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Iraqi Journal of Agricultural Sciences –3:54(5):- 12 Muhi & et al.
1243
THE EFFECT OF HELICOBACTER PYLORI ON INTERLEUKIN-6 IN
PATIENTS WITHISCHEMIC HEART DISEASE
Shahrazad H. Muhi 1 Wisal S. Abd 1 Najah S. Abd 2
Researcher Assist. Prof. Consultant Doctor
1 Dept. of Biotechnology Coll. of Sci. University of Baghdad-Iraq
2 Iraqi Center for Heart Disease, Ghazy AL-hariri Hospital for Surgical Specialities - Iraq
shahrazadhazim@gmail.com wisalimmunology@gmail.com
njh_njh@rocketmail.com
ABSTRACT
Several studies have shown that the presence of Helicobacter pylori (H. pylori) may be a risk
factor for Ischemic Heart Diseases (IHD). Therefore, it is needed to look into the relation
between IHD and H. pylori infection. One hundred Iraqi patients with IHD and 60 healthy
Iraqis as a control group participated in the current study. The ELISA assay was used for the
measurement of the sera levels for anti- H. pylori IgG, IgM, and Interleukin-6 (IL-6). Also, H.
pylori DNA in the human venous blood was investigated by the polymerase chain reaction
(PCR) using 16S rDNA primers. Between IHD patients and the control group, H. pylori-IgG
and IL-6 mean ± SD were (Patient =1.814±0.08, Control = 0.823±0.09, Patient = 49.73±6.60,
Control = 0.21±0.02, respectively), and this results showed a highly significant variation (p <
0.05) in anti-H. pylori IgG and IL-6, while no significant variations (p > 0.05) were found in
anti- H. pylori IgM. It was also found that PCR amplification of Helicobacter pylori DNA using
16S rDNA primers was positive in the venous blood of 81 (81%) IHD patients. The current
study results proposed that there was a relation between H. pylori and Ischemic Heart
Disease.
Keyword: IHD, H. pylori-IgG, H. pylori-IgM, IL-6, PCR, 16S rDNA, ELISA, DNA.
- 5
100
60 ( IL-6 & IgG ,IgM)
(16S rDNA)
IgG IL-6 P=1.814±0.08
C=0.823±0.09 P=49.73±6.60 C=0.21±0.02
(
p
<0.05) (IgG) (IL-6)
(
p
> 0.05) (IgM)
(16S rDNA)
811
Received:11/10/2021 Accepted:9/1/2022
Iraqi Journal of Agricultural Sciences –3:54(5):- 12 Muhi & et al.
1244
INTRODUCTION
Ischemic Heart Disease (IHD) is considered
the most popular among cardiovascular
disorders (26). Sometime, the term coronary
heart disease (CHD) is often used to describe
this syndrome, because the reduction of blood
flow in IHD is caused by coronary artery
atherosclerosis (4). IHD is considered as a
pathological case characterized by reduced
flow in the cardiac blood that results in a
mismatch between myocardial oxygen supply
and demand and it is considered a chronic,
progressive disease (30). IHD causes 30% of
all yearly deaths, which has become the main
death reason and a major economic burden
worldwide (42). Atherosclerosis is the major
pathological process that results in IHD,
atherosclerosis is an artery inflammatory
disease associated with deposition of lipids
and metabolic alterations due to many risk
factors (38). Classical risk factors for
atherosclerosis and consequently
cardiovascular disease (CVD) involve
dyslipidemia, smoking, genetic predisposition,
diabetes mellitus, arterial hypertension,
metabolism disorder, a rich fats diet, and
mental or emotional stress (34). Different
experimental and scientific researches show
that atherosclerosis is significantly affected by
inflammatory situations. A significant
relationship between coronary ischemia and
various infectious agents was shown by many
epidemiological types of research.
