Effects of radix curcumae-derived diterpenoid C on Helicobacter pylori-induced inflammation and nuclear factor kappa B signal pathways

AIM:To study effect of diterpenoid C extracted from radix curcumae on Helicobacter pylori(H.pylori)-infected inflammation,intestinal metaplasia,and nuclear factor kappa B(NF-κB)signaling pathway in vitro.METHODS:We used I-type H.pylori to infect human gastric epithelial gastric epithelium cell line(GES-1)cell lines,and then H.pylori-infected GES-1 cells were treated with radix curcumae(RC)-derived diterpenoid C of different concentrations(5,10,20μg/mL)and amoxicillin.The expression of p65,IκB kinase(IKK)αand IKKγproteins was detected with Western blotting,and the expression of interleukin(IL)-8,IL-6 and IL-4 was determined with enzyme-linked immunosorbent assay method.Data were analyzed using SPSS software ver18.0.For comparisons between groups of more than two unpaired values,one-way analysis of variance(ANOVA)was used.If an ANOVA F value was significant,post hoc comparisons were performed between groups.If results were not normally distributed,the Mann-Whitney U test was used to compare two groups of unpaired values,whereas for comparisons between groups of more than two unpaired values,the Kruskal-Wallis H test was used.Statistical significance was established at P<0.05.RESULTS:The MTT assay results revealed the inhibited rate of GES-1,and indicated that the IC5 of RCderived diterpenoid C and amoxicillin all were 5μg/mL for gastric GES-1 cells.The expression of IL-8 was significantly increased,especially at 12 h time point;and the expression of IL-4 was decreased in H.pyloriinfected GES-1 cells.After H.pylori-infected GES-1 cells were treated with RC-derived diterpenoid C of different concentrations and amoxicillin,the expression of IL-8was decreased at 12,24,48,72 h points(P<0.01),especially in high-concentration diterpenoid C(20μg/mL)group;and the expression of IL-4 was increased,especially in moderate and high-concentration diterpenoid C(10 and 20μg/mL)groups.RC-derived diterpenoid C had the inhibitory effects on H.pylori-induced p65 translocation from cytoplasm into cell nucleus,H.pylori-sti     

Potential effect of chronic Helicobacter pylori infection on glucose metabolism of Mongolian gerbils

AIM: To assess the effect of Helicobacter pylori(H. pylori) infection on metabolic parameters in Mongolian gerbils.METHODS: A total of 40 male, 5- to 8-wk-old, specific-pathogen-free Mongolian gerbils(30-50 g) were randomly allocated into two groups: a control group(n = 20) and an H. pylori group(n = 20). After a two-week acclimation period, the control group was administered Brucella broth and the H. pylori group was challenged intra-gastrically five times every other day with approximately 109/CFU H. pylori ATCC43504(Cag A+, Vac A+). Each group was then divided into two subgroups, which were sacrificed at either 6 or 12 mo. The control and H. pylori subgroups each contained 10 Mongolian gerbils. Body weight, abdominal circumference, and body length were measured, and body mass index(BMI) and Lee's index were calculated. Biochemical assays were used to detect serum indexes, including glucose, glycated hemoglobin(GHb), glycated hemoglobin A1c(Hb A1c), triacylglycerol, and total cholesterol, using an automatic biochemistry analyzer. Inflammatory cytokines, including interleukin(IL)-1β, IL-2, IL-4,IL-10, IL-12, tumor necrosis factor-α(TNF-α) and interferon(IFN)-g, were assayed using ELISA. The expression of insulin and insulin-like growth factor 1(IGF-1) was detected by immunohistochemistry, and islet apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL) assay.RESULTS: At each time point, body weight, abdominal circumference, BMI, and Lee's index were increased after H. pylori infection. However, these differences were not significant. H. pylori infection significantly increased the GHb(5.45 ± 0.53 vs 4.98 ± 0.22, P 0.05) and Hb A1c(4.91 ± 0.61 vs 4.61 ± 0.15, P 0.05) levels at 12 mo. We observed no significant differences in serum biochemical indexes, including fasting blood glucose, triacylglycerol and total cholesterol, at 6 or 12 mo after infection. H. pylori infection significantly increased the expression of IGF-1(P 0.05). Insulin levels from the pancreas and the apoptotic rate of islet β-cells remained unchanged. Also, we observed no significant differences among cytokines levels, including IL-1β, IL-2, IL-4, IL-10, IL-12, TNF-α and IFN-g. IL-4 was the only exception, which increased at 6(44.36 ± 25.17 vs 17.38 ± 3.47, P 0.05) and 12 mo(33.41 ± 10.00 vs 18.91 ± 5.31, P 0.05) after H. pylori infection.CONCLUSION: Long-term H. pylori infection is significantly associated with high levels of Hb A1 c in Mongolian gerbils, indicating a potential role of H. pylori infection in glucose dysregulation.     

