Helicobacter pylori Infection Eradication on Nonalcoholic Fatty Liver Disease: A Randomized Controlled Trial

Abstract

Previous studies have found that Helicobacter pylori (H. pylori) infection is associated with nonalcoholic fatty liver disease (NAFLD). The purpose of the present study was to investigate the effect of H. pylori eradication treatment on NAFLD patients. Two hundred NAFLD patients who tested positive for H. pylori infection were randomized into the H. pylori eradication treatment group or the control group. Metabolic and inflammatory parameters and FibroScan were measured in all subjects at baseline and 1 year after treatment. At 1 year after treatment, the decrease in metabolic indicators, such as fasting blood glucose, glycosylated haemoglobin, homeostasis model assessment of insulin resistance (HOMA-IR), triglycerides, body mass index and controlled attenuation parameter (CAP) value, were more obvious in the treatment group. Moreover, the inflammatory indicators white blood count (WBC) and high-sensitivity C-reactive protein (hs-CRP) and the inflammatory factors Interleukin 6 (IL-6) and Tumor necrosis facter-α(TNF-α) were also significantly decreased. H. pylori eradication can further reduce the metabolic indices of NAFLD and the degree of liver steatosis. H. pylori infection may participate in the occurrence and development of NAFLD through its influence on inflammatory factors. Thus, checking for the presence of H. pylori infection in patients at risk of NAFLD may be beneficial.

Introduction

Helicobacter pylori (H. pylori) causes significant morbidity and mortality in gastric cancer, peptic ulcer and chronic gastritis, which infect more than half of the world’s population¹.The World Health Organization (WHO) has classified H. pylori as a class I carcinogen².

Moreover, H. pylori infection has been defined as an infectious disease^3,4 and considered to be associated with many extragastrointestinal diseases. Studies have suggested that H. pylori infection increases systemic inflammation by producing inflammatory factors, which results in the development of insulin resistance (IR) and metabolic syndrome (MetS)^5–7. Therefore, H. pylori may be a risk factor for cardiovascular disease, diabetes and nonalcoholic fatty liver disease (NAFLD)^8–15.

NAFLD is characterized by excessive fat deposition in liver cells, excluding secondary causes such as viral hepatitis, alcohol or hereditary liver diseases. It is closely related to IR and genetic susceptibility to metabolic stress-induced liver damage and ranges from isolated steatosis, nonalcoholic steatohepatitis (NASH) and cirrhosis^16,17. Current routinely used modalities, such as laboratory tests and ultrasonography, cannot adequately determine the levels of steatosis and fibrosis, and liver biopsy is not accepted by most people. Among the noninvasive tests, transient elastography (FibroScan®, TE) has demonstrated good accuracy in quantifying the levels of liver steatosis and fibrosis in patients with NAFLD^18,19.

To our knowledge, few studies have evaluated the effect of H. pylori eradication treatment on NAFLD patients. Thus, we conducted a randomized controlled trial to investigate the effect of eradicating H. pylori infection on NAFLD patients.

Results

Discussion

In our study, the influence of lifestyle intervention on NAFLD was considered in the design. In addition to the comparison before and after treatment in the treatment group, a control group with only lifestyle intervention was specifically added. In addition, the hepatic steatosis CAP value was used to evaluate the treatment effect, which made the results more objective. Our prospective study showed the significant effect of eradicating H. pylori infection on NAFLD patients. Thus, H. pylori eradication therapy may improve the therapeutic effect of NAFLD by reducing the body's inflammatory indicators.

Although controversy remains, increasing evidence shows that there is an association between H. pylori infection and NAFLD. In addition, NAFLD and H. pylori infection present a high global incidence^20,21, and both increase with age. The potential clinical significance of research into these diseases lies in the fact that if the two are related, then eradicating H. pylori infection may prevent NAFLD from further developing into advanced diseases, such as liver cirrhosis and even hepatic cancer.