Helicobacter pylori (H. pylori) is a spiral
microaerophilic gram-negative bacterium that
causes inflammatory and infectious disease in
humans by forming colonies in the stomach
(12). H. pylori cause many extra digestive
disorders, involving functional vascular
diseases like Raynaud's (primary) and
migraine (primary) and IHD (34). Many
studies attested that there is an association
between IHD and the stomach H. pylori
infection (11). Chronic stimulation of
inflammatory responses as a result of the gut
and gastric organs infection by bacteria causes
the stimulation of dyslipidemia, elevates the
fibrinogen levels, induces the liberation of
CRP, rapidly increases blood leukocyte and
homocysteine, stimulates hypercoagulability,
induces immune cross-reactivity, elevates pro-
inflammatory cytokines (such as Interleukins)
and another cytotoxic agent (27). H. pylori
might result in a chronic infection that could
result in atherosclerosis, chronic inflammatory
responses in atherosclerosis can elevate the
values of various cytokines such as
Interleukin-6 (IL-6), CRP, and fibrinogen. IL-
6 stimulates CRP in the liver, which causes
elevation in CRP values, fibrinogen, and
amyloids A in blood. Arteries thrombosis
could result from stimulated fibrinogen, which
can cause an infarction (7). IL-6 is an essential
cytokine in the inflammatory response, it has
two unique and special signaling pathways.
The first is named classic signaling of IL-6, in
this signaling, IL-6 connects to a membrane-
bound IL-6 receptor (IL-6R). The linkage of
IL-6 to its receptor stimulates the activation
and dimerization of the signal-transducer
glycoprotein 130 (gp130). The non receptor
tyrosine kinase JAK will be recruited by the
active complex, which phosphorylates the
gp130's tyrosine residues. Recruitment sites
for another protein like SHP2/ERK and
STAT1/3 and the activation of many signaling
cascades will be initiated as a result of the
gp130's phosphorylation. In contrast, the
second is named trans-signaling of IL-6, in the
IL-6 trans-signaling, soluble IL-6R (sIL-6R)
was recognized in several body fluids. In
human, sIL-6R was manufactured via
dropping of the membrane-bound IL-6R by
metalloprotease A disintegrin and
metalloproteinase (ADAM10 or ADAM17) or
by alternative splice. In circulation, IL-6 can
interact with sIL-6R and the IL-6/sIL-6R
complex, (also designated Hyper-IL-6), links a
membrane bound gp130, triggering a pathway
identical to the pathway in the classical
signaling that was discussed earlier (42) (21).
The first classic signaling pathway is mainly
active in hepatocytes and lymphocytes, while
the second trans-signaling pathway is active in
almost any cell type (32). The soluble gp130
(sgp130) is available and it is manufactured
either by shedding using ADAM10 and
ADAM17 preferentially or by alternative
splicing. Sgp130 binds with the complex of
IL-6/sIL-6R but does not bind with IL-6 alone.
So, the sgp130 job is to catch the complex of
IL-6/sIL-6R selectively, leading to the
inhibition of trans-signaling of IL-6 with no
disruption to the classical signaling of IL-6. In
Iraqi Journal of Agricultural Sciences –3:54(5):- 12 Muhi & et al.
1245
animal studies, targeted inhibition of trans-
signaling by sgp130 have helpful effects in the
inflammatory disorders and in the
atherosclerosis. It has been suggested that
classical signaling mediates IL-6 anti-
inflammatory and regenerative activities,
while trans-signaling mediates IL-6 pro-
inflammatory actions (42). Researchers stated
that there is a correlation between different
diseases and many different immune markers
(2, 3, 5, 6, 8, 19).
Epidemiological studies have recorded a direct
relation between the values of circulating IL-6
and the danger or severity of CHD (31).
MATERIALS AND METHODS
Subject: This study was done in Baghdad,
Iraq between the period Oct. 2020 to Mar.
2021. Subject included 100 patients with
clinically defined IHD who were admitted to
the Iraqi Center for Heart Disease (male:55
and female:45 aged 40-75 years) and 60
healthy volunteers (male:34 and female:26
aged 39-60 years) as the control group.
Blood sample
The sample collection and laboratory
investigation was done as the following,
peripheral blood was drawn with a 5 ml
sterilized syringe and was discharged in two
aliquots. Three milliliters of blood were
discharged in a gel tube and the tubes were
centrifuged (4000 rpm\10 min.) after clot
formation at room temperature. Eppendorf
tubes were used for the serum storage until the
ELISA measurements were done and the other
2 ml of blood was stored in EDTA tubes until
the nucleic acid measurement was done. All
tubes were stored at 20° C (22).
Measurements of IL-6, IgG, and IgM
Human Interleukin-6 (IL-6) ELISA kit
(Elabscience), human H. pylori IgG (Hp-IgG)
ELISA kit (Shanghai Yehua), and human H.
pylori IgM (Hp-IgM) ELISA kit (Shanghai
Yehua) were used to identify the IL-6 and H.
pylori IgG and IgM values depending on the
protocol of the manufacturer company.