Mutagenic potency of Helicobacter pylori in the gastric mucosa of mice is determined by sex and duration of infection

Helicobacter pylori is a human carcinogen, but the mechanisms evoked in carcinogenesis during this chronic inflammatory disease remain incompletely characterized. We determined whether chronic H. pylori infection induced mutations in the gastric mucosa of male and female gpt delta C57BL/6 mice infected for 6 or 12 mo. Point mutations were increased in females infected for 12 mo. The mutation frequency in this group was 1.6-fold higher than in uninfected mice of both sexes (P < 0.05). A:T-to-G:C transitions and G:C-to-T:A transversions were 3.8 and 2.0 times, respectively, more frequent in this group than in controls. Both mutations are consistent with DNA damage induced by oxidative stress. No increase in the frequency of deletions was observed. Females had more severe gastric lesions than males at 6 mo postinfection (MPI; P < 0.05), but this difference was absent at 12 MPI. In all mice, infection significantly increased expression of IFNγ, IL-17, TNFα, and iNOS at 6 and 12 mo, as well as H. pylori–specific IgG1 levels at 12 MPI (P < 0.05) and IgG2c levels at 6 and 12 MPI (P < 0.01 and P < 0.001). At 12 MPI, IgG2c levels in infected females were higher than at 6 MPI (P < 0.05) and also than those in infected males at 12 MPI (P < 0.05). Intensity of responses was mediated by sex and duration of infection. Lower H. pylori colonization indicated a more robust host response in females than in males. Earlier onset of severe gastric lesions and proinflammatory, Th1-biased responses in female C57BL/6 mice may have promoted mutagenesis by exposing the stomach to prolonged oxidative stress.     

Nucleotide Binding Oligomerization Domain 1 Is an Essential Signal Transducer in Human Epithelial Cells Infected with Helicobacter pylori That Induces the Transepithelial Migration of Neutrophils

Background/Aims

The cytosolic host protein nucleotide binding oligomerization domain 1 (Nod1) has emerged as a key pathogen recognition molecule for innate immune responses in epithelial cells. The purpose of the study was to elucidate the mechanism by which Helicobacter pylori infection leads to transepithelial neutrophil migration in a Nod1-mediated manner.

Methods

Human epithelial cell lines AGS and Caco-2 were grown and infected with H. pylori. Interleukin (IL)-8 mRNA expression and IL-8 secretion were assessed, and nuclear factor κB (NF-κB) activation was determined. Stable transfections of AGS and Caco-2 cells with dominant negative Nod1 were generated. Neutrophil migration across the monolayer was quantified.

Results

Cytotoxin-associated gene pathogenicity island (cagPAI)(+) H. pylori infection upregulated IL-8 mRNA expression and IL-8 secretion in AGS and Caco-2 cells compared with controls. NF-κB activation, IL-8 mRNA expression and IL-8 secretion by cagPAI knockdown strains were reduced compared with those infected with the wild-type strain. NF-κB activation, IL-8 mRNA expression and IL-8 secretion in dominant-negative (DN)-Nod1 stably transfected cells were reduced compared with the controls. The transepithelial migration of neutrophils in DN-Nod1 stably transfected cells was reduced compared with that in controls.

Conclusions

Signaling through Nod1 plays an essential role in neutrophil migration induced by the upregulated NF-κB activation and IL-8 expression in H. pylori-infected human epithelial cells.     

Helicobacter pylori tumor necrosis factor-α inducing protein promotes cytokine expression via nuclear factor-κB

