1 Introduction
Acute pancreatitis is an inflammatory condition that targets the pancreas and could result in an intense systemic inflammatory response leading to multiple-organ dysfunction. Several factors play a role in inducing AP, including gall stones, alcohol abuse, hypertriglyceridemia, and viral infections among others (Jha et al., 2009; Sarshari et al., 2023). Patients with AP show a high mortality rate; however, only symptomatic treatments that aim at supporting the patients are currently utilized, like analgesics, antiemetics, anti-inflammatory drugs and drugs to improve microcirculation (Sandakov et al., 2014).
Despite the fact that the exact mechanisms behind the cause of AP are still controversial, oxidative stress that results from the immense release of reactive oxygen species (ROS) is believed to be the early and central event in AP initiation and prognosis. This can be regarded to the deleterious effect of ROS on macromolecules and their role in activating inflammatory transcriptional factors and hence their damaging effect on pancreatic tissue (Zeybek et al., 2003). It has been also reported that immune response has a major role in the pathogenesis and prognosis of AP both in triggering and regulating these events (Sendler et al., 2013). Damage to pancreatic acinar cells induces leukocyte activation and neutrophils recruitment to the site of inflammation resulting in the release of massive amounts of pro-inflammatory mediators, e.g., TNF and various interleukins. As a result, the balance between the levels of pro-inflammatory and anti-inflammatory molecules is lost in favor of the pro-inflammatory mediators. This promotes the pathogenesis of AP and mediates the systemic inflammatory response (Hegyi et al., 2011). This central role of inflammatory cytokines led to the assumption that an improvement in AP status could be achieved upon regulating the expression of the inflammatory mediators to retain the balance, as several studies reported positive impacts for regulating interleukins (ILs) levels in different inflammatory diseases (Bhatia, 2005; Ni et al., 2013; Scheller et al., 2014).
The L-arginine-induced model of AP represents one of the most commonly utilized animal models to investigate the pathological changes in the pancreas due to its similarity to the human phenotype of the disease (Kui et al., 2014). Its administration induces severe AP, where the severity is dose dependent (Hegyi et al., 2004). The mechanism behind the deteriorative effects of L-arginine on the pancreas is not yet fully elucidated; however, different research groups postulated that L-arginine-induced AP could be attributed to oxidative stress and toxic metabolites, or even metabolic acidosis (Varga et al., 1997; Hegyi et al., 2004; Hardman et al., 2005). In addition, others related its effects to nitric oxide (NO) and inflammatory mediators’ activation (Takacs et al., 1996; Takacs et al., 2002; Rakonczay et al., 2003).
Arginine specifically targets acinar cells in the pancreas without affecting other cellular components of the pancreas (Kui et al., 2015). Its compromising effect on acinar cells induces the release of pro-inflammatory cytokine expressions such as TNF-α and various interleukins (ILs) (Yenicerioglu et al., 2013; Wang et al., 2017).
Dipeptidyl peptidase-4 (DPP-4) is a glycoprotein enzyme found on cell membranes of natural killer (NK) cells, macrophages, B-cells and T-cells among others. It cleaves dipeptides and promotes the breakdown of circulating incretins like glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide, which are responsible for regulating the glucose-dependent insulin release from the pancreas. In addition, DPP-4 possesses other functions that are not related to insulin and glucose metabolism, as it aids extracellular matrix degradation and cytokine production, in addition to stimulating immune responses, and inducing signaling and resistance to anti-mitotic drugs as well (Itou et al., 2013; Shao et al., 2020). Inhibiting DPP-4 via specific inhibitors allows for increasing the availability of incretins (e.g., GLP-1) and gastric inhibitory polypeptide leading to increased insulin release after meals and hence a better regulation of blood glucose levels, which explains the use of such inhibitors for the control of blood glucose levels in diabetic individuals (Zerilli and Pyon, 2007). Sitagliptin is a well-known DPP-4 inhibitor that is used since 2006 to control blood glucose levels in type 2 diabetic patients (Zerilli and Pyon, 2007). However, it has been reported in several studies that sitagliptin, in addition to other DPP-4 inhibitors, exerts additional biological functions unrelated to their blood glucose-lowering effect. The use of DPP-4 inhibitors showed positive effects in cardiovascular disorders, autoimmune diseases, lung disorders and inflammatory conditions via retrieving the endogenous antioxidant capacity that ameliorates oxidative stress-induced damage and modulating immune response (Fan et al., 2003; Eligar and Bain, 2013; Nade et al., 2015; Al-Rasheed et al., 2016; Civantos et al., 2017).