DNA extraction
Extraction of the genomic DNA from
peripheral blood mononuclear cells (PBMCs)
was done by ReliaPrep Blood gDNA Miniprep
System (Promega) (40) (7) to examine DNA
of H. pylori in the human venous blood by
PCR. Nano-Drop device was used to measure
the purity and the concentration of the
extracted DNA (12).
Molecular diagnosis of H. pylori by PCR
For the detection of H. pylori DNA presence
in the blood, DNA of blood samples were
amplified by H. pylori specific 16s rDNA (or
16s rRNA) gene, sequence of primers was: C-
97, 5'- GCT ATG ACG GGT ATC C-3' (276–
291 forward), C-98, 5'- GAT TTT ACC CCT
ACA CCA -3' (681–698 reverse), and the
product size was ~ 400 base-pair (45) (24) (22)
(13). Singleplex PCR program was as
followed: initial-denaturation 94 ˚C and final
extension 72 ˚C were for five minutes and 1
cycle for each, while denaturation, annealing,
and extension were for only one minute but 35
cycles and the temperature was (94, 50, 72) ˚C
respectively. The product was examined by 1
% agarose gel stained by ethidium bromide (1)
and visualized using UV transilluminator (9).
Statistical analysis
The SAS 2012 {Statistical Analysis System}
application was employed to detect the impact
of different groups in the parameters of the
study. The T-test was performed to significate
and compare between the means. A Chi-square
test was employed to significate compare the
percentage (0.05 and 0.01) probability in the
current study.
RESULTS AND DISCUSSION
In this case-control study, many parameters
were examined, including the serum level of
IL-6, Hp-IgG, Hp-IgM, and the presence of H.
pylori DNA in the blood among IHD and
healthy controls. This study was recruited 100
IHD patients and 60 healthy subjects.
Serum levels of IL-6, Hp-IgG, Hp-IgM
The results showed highly significant
differences in the H. pylori IgG antibodies and
IL-6 concentration in the serum of the patients
infected with IHD compared to the healthy
control group (p < 0.0001, for IgG and IL-6).
However, there were no significant differences
(p = 0.279) in the H. pylori IgM antibodies
concentration in the serum of IHD patients
compared to the healthy control group, (Table
1).
Iraqi Journal of Agricultural Sciences –3:54(5):- 12 Muhi & et al.
1246
Table 1. Relation of some immune H. pylori
parameters in IHD patients compared with
healthy individuals
H. pylori infection stimulates the
manufacturing of immune antibodies. Sera-
diagnostic tests give the sensitivity, rapidity,
and specificity for the identification of the
bacterium. The anti-H. pylori IgM
seroprevalence in the current study showed no
significant differences between IHD patients
and healthy individuals. The current study
findings were in accordance with a few studies
which estimated the relation of H. pylori with
acute myocardial infarction and CHD. The
results were in agreement with the study
showed that there was no significant difference
in H. pylori IgM levels between cases and
controls (p = 0.29) (31). Also, Viswanath et
al., reported that H. pylori IgM antibodies
were analyzed and it was noticed that all of the
subjects were negative for IgM antibodies
(43). While a study was done in 2008
disagreed with the current study, Jha et al.,
showed that heavy smokers were significantly
associated with seropositivity (p = 0.044 for
IgM of H. pylori) (28). The results were in
agreement with Jafarzadeh et al., study who
showed that the infection with H. pylori
bacteria might participate in IHD pathogenesis
directly or indirectly, and the study also
reported that the anti- H. pylori IgG
seroprevalence was 58.3 % among healthy
individuals and 89.2 % among patients with
IHD. Anti-H. pylori IgG was shown to be
much more prevalent in patients with IHD
than in healthy individuals (p < 0.0001).
Additionally, the association of H. pylori with
IHD may vary by countries or even within a
country. The variances are largely explained
via variations in age, race, socio-economic
condition, and ethnic origin. Additionally,
variation in the inclusion criteria for patients
and controls, genetic heterogeneity of
Helicobacter pylori, and variations in the
distribution of the IHD risk factors may
account for some of the differences. As a
result, the findings of one study may not be
applicable to other populations, even within
the same area or the same country (26).