AIM:To study the effects of Helicobacter pylori(H. pylori)tumor necrosis factor-α(TNF)inducing protein (Tip-α)on cytokine expression and its mechanism. METHODS:We cloned Tip-αfrom the H.pylori strain 26695,transformed Escherichia coli with an expression plasmid,and then confirmed the expression product by Western blotting.Using different concentrations of Tip-αthat affected SGC7901 and GES-1 cells at different times,we assessed cytokine levels using enzyme-linked immunosorbent assay.We blocked SGC7901 cells with pyrrolidine dithiocarbamate(PDTC),a specific inhibitor of nuclear factorκB(NF-κB).We then detected interleukin(IL)-1βand TNF-αlevels in SGC7901 cells. RESULTS:Western blot analysis using an anti-Tip-α antibody revealed a 23-kDa protein,which indicated that recombinant Tip-αprotein was recombined successfully.The levels of IL-1β,IL-8 and TNF-αwere sig-nificantly higher following Tip-αinterference,whether GES-1 cells or SGC-7901 cells were used(P<0.05).However,the levels of cytokines(including IL-1β,IL-8 and TNF-α)secreted by SGC-7901 cells were greater than those secreted by GES-1 cells following treatment with Tip-αat the same concentration and for the same duration(P<0.05).After blocking NF-κB with PDTC, the cells(GES-1 cells and SGC-7901 cells)underwent interference with Tip-α.We found that IL-1βand TNF-αlevels were significantly decreased compared to cells that only underwent Tip-αinterference(P<0.05). CONCLUSION:Tip-αplays an important role in cyto-kine expression through NF-κB.     

Induction of various cytokines and development of severe mucosal inflammation by cagA gene positive Helicobacter pylori strains

Background—Helicobacter pyloristrains possessing the cagA gene are thought to induceinterleukin 8 (IL-8) in gastric mucosa. However, it is still unclearwhether a relation exists between the cagA gene and theexpression patterns of cytokines other than IL-8.
Aims—To investigate therelation between the cagA gene and the productionof various cytokine proteins using an enzyme linked immunosorbentassay (ELISA).
Patients and methods—In 184 patients,the cagA gene was detected by polymerase chain reaction(PCR), and levels of production of IL-1β, IL-6, IL-7, IL-8, IL-10,and tumour necrosis factor α (TNF-α) in antral biopsy specimenswere measured by ELISA.
Results—Mucosal levels of IL-1β, IL-6,IL-8, and TNF-α were significantly higher in H pyloripositive than in H pylori negative patients.Furthermore, the mucosal levels of IL-1β and IL-8 were significantly higher in specimens infected with cagApositive strains than in those infected with cagAnegative strains. In H pylori positivepatients, the mucosal level of IL-8 was closely correlated withthat of IL-1β (p<0.0001), and the mucosal level of IL-6was closely correlated with that of TNF-α (p<0.0001).
Conclusion—These findings suggest that theability to induce cytokines differs among the strains;cagA⁺ strains induce various kinds ofcytokines and may cause severe inflammation, whereascagA strains induce IL-8 and IL-1β onlyweakly and may cause only mild inflammation. However, as most patientsinfected with the cagA⁺ strains havegastritis, these strains may not be equivalent to ulcerogenic strains.

Keywords:cytokines; Helicobacter pylori; cagA gene; interleukin 8; interleukin 1β

     

Novel role of toll-like receptors in Helicobacter pylori-induced gastric malignancy

Helicobacter pylori(H.pylori)infects the human stomach during infancy and develops into chronic activeinflammation.The majority of H.pylori tend to colonize within the mucous gel layer of the stomach.Thestomach lacks its own immune function,thus innateimmunity as the first line of defense is vital for specificimmunity against H.pylori.We review recent discoveries in the pathophysiologic roles of toll-like receptors(TLRs),mainly TLR2 and TLR4,in H.pylori-induced inflammation.In addition,the TLR pathways activated byH.pylori-induced inflammation have been shown to beclosely associated not only with gastric carcinogenesis,but also with formation of the tumor microenvironmentthrough the production of pro-inflammatory cytokines,chemokines,and reactive oxygen species.Althoughthe correlation between single nucleotide polymorphisms of TLRs and gastric cancer risk remains unclear,a recent study demonstrated that STAT3-driven upregulation of TLR2 might promote gastric tumorigenesis independent of inflammation.Further research onthe regulation of TLRs in H.pylori-associated gastriccarcinogenesis will uncover diagnostic/predictive biomarkers and therapeutic targets for gastric cancer.     

The Effect of Helicobacter pylori on Epidermal Growth Factor Receptor-Induced Signal Transduction and the Preventive Effect of Celecoxib in Gastric Cancer Cells

Background/Aims

Helicobacter pylori infection induces cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) overexpression, and these factors may engage in cross-talk. The aim of the present study was to evaluate the effect of H. pylori on EGFR signaling pathways and to determine whether celecoxib has an inhibitory effect on this pathway.

