Abstract: Helicobacter pylori infection and its eradication may influence plasma levels and gastric production of some peptides, which can affect appetite. Ghrelin, an orexigenic hormone, is primarily produced in and secreted from oxyntic mucosa of the stomach. Ghrelin has been demonstrated to play a central role in appetite, food intake and energy homeostasis. As ghrelin production in human is exclusively gastric origin, it is conceivable and not surprising that any injury to the gastric mucosa will affect plasma ghrelin concentrations. To verify this hypothesis, a total of 135 adult consecutive individuals with normal body mass index including 84 H. pylori-infected and 51 H. pylori-negative subjects were included in a randomized controlled trial. Gastric ghrelin mRNA expression levels were measured in endoscopic biopsy specimens in both groups before and after H. pylori eradication. Also, plasma active n-octanoyl ghrelin and obestatin levels and ghrelin/obestatin ratio were measured in both groups before and after H. pylori eradication. The treatment group (44/84) received triple H. pylori eradication therapy for 7 days and followed up for 6 months. In contrast to obestatin, plasma and gastric ghrelin mRNA expression levels were significantly lower in H. pylori-infected subjects. H. pylori eradication significantly reversed these changes. The decrease in plasma ghrelin concentration in H. pylori-positive subjects was accompanied by depletion of ghrelin mRNA expression. These findings suggest that H. pylori induced chronic gastritis impair gastric ghrelin production and consequently the decrease in plasma ghrelin concentration.
INTRODUCTION
Ghrelin is a 28 amino acid peptide hormone discovered by Kojima et al. (1999) and produced mainly in the stomach. Human ghrelin producing cells, the P/D1 enteroendocrine cells of the gastric mucosa (X/A-like cells in rats), are situated mainly in the corpus and fundus of the stomach. Ghrelin is secreted into the extracellular matrix then to the general circulation through capillary vessels in the gastric lamina propria in its active octanoylated form (Date et al., 2000). It has been demonstrated that ghrelin plays central as well as peripheral roles in appetite modulation, food intake, eating behavior, body-weight regulation, energy homeostasis, gastric motility and acid secretion (Masuda et al., 2000; Nakazato et al., 2001).
Ghrelin O-acyltransferase (GOAT) is responsible for octanoylation of ghrelin before its secretion into blood stream which is essential for biological actions of ghrelin. Only the active acylated form of ghrelin can bind to and stimulate the ghrelin receptors which are growth hormone secretagogue receptors (GHS-R) accounting for many of its biological actions such as appetite stimulation and anti-inflammatory actions (Osawa, 2008; Jeffery et al., 2011).
Ghrelin, to a lesser extent, was found to be expressed in and secreted from multiple tissues like the gut, kidney, lung, pancreas (A cells) and hypothalamic nucleus (Ariyasu et al., 2001). It exerts multiple endocrine, paracrine and autocrine effects. Circulating ghrelin levels have been associated with and responsible for multiple biological effects including appetite and body weight control. Ghrelin stimulates GHS-R and via a GHRH-dependent mechanism will stimulate growth hormone and insulin-like growth factor 1 (ILGF-1) release. The released hormones (GH and ILGF-1) affect a range of physiological processes (Nakazato et al., 2001).
The normal serum ghrelin level in adults of healthy body mass index (BMI) ranges from 145-200 fmol mL-1 with little higher levels in females. Plasma ghrelin secretion is episodic and dimorphic. Ghrelin is considered to be a short term orexigen related to meals as its plasma concentrations rise before and decrease after meals. Collectively, ghrelin regulates somatic and adipose tissue growth thus contributes to long term regulation of body weight. It has been suggested that ghrelin increases acid secretion, stimulates gastric emptying (Cummings et al., 2002) and exerts a gastro-protective actions on the gastric mucosa (Asakawa et al., 2001).
It is intuitive that chronic gastritis and gastric damage with or without atrophy might affect ghrelin production, activation and secretion because of the gastric location of ghrelin producing cells. This will affect appetite and food intake and in turn change body weight and BMI (Jeffery et al., 2011).
Helicobacter pylori is a gram negative curved or S shaped rods and has a worldwide prevalence. More than 50% of the world’s population are infected with higher prevalence in developing countries (Brown, 2000). H. pylori specifically infects the gastric mucosal cells. The stomach and/or heterotopic gastric mucosa are the natural habitat of this well adapted parasite (Hofman et al., 2004).
Several studies have suggested an association between H. pylori infection and various gastric and extra-digestive diseases such as immunological, cardiovascular and a variety of other pathologies (Gasbarrini et al., 1999, 2004). H. pylori infection leads to multiple gastro-doudenal pathologies (Schreiber et al., 2004). H. pylori infection is involved in the pathogenesis of gastritis, gastric and doudenal erosions and/or ulcers; mucosa associated lymphoid tissue lymphoma (MALT-oma), intestinal metaplasia with or without dysplasia or gastric adenocarcinoma (Uemura et al., 2001; Goodwin et al., 1997).
Several sero-epidemiological studies have suggested an association between ghrelin and Helicobacter pylori infection. Many authors reported an increase of plasma ghrelin levels after H. pylori eradication (Nweneka and Prentice, 2011; Nwokolo et al., 2003). On the other hand, others concluded that H. pylori infection and/or its eradication had no effect on ghrelin concentrations (Gokcel et al., 2003). So, it is not known whether alteration of plasma ghrelin levels is the result of compromised gastric ghrelin production caused by H. pylori infection. Consequently, the relationship between gastric ghrelin and H. pylori is still conflicting. The aim of this study was to investigate and assess the effects of H. pylori infection on gastric ghrelin mRNA expression using real-time quantitative RT-PCR in gastric endoscopic biopsies and concomitantly examining plasma ghrelin concentrations.