To the best of our knowledge, no studies have utilized sitagliptin in ameliorating L-arginine-induced AP. Based on this; we aimed in this study at investigating the potential role of sitagliptin in ameliorating L-arginine-induced AP in a rat model and the possible mechanisms behind this effect.
4 Discussion
AP represents a self-limited, emergency condition that can be induced by different triggers, however, it can easily develop into a life-threatening systemic inflammatory condition with multiple organ failures leading to high rates of morbidity and mortality (Whitcomb, 2006). Reactive oxygen species (ROS) have been shown to play a major role in AP by altering the nature of zymogen granules’ membranes, resulting in leakage of digestive enzymes, e.g., amylase, and lipase, and cellular proteins within acinar cells and pancreatic tissue, with the subsequent auto-digestion. This event is accompanied by the early activation of inflammatory pathways via activation of nuclear factor-κB (NF-κB) and its downstream inflammatory mediators (e.g., tumor necrosis factor- α (TNF-α) and IL-6), which represent a main event in AP pathogenesis (Ohmuraya and Yamamura, 2008). These molecular changes were reported in l-arginine-induced AP model, which is one of the most commonly used AP models recently (Hegyi et al., 2004).
These mediators attract immune cells such as macrophages and neutrophils, and induce their infiltration and activation in the pancreas, representing another fundamental event in the evolution of AP (Yu et al., 2002).
Activated macrophages are either M1-type, producing pro-inflammatory mediators (e.g., IL-1β, IL-6, IL-8, IL-12, and TNF-α), or M2-type, producing anti-inflammatory molecules such as IL-4, IL-10, TGF-β, and tissue repair factors (Van Dyken and Locksley, 2013; Zhang et al., 2020). Whereas M1 macrophages propagate the inflammatory response, M2 cells ameliorate inflammation via interrupting downstream signaling pathways of pro-inflammatory mediators allowing better control of the inflammatory response in AP (Virlos et al., 2004).
In the current study, we aimed at exploring the potential effect of the DPP-4 inhibitor sitagliptin on L-arginine-induced AP in a rat animal model. In agreement with previous reports, L-arginine injection induced oxidative stress as observed by the upregulation of different oxidative stress markers including MDA and NO, and depletion of GSH. It also increased MPO-, lipase- and amylase activity and resulted in depletion of serum calcium as well (Condon et al., 1975). An overall increase in various ILs including IL-1α/IL-1β, IL-6, IL-10, IL-12 and IL-15 was observed together with an increase in the level of circulating IFN-γ, TNF-α and LT-α. Histological investigation revealed positive expression signals for the pro-inflammatory cytokines TNF-α and IL-17 in pancreatic tissue sections after induction of AP.
Interestingly, sitagliptin administration reduced MPO-, lipase- and amylase activity. It also normalized calcium levels and ameliorated the signs of L-arginine-induced oxidative stress as it retrieved antioxidant capacity and replenished GSH content which represents a part of the pancreas endogenous antioxidant capacity. It also resulted in a significant reduction in serum levels of pro-inflammatory ILs, IFN-γ, LT-α and TNF-α. In addition, sitagliptin treatment caused a downregulation of IL-17 and TNF-α expression signals in pancreatic sections as observed by histopathological investigation.
In response to increased lipase activity upon autodigestion of pancreatic cells, free fatty acids (FAAs) are liberated from visceral fat into the circulation. These FFAs deplete serum calcium by forming Ca+2-FFA insoluble complexes, resulting in a characteristic reduction of total serum calcium level as observed by us and others (Ahmed et al., 2016).