Methods

The AGS cell line was cocultured with H. pylori G27 and the isogenic cagE- mutant. The expression of COX-2, EGFR, heparin binding-epidermal growth factor (HB-EGF), and transforming growth factor-β (TGF-β) was measured by real time-polymerase chain reaction (RT-PCR). Next, Western blot analyses of COX-2, EGFR, total Akt, phosphorylated Akt (pAkt), and phosphorylated glycogen synthase kinase-3β (pGSK3β) were performed after incubating H. pylori-treated AGS cells for 24 hours with various concentrations of celecoxib (0, 10, 20, and 30 µmol/L).

Results

H. pylori infection upregulated the mRNA levels of COX-2, EGFR, HB-EGF, and TGF-β, as detected by RT-PCR. However, AGS cells treated with cagE- mutants, which have a defective type IV secretion system, did not exhibit EGFR upregulation. Celecoxib had inhibitory effects on the H. pylori-induced overexpression of COX-2 (p=0.015), EGFR (p=0.025), pAkt (p=0.025), and pGSK3β (p=0.029) by Western blot analysis.

Conclusions

H. pylori with an intact type IV secretion system activated the COX-2 and EGFR-Akt pathways in the AGS cell line. As celecoxib exhibited inhibitory effects on the EGFR signaling pathway, the cross-talk of COX-2 and EGFR likely mediates H. pylori-induced gastric cancer.     

MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults

AIM:To evaluate the association between Helicobacter pylori(H.pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability(MSI).METHODS:The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction(MSPPCR) in gastric biopsy samples from uninfected or H.pylori-infected children(n = 50),from adults with chronic gastritis(n = 97) and from adults with gastric cancer(n = 92).MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene(β actin).MSI analysis was performed by screening MSI markers at 4 loci(Bat-25,Bat-26,D17S250 and D2S123) with PCR;PCR products were analysed by single strand conformation polymorphism followed by silver staining.Statistical analyses were performed with either the χ 2 test with Yates continuity correction or Fisher’s exact test,and statistical significance for expression analysis was assessed using an unpaired Student’s t-test.RESULTS:Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children,regardless of H.pylori infection status.The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H.pylori infection(P < 0.05);this region was methylated in 66% of gastric cancer patients,and the difference in the percentage of methylated samples between these patients and those from H.pylori-infected chronic gastritis patients was statistically significant(P < 0.05).MLH1 methylation frequencies among H.pylori-infected and non-infected chronic gastritis adult patients were 13% and 7%,respectively.We observed methylation of the MLH1 promoter(39%) and increased MSI levels(68%) in samples from gastric cancer patients in comparison to samples from H.pylori-infected adult chronic gastritis patients(P < 0.001 and P < 0.01,respectively).The frequency of promoter methylation for both genes was higher in gastric cancer samples than in H.pylori-positiv     

Gastric mucosa in Mongolian and Japanese patients with gastric cancer and Helicobacter pylori infection

AIM: To investigate the characteristics of gastric cancer and gastric mucosa in a Mongolian populationby comparison with a Japanese population.METHODS: A total of 484 Mongolian patients with gastric cancer were enrolled to study gastric cancer characteristics in Mongolians. In addition, a total of 208 Mongolian and 3205 Japanese consecutive outpatients who underwent endoscopy, had abdominal complaints, no history of gastric operation or Helicobacter pylori eradication treatment, and no use of gastric secretion inhibitors such as histamine H2-receptor antagonists or proton pump inhibitors were enrolled. This study was conducted with the approval of the ethics committees of all hospitals. The triple-site biopsy method was used for the histologic diagnosis of gastritis and H. pylori infection in all Mongolian and Japanese cases. The infection rate of H. pylori and the status of gastric mucosa in H. pylori-infected patients were compared between Mongolian and Japanese subjects. Age(± 5 years), sex, and endoscopic diagnosis were matched between the two countries.RESULTS: Approximately 70% of Mongolian patients with gastric cancer were 50-79 years of age, and approximately half of the cancers were located in the upper part of the stomach. Histologically, 65.7% of early cancers exhibited differentiated adenocarcinoma, where as 73.9 % of advanced can cersdisplayed undifferentiated adenocarcinoma. The infection rate of H. pylori was higher in Mongolian than Japanese patients(75.9% vs 4 8. 3 %, P0.0001). When stratified by age, the prevalence was highest among young patients, and tended to decrease in patients aged 50 years or older. The anti-East-Asian Cag Aspecific antibody was negative in 99.4% of H. pyloripositive Mongolian patients. Chronic inflammation, neutrophil activity, glandular atrophy, and intestinal metaplasia scores were significantly lower in Mongolian compared to Japanese H. pylori-positive patients(P 0.0001), with the exception of the intestinal metaplasia score of specimen from the greater curvature of the upper body. The type of gastritis changed from antrumpredominant gastritis to corpus-predominant gastritis with age in both populations.CONCLUSION: Gastric cancer was located in the upper part of the stomach in half of the Mongolian patients; Mongolian patients were infected with non-East-Asiantype H. pylori.     

