Study On The Protective Mechanism Of Bacteroides Fragilis In Inhibiting TNF-α-induced Inflammatory Response In Colon Cells

Objectives:Colorectal cancer(CRC)is the most common malignant tumor of the digestive tract.High morbidity and mortality rate of CRC seriously endanger human health and safety.The prevention and treatment of CRC needs to be solved urgently.Non-enterotoxin producing Bacteroides fragilis(NTBF)is a new generation of probiotics.Our previous research found that the abundance of Bacteroides fragilis was significantly lower in patients with adenomatous colon polyps than healthy people.However,adenomatous colon polyps are the precancerous lesions of CRC.Therefore,this research aims to study the protective role and mechanism of Bacteroides fragilis in the occurrence of CRC on the basis of the previous stage.Methods:1.A"bacterial-cell co-culture" model of Bacteroides fragilis and human colon cancer cells LOVO,normal colonic epithelial cells hcoEPIC was established to detect the adhesion and invasion ability of Bacteroides fragilis to different colon cells.2.Inflammation models of LOVO cells and hcoEPIC cells were established by TNF-α induction in vitro,and grouped as follows:①normal control group;②Bacteroides fragilis treatment group,co-cultured LOVO and hcoEPIC cells with Bacteroides fragilis at a concentration of 1×108 CFU/ml(MOI=100)for 24h;③TNF-α induction group,LOVO and hcoEPIC cells were only treated with 30ng/ml TNF-α for 24h;④ Bacteroides fragilis+TNF-α group,the cells were co-cultured with Bacteroides fragilis for 4 hours,and then TNF-α was added to treat the cells for 24 hours.3.CCK8,FITC-AnnexinV apoptosis assay,LDH release assay,and scratch assay were used to detect cells survival rate,apoptosis,damage,and migration in different treatment groups.4.Western bloting was used to detect the protein expression of TLR2,TLR4,MYD88,phosphorylated NF-κB p65,NF-κB p65,phosphorylated IκBα,IκBα;qPCR was used to detect the expression of TLR2,TLR4,MYD88,NF-κB,IκBαin mRNA levels.Results:1.The "Bacteroides fragilis-cell co-culture" model and the TNF-α-induced in vitro inflammation model of hcoEPIC and LOVO cells were established Successfully.The main manifestation is that Bacteroides fragilis alone has no effect on cell morphology and survival rate for 24h.TNF-α alone treatment after 24 hours,cell morphology shrank and ruptured;cell survival rate decreased,apoptosis increased,and migration slowed;NF-κB signaling pathway related proteins and cytokines were activated.2.The adhesion experiment showed that co-cultivation of Bacteroides fragilis and LOVO and hcoEPIC cells after 4 hours,the number of adhesion was(1.64±0.54)×106CFU/ml and(0.86±0.17)×106CFU/ml(P<0.05)respectively;Invasion experiments showed that Bacteroides fragilis showed no invasive characteristics in two cell lines.3.CCK8 assay and LDH release assay showed that Bacteroides fragilis can inhibit LOVO and hcoEPIC cell damage induced by TNF-α,increase cell survival rate(P<0.05);FITC-AnnexinV apoptosis assay showed that Bacteroides fragilis can inhibit cell apoptosis induced by TNF-α(P<0.05);The scratch assay showed that the migration of LOVO cells and hcoEPIC cells in the TNF-α alone treatment group was significantly inhibited(P<0.05),but this change was reversed by Bacteroides fragilis(P<0.05).4.The expression of TLR2 was down-regulated and TLR4 was up-regulated by Bacteroides fragilis slightly in LOVO cells(P>0.05).In normal epithelial cells hcoEPIC,the result was just the opposite(P<0.05).But both significantly down-regulated the expression of MYD88 in the two cell lines(P<0.05).5.After TNF-α alone treatment for 24 hours,the protein expression of phosphorylated NF-κB p65 and phosphorylated IκBa increased significantly,and the levels of NF-κB mRNA and IκBa mRNA increased(P<0.05),indicating that the NF-κB signaling pathway was activated,but after pretreatment with Bacteroides fragilis and adding TNF-α,the protein expression of phosphorylated NF-κB p65 and phosphorylated IκBα were down-regulated,and the levels of NF-κB mRNA and IκBα mRNA were decreased(P<0.05),indicating Bacteroides fragilis inhibited the activation of NF-κB signaling pathway induced by TNF-α.6.ELISA results showed that Bacteroides fragilis inhibited the release of pro-inflammatory cytokines IL-6,IL-8,IL-1β,and TNF-α induced by TNF-α,and promoted the production of anti-inflammatory cytokine IL-10.Conclusions:1.Bacteroides fragilis has different adhesion abilities to different colon cells,but it does not exhibit invasive properties;Bacteroides fragilis can increase the survival rate of colon cells induced by TNF-α,inhibit cell apoptosis and damage,and promote colonic epithelial cell migration in vitro.2.Bacteroides fragilis may counteract the inflammatory response induced by TNF-αby down-regulating the TLR2/TLR4-M YD8 8-NF-κB signaling pathway,reducing the release of pro-inflammatory cytokines and increasing the production of anti-inflammatory cytokines.