Research On Astragalus Protein Induced Programmed Necrosis In HepG2 Cells

Objective: Proteins are biological macromolecules with rich types and different functions.Some pure protein preparations have been widely used as medicines for treating diseases,such as insulin.Some scholars have found that natural protein substances have anti-tumor,anti-inflammatory and immune effects.Studies have shown that astragalus has abundant effective parts,and astragalus glycoprotein has immunosuppressive effects on certain diseases,but it is unknown whether astragalus protein components have an effect on the growth of tumor cells.Therefore,this article extracts and purifies the astragalus protein components,screens the astragalus protein for in vitro antitumor activity,and further discusses its tumor inhibition and its mechanism.At the same time,combined with transcriptomics RNA sequencing technology,it is hoped that the full utilization of astragalus active ingredients,and provide an experimental basis for the anti-tumor research of protein components.Methods: Astragalus membranaceus extracts and demineralizes water to obtain astragalus protein solutions with different molecular weights.Screen from three different tumor cells,and select the one that were most significantly affected by astragalus protein.Cell counting method were used to calculate the value-added inhibition rate of astragalus protein on tumor cells.Flow cytometry and PI staining were used to observe the tumor cells.Apoptosis and necrosis;Hoechst33342 and PI co-stained under a microscope to distinguish between changes in the number of live and dead cells;Western Blot method were used to detect the phosphorylation levels of apoptosis marker proteins PARP and caspase-3,and the content of necrosis marker protein RIP1;Transcription compare the blank group and the medicated group to analyze the differential expression genes and signal pathway changes after astragalus protein treatment.Real-time quantitative PCR were used to verify in depth the relative changes in m RNA levels of related genes after the metabolic pathway was regulated.Western Blot confirmed it again Pathway protein levels change.Results: The data showed that astragalus protein significantly inhibited the proliferation of Hep G2 cells in a time and concentration dependent manner.Flow cytometry showed that the number of necrotic cells increased with the astragalus protein concentration,and the necrotic rate of the group with 100?g/m L reached 18.78%.By fluorescence microscopy,compared with the blank group,the increased concentration of astragalus protein treatment directly led to the increase of red necrotic cells in PI staining,while the blue normal cells in Hoechst33342 staining significantly decreased.Western Blot results showed that Hep G2 cells treated with astragalus protein had no effect on apoptotic proteins,while the phosphorylation level of necrotic marker protein RIP1 was significantly up-regulated.This suggests that astragalus proteins may inhibit proliferation by inducing hepatocellular carcinoma cell necrosis.Next,to explore the specific mechanism of proliferation inhibition,the treated cell RNA was sequenced and the results were analyzed.Firstly,through the gene volcano map and cluster analysis map,it can be found that after the treatment of astragalus protein,many genes showed significant differences in expression,so the specific pathway changes after the dosing treatment were further explored.KEGG analysis showed that the p53 metabolic pathway after astragalus protein treatment was significantly different from that of the blank control group,and this pathway ranked the top three of all pathways.According to literature investigation,p53 signaling pathway is related to programmed cell necrosis,and p53 change directly lead to some gene changes.Real-time fluorescent quantitative PCR further confirmed that the expression level of genes related to the p53 pathway was consistent with that of transcriptome data.This result shows that the sequencing results are reliable.Western Blot results showed that astragalus protein could significantly up-regulate the expression of p53 protein after the treatment of cells,which proved that astragalus protein caused programmed necrosis of Hep G2 cells by affecting the p53 signaling pathway,thus leading to the inhibition of the growth of liver cancer cells.Conclusion:This study proved that astragalus protein extract has obvious anti-tumor effect,found and proved the molecular mechanism of astragalus protein against liver cancer cells,which not only provides a basis for astragalus protein against liver cancer,but also provides future anti-tumor research of protein compounds.