Peptostreptococcus Anaerobius Promotes Gastric Mucosal Lesion Through TLR4/TRIF/NF-κB Signaling Pathway

Background and objective:The incidence rate of gastric cancer is ranked fifth and the mortality rate ranks third in the world.About half of the new and dead cases of gastric cancer occur in China,which causes a great burden of disease.The occurrence of intestinal-type gastric cancer is a multi-factor and multi-step process.Helicobacter pylori(H.pylori)infection,host factors,and environmental factors jointly determine the occurrence of gastric cancer.H.pylori infection is considered to be the initiating factor in the process of gastric carcinogenesis.The latest consensus indicates that H.pylori eradication in the early stage of gastric mucosal carcinogenesis(before gastric mucosal atrophy and/or intestinal metaplasia)can reverse gastric mucosal carcinogenesis and even eliminate the possibility of gastric cancer.After the atrophy of gastric mucosa,the intragastric environment is not suitable for H.pylori colonization,bacterias other than H.pylori are increased and altered gastric microbiota plays an important role in the process of carcinogenesis.In addition to H.pylori,there are a large number of bacteria in the stomach to participate in gastric carcinogenesis.Our study found that compared with patients with gastritis,the abundance of gastric mucosal Peptostreptococcus anaerobius(P.anaerobius)in patients with gastric cancer was significantly increased,indicating that it may be involved in the process of gastric mucosal carcinogenesis.However,its exact role in gastric carcinogenesis is still unclear.The toll-like receptor(TLR)family is the main receptor for the human to recognize the pathogenic components of various microorganisms.These receptors are located on the cell surface(TLR1,TLR2,TLR4,TLR5,TLR6),or in cells(TLR3,TLR7,TLR8,TLR9)such as the endoplasmic reticulum and lysosome.TLRs are located in different cell subsets,and their localization corresponds to the macromolecular properties of the recognized ligands.TLRs recognize bacterial ligands are expressed on the cell membrane,while the TLRs recognize viral nucleic acids are located in the cell.When TLRs are activated,the cascade of intracellular signal transduction can be initiated through the downstraem Myd88 dependent pathway that is shared by all LTRs and the downstream TRIF dependent pathway that is unique to TLR3 and TLR4.Recently,it was found that P.anaerobius can induce intestinal epithelial cells to produce ROS,promote the proliferation of intestinal epithelial cells and participate in the malignant transformation of the intestinal mucosa by interacting with TLR2/4.The NF-κB signaling pathway is the hub of the downstream signaling pathway after TLRs activation.The NF-κB family includes five transcription factors: P65(Rel A),Rel B,C-Rel,P50(P105),and P52(P100).In the steady-state setting,NF-κB forms dimers in the cytoplasm(mainly P65 and P50)and is replaced by IκB(NF-κB inhibitor)protein isolated in the cytoplasm.TLRs can activate NF-κB by activating the IKK kinase,the activated IKKβ Induction the Phosphorylation of IκB and its subsequent degradation,which leads to NF-κB dimer being released from cytoplasmic inhibition and translocated to the nucleus and drives the transcription of target genes.As s a possible gastric pathogen,what is the role of P.anaerobius in gastric carcinogenesis and whether it can activate TLRs and downstream NF-κB signaling pathway,what is the significance of this process in its pathogenic process,which is worthy of further evaluation.This study intends to use human gastric mucosa samples,transgenic animals,and cells to clarify: whether the abundance of P.anaerobius increases during gastric carcinogenesis;whether P.anaerobius promotes gastric carcinogenesis in vivo and in vitro;what is the pathogenic mechanism of P.anaerobius,what role does TLRs,and NF-κB signaling pathway play in its pathogenesis.Clarifying the above problems may provide new evidence for the involvement of gastric microbiota in the occurrence of gastric cancer and new ideas for the prevention and control of gastric cancer.Materials and methods:P.anaerobius abundance in patients with gastritis,intestinal metaplasia,and gastric cancer1.Collect gastric mucosa samples from patients with gastritis,intestinal metaplasia,and gastric cancer,extract mucosa DNA and detect gastric mucosa microbiota by 16 S r RNA sequencing,and detect significantly different bacteria in gastric mucosa of patients with gastritis and gastric cancer by LEFSe analysis.2.Paraffin embedding gastric mucosa samples from patients with gastritis,intestinal metaplasia,and gastric cancer were collected.The