MATERIALS AND METHODS
Subjects: A total of 161 consecutive adult outpatients, older than 20 years, with functional dyspepsia (classification according to Rome III criteria) (Drossman, 2006) were initially enrolled in this study (from June 2010 through 2011). The study was approved by the Ethical Commission and Institutional Review Board of Mansoura University Hospital in EGYPT. A written informed conscious consent was obtained from all patients before their participation.
Exclusion Criteria included chronic or psychiatric illness, age<20 year; age>70 year; pregnancy; liver disease; renal impairment; cardiopulmonary disease; abnormal macroscopic endoscopic lesions, including gastric ulcers, cancer or duodenal ulcers; previous gastrointestinal surgery; history of H. pylori eradication; smoking, drug or alcohol abuse-defined as consumption of more than two alcoholic drinks per day and NSAIDs or PPIs administration during the 2-4 week prior to the study. Patients with histopathological changes higher than those of non-atrophic gastritis (according to Sydney System Scale update (Dixon et al., 1996) and with a Body Mass Index (BMI) below 18 and above 29 kg m-2 were excluded from the study.
Methods
History and examinations: Initially, all patients completed a detailed questionnaire
regarding diet and habits, submitted to thorough history taking and detailed
physical examinations performed at fasting in the morning. The selected subjects
had their BMI calculated (as body weight in kilograms divided by the square
of their height in meters (Weigle et al., 2003).
The appetites of all subjects were estimated using a Visual Analog Scale (VAS)
of appetite (Kim et al., 2008).
Endoscopy: All the participants underwent routine investigations and upper gastrointestinal endoscopic evaluation at enrolment (a gastroscopy with biopsy; XQ20 or XQ40, Pentax Fibreoptic, Tokyo) performed after an overnight fast between 08:00 and 10:00 a.m. to avoid the effects of diurnal hormone variation.
During endoscopy, the macroscopic picture of the mucosa was assessed in terms of color, mobility, vascular network. The intensity of various features of gastritis was evaluated in accordance with Sydney visual scale of division criteria, with Houston modification for chronic gastritis (Bartuzi et al., 2000).
From each patient, four biopsy specimens were taken from the midportion of the fundus along the greater curvature; four were taken from intact mucosa in the gastric antrum, 2 cm proximal to the pylorus and two from the incisura angularis where maximal degrees of atrophy and intestinal metaplasia are consistently found (Stemmermann, 1994).
One biopsy from each site was used to perform either biopsy urease test (CLO test: Ballard Medical Products, Draper, UT, USA) or culture to detect H. pylori infection.
One biopsy sample from each site was fixed in neutral formaldehyde 10% solution, dehydrated in ascending grades of alcohol then embedded in paraffin for histological assessment.
| Table 1: | Flow chart of the study |
| A total of 161 consecutive adult outpatients with Functional Dyspepsia, Twenty six patients were excluded according to exclusion criteria, The remaining 135 patients were assigned to the following groups | |
The remaining two specimens, taken from the anterior and posterior wall of the antrum or corpus were transferred to tubes containing TRIzol® (Gibco, Long Island, NY, USA) and stored immediately at -70°C until assayed for gastric ghrelin concentrations.
Groups: A total of 161 consecutive adult outpatients with Functional Dyspepsia (FD) were initially enrolled in this study. From these patients, eleven with severe oesophagitis, nine with peptic ulcer, one with gastric cancer and five with atrophic gastritis were excluded. The remaining 135 patients (Group I:84 H. pylori positive and Group II:51 H. pylori negative) were assigned to the following groups (Table 1):
Group Ia: treatment group: This group comprised 44 H. pylori positive patients (females/males 18/26; median age 44 (29-55 years) with a diagnosis of chronic H. pylori gastritis. They were re-evaluated one and six months after appropriate eradication therapy which included; Lansoprazole (30 mg bid), Metronidazole (500 mg bid), Amoxicillin (1000 mg bid) and Clarithromycin (500 mg bid) for seven days. This 7-day regimen of quadruple therapy was proved highly (98%) effective eradication therapy (De Boer et al., 1996).
Group Ib: Non-treated control group: This group comprised 40 H. pylori positive patients (females/males 18/22; median age 44 (29-63 years) with a diagnosis of chronic H. pylori gastritis. This control group did not receive any prescription and was directed not to take any medication.
Group IIa: H. pylori negative patients without chronic gastritis: This group comprised 35 patients (females/males 16/19; median age 45 (30-57 years) patients with a diagnosis of H. pylori negative and a normal gastric histology.
Group IIb: H. pylori negative patients with chronic gastritis: This group comprised 16 patients (females/males 6/10; median age 46 (36-57 years) patients with a diagnosis of H. pylori negative but with chronic gastritis.
All groups underwent a repeated endoscopy one month later. The eradicated subjects (39/44) were followed up for further six months. All of the procedures were repeated at the time of repeated endoscopy including the determination of gastric and plasma active ghrelin and obestatin concentrations.
| Table 2: | Definition and grading guidelines for each of the histological features to be graded according to the Sydney classification Kim and Baek (1999) Mild if gastritis score is ≤10, Moderate if gastritis score is 10-20, Severe if gastritis score is ≥20 |
Histology: The biopsy samples were treated in a standard manner. The sections were stained with haematoxylin and eosin for conventional histopathological examination and preliminary assessment of presence of H. pylori using oil immersion lens. H. pylori infection was further evaluated with (modified) Giemsa staining and/or Cresyl violet acetate stain (Stevens and Francis, 1996). Two paraffin-embedded tissue blocks (from the antrum and corpus) were used for microscopic section preparation. From each paraffin block, two sections were obtained.
