4. Discussion
Intra-rectal administration of 4% acetic acid (A-A) is a well-known
experimental model to induce acute colitis chemically in mice. A-A
simulates mucosal injury and ulcerations, enhances vaso-permeability and
neutrophil infiltration and up-regulates inflammatory mediators
similarly to those seen in human IBD [19, 20].
NFκB is a transcription factor controlling the transcription of DNA,
cytokine production, and cell survival. In inactivated immune cells,
most of the NFκB dimers are inactivated and retained in the cytoplasm
associated with small inhibitory molecules (IκBa, IκBb and IκB) [21,
22]. If there is a defect in the intestinal barrier, bacterial
antigens can get access to the antigen-presenting cells (APC) in the
intestinal lamina propria [23, 24]. These cells then present the
antigens to CD4+ lymphocytes and macrophages that will
start the NFκB signaling pathway. Activation of the NFκB signalling
pathway results in the release of NFκB from its inhibitory molecules and
its nuclear localization, thereby inducing the expression of NFκB target
genes [25, 26]. The increased NF-κB expression in the intestinal
mucosa results in an increased capacity of these cells to produce and
secrete pro-inflammatory cytokines such as TNF-α, IL-1β, IL-13, IL-21,
IL-22, IL-6. NFκB is also able to regulate the expression of IL-12 and
IL-23 that are directly involved in the mucosal damage typically seen in
IBD. NFκB is simultaneously activated by TNF-α thereby providing a kind
of positive feedback effect, which maintains the chronicity of the IBD
[27, 28].
Sulfasalazine is a well-known drug used in treatment of IBD [29].
Wahl et al. (1998) studied the effect of sulfasalazine on NFκB and found
that sulfasalazine can inhibit both activation and nuclear translocation
of NFκB in response to three different stimuli (TNF-α,
lipopolysaccharide (LPS), or phorbol ester). These data suggest that the
anti-inflammatory activity of sulfasalazine may rely on its ability to
modulate the actions of NFκB signalling [30].
In this study, experimentally-induced colitis led to a significant
increase in the expression of the transcription factor, NFκB and the
pro-inflammatory marker, TNF-α in the colon of mice as compared to the
control group. This is in consistent with study of Schottelius and
Baldwin (1999) that pointed out that the increased expression of NFκB is
crucial for the initiation and conserving of chronic intestinal
inflammation [31]. In the same context, Daneshmand et al. (2009) and
Abdel-Daim et al. (2015) concluded that the exaggerated release of TNF-α
in A-A model of colitis mediates the intestinal damage observed in IBD
[19, 20].
Moreover, in the present study, liraglutide and sitagliptin, either
alone or combined to sulfasalazine reduced colonic levels of NF-κB and
TNF- α in mice. This can be explained by the ability of GLP-1R agonist
to down-regulate NF-κB phosphorylation and nuclear translocation that
results in reduction in the expression of the pro-inflammatory
cytokines, such as TNF- α that is influential in IBD pathophysiology
[25]. Similarly, DPP-IV inhibitors can partially ameliorate the IBD
pathology through increasing the endogenous levels of GLP-1 and GLP-2.
Tang et al. (2016) reported that the elevated levels of GLP-1 seen with
DPP-IV inhibitors, subsequently suppressed the NF-κB/IκBα set-up that is
responsible for NF-κB activation [32]. Furthermore, the correlation
between GLP-2 and inflammation was investigated in a study by Xia et al.
(2014) that studied the effect of GLP-2 on LPS-induced inflammation in
macrophages. GLP-2 was found to inhibit LPS-induced NFκB translocation,
IκB-degradation and IκB-α phosphorylation and thus attenuates the
inflammatory cascade [33]. Moreover, Broxmeyer et al. (2012) showed
that DPP-IV inhibitors have important immunomodulatory actions through
the recruitment of immune cells (especially T lymphocytes) and the
inhibition of the NFκB-dependent transcription of pro-inflammatory
cytokines [34].
Lipid peroxidation evidenced by the high MDA level in UC patients is one
of the pathogenic mechanisms of UC. Oxidants play an important role in
the chronicity of IBD by increasing the number of neutrophils and
macrophages that induce a self-sustaining activation loop [35]. It
is famed that sulfasalazine and its metabolites are highly effective
reactive oxygen metabolite scavengers that decrease MDA levels [36].
On the other hand, cytokines associated with IBD activity (IL-6, TNF-α,
and IL-1 β) stimulate the production of CRP over baseline levels, which
are typically less than 1 mg/L [37]. Additionally, Huh et al. (2019)
mentioned in a study that evaluated some risk factors and predictors for
hospitalization of patients with IBD, that in UC, a serum CRP level
> 0.5 mg/dL was the only independent risk factor to predict
hospitalization [38]. Therefore, CRP is considered a good predictor
of disease remission and response.