FISH experiment was performed to detect the colonization of P.anaerobius in gastric mucosa of patients with gastritis,intestinal metaplasia,and gastric cancer.Effect of P.anaerobius on the biological behavior of gastric epithelial cellsThe AGS cells co-cultured with P.anaerobius in vitro model were constructed and the following experiments were performed:1.Electron microscope was used to observe the direct effect of P.anaerobius on AGS cells.2.CCK-8 and EDU experiments were used to detect the cell proliferation ability after co-cultured with P.anaerobius.3.Detect the clone formation ability of cells after co-cultured with P.anaerobius.4.Detect the cell invasion,migration,and wound healing ability of cells after co-cultured with P.anaerobius.5.q RT-PCR assay was used to detect the cellular inflammatory factor(TNF-α,IL-1β,IL-8,IL-10,IL-18)after co-cultured with P.anaerobius.P.anaerobius promotes gastric mucosal inflammation through TLR4/TRIF/NF-κB signaling pathwayThe AGS and GES-1 cells co-cultured with P.anaerobius in vitro model were constructed and the following experiments were performed:1.The m RNA of AGS and GES-1 cells were extracted,and transcriptome sequencing was performed to find differential expressed genes related to P.anaerobius infection.2.Cell protein was extracted and western blot was used to detect the protein expression of key proteins of NF-κB signaling pathway(p-P65(Ser536),P65,IKK,p-IKK(Ser176/180),IκBα).3.Extract cell m RNA and detect the m RNA expression of TLRs(TLR1,TLR2,TLR4,TLR5),TRIF,and Myd88,and the protein expression was verified by Western blot.4.Transfection of TLR4 si RNA to inhibit the expression of TLR4 in AGS cells.The effect of P.anaerobius infection on the protein expression of key proteins of the NF-κB signaling pathway(p-P65(Ser536),P65,IKK,p-IKK(Ser176/180),IκBα),and TLR4,TRIF Myd88 after inhibiting TLR4 was detected by western blot.5.TLR4 si RNA,TLR4 inhibitor Tak-242,and TRIF si RNA were used to inhibit the expression of TLR4 and TRIF.The effect of P.anaerobius infection on cellular inflammatory factor(TNF-α,IL-1β,IL-8,IL-10,IL-18)expression after the inhibition of TLR4 and TRIF was detected by q RT-PCR.6.Construct the fusion plasmid of the flag and target gene TLR4,and construct the P.anaerobius and TLR4 overexpression cell co-culture model.Flag antibody was used for the exogenous immunoprecipitation(IP)experiment to pull down the bacterial protein that bound to TLR4,and the protein components were identified by mass spectrometry.Effects of P.anaerobius infection on gastric mucosal lesions in INS-GAS miceP.anaerobius infected INS-GAS mice model was established by intragastric administration of P.anaerobius every other day for 3 months.The mice were euthanased one month after intragastric administration,and the feces and gastric mucosa samples of mice were collected for the following experiments:1.PCR experiment and FISH experiments were performed to detect the colonization of P.anaerobius.2.H&E staining was performed to detect the pathology of gastric mucosa and alcian blue nuclear solid red staining was performed to detect the metaplasia of gastric mucosa.3.Immunohistochemistry experiment was performed to evaluate the expressions of CD45 and Ki67 in mouse gastric mucosa.4.QRT-PCR was performed to detect the inflammatory factors(TNF-α,IL-1β,IL-8,IL-10,IL-18)expression of mouse gastric mucosa.5.Western blot,immunohistochemistry,and immunofluorescence experiments were performed to detect key proteins of the NF-κB signaling pathway(p-P65(Ser536),P65,IκBα),and TLR4,TRIF Myd88 protein expression.Results:P.anaerobius abundance in patients with gastritis,intestinal metaplasia,and gastric cancerWe collected the gastric mucosa samples of patients with gastritis,intestinal metaplasia,and gastric cancer for 16 S r RNA sequencing,and screened the gastric mucosal bacteria with significant differences between patients with gastritis and gastric cancer by LEFSe analysis.The results showed that the abundance of Peptostreptococcus in patients with gastric cancer was significantly higher than that of gastritis patients(P<0.05).Further analysis showed that the relative abundance of Peptostreptococcus was gradually increased during gastritis,intestinal metaplasia,and gastric cancer process.To confirm the sequencing results,we collected paraffin embedding gastric mucosa samples from patients with gastritis,intestinal metaplasia,and gastric cancer,and FISH experiment was performed to detect P.anaerobius colonization.The results were consistent with the sequencing results that the abundance of P.anaerobius was gradually increased in gastric mucosa of patients with gastritis,intestinal