The microscopic gastric mucosa assessment, H. pylori density, polymorph-nuclear neutrophil activity (PMN) and chronic inflammation (mononuclear cell; MNC) were scored on a scale of 0 to 3 to each graded variable: 0 = absence, 1 = mild, 2 = moderate and 3 = severe degree (Table 2) based on criteria according to the updated Sydney System Classification. The scores from the body were calculated to give a total "corporal gastritis score" for each patient which ranged from 0-15. Similarly, figures from gastric antrum were calculated to give a total "antral gastritis score" for each patient. If the difference between both scores was more than two points, it was considered either antral-predominant or corpus-predominant gastritis. Atrophy and metaplastic changes in gastric glands were excluded (Dixon et al., 1996).
Histological evaluations were performed by a well-trained Histopathologist who was blind to the treatment, endoscopic diagnosis, H. pylori status and subject group identity.
Hormonal assays: Venous blood samples were collected after an over-night fast into the Lavender Vacutainer tubes (#VT-6450) which contain EDTA-2Na and can collect up to 7 mL blood/tube. Gently rock the Lavender Vacutainer tubes several times immediately after collection of blood for anti-coagulation. Transfer the blood from the lavender vacutainer tubes to centrifuge tubes containing aprotinin (0.6 TIU mL-1 of blood) and gently rock several times to inhibit the activity of proteinases. Centrifuge the blood at 1,600xg for 15 min at 4°C and collect the plasma. Plasma stored at -70°C until assayed. 1 TIU (Trypsin Inhibitor Unit) = 1025~1300 KIU (Kallikrein Inhibitor Unit) (Catalog No.: RK-APRO).
Plasma levels of active octanoylated ghrelin were determined with Human Ghrelin (Active) competitive radioimmunoassay (RIA) kit which measures only acylated ghrelin (Linco/Millipore Research, St. Charles, MO, Bio-manufacturing and Life Science Research, Catalogue Number: GHRA-88 HK). The sample designated for ghrelin analysis was acidified with 50 μL of 1 NHCl and 10 μL of phenylmethylsulfonyl fluoride (PMSF) were added at 1 mL of plasma. These samples were measured in duplicate. Standard Curve Range: 10-2000 pg mL-1. Intra-assay coefficients of variation (CV) were 7.3 and 7.8% at 78 and 538 pg mL-1, respectively. Inter-assay CV was 9 and 12% at 40 and 356 pg mL-1. Accuracy was 100%. Assay sensitivity is 7.8 pg mL-1 when using a 0.1 mL sample size. Assay specificity is as follows: human ghrelin, 100%; ghrelin 1-10, 100%.
The plasma obestatin was measured using a radioimmunoassay (RIA) kit (Phoenix Pharmaceuticals, INC.). Catalog # RK-031-92 Range: 5-640 pg tube-1 in 100 μL of Standard solution = 50-6400 pg mL-1. Lowest Detection Limit: 10 pg mL-1. The inter-and intra-assay coefficients of variation were less than 11% and less than 5%, respectively (Patrono and Peskar, 1987).
Gastric mRNA detection
RNA isolation: Total RNA was isolated from the biopsy specimen with
ISOGEN (Nippon Gene, Tokyo, Japan) using a standard reported procedure. The
isolated RNA used for RT-PCR by using random nanomers and reverse transcriptase
according to the manufacturer’s protocol (Mackey and
Chomczynski, 1996).
RNA isolation protocol: The protocol includes the following steps:
| • | Homogenization: ISOGEN 1 mL (50~100 mg tissue) using a glass-Teflon or Polytron homogenizer |
| • | Phase separation: Homogenate+0.2 mL chloroform centrifuge at 12000 g (max.) |
| • | RNA precipitation: Aqueous phase+0.5 mL isopropanol. Precipitate RNA from the aqueous phase by mixing with 0.5 mL of isopropanol per 1 mL of ISOGEN. RNA precipitate (often invisible before centrifugation) forms a gel-like pellet or white pellet on the side and bottom of the tube |
| • | RNA wash: Ethanol 75% 1mL. Remove supernatant and wash the RNA pellet (by vortexing) with 75% ethanol and subsequent centrifugation at 7500 g (max.) at 4~25 °C for 5 min |
| • | RNA solubilization: SDS or water 0.5%. Remove the ethanol and briefly air-dry the RNA pellet for 3~5 min. Dissolve RNA in water or 0.5% SDS by passing the solution several times through a pipette and incubate at 55~60°C for 10~15 min. Water or 0.5% SDS solution used for RNA solubilization were made RNase-free by diethylpyrocarbonate (DEPC) treatment |
The final preparation of total RNA was highly pure, free of DNA and proteins and had a 260/280 ratio 1.6-1.9. For optimal spectrophotometric measurements, RNA aliquots were diluted with water or buffer with pH>7.5. The concentration and purity of the RNA were determined using spectrophotometry (Ultrospec® 1100 Pro; Amersham Pharmacia Biotech, Buck, UK) to measure the optical density ratio at 260 and 280 nm. The isolated purified RNA is ready to use in, real-time RT-PCR.