As shown in figure 3C and figure 3D , our data
demonstrates that Liraglutide, CLS and CSS groups showed significant
reduction in MDA levels. While Sitagliptin, CLS and CSS groups decreased
the CRP levels in comparison to A-A group with no significant difference
than the control group. In a study investigating the effect of
liraglutide on peripheral neuropathy in diabetic rats, treatment with
liraglutide normalized MDA levels and increased superoxide dismutase
level in sciatic nerve [39]. Similarly Varanasi et al. (2012)
documented a decline in the mean CRP concentration in patients with T2DM
treated with liraglutide [40]. On the other hand, various studies
have investigated the anti-oxidant effect of DPP-IV inhibitors. Mega et
al. (2011) and Omolekulo et al. (2019) supported the anti-oxidant effect
of DPP-IV inhibitors, particularly by sitagliptin in animal models of
diabetic nephropathy and insulin resistance respectively that showed
significant reduction in plasma MDA levels [41, 42]. While Tremblay
et al., (2014) has reported that Sitagliptin, most likely by increasing
plasma GLP-1 levels and improving glucose-insulin homeostasis, can
down-regulate CRP concentration [43] which may explain the lower CRP
levels observed with GLP based therapy [35].
Additionally, several preclinical studies have reinforced the potential
anti-inflammatory effects of GLP-1 as regards IBD through regulating
invariant natural killer T cells (iNKT) activity, decreasing macrophage
propagation, and suppressing lymphocyte maturation and differentiation
[44]. In support of these data, Yusta et al. (2015) has reported
that GLP-1 receptor agonists can significantly cause a reduction in the
expression of epidermal growth factor receptor (EGF), transforming
growth factor (TGF)-β1, keratinocyte growth factor (KGF) and the
interleukins; IL-6, IL-1 β, and IL-2, that are major constituents of the
innate immunity and are involved in mucosal repair [45]. Moreover,
Anbazhagan et al. (2017) showed that treatment of dextran sodium sulfate
(DSS)-induced colitis with GLP-1 coated with sterically stabilized
phospholipid micelles has significantly ameliorated the progress of
colitis with subsequent improvement in the epithelial architecture
[46]. In parallel, Bang-Berthelsen et al. (2016) found that
liraglutide can improve IBD activity endpoints that include colon length
and weight as well as colonic tissues histological changes [8].
Moreover, sitagliptin can potentiate the intestine-tropic effects of the
endogenous GLP-2. The later was found to improve the intestinal mucosal
tight junctions, decreases the internalization of enteric microbiota and
to decrease plasma LPS concentration together with a significant
reduction in macrophage migration and the production of oxidative stress
markers; iNOS and NADPHox. While lacking of GLP-2 effects was associated
with increased liability to gastrointestinal inflammation [47-49].
In the same context, Moran et al. (2012) hypothesized that the reduced
activity of DPP-IV enzymes reported in patients with active CD may
represent the body’s venture to increase the intestino-trophic and
anti-inflammatory effects of endogenous GLP-2 and GLP-1 respectively
[50]. Furthermore, in an experimental model of colitis in mice,
EMDB-1, a novel DPP IV inhibitor showed remarkable anti-inflammatory
effect that may be explained by the upregulation of endogenous GLP-1 and
GLP-2 levels [51]. Moreover, Higashijima et al. (2015) has reported
that DPP-IV inhibitors can adjust the immune response in a rat model of
nephritis by reducing macrophage infiltration [52]. Yazbeck et al.
(2010) as well, disclosed that sitagliptin has altered the secretion of
pro-inflammatory cytokines (IFN-c and IL-6), besides having the ability
to regulate the production of TGF-β which modulates immune cells
differentiation, maturation, apoptosis and actions [53].
Contrarily Abrahami et al. (2018) suggested a probable alliance between
inhibition of DPP-IV enzyme and IBD sequel [54]. However, a
meta-analysis carried out by Radel et al. (2019) has invalidated
Abrahami’s findings and pointed out that DPP-IV inhibitors are not
correlated with IBD incidence [55], but the need for long-term
clinical trials designed to identify the role of DPP-IV inhibitors in
IBD is a limitation to end this argument.
Nevertheless, while hypoglycemia is one of the adverse effects reported
for sulfasalazine, (possibly through its sulfapyridine component which
is structurally similar to glyburide which is a member of the
hypoglycemic sulfonylurea group) [56, 57], this study has supported
the euoglycemic effect of GLP-1 and DPP-IV based therapy. As liraglutide
and sitagliptin are well-known to have a glucose-dependent action,
therefore they are essentially well tolerated and are not familiar to
cause hypoglycemia unless combined with other oral hypoglycemic drugs or
insulin [58].