Effect of Helicobacter pylori and its Virulence Factors on Portal Hypertensive Gastropathy and Interleukin (IL)-8, IL-10, and Tumor Necrosis Factor-alpha Levels

Background/Aim:

We aimed to assess the influence of Helicobacter pylori and its virulent factors, cytotoxin associated gene (cag) A and E, on portal hypertensive gastropathy (PHG) and the levels of interleukin (IL)-8, IL-10, and tumor necrosis factor-alpha (TNF-α).

Patients and Methods:

The patients with cirrhosis underwent screening endoscopy and the lesions related to PHG were graded. Biopsies were obtained for histology, and polymerase chain reaction (PCR) of H. pylori 16S rRNA, cagA, cagE, and tissue cytokine levels was carried out. Absent or mild PHG was compared with moderate to severe PHG.

Results:

One hundred and forty patients with cirrhosis were studied; males numbered 92 and the mean age of the patients was 50.3 ± 12.0 years, H. pylori positivity in 87 (62.1%) patients was associated with male gender (P = 0.032), younger age (P = 0.029), hepatitis D etiology (P = 0.005), higher serum albumin (0.000), lower Child Pugh score (P = 0.001), and lower portal vein diameter (P = 0.001). There was no significant difference in the levels of TNF-α and IL-8. However, a decrease in the anti-inflammatory cytokine IL-10 was noted with moderate to severe gastropathy. Four H. pylori strains were positive for both cagA and cagE, while four were positive for cagA only. All the four patients with both virulent factors had mild gastropathy only.

Conclusion:

The presence of H. pylori infection neither affected the severity of PHG nor augmented the IL-8 and TNF-α levels. There was a decline of virulent H. pylori strains and IL-10 levels in patients with advanced PHG.     

Helicobacter pylori-negative gastric mucosa-associated lymphoid tissue lymphomas: A review

Since Isaacson and Wright first reported on the extranodal marginal zone B-cell lymphoma of the stomach in 1983,following studies have clarified many aspects of this disease.We now know that the stomach is the most affected organ by this disease,and approximately90% of gastric mucosa-associated lymphoid tissue(MALT) lymphomas are related to Helicobacter pylori(H.pylori) infection.This implies that approximately 10% of gastric MALT lymphomas occur independent of H.pylori infection.The pathogenesis of these H.pylori-negative gastric MALT lymphomas remains unclear.To date,there have been several speculations.One possibility is that genetic alterations result in nuclear factor-kappa B(NF-κB) activation.Among these alterations,t(11;18)(q21;q21) is more frequently observed in H.pylori-negative gastric MALT lymphomas,and such translocation results in the synthesis of fusion protein API2-MALT1,which causes canonical and noncanonical NF-κB activation.Another possibility is infection with bacteria other than H.pylori.This could explain why H.pylori eradication therapy can cure some proportions of H.pylori-negative gastric MALT lymphoma patients,although the bacteria responsible for MALT lymphomagenesis are yet to be defined.Recent advances in endoscopy suggest magnifying endoscopy with narrow band imaging as a useful tool for both detecting gastric MALT lymphoma lesions and judging the response to treatment.A certain proportion of H.pylori-negative gastric MALT lymphoma patients respond to eradication therapy; hence,H.pylori eradication therapy could be considered as a first-line treatment for gastric MALT lymphomas regardless of their H.pylori infection status.     