REAL-time RT-PCR of ghrelin mRNA: The expression level of ghrelin mRNA was evaluated by using a real-time quantitative RT-PCR method. One microgram of total RNA was used as a template to generate cDNA by using M-MLV (Moloney murine leukemia virus) reverse transcriptase (Super Bio, Suwon, Korea, Cat. No. 04655885001) with random hexamer priming (Car. No. 11034731001). The resultant cDNA was amplified using an Exicycler 96 Real-time Quantitative Thermal Block (Bioneer, Seoul, Korea) then used as a template for RT-PCR. Real-time PCR analysis was carried out with SYBR® Premix Ex Taq™ (TaKaRa Bio, Tokyo, Japan, Cat. No. RR041A) and gene specific primers retrieved from Primer Bank.
The sequences of primers were as follows: Ghrelin: 5'-ATG CTC CTG TGG GAC TTG-3'(sense) and 5'-TCT GCT TGA CCA CCT TCT T-3'(antisense; Genebank; Accession number NM 016362; ACC No. NM 016362; product size, 155 bp) The PCR specificity is examined by 3% Ready Agarose Precast Gel (Bio-Rad). Analyze the real-time PCR result with the SDS 7000 software. The gene mRNA levels were corrected using β-actin.
Determinations of intragastric pH: At gastroscopy, immediately after passing the endoscope into the stomach, a sterile Teflon catheter was passed through the biopsy channel and starting from the mid-body along the greater curve approximately 5 mL of gastric juice were aspirated avoiding contamination with blood and collected in a sterile tube containing EDTA. Gastric juice pH values were immediately measured with a glass electrode pH meter.
Statistical analysis: Data were analyzed using SPSS software (Version 19.0). Quantitative data were expressed as (Mean±SD) while qualitative data were expressed as number and percentage. Continuous data are expressed as median (range) and were evaluated by appropriate statistical tests; t test (for paired data e.g. to compare ghrelin/obestatin ratio in the plasma and gastric mRNA expression measured before and after treatment). Proportions were compared by means of Fisher’s exact test. Correlations were evaluated using the Spearman rank correlation coefficient test. Kruskal-Wallis one way analysis of variance (ANOVA) compares more than two groups.
Multiple regression analysis was used to assess the effects of H. pylori eradication on ghrelin production by the gastric mucosa, adjusted by the weight change and change in neutrophil or mononuclear cell infiltration. Subgroups (percentages of patients) were compared by using the McNemar test. A value of p≤0.05 was considered statistically significant. Sensitivity, specificity and predictive values were calculated to study the overall predictability of other techniques (histopathology, urease test …) according to the following formulae:
RESULTS
Table 1 shows the flow chart of the study. Twenty six patients of a total of 161 consecutive adult outpatients with Functional Dyspepsia (FD) were excluded according to exclusion criteria.
| Table 3: | Histological Criteria of Gastritis in H. pylori+ve Subjects (Group I) Mild if gastritis score is ≤10; Moderate if gastritis score is 10-20; Severe if gastritis score is ≥20; Predominance: if the differences between antral and corporal gastritis scores is >2 |
The remaining 135 patients were assigned to the following groups: group Ia: H. pylori+ve (84 (♀/♂: 36/48) and group Ib: H. pylori-ve (51(♀/♂22/29). Only 39 patients in group Ia (13/26) were followed up to 6 ms (two were not eradicated and three received drugs which affect appetite). Five of forty in the control groups were excluded from follow up; three received eradication therapy for H. pylori and two received drugs which affect appetite.
Table 2 Shows the Definition and Grading Guidelines for each of the Histological Features Graded According to the Sydney Classification.
Table 3 shows the Histological Criteria of Gastritis in H. pylori+Ve Subjects (the type, severity, extent and topography of gastritis and Density of H. pylori colonization). All patients of group I had chronic superficial, non atrophic, H. pylori-induced gastritis. The majority of patients had diffuse pan gastritis (70/84; 83%) with corporal predominance while only 2/84 had antral restricted gastritis and the remaining 12/84 (14.3%) had corporal restricted gastritis. Most patients had moderate (33/84; 39%) to severe (40/84; 47.6%) H. pylori colonization from which only 12/84 (14.3%) had severe H. pylori gastritis.
Table 4 shows some characteristics (Mean±SD) of all patients groups including the age, sex, BMI, VAS, total gastritis scores, H. pylori density in the corpus, PMN, MNC.
Table 5 shows some characteristics (Mean±SD) of all patients groups including plasma ghrelin and obestatin levels, ghrelin/obestatin ratio, gastric ghrelin mRNA expression level and intragastric pH.
Comparative analysis of the different characteristics in different groups (p-values and ANOVA are significant values if p≤0.05) are given in both Table 4 and 5. There were no significant differences between the H. pylori+ve and H. pylori-ve groups were observed with regard to age, sex, BMI, VAS (P1≤0.05).While, there were highly significant differences between the two groups with regard to baseline characteristics including plasma ghrelin and obestatin levels, ghrelin/obestatin ratio, gastric ghrelin mRNA expression levels, neutrophil (PMN), mononuclear cell (MNC) infiltration, HP density, total gastritis score and intragastric pH (P1≤0.01). The increase in gastritis scores (total, H. pylori, PMN, MNC) was associated with significant decrease in plasma and gastric ghrelin levels and ghrelin/obestatin ratio but was associated with significant increase in plasma obestatin level and intragastric pH.
