at 800～1600μg/m L.The apoptosis of MFC cells was significantly promoted at 400～1600μg/m L.（3）Based on transcriptome sequencing technology,the differential expression of genes（DEGs）between the control group and DDP-3 treatment group was compared.The results showed that DEGs in the control group and low or high dose groups were 2333 and 1882,respectively.The number of up-regulated DEGs were 1454 and 1388,and the number of down-regulated DEGs were 879 and 494,respectively.The total number of DEGs between low-dose group and high-dose group was 165,including 47 up-regulation genes and 118down-regulation genes,respectively.Go enrichment and KEGG analysis of differentially expressed genes showed that the anti hepatoma activity of Dobinea delavayi polysaccharides was mainly related to MAPK signaling pathway,p53 signaling pathway and TGF-βSignaling pathway,apoptosis and other pathways.Five key genes were screened by protein interaction network analysis:BCL2A1,GADD45B,MDM4 and TGF-β1 and MAP2K6.q RT-PCR results showed that compared with the control group,the mRNA expression of GADD45B,MAP2K6and BCL2A1 in the administration group decreased and TGF-β1 and MDM4 were increased.In this study,it was found that the polysaccharides of Dobinea delavayi has obvious antitumor activities in vitro.The anticancer mechanism of D.delavayi polysaccharides was preliminarily explored.The results showed that it can play an antitumor role by reducing the activity of Hep G2 and MFC cells,arrest cell cycle and promoting apoptosis.In addition,transcriptomic studies showed that DDP-3 regulated MAPK and p53 signaling pathways to affect the proliferation and apoptosis of Hep G2 cells.This lays a theoretical foundation for exploring the medicinal potential of Dobinea delavayi and promoting it to become a medical and health care product.