Regulatory Effect Of Geniposide On Endocytosis And Polarization Of RAW264.7 Cells

Objective To observe the effect of geniposide on the endocytosis of macrophage RAW264.7 and its related mechanism.To study the effect of geniposide on the phenotypic polarization of RAW264.7 cells.Methods1.Effect of Geniposide on Endocytosis of RAW246.7 Cells:CCK-8 assay was used to observe the effect of geniposide with different concentrations and co-cultured time on the proliferation of RAW264.7 cells.According to the results of CCK-8 assay,the concentration and the co-cultured time were determined.The blank control group and the geniposide experimental groups with different concentrations were set up.Then test the pinocytosis of RAW264.7 cells by neutral red solution method.According to the results of pinocytosis test,the concentration in the phagocytic experiment was further selected.Stained Pseudomonas aeruginosa phagocytosed by RAW264.7 cell was observed by fluorescence microscope,and intracellular survival experiment was conducted to count the intracellular bacteria released from RAW264.7 cell at different time points.2.Experiments on the signaling pathways mechanism affecting the endocytosis of macrophages:Set up five different groups:blank control group,geniposide group(RAW264.7 cells cultured with 2.0 mg/m L geniposide for 24 h),LPS group(RAW264.7 cells cultured with 100 ng/ml LPS for 4 h),geniposide treatment group(treat LPS group with 2.0 mg/m L geniposide for 24 h)and geniposide prevention group(treat geniposide group with 100 ng/m L LPS for 4h).The m RNA expressions of m Tor-Rho GTPases signaling pathway related genes(Cdc42,Rho A,Rac1)were detected by RT-PCR,and the expression levels of related proteins(m Tor,Rho A,Rac1,Cdc42)and their phosphorylation were detected by Western Blot.3.Experiments on phenotypic polarization of RAW264.7 cells:Set up five different groups:blank control group,geniposide group,LPS group,geniposide treatment group and geniposide prevention group.Flow cytometry was used to detect the expression of i NOS~+and CD206~+of RAW264.7 cells,the phenotype was determined according to the result.ELISA was used to detect the levels of pro-inflammatory cytokines(TNF-α,IL-1)and anti-inflammatory cytokines(IL-10)in the supernatant of each group,to further verify the phenotype changes of RAW264.7 cells.Results1.The results of CCK-8 assay showed that,within the concentration range of 2.0-8.0mg/m L,geniposide had no significant inhibitory effect on the growth of RAW264.7cells after co-cultured for 48 h.After 24 h co-incubation with 2.0 mg/m L of geniposide,RAW264.7 cells significantly increased their ability to swallow neutral red compared with the blank control group(P<0.01).2.Fluorescence microscope was used to observe the phagocytosis of RAW264.7 cells to stained Pseudomonas aeruginosa.The results showed that a large number of stained PA were swallowed in RAW264.7 cells in the blank control group,while a small amount of stained PA was observed in RAW264.7 cells of the geniposide group.The results of bacterial intracellular survival test showed that there were differences in the geniposide groups at 0 h and 2 h(P<0.01).Compared with the blank control group,the RAW264.7 cells in the geniposide group had less phagocytosis of PA at 0 h(P<0.05)and more intracellular bacteria at 2 h(P<0.01).3.The RT-PCR results showed that there was no difference in the expression of m TOR,Cdc42,Rac1,Rho A m RNA between the blank control group and the geniposide group(P>0.05).After the effect of LPS,the expression of the four gene all significantly increased(P<0.01).In the geniposide treatment and preventive group,the expression of the above four genes decreased significantly(P<0.01),and the decline was more significant in the preventive administration group(P<0.01).Western Blot analysis showed no significant changes in the protein expression of m Tor,Cdc42,Rac1 and Rho A in the five groups(P>0.05).The phosphorylation levels of p-m Tor,p-Cdc42/Rac1 and p-Rho A showed no significant difference in the blank control group and the geniposide group(P>0.05).In LPS inflammatory model group,phosphorylated protein expression increased significantly(P<0.01).Geniposide can inhibit the increase of the phosphorylated protein induced by LPS in both the treatment and prevention groups(P<0.01),and the inhibition is more significant in the prevention group than the treatment group(P<0.01).4.The results of flow cytometry showed that the M1 cell ratio in the LPS inflammatory model group was significantly increased(P<0.05),while that in the geniposide treatment group and the preventive treatment group was significantly decreased(P<0.05,P<0.01,respectively).The M2 ratio in the LPS inflammatory model group was significantly lower than that in the blank control group and the geniposide group(P<0.01),while the M2 ratio in the geniposide treatment group and the preventive treatment group was significantly higher than that in the blank control group and the geniposide group(P<0.01),and the increase in the geniposide treatment group was more significant than that in the prevention group(P<0.01).ELISA results showed no statistically differences in TNF-α,IL-1,IL-10between the blank control group and the geniposide group(P>0.05).In the LPS inflammatory model group,all the three cytokines were increased(P<0.01).Compared with the LPS inflammatory model group,TNF-αand IL-1 in the geniposide treatment group decreased significantly(P<0.01,P<0.05,respectively),and the decline of TNF-αand IL-1 in the prevention group was more significant than that in the treatment group(P<0.01).Compared with the LPS model group,IL-10increased in both the treatment group and the prevention group(P<0.01),and the increase was more significant in the prevention group(P<0.01).Conclusion1.Geniposide can promote the pinocytosis activity of macrophage RAW264.7 to neutral red,and inhibit the phagocytosis and intracellular digestion,bactericidal ability of PA.The mechanism of inhibiting macrophage phagocytosis ability may be related to the decreased expression of genes and proteins related to the m Tor-Rho GTPase pathway.2.Geniposide can induce LPS-induced M1 RAW264.7cells to the M2 phenotype.