metaplasia,and gastric cancer.The above results suggest that P.anaerobius may be involved in the gastric carcinogenesis process.Effect of P.anaerobius on the biological behavior of gastric epithelial cellsThe above results suggest that P.anaerobius may be a gastric pathogen.To clarify the effect of P.anaerobius on the biological behavior of gastric epithelial cells,we further constructed an in-vitro cell model of P.anaerobius co-cultured with gastric epithelial AGS cells.The results of electron microscopy showed that P.anaerobius bacteria could directly contact the cell surface,and P.anaerobius bacteria could enter the cell also.CCK-8 and EDU experiments showed that P.anaerobius bacteria infection could significantly promote the proliferation of gastric epithelial cells(P<0.05).Transwell invasion and migration experiments showed that P.anaerobius could significantly promote cell invasion and migration ability(P<0.01).In addition,P.anaerobius infection also significantly up-regulated cell inflammatory factors TNF-α,IL-1β,IL-8,IL-10,IL-18 and thus promotes cell inflammation(P<0.001).P.anaerobius promotes gastric mucosal inflammation through TLR4/TRIF/NF-κB signaling pathwayWe further constructed P.anaerobius infected AGS and GES-1 cell models,extracted the cell m RNA,and performed the transcriptome sequencing.Analysis showed that the NF-κB signal pathway is significantly enriched after P.anaerobius infection.We speculate that P.anaerobius may promote gastric mucosal inflammation through the NF-κB signaling pathway.It was found that P.anaerobius bacteria could up-regulate the expression of p-P65(Ser536)and p-IKK(Ser176/180)and down-regulate IκBα expression,promoting P65 into the nucleus(P<0.05).TLRs,TRIF,and Myd88 are upstream of the NF-κB signaling pathway,we found that P.anaerobius infection significantly promoted the expression of TLR4,TLR5,and TRIF(P<0.05),but there was no significant change in the expression of TLR1 and Myd88.Therefore,we speculate that P.anaerobius may activate the NF-κB signaling pathway through TLR4/TRIF axis and promotes gastric mucosal inflammation.The results showed that the inhibition of TLR4 expression by TLR4 si RNA could inhibit P.anaerobius-mediated NF-κB signal pathway activation.The inhibition of TLR4,TRIF by si RNA and inhibitor significantly reduced cellular inflammatory response induced by P.anaerobius(P<0.01).Effects of P.anaerobius infection on gastric mucosal lesions in INS-GAS miceTo determine whether P.anaerobius infection induces gastric mucosal lesions in vivo,we constructed a P.anaerobius-infected transgenic INS-GAS mice model.Firstly,the colonization of P.anaerobius was detected.The result showed that there was no P.anaerobius-specific amplification band in the fecal DNA of the uninfected group,and there were obvious P.anaerobius-specific amplification bands in 4 of the6 mice in the P.anaerobius-infected mice.The gastric mucosal FISH experiment showed that P.anaerobius colonized the stomach of infected mice.The above results showed that P.anaerobius was successfully colonized in the stomach of INS-GAS mice.H&E staining of mice gastric mucosa showed that the gastric mucosa of P.anaerobius infected mice was significantly thickened,with the cystic expansion of glands and obvious inflammatory cell infiltration at the base of mucosa.The inflammatory score was significantly higher than that of the control(P<0.05).The score of CD45 immunohistochemical staining of P.anaerobius-infected mice was significantly higher than that of the control(P<0.05).In addition,P.anaerobius infection could significantly up-regulate inflammatory factors TNF-α,IL-1β,IL-18 expression of INS-GAS mice mucosa(P<0.05).Alcian blue nuclear fixation red staining of gastric mucosa showed that there were large numbers of blue acid-secreting mucus glands in the gastric mucosa of P.anaerobius infected mice,indicating that P.anaerobius infection was related to mucosal intestinal metaplasia.We further verified the effect of P.anaerobius infection on the NF-κB signaling pathway activation,TLR4/TRIF expression in the animal model.The results showed that P.anaerobius infection significantly up-regulate the expression of TLR4,TRIF,and p-P65(Ser536),promoting the entry of P65 into the nucleus,and down-regulate the expression of IκBα in gastric mucosa(P<0.05).Conclusion:1.The P.anaerobius infection induces biological behavior change of gastric epithelial cells,and gastric mucosal lesions in INS-GAS mice,and promotes inflammation in vivo and in vitro.2.P.anaerobius promotes gastric mucosal inflammation through TLR4/TRIF/NF-κB signaling pathway,and inhibition of TLR4 and TRIF alleviates the pro-inflammatory effect of P.anaerobius.