Effects of active and latent H. pylori infection coupled with chronic alcohol ingestion on cytokine profiles and markers of oxidative balance in men seropositive for H. pylori CagA Ab: An observational study.

This study aimed to explore the effects of active and latent Helicobacter pylori infection coupled with alcohol consumption on cytokine profiles and markers of oxidative balance in men seropositive for H. pylori CagA Ab.The 100 male subjects were divided into groups with active H. pylori infection and H. pylori CagA Ab coupled with chronic alcohol ingestion (group A, n = 38), latent H. pylori infection with H. pylori CagA Ab coupled with chronic alcohol ingestion (group B, n = 30), and latent H. pylori infection with H. pylori CagA Ab without chronic alcohol ingestion (group C, n = 32).No differences in serum levels of CRP, IL-10, ADP, E-selectin, MDA, or SOD were detected between the 3 groups or between any 2 groups (all P > .05). The serum IL-6 and TNF-α concentrations in groups A and B were significantly lower than those in group C (P = .004, P = .005, P = .009, and P = .023). However, there were no differences in serum IL-6 and TNF-α between group A and group B (all P > .05).In conclusion, active or latent H. pylori infection coupled with chronic alcohol ingestion may decrease certain cytokines, that is, IL-6 and TNF-α, in men with H. pylori CagA Ab seropositivity. However, there was no difference in the detected cytokine profile between active and latent H. pylori infection coupled with chronic alcohol ingestion, and no changes were detected in markers of oxidative balance in men with H. pylori CagA Ab.

Introduction

Helicobacter pylori (H. pylori) infection is one of the most common chronic bacterial infections affecting humans worldwide.[H. pylori has received much attention over the last 2 decades after its recognition as a pathogen that infects a significant proportion of the human population.[ Low-to-moderate alcohol consumption is known to reduce the risk of cardiovascular disease; however, chronic high-dose alcohol consumption is associated with cardiovascular diseases, such as hypertension.[ Previous studies have demonstrated that H. pylori and chronic ethanol intake are associated with increased incidences of a variety of illnesses, including atherosclerosis-related diseases such as cardiovascular and cerebrovascular diseases, metabolic diseases such as type 2 diabetes mellitus, fatty liver disease, gastrointestinal diseases, and cancers. The impact of chronic ethanol intake on H. pylori infection remains unknown. A previous study using a multiple logistic model demonstrated that alcohol consumption and related pathologies (i.e., active gastritis) are associated with H. pylori infection, establishing a link between alcohol consumption and this infection.[ Chronic alcohol abuse appears to favor colonization by H. pylori, resulting in the production of ammonia that promotes the development of chronic gastritis.[ Other studies have demonstrated an inverse relationship between ongoing moderate alcohol consumption and the presence of H. pylori infection, suggesting that alcohol consumption may facilitate the elimination of this chronic infection.[ Daily alcohol consumption appears to have an additive effect on this eradication therapy,[ and alcohol intake has also been suggested to promote elimination of H. pylori infection in adults.[ For example, moderate consumption of wine and beer (approximately 7 units/wk) appears to protect against H. pylori infection, presumably by facilitating eradication of the organism.[ The consumption of alcohol, particularly of wine, may reduce the risk of developing an active H. pylori infection.[ Furthermore, heavy alcohol intake may be associated with reductions in the prevalence and severity of H. pylori infection.[ However, contradictory results have demonstrated that the type of alcohol consumed does not affect the age-adjusted risk of H. pylori infection[ and that smoking habits and alcohol consumption do not affect this infection in the stomach.[ Our previous study[ revealed that H. pylori CagA expression may lead to significantly higher levels of several inflammatory markers in both chronic alcohol users and nonconsumers. Chronic alcohol ingestion coupled with H. pylori CagA positivity does not result in significant changes in a subject's cytokine profile. However, it remains unclear whether male subjects presenting with H. pylori infection coupled with H. pylori CagA antibody positivity and chronic alcohol consumption exhibit cytokine profile changes or alterations in oxidative balance. We therefore sought to explore whether active and latent H. pylori infection with H. pylori CagA antibody positivity accompanied by chronic alcohol consumption might affect cytokine profiles and oxidative balance by measuring the levels of cytokines and oxidative markers.

Materials and methods

Ethics statement

Written informed consent was obtained from all the enrollees. The protocol was approved by the clinical research ethics committee of Taishan Hospital in Shandong Province.