Lactobacillus plantarum B7 inhibits Helicobacter pylori growth and attenuates gastric inflammation

AIM:To determine the anti-Helicobacter property of Lactobacillus plantarum B7(L.plantarum)B7 supernatants in vitro and the protective effects of L.plantarum B7 on serum tumor necrosis factor-alpha(TNF-?),gastric malondialdehyde(MDA)level,apoptosis,and histopathology in Helicobacter pylori(H.pylori)-induced gastric inflammation in rats. METHODS:In vitro,the inhibition of H.pylori growth was examined using L.plantarum B7 supernatants at pH 4 and pH 7 and at the concentration of 1×,5×and 10×on plates inoculated with H.pylori.The inhibitory effect of H.pylori was interpreted by the size of the inhibition zone.In vitro,male Sprague-Dawley rats were randomly divided into four groups including group 1(control group),group 2(H.pylori infected group), group 3(H.pylori infected with L.plantarum B7 106 CFUs/mL treated group)and group 4(H.pylori infected with L.plantarum B7 1010 CFUs/mL treated group).One week after H.pylori inoculation,L.plantarum B7 106 CFUs/mL or 10 10 CFUs/mL were fed once daily to group 3 and group 4,respectively,for one week.Blood and gastric samples were collected at the end of the study. RESULTS:In vitro,at intact pH 4,mean inhibitory zone diameters of 8.5 mm and 13 mm were noted at concentrations of 5×and 10×of L.plantarum B7 supernatant disks,respectively.At adjusted pH 7, L.plantarum B7 supernatants at concentrations of 5 ×and 10×yielded mean inhibitory zone diameters of 6.5 mm and 11 mm,respectively.In the in vitro study, in group 2,stomach histopathology revealed mild to moderate H.pylori colonization and inflammation.The level of gastric MDA and epithelial cell apoptosis were significantly increased compared with group 1.The serum TNF-??level was significant decreased in group 3 compared with group 2(P0.05).In addition,L.plantarum B7 treatments resulted in a significant improvement in stomach pathology,and decreased gastric MDA level and apoptotic epithelial cells. CONCLUSION:L.plantarum B7 supernatant inhibits H.pylori growth.This inhibition was dose-dependent and greater at pH 4.Moreover,L.plantarum B7 attenuated H.pylori-induced gastric inflammation.     

Hypermethylation of TGF-β1 gene promoter in gastric cancer

AIM:To examine transforming growth factor-β1(TGF-β1)promoter methylation in gastric cancer and to determine if Helicobacter pylori(H.pylori)or interleukin(IL)-1β could induce TGF-β1 hypermethylation in vitro.METHODS:We examined the frequency and extent of TGF-β1 promoter methylation using methylationspecific PCR in the gastric tissues from 47 gastric cancer patients and 39 non-gastric cancer subjects.H.pylori infection was confirmed by a positive result from either a serological test,histological analysis or C13urea breath test.GES-1 and MKN-45 cells co-cultured with H.pylori or treated with IL-1β for 12,24 and 48 h in vitro tested the effects of H.pylori or IL-1β on TGF-1β.RESULTS:Twenty-four/forty-seven(51%)cases of gastric cancer(GC)tissues showed TGF-β1 promoter methylation,15/47(31.9%)cases of matched noncancerous gastric mucosa tissues from the GC patients,and 11/39(28%)case of the normal gastric mucosa tissues from non-GC subjects showed TGF-β1 promoter methylation(51%vs 28%,P<0.05).Significantly higher levels of methylation of TGF-β1 were found in the tumor tissues than in non-tumor tissues from GC patients(0.24±0.06 vs 0.17±0.04,P<0.05)and normal gastric tissues from non-GC subjects(0.24±0.06 vs 0.15±0.03,P<0.05).TGF-β1 methylation was found in 48.3% of H.pylori-positive gastric mucosal tissues whereas only 23.1% of H.pylori-negative gastric mucosal tissues showed TGF-β1 methylation(48.3%vs 23.1%,P<0.05).IL-1β appeared to induce a dose-dependent methylation of TGF-β1 and the strongest methylation was observed in GES-1 cells treated with 2.5 ng/mL of IL-1β for 48 h.Further studies showed that pre-treatment of GES-1 cells with 20ng/mL IL-1RA for 1 h could partially abolish the effect of IL-1β on TGF-β1 methylation.Infection of GES-1cells by H.pylori was not found to induce significant TGF-β1 promoter methylation.CONCLUSION:Our data revealed that TGF-1 promoter is methylated in GC patients.IL-1β may be an important mediator for H.pylori induced gene methylation during GC     

Association between TNF-a and IL-1β genotypes vs Helicobacter pylori infection in Indonesia