Ismael et al., suggested that there was a
relationship between coronary artery disease
and H. pylori infection, and reported that 78.57
% (55/70) of CAD patients had a positive
result for anti- H. pylori IgG and these results
showed great significant differences between
the studied groups (p < 0.01). This study and
others support the hypothesis that H. pylori
can be correlated with CAD or even be
considered a risk factor for atherosclerosis
plaque formation. H. pylori infection has been
implicated in the development of
atherosclerotic changes in coronary arteries,
implying that this pathogen or its products
(cytokines, cytotoxins, endotoxins, and several
other virulence factors) have a detrimental
effect on the coronary endothelium (23). An
Egyptian study that targeted the relation
between acute coronary syndrome (ACS) and
H. pylori chronic infection. Ghaleb et al.,
reported that ACS was assured in 94 % of the
entire study sample with H. pylori IgG
antibodies (mean 2.36 U/mL). The danger of
ACS development in patients with greater H.
pylori IgG was 1.6 times (95% CI: 0.662-
1.704) (18). Also, Afsharpooyan and
Mohammadian., showed that the
predominance of H. pylori infection was 68.04
percent, and the mean level of the IgG was
67.35 percent (7). While the result of the
current study disagreed with Mohammed et
al., who revealed that there were no significant
differences between the patient and healthy
individuals in the IgG levels (p = 0.53) (31).
Also, EL-Ageery et al., showed that H. pylori
IgG was high in both case and control groups
(p = 0.346). However, there have been
conflicting reports regarding the relationship
between serology and the occurrence of CAD.
While some researchers found no correlation
between H. pylori seropositivity and CAD,
others found a significant correlation. These
contradictory reports may be explained by the
inability of antibody testing to distinguish
between current and past infection (15). The
IL-6 levels in healthy people’s blood are
between 1–5 pg/ml. IL-6 values rise several
thousand folds during inflammatory conditions
Variables
Patients
Controls
Mean of H. pylori
IgM conc. (ug/ml)
5.97±0.37
5.34±0.43
Mean of H. pylori
IgG conc. (ug/ml)
1.814±0.08
0.823±0.09
Mean of IL-6
conc.(pg/ml)
49.73±6.60
0.21±0.02
Iraqi Journal of Agricultural Sciences –3:54(5):- 12 Muhi & et al.
1247
(34). Numerous investigations established the
function of interleukin-6 in the association
between systemic inflammation and
cardiovascular disease. The current study
results agree with the results of a study in
Saudi Arabia on a group of 100 patients which
showed that there was a significant increase in
the levels of IL-6 in the serum of patients with
IHD than in the serum of the control group.
The increase in IL-6 levels could be a result of
the development and instability of
atherosclerotic plaques via activation of
leukocytes and endothelial cells, or it could be
a result of the induction of various cytokines
(25). Tomas et al., reported that there is a
significant variation (p < 0.001) in the levels
of IL-6 of people suffered from IHD (median
of 33 pg/mL) from individuals without IHD
(median of 3.8 pg/mL). As an inflammatory
cytokine, IL-6, are known to increase the risk
of CAD via the stimulation of the acute phase
response and hepatic production of acute-
phase proteins like the production of CRP and
fibrinogen. This elevates blood viscosity and
stimulates the proliferation and activity of the
platelets. Following that, autocrine and
paracrine activation of monocytes via IL-6
promotes fibrinogen deposition in the vessel
wall. Then, IL-6 induces a reduction in the
action and levels of plasma of the monomeric
lipoprotein lipase, resulting in an increase in
macrophage lipid uptake. As well as, IL-6
stimulates the hypothalamic-pituitary-adrenal
axis. Obesity, hypertension, and insulin
resistance are all associated with this (41).
Also, Furuto et al showed that H. pylori
increase the levels of many inflammatory
cytokines including IL-6 that directly or
indirectly damage the vascular walls, thereby
causing arteriosclerosis (14). Yildirim et al.,
revealed that IL-6 levels were significantly
higher in H. pylori-positive patients than in
control (p < 0.05). Infection with Helicobacter
pylori results in the infiltration of macrophages
into the gastric mucosa and subsequent
secretion of the B-cell stimulatory cytokine IL-
6. Additionally, they suggested that
phagocytosis of the bacteria appears to be a
requirement for induction of IL-6 in these cells
(46). The current study disagrees with
Afsharpooyan and Mohammadian., who
indicates that there was no relationship
between H. pylori-infected people and CVD
because the IL-6 expression by the H. pylori
effect has reported that the bacterium had no
significant impact on it, and there were no
main variations between the titer of H. pylori-
positive and H. pylori-negative individuals and
IL-6 (10).