Only a few studies have evaluated the therapeutic effect of eradicating H. pylori infection on NAFLD. Polyzos et al.¹¹ conducted a 12-month prospective study, selected NAFLD patients confirmed by liver biopsy, and divided them into an H. pylori-positive group and an H. pylori-negative group. The H. pylori-positive group was treated with eradication therapy, and hepatic steatosis, NAFLD fibrosis score and HSENSI (homocysteine, serum glutamic oxaloacetic transaminase, erythrocyte sedimentation rate, nonalcoholic steatohepatitis index) were used to evaluate the efficacy of H. pylori eradication treatment on NAFLD. The results showed that eradicating H. pylori had no long-term effect on hepatic steatosis but improved the NAFLD fibrosis score and HSENSI index. However, another randomized open-label trial²² compared lifestyle modification alone and lifestyle modification combined with the eradicating H. pylori eradication therapy in NAFLD patients. After follow-up for 6 months, both groups showed improved liver function (AST, ALT and r-GT), liver fat content and HOMA-IR; however, the values did not differ between the two groups and only dyspeptic NAFLD patients were included in the study.

A recently published study²³ further established the relationship between H. pylori infection and NAFLD and provided evidence for eradicating H. pylori in patients with NAFLD. This is a multicentre cohort study of 369 adults without NAFLD at baseline who were followed up for 2 years. H. pylori infection was detected by stool antigen, and the patients were divided into positive and negative groups. During the follow-up period, the researchers observed a higher incidence of NAFLD in H. pylori-positive individuals and no NAFLD in the H. pylori-negative group. After 2 years of follow-up, the H. pylori-positive NAFLD patients were further treated with eradication therapy based on lifestyle modification. It was found that eradicating H. pylori treatment can reduce HIS and NAFLD liver fat scores. During the 2-year follow-up, 23 new NAFLD patients were diagnosed, 18 (78.3%) NAFLD patients achieved remission after H. pylori eradication treatment, and only 5 remained unchanged (21.7%). More importantly, the HOMA-IR, adipokines, and inflammatory markers, which are considered to be key indicators involved in the occurrence and development of NAFLD, were also significantly improved.

Studies have found that due to low-grade inflammation in obese and type 2 diabetic patients, the tissue and serum IL-6 levels are abnormally increased, which promotes the occurrence and development of chronic inflammation²⁴. IL-6 can inhibit insulin-mediated lipolysis in white adipose tissue, thereby increasing the delivery of FFAs to the liver and promoting the occurrence and development of NAFLD²⁵. Studies have found that the use of anti-IL-6 antibodies to treat obese mice can increase insulin sensitivity, indicating that IL-6 is involved in the pathogenesis of liver IR²⁶. Similarly, elevated levels of TNF-α can disrupt insulin signalling through serine phosphorylation, thereby inducing insulin resistance in adipocytes and surrounding tissues and leading to the development of T2DM²⁷.

WBC and hs-CRP are the most commonly used inflammatory indicators in clinical practice, and their detection is simple, easy and inexpensive. Many studies^28–30 have found that the WBC level is positively correlated with NAFLD. In addition, other studies have found that hs-CRP is also a promising biomarker for screening cardiovascular metabolic diseases and NAFLD^31,32. At the same time, a retrospective cohort study by Lee et al.³² found that even within the normal range, a higher level of hs-CRP was still an independent risk factor of NAFLD.

The results of our study showed that after only one year of treatment with correct lifestyle changes, the control group presented decreases in various metabolic indices and hepatic steatosis CAP values, and the difference was significant. This showed that simply relying on lifestyle intervention has a therapeutic effect on NAFLD. Similarly, we found that 1 year after H. pylori eradication treatment with lifestyle intervention, the metabolic indices and hepatic CAP values were also significantly lower than those before treatment, and the difference was significant.

