Study Of The Activities Of Polysaccharides From Pegamum Harmala To Induce Autophagy And Regulate Macrophage Immunity

Objective: To investigate the autophagy induced by Peganum harmala polysaccharide(LTP)and regulate the immunological activity of macrophages.Methods: 1)The GFPLC3 fusion protein stably expressing NRK cells treated with Different concentrations of LTP polysaccharide and the optimal concentration and timepoint of LTP polysaccharide induced autophagy were selected.2)Western blotting was used to further detect the expression of autophagosome marker proteins LC3 and p62 after treated with LTP polysaccharide.The number of autophagosomes in cells was observed by electron microscopy.Immunofluorescence was used to observe the colocalization of autophagosomes and lysosomes in LTP polysaccharides.3)After treated with LTP polysaccharide Western blotting was used to detect the changes of key molecules p70s6 k and AMPK in autophagy signaling pathway,and combined with autophagy inhibitors and inducers,Western blotting was used to detect the changes of LC3 and p62 expression levels.4)1μg/mL lipopolysaccharides(LPS)were treated RAW264.7 cells in vitro to establish an inflammatory cell model.Cell viability was detected by CCK-8 method,apoptosis was observed by Hoechst and PI double staining,and inflammatory factors were detected by ELISA.The levels of IL-1β,IL-6 and TNF-a were used to identify inflammatory cell models;5)After LTP polysaccharide treated the cell viability,apoptosis and inflammatory factors were detected,and autophagy inhibitors 3-MA and lysosomes inhibitors BafA1 were treated with LTP,Western blotting method was used to detect the changes of LC3 and p62 protein expression levels in cells;Results:1)Different concentrations of LTP polysaccharide could induce autophagy production.The content of autophagosome in cells increased significantly after treatment with 100 ug/ml LTP polysaccharide for 12 h(P<0.05).and the expression level of p62 protein was significantly lower than that of the control group(P<0.05).The results of immunofluorescence showed that the autophagy and lysosome colocalization after 100 ug/ml LTP polysaccharide intervention.The control group increased significantly(P<0.05).2)Western blotting results showed that the expression of p-AMPK protein was significantly higher than that of the control group after LTP polysaccharide intervention(P<0.05),and the expression level of p-p70s6 k protein was significantly different from the control group(P<0.05).There was no significant decrease in the STA group and the rapamycin group.3)After treatment of RAW264.7 cells with LPS,the cell viability did not change significantly,the apoptotic rate was significantly higher than the control group(P<0.05),and the levels of IL-6,IL-1β and TNF-a were significantly higher than those of the control group(P<0.05),there was no significant change in cell viability after LTP polysaccharide treated,and the apoptotic rate was significantly lower than the model group(P<0.05).The levels of IL-6,IL-1β and TNF-a were significantly lower than the model group(P< 0.05);4)Western blotting results showed that after LTP polysaccharide treated LPS-treated RAW264.7 cells,the expression level of LC3 protein was significantly higher than that of the control group(P<0.05),and the expression level of p62 protein was significantly lower than that of the control group(P< 0.05);Conclusion: LTP polysaccharide can induce autophagy and may play an immunomodulatory role on macrophages through autophagy pathway.