Study population

A health examination-based cross-sectional study was conducted on 100 male subjects from January 2012 to May 2015. All of the subjects were evaluated for chronic alcohol consumption by completing a relevant questionnaire, and active H. pylori infection was assessed with the 13C-urea breath test.[H. pylori CagA positivity, in which a serum H. pylori CagA Ab concentration ≥80 pg/mL was considered positive (CagA Ab+), was analyzed in patients at Taishan Hospital, Shandong Province. Chronic alcohol ingestion for men was defined as a daily ethanol intake of greater than 40 g for a period of >5 years.[ The subjects enrolled in this study were divided into the following 3 groups: active H. pylori infection with H. pylori CagA Ab positivity coupled with chronic alcohol ingestion (group A, n = 38), latent H. pylori infection with H. pylori CagA Ab and chronic alcohol ingestion (group B, n = 30), and latent H. pylori infection with H. pylori CagA Ab without chronic alcohol ingestion (group C, n = 32). This study was conducted to evaluate the correlations between alcohol consumption and cytokine profiles and oxidative balance in men with active and latent H. pylori infection coupled with H. pylori Ab positivity by measuring serum levels of H. pylori CagA Ab, C-reactive protein (CRP), interleukin (IL)-6, IL-10, adiponectin (ADP), E-selectin, tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), and superoxide dismutase (SOD) by enzyme-linked immunosorbent assay (ELISA). The clinical protocol conformed to the Declaration of Helsinki. Subjects were excluded based on the following criteria: a positive smoking status, the presence of a fever or infectious disease, the use of anti-inflammatory drugs, antibiotics or medication toxic to H. pylori, diagnosis of primary or secondary liver, kidney, heart, nerve, endocrine, autoimmune or hematological disease, the presence of an electrolyte or acid-base balance disorder, cancer, or mental disorders.

Experimental setup and reagents

The experimental equipment included an Enzyme Standard Instrument (Type ANTHOS 2010, Austria), and the reagents used in this study included ELISA kits for H. pylori CagA Ab, CRP, IL-6, IL-10, ADP, E-selectin, TNF-α, MDA, and SOD (Shanghai Enzyme-Linked Immune Co. Ltd., made by R&D Systems, USA).

Blood collection and handling

On the same day as the general health examinations were performed, peripheral venous blood samples were collected from the patients after fasting overnight for at least 10 hours. To measure serum levels of H. pylori CagA and CRP, IL-6, IL-10, ADP, E-selectin, TNF-α, MDA, and SOD, the samples were collected in ice-cold tubes containing EDTA (1 mg/mL) and centrifuged at 3000 rpm for 10 minutes. The plasma was then stored at −70°C until analysis. To ensure the reliability of the experimental results, all of the serum samples were carefully preserved, and repeated freeze-thaw cycles were avoided. Finally, all of the parameters were measured by ELISA according to the manufacturer's instructions.

Statistical analysis

The SPSS statistical package (version 17.0 for Windows; SPSS Inc., Chicago, IL) was used for all statistical analyses. All of the data for the 3 groups were expressed as the median and range; Kruskal–Wallis ANOVA was used for comparisons among the 3 groups, and the Mann–Whitney U test was used for comparisons between 2 groups. Differences with a P value of <.05 were considered statistically significant.

Results

Age, BMI, mean duration of alcohol use, and mean daily alcohol consumption are listed in Table 1. No differences in age were detected among the 3 groups or between any 2 groups, and no differences in the mean duration of alcohol use or mean daily alcohol consumption were detected between any 2 groups (all P > .05). No significant differences in BMI were detected among the 3 groups or between any 2 groups (P > .05).

Table 1

Comparison of age, BMI, mean duration of alcohol use, and mean daily alcohol consumption among 3 or 2 groups (medians with ranges).

Serum CRP, IL-6, and IL-10 concentrations from the subjects in each of the 3 groups are listed in Table 2. No differences in serum CRP and IL-10 levels were detected among the 3 groups or between any 2 groups (all P > .05). Significant differences in serum IL-6 concentrations were observed among the 3 groups (P = .004); the IL-6 concentrations in groups A and B were significantly lower than that in group C (P = .004, P = .005, respectively), and no difference in serum IL-6 was detected between group A and group B (P > .05). Serum ADP, E-selectin, and TNF-α concentrations in the 3 groups are listed in Table 3. No differences in serum ADP or E-selectin levels were detected among the 3 groups or between any 2 groups (all P > .05). Significant differences in serum TNF-α were observed among the 3 groups (P = .017); TNF-α levels were significantly lower in groups A and B than in group C (P = .009 and P = .023, respectively), and no difference in serum TNF-α was detected between group A and group B (P >.05).