Tissues utilize endogenous GSH, catalase, superoxide dismutase (SOD), thioredoxin and thiol-proteins to evade the deleterious effects of ROS. As mentioned before, L-arginine induces oxidative stress via superoxide, NO and peroxynitril radicals, which easily depletes the endogenous antioxidant reserves, as observed in the current study and reported by others (Zhang et al., 2010). In addition, the acute inflammatory reaction induced by L-arginine induces Neutrophils to be recruited and activated within the inflamed pancreas resulting in upregulation of pancreatic MPO activity; an enzyme that possesses potent pro-inflammatory properties and can be used as a valuable diagnostic factor for AP in animal models and patients as well (Rayner et al., 2014).
One of the hallmarks f acute pancreatitis is the increased levels of the proinflammatory cytokine IL-6, which is released by macrophages that become activated within the inflamed pancreas. Consequently, IL-6 induces the release of IL-17, IFN-γ and IL-12 from differentiated T-cells, and the release of IL-1β, IL-6 and TNF-α from infiltrating macrophages under the control of NFκB (Watanabe et al., 2017).
It is to be noted that IL-17, IL-12 and IL-15 areall pro-inflammatory cytokines that are upregulated in response to L-arginine-induced oxidative stress in AP and play major roles in inducing the production and release of multiple cytokines. Within the inflamed pancreas, IL-17 is produced mainly by T helper-17 cells (Th-17) and injured acinar cells in addition to other cells (Ni et al., 2013), inducing the release of TNF-α, IL-1β, IL-6 and IL-8 (Ni et al., 2013).
On the other hand, IL-12 and IL-15 induce transcription and release of IFN-γ by active NK cells (de Weerd and Nguyen, 2012). Generally, IFN-γ released by NK cells has multiple physiological roles, in addition to its role in tumor genesis and inflammation as it stimulates the release of variable pro-inflammatory cytokines (de Weerd and Nguyen, 2012). Reports have shown that IFN-γ serum levels are upregulated in patients with AP (Bhatnagar et al., 2003).
Notably, multiple inflammatory mediators, i.e., IL-17, IL-6 and IL-10 as well as IL-12 and IL-15 have been shown to play a main role in the pathogenesis of pancreatitis, and their serum levels have been reported to correlate with disease severity in AP patients (Pezzilli et al., 1999; Kamei et al., 2010; Li et al., 2021).
Both TNF-α and TNF-β (recently known as Lymphotoxin-α, LT-α) are inflammatory mediators belonging to the same family, TNF superfamily and become upregulated in pancreatitis. TNF-α is involved as a main player in the pathogenesis of AP and becomes upregulated during the AP-induced inflammatory response, however, its serum level doesn’t usually correlate with the severity of the inflammation in AP due to its prompt hepatic clearance (Grewal et al., 1994; Malleo et al., 2007). LT-αwhich is produced mostly by T-cells, CD+8 cells and NK cells, is linked to cell differentiation and apoptosis as well as various diseases including autoimmune pancreatitis (Seleznik et al., 2012).
As mentioned before, recruited macrophages in AP release IL-1β, which acts as a chemotactic factor allowing neutrophil infiltration into the pancreas with the consequent release of further chemokines and pro-inflammatory cytokines leading to aggravating the inflammatory response (Garlanda et al., 2013). Both IL-1α and IL-1β are inflammatory mediators that are shown to be upregulated in AP and bind the same receptor, resulting in activation of downstream NFκB signaling followed by transcription of its downstream pro-inflammatory cytokines, including IL-6 and TNF-α. It is to be mentioned that IL-1α is over-expressed in response to oxidative stress and pro-inflammatory cytokines, acting as a danger signal upon cell necrotic injury in several pathological conditions including AP (Fink and Norman, 1997; Dinarello, 2011; Malik and Kanneganti, 2018).