AIM:To investigate the correlation between the Helicobacter pylori(H.pylori)infection and host genetic background of healthy populations in Indonesia.METHODS:In March 2007,epidemiological studies were undertaken on the general population of a city in Indonesia(Mataram,Lombok).The participants included 107 men and 187 women,whose ages ranged from6 to 74 years old,with an average age of 34.0(±14.4)(±SD).The H.pylori of subject by UBT method determination,and through the polymerase chain reaction with confronting two-pair primers(PCR-CTPP)method parsing the single nucleotide polymorphism of interleukin(IL)-8,IL-4,IL-1β,CD14,tumor necrosis factor(TNF-a)and tyrosine-protein phosphates non-receptor type 11(PTPN11)genotypes.The experimental data were analyzed by the statistical software SAS.RESULTS:The H.pylori infection rates in the healthy Indonesian population studied were 8.4%for men and12.8%for women;no obvious differences were noted for H.pylori infection rates by sex or age.TC genotypes of IL-4,TC and CC genotypes of TNF-a,and GA genotypes of PTPN11,were higher in frequency.Both CC and TC genotype of TNF-a T-1031C loci featured higher expressions in the healthy Indonesian population Indonesia studied of(OR=1.99;95%CI:0.67-5.89)and(OR=1.66;95%CI:0.73-3.76),respectively.C allele of IL-1βT-31C gene locus was at a higher risk(OR=1.11;95%CI:0.70-1.73)of H.pylori infection,but no statistical significance was found in our study.CONCLUSION:We reveal that the association between the TNF-a and IL-1βgenotypes may be the susceptibility of H.pylori in the studied population.     

Helicobacter pylori infection is associated with gallstones: Epidemiological survey in China

AIM: To elucidate the prevalence and risk factors for gallstones, primarily focusing on Helicobacter pylori(H. pylori) infection. METHODS: A total of 10016 Chinese subjects, who had undergone physical examination, fasting 13 C urea breath test and abdominal ultrasonography, had sufficient blood test data, and had finished a questionnaire, were included in this cross-sectional study. Participants(n = 1122) who had previous eradication of H. pylori were studied separately. RESULTS: Gallstones were discovered in 9.10% of men and 8.58% of women, with no significant sex difference. Multivariate analyses displayed that age, aspartate aminotransferase, total cholesterol, H. pylori infection, hepatitis C virus(HCV) infection, and fattyliver had a significant association with gallstones(P 0.05). Successive multiple logistic regression analysis including index of odds ratio(OR) and standardized coefficient(β) indicated that older age(OR/β = 1.056/0.055), H. pylori infection(OR/β = 1.454/0.109), HCV infection(OR/β = 1.871/0.123), and fatty liver(OR/β = 1.947/0.189) had a significant positive association with gallstones. After age stratification, H. pylori infection and fatty liver still had a significant positive association with gallstones in any age-specific groups, whereas HCV infection had a significant positive association in patients aged 40 years. The prevalence of gallstones among H. pylori-positive, H. pylori-eradicated, and H. pylori-negative subjects was 9.47%, 9.02%, and 8.46%, respectively. The matched analysis showed that gallstones among H. pylori eradicated subjects was significantly lower compared with H. pylori-positive subjects(P 0.05).CONCLUSION: H. pylori infection and fatty liver have a significant positive association with gallstones. H. pylori eradication may lead to prevention of gallstones.     

Connexin 32 and 43 promoter methylation in Helicobacter pylori-associated gastric tumorigenesis

AIM: To explore the mechanism of abnormal Connexin (Cx) 32 and Cx43 expression in the gastric mucosa after Helicobacter pylori (H. pylori) infection.METHODS: Biopsy specimens of gastric mucosa in different gastric carcinogenesis stages with H. pylori infection, that is, non-atrophic gastritis (NAG; n = 24), chronic atrophic gastritis (CAG; n = 25), intestinal metaplasia (IM; n = 28), dysplasia (DYS; n = 24), and gastric cancer (GC; n = 30), as well as specimens of normal gastric mucosa without H. pylori infection (NGM; n = 25), were confirmed by endoscopy and pathological examination. Cx32 and Cx43 mRNA expression was detected by real-time polymerase chain reaction (PCR). Cx32 and Cx43 promoter CpG island methylation status was determined by methylation-specific PCR (MSP), bisulfite PCR sequencing (BSP) and MassArray methods.RESULTS: The relative mRNA expression levels in the gastric mucosa of patients with NGM, NAG, CAG, IM, DYS and GC were 0.146 ± 0.011, 0.133 ± 0.026, 0.107 ± 0.035, 0.039 ± 0.032, 0.037 ± 0.01 and 0.03 ± 0.011 for Cx32; and 0.667 ± 0.057, 0.644 ± 0.051, 0.624 ± 0.049, 0.555 ± 0.067, 0.536 ± 0.058 and 0.245 ± 0.121 for Cx43, respectively, which were gradually decreasing and significantly different (GC vs NGM: P < 0.001 for Cx32, P < 0.001 for Cx43). The promoter methylation levels in the gastric mucosa from NGM to GC stages by MSP were 38.8% ± 9.0%, 43.1% ± 9.4%, 56.5% ± 3.1%, 64.4% ± 9.7%, 72.5% ± 4.2% and 79.6% ± 6.8% for Cx32; and 49.0% ± 3.9%, 58.1% ± 5.0%, 66.5% ± 7.9%, 74.0% ± 8.8%, 78.3% ± 3.6% and 88.7% ± 6.2% for Cx43, respectively, which were gradually increasing and significantly different (P = 0.039, P = 0.019). The promoter methylation levels by BSP and MassArray exhibited similar trends. Cx32 and Cx43 mRNA expression was negatively correlated with promoter methylation status and gastric carcinogenesis stages (P < 0.001, P = 0.016).CONCLUSION: Cx32 and Cx43 mRNA expression decreased gradually during H. pylori infection-associated gastric carcinogenesis, and it is associated with hypermethylation of these genes’ promoter.     