No initial differences between the treatment (Ia) and control (Ib) groups were observed with regard to all baseline characteristics (P2≤0.05).
| Table 4: | Clinical, demographic data and histological criteria of gastritis in all groups of patients |
| MNC: Mononuclear cells, PMN: Polymorphonuclear cells, VAS: Visual analogue scale, BMI: Body mass index, P1: Compares baseline characteristics of HP+ve and HP-ve groups, P2: Compares baseline characteristics of group Ia (treatment) and group Ib (control), P3: Compares characteristics of group Ia (treatment) and group Ib (control) after one month. P4: Compares characteristics of group Ia (treatment) at baseline and after one month, P5: Compares characteristics of group Ib (control) at baseline and after one month, Anova 1: Compares characteristics of treatment group at baseline, one month and 6 months, Anova 2: Compares baseline characteristics of HP+ve group according to density of H. pylori, Significance if p≤0.05 | |
After one month follow up, there were highly significant differences between the treatment (Ia) and control (Ib) groups with regard to characteristics including plasma ghrelin and obestatin levels, ghrelin/obestatin ratio, gastric ghrelin mRNA expression levels, PMN, HP density, total gastritis score and intragastric pH (P3≤0.01). Whereas, no differences between the two groups were observed with regard to age, sex, BMI, VAS and MNC infiltration (P3≤0.05).
| Table 5: | Laboratory parameters in all groups of patients |
| P1: Compares baseline characteristics of HP+ve and HP-ve groups, P2: Compares baseline characteristics of group Ia (treatment) and group Ib (control), P3: Compares characteristics of group Ia (treatment) and group Ib (control) after one month, P4: Compares characteristics of group Ia (treatment) at baseline and after one month, P5: Compares characteristics of group Ib (control) at baseline and after one month, Anova 1: Compares characteristics of treatment group at baseline, one month and 6 months, Anova 2: Compares baseline characteristics of HP+ve group according to density of H. pylori, Significance if p≤0.05 | |
After one month follow up, no statistically significant differences between the subjects of control (Ib) groups were observed with regard to all characteristics (P5≤0.05).While, in treatment group, at one month after eradication therapy, gastritis scores significantly improved (total, H. pylori, PMN) and consequently plasma and gastric ghrelin levels and ghrelin/obestatin ratio were significantly increased while plasma obestatin level and intragastric pH were significantly decreased (P4≤0.01).
Also, by comparing the changes in treatment groups at baseline, one month and six months after eradication therapy, highly significant changes between the three groups were observed with regard to all characteristics except age, sex (ANOVA 1:p≤0.05). There were marked improvement (increase of plasma and gastric ghrelin levels and ghrelin/obestatin ratio; decrease of obestatin and intragastric pH) especially after six months.
In H. pylori+ve group, patients are divided into mild, moderate, severe H. pylori density and colonization.
| Table 6: | Spearman correlations (r) in group I (H. pylori+ve) at baseline; significance if p≤0.05 (2-tailed) |
No initial significant differences between the three subgroups were observed with regard to age, sex and VAS (ANOVA 2:p≤0.05) but there were highly significant changes between the three subgroups with regard to other characteristics including plasma ghrelin and obestatin levels, ghrelin/obestatin ratio, gastric ghrelin mRNA expression levels, total gastritis score, MNC infiltration, BMI and intragastric pH (ANOVA 2:p≤0.05). The more H. pylori density and colonization in both gastric compartments especially the corpus, the more depletion in gastric ghrelin mRNA expression levels and consequently reduction in plasma ghrelin concentrations.
Table 6 shows the spearman correlations (r) in group I (H. pylori+ve) at baseline; significance only if p≤0.05 (2-tailed). In patients of Group I, gastric ghrelin mRNA expression as well as plasma ghrelin and obestatin levels, ghrelin/obestatin ratio and BMI were significantly negatively correlated with total gastritis scores (esp. corporal score, H. pylori density, PMN) (r = -0.714, p = 0.000) and intragastric pH (r = -0.564, p = 0.000). On the other hand, gastric ghrelin mRNA expression was positively correlated with plasma ghrelin and obestatin levels, ghrelin/obestatin ratio and BMI (r = 0.907, 0.485, 0.541 and 0.520; p = 0.000, 0.000, 0.000 and 0.000, respectively). As regard to intragastric pH, there was significantly positively correlated with total gastritis scores. (r = 0.590, p = 0.000).
Figure 1 shows the mean values of plasma ghrelin and obestatin levels and gastric ghrelin mRNA expression level in H. pylori+ve and H. pylori-ve subjects. Bars indicate mean values. In HP+ve group, the plasma ghrelin levels and gastric ghrelin mRNA expression level were significantly lower compared with HP-ve group while plasma obestatin level was significantly higher in H. pylori+ve group (p<0.001).
Figure 2 shows the mean values of plasma obestatin, gastric ghrelin mRNA expression level and total gastritis scores in H. pylori+ve subjects in relation to plasma ghrelin concentrations.
| Fig. 1: | Mean values of plasma ghrelin and obestatin levels and gastric ghrelin mRNA expression level in H. pylori+ve and H. pylori-ve subjects, Bars indicate mean values |
| Fig. 2: | Mean values of plasma obestatin, gastric ghrelin mRNA expression level and total gastritis scores in subgroups of group I (H. pylori+ve subjects) according to plasma ghrelin concentrations. Bars indicate mean values |
Bars indicate mean values. H. pylori+ve subjects with low plasma ghrelin levels<200 pg mL-1 had significantly lower gastric ghrelin mRNA expression level, plasma obestatin level and ghrelin/obestatin ratio and significantly higher gastritis scores (total, PMN, MNC) (p<0.001) compared with higher ghrelin subgroup (ghr.≥200 pg mL-1).