Then, we further analysed the difference in the final efficacy between the two groups. The results of our study found that compared with the control group, the treatment group had a more significant decrease in metabolic indices and hepatic steatosis CAP values. This demonstrated that on the basis of lifestyle intervention, the eradicating H. pylori treatment can significantly improve the therapeutic effect on NAFLD patients. Moreover, the inflammatory indicators WBC and hs-CRP and inflammatory factors IL-6 and TNF-α all decreased significantly in the treatment group, with significant difference observed compared with the control group. Therefore, we believe that eradicating H. pylori infection helps to delay the occurrence and development of NAFLD by decreasing inflammatory factors. These results can be reflected in the clinical routine inflammation indicators WBC and hs-CRP at the same time; therefore, the therapeutic effect of NAFLD can be followed-up by monitoring the changes in WBC and hs-CRP in the clinic.

This study has some limitations. First, we diagnosed NAFLD by ultrasonography, which was not as accurate as biopsy but was easily accepted by patients. We also used the hepatic steatosis parameter CAP value to evaluate the treatment effect. Second, patients with obvious abnormal liver function are not suitable for simultaneous H. pylori eradication therapy. The enrolled subjects were selected based on abnormal liver enzyme index values less than 2 times the UNL, and those with hypertension, diabetes, and hyperlipidaemia under treatment were excluded. Thus, selection bias may have occurred, which may explain the lack of significant differences in the changes in liver function indices between the two groups.

In conclusion, our study found that for patients subject to the same lifestyle management, treatment to eradicate H. pylori infection can further reduce the metabolic indices of NAFLD and the degree of liver steatosis. H. pylori infection may participate in the occurrence and development of NAFLD through its influence on inflammatory factors in the body. Clinically, the therapeutic effect of NAFLD can be followed-up by monitoring changes in WBCs and hs-CRP. We hope that additional well-designed large randomized controlled studies with longer follow-up durations are performed to further explore the relationship between H. pylori infection and NAFLD patients.

Methods

Ethics approval and consent to participate

All participants provided written informed consent before the examination. The present study was reviewed and approved by the Ethics Committee of the 2^nd Affiliated Hospital, School of Medicine, Zhejiang University (2016-322). 

Registration

The trial was registered on Chinese Clinical Trials Registry, with the name of the registry being “Helicobacter pylori Infection Eradication on Nonalcoholic Fatty Liver Disease: A Randomized Controlled Trial.” The trial registration number was ChiCTR2200061243.  

Questionnaires

The medical history of each participant was obtained using a questionnaire that included the history of the present illness, previous diagnoses of H. pylori infection, history of anti-H. pylori therapy, history of gastric surgery, history of significant mental or neurological disorders, history of cancer(s), use of bismuth, antibiotics, PPIs or H₂ blockers within the previous 4 weeks, alcohol intake, and cigarette smoking.

Diagnosis of H. pylori infection

After fasting for at least 2 hours, all participants underwent ¹³C-UBT at our centre. After a baseline breath sample was collected, the participants ingested a ¹³C-urea reagent that was dissolved in water. The second breath sample was collected 30 minutes later and analysed. A delta over baseline (DOB) value ≥4.0 indicated a positive result for H. pylori infection.

Definition of NAFLD

NAFLD was defined according to the guidelines published in 2012 by the American Association for the Study of Liver Diseases (AASLD), American College of Gastroenterology (ACG), and American Gastroenterological Association (AGA)³³. In this study, the diagnosis of NAFLD required (1) hepatic steatosis detected by ultrasonography; (2) no significant alcohol consumption (to strictly exclude the influence of alcohol, we chose individuals with alcohol consumption of less than 3 drink units per week); and (3) no coexisting causes of chronic liver disease, such as hepatitis C, medications, parenteral nutrition, Wilson’s disease or severe malnutrition.