Table 2

Comparison of serum CRP, IL-6, and IL-10 levels among the 3 groups (ng/L, medians with ranges).

Table 3

Comparison of serum ADP, E-selectin, and TNF-α levels among the 3 groups (ng/L, medians with ranges).

Serum MDA and SOD concentrations in the 3 groups are listed in Table 4. No differences in serum MDA and SOD were detected among the 3 groups or between any 2 groups (all P > .05).

Table 4

Comparison of serum MDA and SOD levels among the 3 groups (medians with ranges).

Discussion

Active H. pylori infection and alcohol consumption may induce changes in inflammatory markers levels. However, this viewpoint remains controversial. Previous studies have examined the association between H. pylori infection and H. pylori CagA positivity, but studies examining the relationship between active H. pylori infection and cytokine profile in patients with chronic alcohol consumption are lacking. Our present study demonstrated that active and latent H. pylori infection coupled with chronic alcohol ingestion may decrease the levels of certain cytokines, that is, IL-6 and TNF-α, in men with H. pylori CagA Ab seropositivity. However, there was no difference in the detected cytokine profile between active and latent H. pylori infection in patients with chronic alcohol ingestion, suggesting that chronic alcohol consumption may result in significantly lower levels of certain inflammatory mediators in men with active and latent H. pylori CagA Ab positivity, consistent with a previous study.[H. pylori infection has been reported to significantly and independently contribute to insulin resistance in a large asymptomatic population.[H. pylori is a highly pathogenic microorganism equipped with various strategies to evade human immune responses,[ as supported by studies demonstrating that H. pylori infection is associated with increased serum TNF-α.[ In contrast to previous findings, the presence of H. pylori infection was not found to affect the severity of portal hypertensive gastropathy or to augment IL-8 or TNF-α levels. A decrease in the abundance of virulent H. pylori strains has been associated with IL-10 levels in patients with advanced portal hypertensive gastropathy.[ In addition, a study on serum cytokines revealed that H. pylori infection in children does not affect systemic cytokine secretion, in contrast with adults.[ The results of another study suggested that the TNF-857T/T genotype may confer protection against chronic H. pylori infection.[Therefore, the changes in systemic cytokine levels that have been observed in patients with various diseases occur differently in patients with H. pylori infection. Other previous studies have revealed that chronic infection with H. pylori expressing CagA is correlated with high circulating levels of IL-6,[ TNF-α,[ and ADP.[ Similarly, alcohol consumption has been reported to alter the levels of certain cytokines, such as IL-6, IL-8, IL-10, IL-12, TNF-α,[ and E-selectin.[ Subjects who harbor CagA(+) strains of H. pylori exhibit more severe mucosal damage, increased bacterial colonization, an increased probability of developing duodenal ulcers, and increased serum TNF-α compared with subjects infected with CagA(−) strains.[ In additionally, seropositivity for H. pylori was found to be strongly associated with CAG incidence, whereas advanced age and H. pylori infection are key risk factors for the development of CAG.[ However, contradictory findings have suggested that H. pylori CagA is not associated with upregulation of IL-8 in gastric epithelial cells[ and that host inflammatory responses in the gastric mucosa are not correlated with CagA expression.[ Notably, different levels of alcohol ingestion can have distinct effects on the human body. Four weeks of moderate alcohol consumption can result in alterations in immune responses and lipid metabolism.[ Chronic ethanol consumption is associated with increased incidences of a variety of illnesses, including cancer.[ However, the subjects who slept well and consumed a moderate amount of alcohol exhibited the lowest IL-6 concentrations compared with the other 3 groups who consumed alcohol. Moderation and regularity in the practice of certain health behaviors, including sleep, have been associated with lower plasma levels of inflammatory markers in older adults.[ Alcohol may modulate the inhibitory effect of TNF-α on ADP production and thus increase its plasma concentration.[ Alcohol may specifically improve insulin sensitivity by increasing the expression of anti-inflammatory genes.[ In agreement with previous studies, our study has demonstrated that active H. pylori infection, H. pylori CagA(+), and chronic alcohol consumption can affect the cytokine profile. In addition, H. pylori CagA positivity may be the main cause of changes in serum cytokines. However, additional studies are required to more deeply investigate the relationship between infection with CagA positive H. pylori and patient cytokine profile to determine the urgency in eradicating H. pylori infection and preventing the inflammatory reaction associated with H. pylori infection in patients displaying chronic alcohol consumption.

