Mechanism Of Soybean Oligopeptide QRPR Activates Autophagy In Inhibiting Inflammation Of RAW264.7 Cells

Autophagy is widely involved in body growth,renewal,and immune defenses.Although the inflammatory response can congenitally protect the body from infection or tissue damage,studies have found that autophagy and inflammatory response are closely related to each other and can protect cells from excessive inflammation by directly inhibiting inflammatory complexes and indirectly removing damaged inflammatory stimulators such as organelles or pathogenic microorganisms.Cytokines can also induce autophagy.Some immunologically active substances may have an effect on autophagy and inflammation.Soybean can be extracted with immunologically active soybean peptides.In this paper,we use immunologically active soybean oligopeptide QRPR(Gln-Arg-Pro-Arg)as raw material,and apply mouse macrophage cell line RAW264.7 to establish cell inflammation model.Using in vitro chemical experiments and molecular biology related techniques to conduct anti-inflammatory research and immunological activity evaluation of immunoreactive soybean oligopeptide QRPR,and explore the mechanism of soybean oligopeptide QRPR attenuating inflammatory response by modulating autophagy in RAW264.7 cells.First,establish a model of cell inflammation,establish a model of inflammation using lipopolysaccharide(LPS)on mouse macrophages(RAW264.7),determine the LPS concentration of 100ng/ml by enzyme-linked immunosorbent assay,and the treatment time is 16 hours.The optimal modeling conditions were to use soybean oligopeptide QRPR to act on RAW264.7 cell inflammation model.It was found that QRPR can reduce the content of cytokines TNF-α and IL-6 in a dose-dependent manner.Then,in order to prove whether the anti-inflammatory effect of QRPR is related to autophagy,cells were treated with autophagy inhibitor 3-methyladenine(3MA)and then co-stimulated with soybean oligopeptides QRPR and LPS for 16 hours.The results showed that the secretion of IL-6 and TNF-α was increased in the RAW264.7 inflammation model of the autophagy inhibitor 3MA group,and the anti-inflammatory effect of the QRPR peptide was significantly decreased after the addition of the autophagy inhibitor 3MA.In addition,the knockdown of autophagy-specific gene Atg5,QRPR inhibition of inflammatory factors decreased,which shows that autophagy was inhibited or blocked after the anti-inflammatory effect of QRPR peptide weakened,indicating that QRPR on Raw264.7 cells inflammation protection is regulated by autophagy.Western blotting results showed that with the increase of QRPR concentration,the expression level of autophagy marker protein LC3 II increased gradually and the expression of downstream protein SQSTM1/P62 decreased,indicating that QRPR could activate autophagy of RAW264.7 cells and showed obvious dose-dependent.The staining of MDC was observed under a fluorescent microscope.The QRPR peptide treatment group showed stronger fluorescence brightness,further demonstrating the induction effect of the QRPR peptide on autophagy.However,the mechanism of specific regulation of autophagy by QRPR peptide remains to be further explored.Finally,the mechanism of autophagy activation by QRPR was studied.The protein expression of PI3 K,AKT and mTOR was detected by western-blot.It was found that QRPR could inhibit the expression of AKT and mTOR,but had no significant effect on the expression of PI3 K.Real-time fluorescence quantitative PCR was used to detect the expression of PI3 K,AKT and mTOR genes.It was found that the expression levels of PI3 K,AKT and mTOR all decreased.At the same time,the internal structure of the cells was observed by transmission electron microscopy.It was found that after the addition of QRPR,the autophagy bodies and autolysosomes were observed in the interior of the cells.It is concluded that QRPR inhibits the pathway of PI3K/AKT/mTOR to a certain extent and activates the occurrence of autophagy.The effect of adding QRPR on the cell cycle of RAW264.7 cells was observed by flow cytometry.The number of cells in G1 phase of RAW264.7 cells increased,and the number of cells in S phase decreased(P<0.05).QRPR delayed the cell entry from G1 to the trumpet phase.Cycle run was inhibited consistent with western blot and RT-PCR results.In conclusion,the above experiments demonstrate that soy oligopeptide QRPR activates autophagy by modulating PI3K/AKT/mTOR pathway,weakens the inflammatory response in RAW264.7 cell model,and provides a certain theory basis for the application of soybean immunoreactive peptides in anti-inflammatory and immune applications.