Olfactomedin 4 down-regulates innate immunity against Helicobacter pylori infection

Olfactomedin 4 (OLFM4) is a glycoprotein that has been found to be up-regulated in inflammatory bowel diseases and Helicobacter pylori infected patients. However, its role in biological processes such as inflammation or other immune response is not known. In this study, we generated OLFM4 KO mice to investigate potential role(s) of OLFM4 in gastric mucosal responses to H. pylori infection. H. pylori colonization in the gastric mucosa of OLFM4 KO mice was significantly lower compared with WT littermates. The reduced bacterial load was associated with enhanced infiltration of inflammatory cells in gastric mucosa. Production and expression of proinflammatory cytokines/chemokines such as IL-1β, IL-5, IL-12 p70, and MIP-1α was increased in OLFM4 KO mice compared with infected controls. Furthermore, we found that OLFM4 is a target gene of NF--κB pathway and has a negative feedback effect on NF-κB activation induced by H. pylori infection through a direct association with nucleotide oligomerization domain-1 (NOD1) and -2 (NOD2). Together these observations indicate that OLFM4 exerts considerable influence on the host defense against H. pylori infection acting through NOD1 and NOD2 mediated NF-κB activation and subsequent cytokines and chemokines production, which in turn inhibit host immune response and contribute to persistence of H. pylori colonization.     

Medicinal plant activity on Helicobacter pylori related diseases

More than 50%of the world population is infected with Helicobacter pylori(H.pylori).The bacterium highly links to peptic ulcer diseases and duodenal ulcer,which was classified as a groupⅠcarcinogen in 1994 by the WHO.The pathogenesis of H.pylori is contributed by its virulence factors including urease,flagella,vacuolating cytotoxin A(VacA),cytotoxin-associated gene antigen(Cag A),and others.Of those virulence factors,VacA and CagA play the key roles.Infection with H.pylori vacA-positive strains can lead to vacuolation and apoptosis,whereas infection with cagA-positive strains might result in severe gastric inflammation and gastric cancer.Numerous medicinal plants have been reported for their anti-H.pylori activity,and the relevant active compounds including polyphenols,flavonoids,quinones,coumarins,terpenoids,and alkaloids have been studied.The anti-H.pylori action mechanisms,including inhibition of enzymatic(urease,DNA gyrase,dihydrofolate reductase,N-acetyltransferase,and myeloperoxidase)and adhesive activities,high redox potential,and hydrophilic/hydrophobic natures of compounds,have also been discussed in detail.H.pylori-induced gastric inflammation may progress to superficial gastritis,atrophic gastritis,and finally gastric cancer.Many natural products have anti-H.pylori-induced inflammation activity and the relevant mechanisms include suppression of nuclear factor-κB and mitogen-activated protein kinase pathway activation and inhibition of oxidative stress.Anti-H.pylori induced gastric inflammatory effects of plant products,including quercetin,apigenin,carotenoids-rich algae,tea product,garlic extract,apple peel polyphenol,and finger-root extract,have been documented.In conclusion,many medicinal plant products possess anti-H.pylori activity as well as an anti-H.pylori-induced gastric inflammatory effect.Those plant products have showed great potential as pharmaceutical candidates for H.pylori eradication and H.pylori induced related gastric disease prevention.