Substantial evidence indicates that H. pylori infection and chronic alcohol intake influence markers of oxidative stress. The results of the present study demonstrated that regardless of H. pylori infection or chronic alcohol ingestion, the subjects with H. pylori CagA Ab positivity presented with significant increases in the levels of MDA and SOD. H. pylori CagA Ab may therefore contribute to increased oxidative stress in men. Previous studies have demonstrated a close association between high MDA levels and H. pylori infection.[ Oxidative stress has been shown to promote tissue damage in H. pylori-infected children.[ In addition, moderate wine consumption may be advantageous for protection against this infection. For example, diabetic patients consuming a moderate amount of red wine and a polyphenol-enriched diet showed slower progression to diabetic nephropathy.[ However, a contradictory study indicated that moderate alcohol consumption promotes oxidative stress in chronic hepatitis C (CHC) patients, suggesting a role of oxidative damage in CHC progression due to alcohol.[ In addition, other studies have demonstrated that ethanol consumption may result in an oxidative stress imbalance[ and that “adaptive cytoprotection” induced by chronic alcohol intake may increase the activities of gastric antioxidants to reduce mucosal damage.[ Hence, alcohol may accelerate oxidative mechanisms directly by increasing the production of reactive oxygen species and indirectly by impairing protective mechanisms against these molecules.[ In addition, increased levels of reactive oxygen species generated from acetaldehyde oxidation may contribute to oxidative stress damage.[ Antioxidants and defense enzymes appear to confer protection as a consequence of chronic adaptation in alcoholics.[ Research has shown that because antioxidant supplementation decreased the alcohol-induced pMMP-9 levels, oxidative stress could be one of the mediators of the generation of MMP-9.[ Components in alcoholic and nonalcoholic wine, particularly polyphenols, may influence oxidative balance and endothelial function.[ Furthermore, the roles of oxidative stress and H. pylori infection with CagA positivity in chronic alcohol ingestion and related diseases remain unknown. Previous studies have revealed that a low prevalence of H. pylori infection is associated with moderate alcohol consumption, suggesting a protective mechanism of adequate alcohol consumption mediated by “adaptive cytoprotection,” which reduces the risk of H. pylori infection.[ However, our previous study demonstrated no association between oxidative balance and H. pylori infection in patients with chronic alcohol consumption;[ The present study detected no difference in markers of oxidative balance between active and latent H. pylori infection with H. pylori CagA Ab positivity and chronic alcohol ingestion. Hence, further studies are required to more rigorously evaluate these mechanisms.

Our study has several limitations. First, it is a prospective observational study rather than a randomized controlled study, and the number of subjects was low. Second, the roles of different types of alcohol in H. pylori infection with or without active H. pylori CagA Ab positivity were not examined in our study. In addition, the study samples were small and biased in that they only contained male subjects. This study was unable to control for the potential effects of other factors, such as other drugs.

In conclusion, active and latent H. pylori infection coupled with chronic alcohol ingestion may reduce levels of certain cytokines, that is, IL-6 and TNF-α, in men with H. pylori CagA Ab positivity. However, there was no difference in the cytokine profile between active and latent H. pylori infection in chronic alcohol users, and no changes were detected in markers of oxidative balance in men positive for H. pylori CagA Ab. These data suggest that chronic alcohol ingestion may help reduce the inflammatory response in men with active or latent H. pylori infection and CagA Ab positivity; we speculate that a curative treatment for H. pylori is not urgent in such populations. However, the number of subjects was low in this study, and more studies in the larger population are needed to confirm these findings.

Acknowledgments

We thank the technical assistance of Yisheng Sun, WAND Yanbo, and Hong Wang. The article was edited by American Journal Experts.

Author contributions

Conceptualization: Zhongdong Wang.

Data curation: Baoge Qu, Yiguo Jia, Zhongdong Wang, Xinghai Han, Guangying Ren.

Funding acquisition: Baoge Qu.

Investigation: Baoge Qu, Yiguo Jia, Zhongdong Wang, Xinghai Han, Guangying Ren.

Methodology: Baoge Qu, Yiguo Jia.

Project administration and supervision: Baoge Qu.

Investigation and data collection: Yiguo Jia, Zhongdong Wang, Xinghai Han, Guang ying Ren.

Statistical analysis: Baoge Qu.

Writing – original draft: Yiguo Jia, Baoge Qu.

Writing – original draft: Yiguo Jia.

Writing – review and editing: Baoge Qu.