Figure 3 shows the mean values of plasma ghrelin, obestatin and gastric ghrelin mRNA expression level in H. pylori+ve subjects in subgroups of group I (H. pylori+ve subjects) according to the severity of gastritis and H. pylori density and colonization. Bars indicate mean values.
| Fig. 3: | Mean values of plasma ghrelin, obestatin and gastric ghrelin mRNA expression level in H. pylori+ve subjects in subgroups of group I (H. pylori+ve subjects) according to the severity of gastritis and H. pylori density and colonization. Bars indicate mean values |
| Fig. 4: | Changes in plasma ghrelin, obestatin, gastric ghrelin mRNA expression levels and BMI in treatment group (group Ia) at baseline, one month and six months after eradication therapy |
In H. pylori+ve subjects, the more severity of gastritis in both gastric compartments especially the corpus (H. pylori density and colonization and PMN infiltrations), the more depletion in gastric ghrelin mRNA expression levels and consequently reduction in plasma ghrelin concentrations and the more depletion in plasma obestatin levels (Anova 2:p≤0.05).
Figure 4 shows the changes in plasma ghrelin, obestatin, gastric ghrelin mRNA expression levels and BMI in treatment group (group Ia) at baseline, one month and six months after eradication therapy. There were significant changes in plasma and gastric ghrelin levels being increased after eradication (Anova 1; p≤0.01) BMI increased also but significant only after six months (Anova 1; p≤0.01). The more increase in gastric ghrelin mRNA expression level, the more increase in plasma ghrelin level (positive correlations) (Anova 1; p≤0.01).
| Fig. 5: | Changes in plasma and gastric ghrelin mRNA expression levels in treatment and control groups at baseline and one month later after eradication therapy |
| Fig. 6: | Endoscopic macroscopic picture of the fundus showing gastritis in the form of hyperemia of the fundic mucosa with some superficial erosions |
Figure 5 shows the changes in plasma and gastric ghrelin mRNA expression levels in treatment and control groups at baseline and one month later after eradication therapy. In treatment group, there were significant changes in plasma and gastric ghrelin levels being increased after eradication (p4≤0.01) while in the control group, these parameters were decreased significantly one month from the baseline (p5≤0.01).
Figure 6 shows the endoscopic macroscopic picture of the fundus showing gastritis in the form of hyperemia of the fundic mucosa with some superficial erosion.
Figure 7 gastric mucosa showing moderate number of H. pylori (arrows) on luminal surface of epithelial cells of gastric pits (crossed arrow). (H and Ex1000).
Figure 8 gastric mucosa showing neutrophils among the rest of inflammatory cells with invasion of a gastric gland (crossed arrow) by a neutrophil (arrow). (H and Ex1000).
Figure 9 gastric mucosa showing infiltration of the lamina propria of mucosa by lymphocytes (arrowheads), plasma (arrows) cells and neutrophils (crossed arrows). (H and Ex1000).
| Fig. 7: | Gastric mucosa showing moderate number of H. pylori (arrows) on luminal surface of epithelial cells of gastric pits (crossed arrow) (H and Ex1000) |
| Fig. 8: | Gastric mucosa showing neutrophils among the rest of inflammatory cells with invasion of a gastric gland (crossed arrow) by a neutrophil (arrow), (H and Ex1000) |
| Fig. 9: | Gastric mucosa showing infiltration of the lamina propria of mucosa by lymphocytes (arrowheads), plasma (arrows) cells and neutrophils (crossed arrows). (H and Ex1000) |
Figure 10 gastric mucosa showing infiltration of the lamina propria of mucosa by mononuclear (MNC) inflammatory cells (arrowheads) (H and Ex400).
Figure 11 gastric mucosa showing infiltration of the lamina propria of mucosa by excess aggregates of inflammatory cells (arrow) (H and Ex400).
| Fig. 10: | Gastric mucosa showing infiltration of the lamina propria of mucosa by mononuclear inflammatory cells (arrowheads) (H and Ex400) |
| Fig. 11: | Gastric mucosa showing infiltration of the lamina propria of mucosa by excess aggregates of inflammatory cells (arrow) (H and Ex400) |
| Fig. 12: | Gram's stain film of Helicobacter pylori in culture showing: Gram negative curved and spiral rods as seen with oil immersion lens (Gram's stainx1000) |
Figure 12 Gram's stain film of Helicobacter pylori in culture showing: Gram negative curved and spiral rods as seen with oil immersion lens (Gram's stainx1000).
DISCUSSION
Helicobacter pylori is a Gram negative curved or S shaped rods. It has a worldwide prevalence. More than 50% of the world’s populations are infected with H. pylori. Infection with multiple strains is quite common and occurs more frequently and with higher prevalence in developing countries (Brown, 2000). H. pylori, if not treated will persist throughout life. It has specificity of tissue involvement. It is exclusively inhabits mucosal cells of gastric glands. It infects and lives only in the human stomach. The enzymatic pathways needed for H. pylori survival in the harsh milieu of stomach are continuously switched on (Hofman et al., 2004).
H. pylori is a uncommonly well adapted parasite which persists indefinitely in the highly cruel environment of the stomach, despite the robust humoral and cellular immune response mounted against it. In addition, H. pylori is associated with specific gastroduodenal pathology and various extra-digestive diseases such as cardiovascular, immunological and a variety of other pathologies (Gasbarrini et al., 1999, 2004).
The assumed pathogenic determinants of H. pylori include virulence factors, which mediate the pathogenetic effects of the bacterium and maintenance factors, which allow the bacterium to colonize and persist indefinitely within the host. Virulence factors contribute to the three major pathogenic effects of H. pylori: gastric inflammation, disruption of the gastric mucosal barrier and alteration of gastric physiology (Hofman et al., 2004).
