Esophageal Helicobacter pylori colonization aggravates esophageal injury caused by reflux.

AIM: To investigate esophageal Helicobacter pylori (H. pylori) colonization on esophageal injury caused by reflux and the related mechanisms. METHODS: An esophagitis model, with acid and bile reflux, was surgically produced in male rats. The rats were randomly divided into either: (1) an esophagogastroduodenal anastomosis (EGDA) group; (2) an EGDA with H. pylori infection group; (3) a pseudo-operation with H. pylori infection group; or (4) a pseudo-operation group. All rats were kept for 36 wk. Based on the location of H. pylori colonization, the EGDA rats with H. pylori infection were subdivided into those with concomitant esophageal H. pylori colonization or those with only gastric H. pylori colonization. The esophageal injuries were evaluated grossly and microscopically. The expressions of CDX2 and MUC2 were determined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Ki-67 antigen expression was determined by immunohistochemistry. The mRNA levels of cyclin D1, c-Myc, Bax and Bcl-2 were determined by RT-PCR. Cell apoptosis was evaluated using the TdT-mediated dUTP nick-end labeling method. RESULTS: Esophagitis, Barrett's esophagus (BE), and esophageal adenocarcinoma (EAC) developed in rats that underwent EGDA. When comparing rats with EGDA and concomitant esophageal H. pylori colonization to EGDA-only rats, the severity of injury (87.9 ± 5.2 vs 77.2 ± 8.6, macroscopically, 92.5 ± 8.0 vs 83.8 ± 5.5, microscopically, both P < 0.05) and the incidences of BE (80.0% vs 33.3%, P = 0.055) and EAC (60.0% vs 11.1%, P < 0.05) were increased. These increases were associated with upregulation of CDX2 and MUC2 mRNA (10.1 ± 5.4 vs 3.0 ± 2.9, 8.4 ± 4.6 vs 2.0 ± 3.2, respectively, Ps < 0.01) and protein (8.1 ± 2.3 vs 3.3 ± 3.1, 7.3 ± 4.0 vs 1.8 ± 2.7, respectively, all P < 0.05). The expression of Ki-67 (8.9 ± 0.7 vs 6.0 ± 1.7, P < 0.01) and the presence of apoptotic cells (8.3 ± 1.1 vs 5.3 ± 1.7, P < 0.01) were also increased significantly in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only. The mRNA levels of cyclin D1 (5.8 ± 1.9 vs 3.4 ± 1.3, P < 0.01), c-Myc (6.4 ± 1.7 vs 3.7 ± 1.2, P < 0.01), and Bax (8.6 ± 1.6 vs 5.1 ± 1.3, P < 0.01) were significantly increased, whereas the mRNA level of Bcl-2 (0.6 ± 0.3 vs 0.8 ± 0.3, P < 0.01) was significantly reduced in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only. CONCLUSION: Esophageal H. pylori colonization increases esophagitis severity, and facilitates the development of BE and EAC with the augmentation of cell proliferation and apoptosis in esophageal mucosa.

Let-7b is involved in the inflammation and immune responses associated with Helicobacter pylori infection by targeting Toll-like receptor 4.

OBJECTIVES: Toll-like receptors (TLRs) are important initiators in native immune responses to microbial infections. TLR4 is up-regulated in response to H.pylori infection in gastric epithelial cells. However, the regulatory mechanisms for the expression of TLR4 in H.pylori infection have not been clearly defined. The aims of this study are to present the evidence that microRNA let-7b directly regulates TLR4 expression in human gastric epithelial cells, and subsequently influences the activation of NF-κB and the expression of the downstream genes in H.pylori infection. METHODS: The expression of let-7b was determined in gastric mucosa specimens and in two gastric epithelial cell lines using quantitative RT-PCR. The expression of TLR4 was determined by immunohistochemistry staining and RT-PCR. The potential target of let-7b was identified by luciferase reporter assay and Western blot. Let-7b mimics and inhibitors were used to examine the effects of let-7b on NF-κB activity. The expression of the downstream genes of NF-κB was also determined in cells infected with H.pylori 26695. RESULTS: Let-7b was significantly decreased in gastric mucosa specimens and in gastric epithelial cell lines (AGS, GES-1) infected with H.pylori 26695 (cagA+). Let-7b was complementary to the 3'-UTR of TLR4 mRNA and regulated TLR4 expression via post-transcriptional suppression in gastric epithelium. Infection of H.pylori induced the expression of TLR4 and activated NF-κB in AGS and GES-1 cells. Overexpression of let-7b by mimics downregulated TLR4, and subsequently attenuated NF-κB, MyD88, NF-κB1/p50, RelA/p65. The expression of IL-8, COX-2 and CyclinD1 was inhibited in H.pylori infected cells with let-7b overexpression. Both TAK-242 (TLR4 inhibitor) and SN50 (NF-κB inhibitor) significantly inhibited the H.pylori induced downregulation of let-7b. CONCLUSIONS: Let-7b targets at TLR4 mRNA, and regulates the activation of NF-κB and the expression of the downstream genes related to the inflammation and immune responses in H.pylori infection.