Treatments

The enrolled NAFLD subjects were randomized to H. pylori eradication treatment or untreated groups based on standard instructions for diet and exercise according to the guidelines. The H. pylori eradication treatment consisted of 14 days of quadruple therapy (Esomeprazole 20 mg given twice daily + colloidal bismuth pectin 200 mg twice daily + amoxicillin 1000 mg twice daily + furazolidone 100 mg twice daily). If the patient was allergic to amoxicillin, it was substituted with clarithromycin (500 mg twice daily). Eradication was assessed by ¹³C-UBT 4 weeks after the end of treatment. Eradication was considered successful when the UBT was negative.

The untreated groups only accepted health education and lifestyle guidance.

Health education and lifestyle guidance

Two general practitioners (unknown about the research group) provided full-time guidance and monthly telephone or network follow-up supervision.

Specific content was based on the recommendations of the guidelines.

1)         Moderate-intensity calorie restriction and personalized daily calorie intake according to the body mass index are recommended;

2)         Changes in the diet composition to a low-sugar and low-fat balanced diet and reductions of sucrose beverages and saturated fat and intake of trans fats and increase fibre content are recommended;

3)         A moderate amount of aerobic exercise is recommended 5 times a week, and the cumulative exercise time should be at least 150 min.

Data collection

Blood pressure measurements were obtained after at least 10 minutes of rest. Body mass index (BMI) was defined as weight divided by height squared (kg/m²). The waist circumference (WC) was measured while standing with a measuring tape midway between the lowest rib and the iliac crest. The fasting plasma WBC, hs-CRP, fasting blood glucose, glycosylated haemoglobin (HbA1c), alanine aminotransferase (ALT), γ-glutamyltranspeptidase (γ-GT), aspartate aminotransferase (AST), total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), and triglyceride (TG) levels were measured after an 8-hour overnight fast (Beckman Coulter AU 5400). The HOMA-IR was calculated according to the following formula: HOMA-IR=[FINS (µIU/mL)×FPG (mmol/L)]/22.5²⁵.

IL-6 and TNF-α

Venous blood (4 ml) was collected into a serum separation tube. After the blood clot formed, the sample was centrifuged, and the serum was collected. The total IL-6 and TNF-α contents in plasma were determined using ELISA (Abcam, USA).

FibroScan

Liver stiffness was evaluated using a FibroScan-502 touch (Echosense, Paris, France). The operation was carried out according to the user manual by two specially trained doctors who obtained the FibroScan operator certificate.

1. Fasting patients were in the supine position, and they held their head with their right hand to maximize the expansion of the intercostal space.
2. The probe was placed on the right lobe of the liver in the intercostal position.
3. The probe was kept perpendicular to the skin, and when the pressure indicator displayed in green, the M waveform on the display screen was consistent and evenly distributed and the A waveform was linear; subsequently, the detection process started.
4. The unit of liver steatosis was dB/m, which was achieved in 10 replications with a success rate of 60% and an interquartile range of <30%.

Statistical analysis

The statistical analysis was performed using SPSS 13.0 software. Measurement data are expressed as the mean ± standard deviation. A paired t test was used for comparisons before and after treatment within groups; a single factor analysis of variance was used for multigroup comparisons, and SNK was used for pairwise comparisons between groups. All p values were based on a two-sided test of statistical significance. Significance was accepted at the level of p<0.05.

Declarations

AcknowledgEments

Funding: This work was supported in part by funding from the Zhejiang Provincial Natural Science Foundation of China (LGF19H030016), and the Medical and Health Technology Projects of Zhejiang Province (2018KY413, 2022KY815). 

Author contributions

Y Yu, L Wu and X Yu. designed the study, performed the experiment, supervised the fndings of this work, and wrote the manuscript. Y Tong. provided patients for the study, monitored the medical conditions of patients, helped in the design and interpretation of data, and critically reviewed the work. All the authors reviewed and approved the final manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

DATA AVAILABILITY STATEMENT

The full trial protocol can be found at Chinese Clinical Trials Registry. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 

ADDITIONAL INFORMATION 

Competing interests

The authors declare no competing interests.

Correspondence and requests for materials should be addressed to Y Yu.

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