H. pylori is most commonly and specifically associated with chronic superficial gastritis which characterized by mononuclear inflammatory cell (MNC) infiltration associated with polymorphonuclear neutrophilic infiltration (PMN) of the epithelium. H. pylori is not specifically associated with metaplastia, granuloma formation, or fundic gland atrophy (Kim and Baek, 1999). H. pylori infection of the gastric mucosa in humans also has been shown to alter the normal gastric physiology (Del Giudice and Michetti, 2004).
Ghrelin, a 28 amino acid peptide, is an orexigenic and appetite-modulating hormone discovered by Kojima et al. (1999) and primarily produced in and secreted from specific enteroendocrine cells of the oxyntic mucosa located mainly in the corpus and fundus of the stomach (Kojima et al., 1999). Ghrelin is secreted into the extracellular matrix and then into the blood via a capillary network in the gastric lamina propria. Previous studies have demonstrated that ghrelin is involved in appetite, food intake, body-weight regulation and energy homeostasis (Bellone et al., 2003).
Ghrelin is also expressed in multiple tissues including small and large intestines (enteroendocrine cells distributed throughout the gut), lung, kidney, hypothalamic nucleus and pancreatic A cells (Ariyasu et al., 2001). It exerts endocrine, paracrine and autocrine effects. It has been demonstrated that several clinical factors including BMI, food intake and serum insulin levels have been associated with plasma ghrelin levels.
Ghrelin targets central nervous system GHS-R and exerts endocrine, paracrine and autocrine effects. Ghrelin exerts a complex interplay with other appetite-modulating peptides such as leptin, obestatin, neuropeptide Y, agouti-related protein, pro-opiomelanocortin and adiponectin, Ghrelin stimulates GHS-R present mainly in the pituitary and hypothalamic nuclei and via a GHRH-dependent mechanism, will stimulate growth hormone and insulin-like growth factor 1 (ILGF-1) release. The released hormones (GH and ILGF-1) affect a range of physiological processes. It has been suggested that ghrelin exerts a gastro-protective actions on the gastric mucosa (Slomiany and Slomiany, 2011a, b). In the alimentary tract, ghrelin increases acid secretion and stimulates gastric emptying via vagal activation (Locatelli et al., 2005). Ghrelin levels change significantly depending on the body’s energy requirements (Tschop et al., 2000).
H. pylori infection is considered as an important factor in reduced appetite. Additionally, some authors reported that H. pylori eradication resulted in improvement in dyspeptic symptoms among patients with non-ulcer dyspepsia (Konturek et al., 2006). So, it perhaps not surprising that ghrelin changes induced by H. pylori infection and/or eradication may also contribute to the changes of appetite or dyspeptic symptoms in those people with H. pylori infections.
Studies are being conducted to determine the effect of sociological and environmental factors on ghrelin release. The plasma ghrelin concentrations in gastrectomized patients still remain about one third of those in normal subjects (Cummings et al., 2002). In this study, the gastric mucosa was focused on to better understand the effects of H. pylori infection on the alterations of ghrelin expression.
It is hypothesized that H. pylori infection may attenuate ghrelin production in the stomach and consequently may reduce plasma ghrelin concentrations in H. pylori-positive subjects. The present study was specifically designed to evaluate the impact of H. pylori infection and its eradication on the plasma levels and gastric mRNA expression of ghrelin, obestatin and ghrelin/obestatin ratio.
In agreement with previous studies (Osawa et al., 2005), a significant association between ghrelin and H. pylori-associated chronic gastritis were found in current study. Plasma ghrelin concentrations and gastric ghrelin mRNA expression levels were significantly lower in H. pylori infection, colonization and its associated chronic gastritis than H. pylori-ve subjects. The presenting data also showed that in patients with H. pylori gastritis, eradication of the infection reversed these negative effects.
An interesting result obtained in this study was the finding that gastric ghrelin mRNA expression and circulating plasma ghrelin levels were significantly negatively correlated with density of H. pylori colonization and histological degree of gastritis according to PMNs, MNCs infiltrations of both corporal and antral mucosa (especially the corporal one).
These results are matched with those obtained by some authors reporting that H. pylori colonization has been negatively associated with circulating ghrelin levels and that H. pylori eradication can reverse these effects (Shiotani et al., 2005). In addition, this study demonstrated that patients with histologically higher degrees of corporal gastritis in the H. pylori-positive subjects tend to have lower plasma ghrelin concentrations than antral gastritis.
There were no statistically significant differences between gastritis scores in H. pylori-negative subjects with chronic gastritis and H. pylori-positive subjects with chronic gastritis. However, H. pylori-negative subjects with chronic gastritis tend to have significantly higher plasma and gastric ghrelin concentrations, ghrelin/obestatin ratio and higher obestatin levels than H. pylori-positive subjects. Also, at one month follow up after H. pylori eradication, the plasma and gastric ghrelin concentrations was recognized to be increased significantly but with insignificant change in gastritis scores (MNC infiltrations).
These contradictory results could suggest that there is another factor rather than chronic gastritis which depleted gastric ghrelin expression and secretion and consequently reduced plasma ghrelin levels. H. pylori was supposed to be that factor. This may be explained as H. pylori induced corporal gastritis may reduce the number and/or the function of ghrelin-producing cells in the fundic mucosa. Virulence factors of H. pylori contribute to three major pathogenic effects:gastric inflammation, alteration of gastric physiology and disruption of the gastric mucosal barrier (Hofman et al., 2004).
As all patients with atrophic body gastritis were excluded, it could be supposed that H. pylori infection resulted in reduction of the function of ghrelin-producing cells in the fundic mucosa and that eradication of H. pylori infection resulted in rapid and considerable recovery of ghrelin-secretory function before any apparent resolution of inflammation or restoration of ghrelin producing cell numbers.
