| Objective:Colorectal cancer(CRC)is the third most common cancer in the world.According to recent studies,tumor microenvironment and fecal samples of patients with colorectal cancer are rich in Fusobacterium nucleatum.There are a large number of stress-related catecholamines in the human gastrointestinal tract,which can affect the growth of intestinal bacteria.However,there is no study on the effect of catecholamines on Fusobacterium nucleatum and the effect and the molecular mechanism of Fusobacterium nucleatum Autoinducer-2(AI-2)infection on human immune cells.In order to further explore the effect of catecholamine hormones on Fusobacterium nucleatum and the mechanism of Fusobacterium nucleatum participating in colorectal cancer,this experiment studied the effect of catecholamine hormones on the growth of Fusobacterium nucleatum and the effect of Fusobacterium nucleatum on dendritic cells,and expounded its potential mechanism.Methods:(1)The effect of catecholamines on the expression of virulence factor FadA of F.nucleatum was detected by qPCR;(2)The effect of catecholamines on the growth of F.nucleatum was detected by bacterial plate counting;(3)The effect of catecholamines on Fusobacterium nucleatum AI-2 was detected by co culture of Vibrio Harvey and purified Fusobacterium nucleatum AI-2 treated with catecholamines to induce bioluminescence;(4)The effect of Fusobacterium nucleatum AI-2 on macrophage migration and the effect of Fusobacterium nucleatum AI-2 inhibitor D-ribose on macrophage migration induced by AI-2 were detected by transwell assay;(5)MTT assay was used to detect the effect of F.nucleatum AI-2 on the proliferation of T lymphocytes,neutrophils and dendritic cells;(6)CCK8 test was used to detect the effect of F.nucleatum AI-2 on NK cell killing activity;(7)The effect of F.nucleatum AI-2 on the phagocytosis of dendritic cells was detected by flow cytometry;(8)Luminex liquid suspension chip was used to detect the effect of F.nucleatum AI-2 on the secretion of common cytokines by dendritic cells;(9)Quantitative proteomics experiment further screened the AI-2 interacting proteins in dendritic cells,bioinformatics analysis of the data,screened out the signal pathway and verified it by cellular immunofluorescence in vitro.Results:(1)Under the action of epinephrine,norepinephrine and dopamine,compared with the control group,the expression of virulence gene FadA,the number of Fn colonies and the concentration of AI-2 secreted by Fn in Fn bacteria in the catecholamine group were significantly higher than those in the control group.The expression of FadA and the number of Fn colonies decreased significantly in the corresponding inhibitor group(P<0.05).(2)Under the action of epinephrine,norepinephrine and dopamine,the absorbance of colon cancer cells in the group with catecholamine hormone was higher than that in the control group at the same time(P<0.05).(3)Compared with the untreated control group,Fn AI-2(400 μM)and(50μM)significantly induced macrophage migration,and AI-2 inhibitor D-ribose significantly inhibited AI-2-induced migration(P<0.01).(4)Compared with the untreated control group,Fn AI-2(400μM)and(50μM)significantly inhibited the proliferation of T lymphocytes and promoted the proliferation of neutrophils(P<0.001)and dendritic cells(P<0.001).AI-2 inhibitor D-ribose significantly inhibited the proliferation induced by AI-2.(5)Compared with the untreated control group,Fn AI-2(400μM)and(50μM)significantly enhanced the killing ability of human NK cells,and AI-2 inhibitor D-ribose significantly inhibited the cytotoxicity of NK cells induced by AI-2(P<0.05).(6)Compared with the untreated control group,the phagocytic function of dendritic cells was significantly enhanced by Fn AI-2(400μM)and(50μM),and the phagocytosis of dendritic cells induced by AI-2 was significantly inhibited by AI-2 inhibitor D-ribose(P<0.001).(7)Treatment of dendritic cells with Fn AI-2 could significantly promote the secretion of 18 cytokines(P<0.05),among which the promotion effect on GM-CSF was the strongest,up to 6.58 times;(8)After dendritic cells were treated with Fn AI-2,the expression level of 51 genes was>2.0 times.45 proteins were up-regulated and 6 proteins were down regulated.AI-2 infected dendritic cells significantly activated 15 pathways(P<0.05).NF-κB pathway was selected for verification in vitro.After dendritic cells were treated with F.nucleatum AI-2,the expression of NF-κB increased significantly(P<0.05).Conclusion:(1)Catecholamines promote AI-2 secretion,virulence and proliferation of F.nucleatum.(2)Catecholamines promote the proliferation of colon cancer cells.(3)F.nucleatum AI-2 promotes macrophage migration,inhibits T lymphocyte proliferation,promotes neutrophil proliferation,promotes NK cell killing function,promotes dendritic cell proliferation and phagocytosis.(4)In vitro experiments showed that F.nucleatum AI-2 may pass through specific signal transduction pathway NF-κB affects dendritic cells. |
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