In contrast to the aforementioned cytokines, IL-10 is an anti-inflammatory cytokine that is produced by M2 macrophages, T-cells, B-cells and dendritic cells to combat the production of inflammatory mediators via inhibiting NFκB activation, and aid the regeneration phase of the pancreas after AP (Mahony et al., 2016). In agreement with the data presented in the current study, serum levels of IL-10, along with IL-1 and IL-6 have been previously shown to be associated with severe AP (Vasseur et al., 2014). In fact, IL-10 can be released from T-cells in response to IL-6, modulating the inflammatory and the immune responses (Ouyang and O'Garra, 2019). This pivotal role of IL-10 enables it to be a main player in inflammatory conditions, as it becomes over-expressed and released by the tissues involved in the inflammatory response in an attempt to circumvent the propagation of the inflammatory response (Murray, 2006). It has been reported to counteract the release of the pro-inflammatory cytokines IL-1α/β, IL-6, IL-12 and TNF-α from various immune cells, indicating its role in resolving the inflammatory status by manipulating the immune response (Ouyang and O'Garra, 2019).
Sitagliptin is a DPP-4 inhibitor that is used as an anti-diabetic drug and was shown to exert antioxidant and anti-inflammatory effects in different pathological conditions in kidneys, liver and cardiovascular system (Nade et al., 2015; Al-Rasheed et al., 2016; Civantos et al., 2017). It acts mainly by increasing insulin secretion from the pancreas via inducing the interaction of GLP-1 with its receptor (GLP-1R). This interaction activates a cAMP-mediated signaling pathway which is involved in regulating various activities including inflammatory response and blood pressure (Hou et al., 2016). It has also been reported to suppress macrophages infiltration in murine nephritis model and to interfere with NFκB signaling and hence preventing inflammation, in addition to reducing the levels of circulating TNF-α and other inflammatory mediators (Satoh-Asahara et al., 2013; Higashijima et al., 2015).
These anti-inflammatory effects of sitagliptin resulted in the observed downregulation in serum levels of pro-inflammatory ILs, IFN-γ, LT-α and TNF-α which are mainly released by these cells. Reduced expression of inflammatory mediators adversely affects the activity of NO synthesis, alleviating the oxidative stress and allowing the replenishment of the endogenous antioxidant defense mechanisms. Within the pancreas, the antioxidant effect of sitagliptin allows it to stabilize and protect biological membranes against peroxidation via scavenging excessive ROS. This prevents the premature activation and leakage of digestive enzymes, including lipase, from the pancreatic zymogen granules and hinders auto-digestion and lipolysis and the subsequent hypocalcemia as mentioned before, explaining the observed results regarding these parameters in the current study (Civantos et al., 2017).
Despite the effectiveness of DPP-4 inhibitors as blood glucose-lowering agents with positive impact on the cardiovascular and renal system in diabetic patients, a wide controversy is detected among researchers regarding the effect of this drug family on pancreas integrity. However, more studies are needed to get a conclusive outcome regarding these drugs.
On one side, some Meta analysis studies identified a minimal risk of pancreatic events in diabetic patients chronically treated with DPP-4 inhibitors (Pinto et al., 2018). This drawback of DPP-4 use was in part explained relying on the mechanism of action of these drugs, as they increase insulin secretion and suppresses glucagon secretion, resulting in pathological alterations within the pancreatic tissue and increasing risk of pancreatitis and pancreatic cancer as well (Matveyenko et al., 2009; Andersen et al., 2013), taking into account that these findings were detected after chronic use of DPP-4 inhibitors.
On the other side, other research groups have reported no significant association between the use of DPP-4 inhibitors and pancreatitis, suggesting their safety on the pancreas and the lack of association between their use and pancreatitis (Buse et al., 2017; Tseng et al., 2017; Dicembrini et al., 2020).
As observed here, sitagliptin treatment alleviated AP symptoms on the cellular and molecular level possibly by interrupting signaling pathways that could include but not limited to the earlier identified signaling pathway for sitagliptin, NFκB (Satoh-Asahara et al., 2013; Higashijima et al., 2015), resulting in a global modulation of the inflammatory cytokines’ profile. This proposes that sitagliptin may exert its effect in AP via retrieving the intricate balance between pro-inflammatory and anti-inflammatory mediators.