Detection by molecular techniques
DNA quality and integrity were estimated
through electrophoresis on 1% agarose for 1
hour. The bands appear sharp, single, not
diffused and have no smear as shown in
(Figure 1)
Figure 1. Gel electrophoresis of genomic DNA of blood samples
As mentioned below in (Table 2) results shows
that there were a highly significant differences
in PCR results between patients and healthy
individuals (p = 0.0001), DNA of H. pylori
was recognized in the venous blood of 81
(81%) of 100 IHD patients by PCR using
H.pylori specific 16S rDNA (16s rRNA)
primers and the amplified PCR products were
~ 400 bp (Figure 2). None of the healthy
individuals gave a positive result for the H.
pylori 16S rDNA gene.
Iraqi Journal of Agricultural Sciences –3:54(5):- 12 Muhi & et al.
1248
Table 2. Distribution of sample study
according to PCR results in patients and
control.
PCR
Group
p-value
Patients
No. (%)
Control
No. (%)
Positive
81
(81 %)
0
(0 %)
0.0001 *
Negative
19
(19 %)
60
(100 %)
0.0001 *
p-value
0.0001 *
0.0001 *
-----
* (p≤0.01).
A blood sample analysis by PCR is a faster
and more accurate way to detect bacteremia
than a blood culture. A bacterium can be
recognized by looking for certain consensus
sequences in its bacterial DNA, like the 16S
rDNA gene. Therefore, Huang et al.,
investigated H. pylori DNA in venous
peripheral blood and gastric mucosa of
individuals with chronic gastritis or peptic
ulcer using PCR. They recognized the DNA of
H. pylori in blood in Three (15%) out of 20
patients while they recognized H. pylori in
fifteen (75%) out of twenty gastric specimens,
and Nine (45%) of those samples were
positive for H. pylori culture. None of the
healthy individuals were positive for
Helicobacter genes. It's believed that H. pylori
infection raises the risk of a variety of extra-
intestinal diseases, such as coronary heart
disease and autoimmune thrombocytopenia, as
well as skin and hepatobiliary diseases (21).
Abd-Alrahman et al., used PCR technique to
detect 16S rRNA for H. pylori in the DNA
samples of whole human blood and they
reported that 66.67 % of chronic urticarial
patients (CU) and 70.37 % of atopic dermatitis
patients (AD) were positive to 16S rRNA.
Only 1 person out of 22 healthy persons gave a
positive result to 16S rRNA. A highly
significant difference (p < 0.01) was shown
through the statistical analysis (6).
Figure 2. PCR result of the H. pylori 16S rDNA (16S rRNA) gene in IHD patient blood
samples
When infected with H. pylori, it can reduce the
production of gastric mucus, cause a
significant inflammatory response, and alter
microcirculatory variables like blood flow and
leukocyte activities. It can also alter vascular
endothelial lining. As a result of these actions,
the stomach mucosal membrane may be
compromised, which could lead to mucosal
damage leading to H. pylori penetrating the
bloodstream. Several circumstances are
expected to cause the enhanced translocation
of microorganisms over the gastro-intestinal
barrier. These involve disturbance of the
ecology of the indigenous gut microbiome
resulting in bacterial overgrowth, reduced
immunity of the host, and physical disruption
of the intestinal mucosal lining via direct
chemical damage, hemorrhagic shocks, and
endotoxin (21). AL-Jobori et al., when using
the PCR for detection 16S rRNA gene which
was specific for H. pylori, reported that 20
(100 %) of patients were positive for PCR
from peripheral blood (13). The study result
was in agreement with saeideh et al., who
Iraqi Journal of Agricultural Sciences –3:54(5):- 12 Muhi & et al.
1249
revealed that H. pylori can secrete DNA in
peripheral blood besides its existence in gastric
mucosa, and it is possible for the infection of
H. pylori to cause bacteremia or inject DNA to
blood by type IV secretory system. They tested
H. pylori DNA in (18%) of all 100 serum
samples of peptic ulcer and gastritis patients
by PCR. Septicemia is not promoted by H.
pylori but it does enable secretion of DNA into
blood and host cells (38). The existence of
Helicobacter pylori DNA has been proved in
atherosclerotic plaques and aortic tissues of
most of CHD patients. Of the 40 aortic wall
samples from coronary patients, 32 (80%)
were positive for H. pylori, whereas 17 of 20
(85%) specimens from valve patients were
positive for H. pylori. This provides important
evidence for the direct involvement of bacteria
in disease pathogenesis. As a result,
Helicobacter pylori can cause a direct effect
on the induction of the inflammation within
atherosclerotic plaques (36).
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