In H. pylori positive patients (group I), gastric ghrelin mRNA expression as well as plasma ghrelin and obestatin levels, ghrelin/obestatin ratio and BMI were significantly negatively correlated with total gastritis scores (esp. corporal score, H. pylori density, PMN). On the other hand, gastric ghrelin mRNA expression was positively correlated with plasma ghrelin and obestatin levels, ghrelin/obestatin ratio and BMI.
Conceding with the results obtained in this study, previous studies suggested that inflammation of the gastric mucosa is the one of the important mechanisms of H. pylori induced change of ghrelin production (Isomoto et al., 2005). In this study, that greater inflammation in an H. pylori-infected gastric mucosa resulted in less ghrelin production.
H. pylori-mediated gastric inflammation critically depends on the efficient recruitment and activation of macrophages, with appropriate activation of nuclear factor NF-κB (Yanai et al., 2008). NF-κB is a transcription factor which plays an important role in activation and expression of pro-inflammatory cytokines (TNF-α). It is known that activation of NF-κB and consequently expression of TNF-α is significantly inhibited by exogenous ghrelin administration (Konturek et al., 2006) thus decreasing the gastric inflammation (a gastro-protective action) (Jeffery et al., 2011).
Thus, it could be supposed that ghrelin exerts gastro-protective actions. In this study, greater inflammation in an H. pylori-infected gastric mucosa resulted in less ghrelin production thus less inhibition of proinflammatory cytokines and more inflammation and ghrelin has potent gastro-protective actions.
This assumption is not able to answer why suppression of gastric ghrelin mRNA expression does not develop in all infected subjects. However, it is possible that the presence of specific pathogenic bacterium strains could play a striking role in decreased gastric ghrelin mRNA expression. So, further studies are needed to assess the effects of pathogenic bacterial strains on gastric ghrelin mRNA expression.
Obestatin, an anorectic hormone, is a 23-amino acid peptide hormone identified in 2005. It is derived from preproghrelin, the same peptide precursor as ghrelin. In contrast to ghrelin, obestatin acts as an anorectic hormone, decreasing food intake and reducing body weight gain. The stomach is considered an important organ for obestatin secretion. Obestatin can be purified and extracted from the stomach and demonstrated to be lower in gastrectomy patients (Huda et al., 2008; Lacquaniti et al., 2011). It is also not surprising that H. pylori infection or its treatment can affect gastric obestatin production.
In contrary to a previous study conducted by Lee et al. (2010), this work could demonstrate a clear association with plasma obestatin level being increased significantly in H. pylori infected subjects and fell significantly after its eradication. Also, in the present study, the post-treatment plasma ghrelin/obestatin ratio significantly increased.
Eradication of Helicobacter pylori is accompanied by an array of metabolic and hormonal changes in the host. Weight gain following H. pylori eradication is a poorly understood phenomenon and probably results from an interaction between multiple factors. Ghrelin and obestatin are involved in the regulation of food intake and appetite and may account for some of these changes. Weight gain following H. pylori eradication may be attributable to changes in plasma and gastric ghrelin and obestatin (Azuma et al., 2002).
To investigate this hypothesis, the changes in ghrelin and obestatin production were evaluated at 4 weeks and 6 months after H. pylori eradication. Plasma and gastric ghrelin mRNA expression and ghrelin/obestatin ratio were found to be significantly increased, whereas plasma obestatin was decreased after H. pylori eradication. In parallel with these changes, the visual analog scales for hunger (VAS) and prospective food consumption were significantly increased after H. pylori eradication (only after 6 moths). Four weeks duration was too short to evaluate the changes in body weight and BMI after H. pylori eradication. In contrast, the body weight and BMI were increased significantly 6 months after H. pylori eradication.
CONCLUSION
The present study indicates that plasma and gastric ghrelin mRNA levels were much lower in H. pylori-positive patients than in H. pylori-negative controls using real-time quantitative RT-PCR. In contrast, plasma obestatin levels were much higher in H. pylori-positive patients than in H. pylori-negative controls. Moreover, ghrelin/obestatin ratio was in parallel with their gastric mRNA expression levels in H. pylori-positive patients. Therefore, the attenuation of the ghrelin production in the gastric mucosa may account for the decrease in the plasma ghrelin concentrations in H. pylori-positive individuals.
This study also demonstrated that alterations in gastric ghrelin mRNA expression and plasma ghrelin, obestatin and ghrelin/obestatin ratio reversed after H. pylori eradication. These alterations of the plasma gastric originated appetite-controlling hormones may not be due to H. pylori per se but may be related to the degree of gastric inflammation present.
It is the matter of corporal pathology rather than antral pathology which determines the alterations in the intragastric milieu and consequently the changes in ghrelin/obestatin balance in H. pylori infected subjects.
Collectively, these observations provide novel insights for understanding the physiological function of ghrelin and obestatin and their relations to various diseases. The ghrelin/obestatin balance may be a key factor which determines an individual’s response to H. pylori infection and weight gain following H. pylori eradication.
RECOMMENDATIONS
These observations may be used in future research to study the appetite-modulating peptides release and to discuss the effect of selected factors on their release process. Further studies are needed to assess the effects of pathogenic bacterial strains on gastric ghrelin mRNA expression and to delineate the role of gastric ghrelin in pathological processes induced by H. pylori. More studies are required to explicate the determinants of plasma ghrelin levels and the complex interplay between factors determining weight gain following H. pylori eradication.