Study On The Effect And Mechanism Of Soybean Oligopeptides(QRPR And HCQRPR) On Hep G2 Cell Apoptosis

As a kind of food-borne active peptides,soybean peptide has various functional activities such as anti-oxidation,blood pressure lowering and anti-inflammatory.Food-borne active peptides can protect the function and nutritional needs of human body and interfer with cancer cell growth.Primary liver cancer is one of the most common malignancies,especially hepatocellular carcinoma(HCC).There are few reports on the consumption of food-borne active peptides in cancer patients.In this paper,the immunologically active soybean oligopeptides QRPR(Gln-Arg-Pro-Arg)and HCQRPR(His-Cys-Gln-Arg-Pro-Arg)were used as the research objects,and Hep G2 human hepatoma cells were used as model cells,from the perspective of apoptosis,the mechanism of inhibition of Hep G2 cell proliferation by soybean oligopeptide(QRPR and HCQRPR)was investigated at the molecular level;starting from the relationship between structure and efficacy,we explored the role of soybean oligopeptide(QRPR and HCQRPR)in the proliferation of Hep G2 cells.The main research contents and results of this thesis are as follows:(1)The effect of soybean oligopeptide(QRPR and HCQRPR)on the viability of Hep G2 cells was evaluated,and Hep G2 cells were used as model cells.The results of MTS assay showed that soybean oligopeptide(QRPR and HCQRPR)inhibited the proliferation of Hep G2 cells.The combination of the treatments,soybean oligopeptide QRPR and HCQRPR,had higher inhibition rate than the two treatments alone,which significantly inhibited Hep G2 cell proliferation(p < 0.05),and showed concentration dependenceand time dependence,the best treatment conditions were combined with a treatment concentration of 800 ?M and a treatment time of 72 h.The results of real-time PCR demonstrared that the expression of PTEN gene enhanced significantly with the increase of concentration and time of soybean oligopeptide QRPR and HCQRPR,which increased the number of cells in Hepatocyte G1 phase(p < 0.05),and delayed Hep G2 cell enters the S phase from the G1 phase,which affected the cell cycle of the tumor cell Hep G2,and then inhibited the proliferation of Hep G2 cells.(2)The effect function and mechanism of soybean oligopeptide(QRPR and HCQRPR)on apoptosis of Hep G2 cells and its mechanism were investigated by in vitro cell culture experiments.The transmission electron microscopy results showed that the ultrastructure of Hep G2 cells were changed after the combination treatments of soybean oligopeptide QRPR and HCQRPR,and apoptotic bodies were produced inside the cells.The results of Annexin V-FITC/PI double staining demonstrated that soybean oligopeptides QRPR and HCQRPR could induce the apoptosis of Hep G2 cells,and the combination treatment of the two treatments had higher apoptotic rate than that of single treatment,and it indicated concentration-dependent and time-dependent.The highest inhibition rate was 54.06 ± 1.38%,which suggests that soybean oligopeptides QRPR and HCQRPR can induce apoptosis in Hep G2 cells.The further experiments were investigated the mechanism of apoptosis induced by soybean oligopeptides QRPR and HCQRPR throughenzyme-linked immunosorbent assay to detect the expression of TNF-?,real-time qPCR and Western-blot expression of caspase-8 and caspase-3 test.The results showed that the soybean oligopeptides QRPR and HCQRPR acted on Hep G2 cells,which promoted the binding of TNF-? to TNFRI and the expression of TNF-? was significantly decreased(p < 0.05),thereby caspase-8 and caspase-3 expression were activated.It indicates that soybean oligopeptides QRPR and HCQRPR can induce apoptosis of Hep G2 cells through TNF cell pathway.(3)The soybean oligopeptides QRPR and HCQRPR were determined to be irregularly coiled by circular Fourier and Fourier transform infrared(FTI)spectroscopy.The ANTHEPROT and Discovery Studio software predicted that the soybean oligopeptides QRPR and HCQRPR were both positively charged,and both have a spatial structurewith hydrophilic amino acids,hydrophobic amino acids,and a molecular conformational map of their spatial structure,andthe structure-activity relationship of soybean oligopeptide(QRPR and HCQRPR)was explored.In summary,this article had studied the biological activity and conformation of soybean oligopeptides QRPR and HCQRPR,which providing the theoretical and scientific basis for the development of soy active peptides as functional foods.It provides new method to further improving the utilization rate of soybean products and increasing the extra value of soybean products.