Mechanistic insights into the pathogenesis and management of acute pancreatitis

Acute pancreatitis (AP) is an inflammatory disorder of the pancreas; its incidence rate is increasing worldwide; it is around 34 cases per 100,000 persons /year. It may range from mild to severe cases and may be associated with morbidity and mortality mainly due to multiple organ dysfunction syndromes (MODS). Till now, there is no specific therapy for the disease and the treatment of AP is mainly supportive. Moreover, the underlying mechanisms included in its pathogenesis are not fully clear. However, it may include oxidative stress and inflammatory response, including critical mediators, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), toll-like receptor-4 (TLR-4), nuclear factor-kappa B (NF-κB), and high-mobility group box protein1 (HMGB1). Thus, there is a pressing need for continuous search in this era to clarify different pathogenesis and the development of new treatment options for AP, also understanding the disease. While research on the human pancreas remains challenging, animal models of AP may help to elucidate the disease pathophysiology & to discover new target options for the development of new therapies. This review aims to revise several aspects related to AP diagnosis and management and to summarize different animal models of AP.


Introduction
Acute pancreatitis (AP) is an inflammatory disorder of the pancreas; it has substantial morbidity and mortality [1].AP may range from mild self-limiting to severe cases, which are frequently associated with complications and a high mortality rate [2, 3].Most cases are mild but about 20% develop multiple organ dysfunction syndrome (MODS) which is the main cause of high mortality [4].AP is considered an essential cause of hospitalization due to gastrointestinal disease [5].Previous data show that the AP mortality rate ranges from 1% to 5% which was reduced due to improvement in diagnosis & care while the hospitalization rate & cost increased [6], but recent data showed that Mortality for AP is approximately 1% overall; however, in case of AP associated with organ failure, mortality may be as high as 30%-40%.5 [7].There are continuous advances in the supportive management of AP, but to date, no specific effective drug is available to treat or prevent AP.Nevertheless, important advances for the Review Article Pharmacology and Toxicology identification of new targets for potential drug development are ongoing, for instance: studying calcium signaling pathways in AP resulted in the discovery of calcium release-activated channels and mitochondrial permeability transition pores that could be promising targets [1].However, the overall development of new therapies with new potential targets is still urgently needed.

Prevalence & etiology
AP has an increasing incidence rate worldwide, approximately 34 cases per 100,000 persons/year [1].This may be due to the increase in gallstone disease or may be due to the increase in routine testing of pancreatic enzymes in patients having abdominal pain at admission to the emergency department [8].It is important to identify the etiology of acute pancreatitis for early and effective management of AP and prevention of recurrence [8].Many studies revealed that gallstones are the most common cause of AP, followed by alcohol consumption [9].Those causes comprise around 75%-80% of the cases [1, 10].Gallstone AP was found to be more common in female subjects while alcohol pancreatitis was more common amongst men and idiopathic pancreatitis was similar in both sexes [8].There are other uncommon causes for AP, such as hypercalcemia, drugs, and tumors as shown in Table 1.Furthermore, the cause of AP is unknown in nearly 10% of the cases [8].

Pathogenesis
The pathogenesis of AP is mainly due to the inappropriate activation of trypsinogen to trypsin which autodigests pancreatic tissues leading to necrosis of acinar cells and pancreatic islets [8].
The actual mechanisms of acute pancreatitis are not fully clarified but there are many hypotheses.Despite continuous investigations, the pathogenesis of AP is not completely unraveled [12]; however, it may include oxidative stress [13, 14] and inflammatory response [15].
Several theories explain how stone causes AP.One theory assumes that a stone may create a channel behind it that allows bile reflux and injure the gland to cause pancreatitis.Another theory assumes that the stone passing reduces the efficiency of the sphincter and causes the reflux of duodenal juice with pancreatic enzymes that can reflux through the inefficient sphincter into the pancreatic ductal system.Another theory assumes that the stone passage obstruction may cause inflammation and edema eventually leading to continuous secretion into the pancreatic ductal system [16].AP caused by gallstones can be explained as the gallstone may lodge the sphincter of Oddi [17].
The resulting duct obstruction may lead to an increase in the pressure in the pancreatic duct that may lead to acinar cell damage and digestive enzyme activation [8].The second most common cause of AP is alcohol consumption [9].The main mechanism of pancreatitis caused by alcohol consumption is unknown but it can be explained by a combination of genetic and environmental factors [8].Many processes were assumed to be involved in the pathogenesis of AP, such as early trypsinogen activation, impaired autophagy, mitochondrial dysfunction, and endoplasmic reticulum stress [1].Oxygenderived free radicals and many cytokines (e.g.TNF-α, interleukin [IL]-1, IL-6, IL-8) also have important roles in AP [10].In the ceruleininduced AP model, it was revealed that there were very high levels of pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6 in the serum.They were released from acinar cells as a result of damage to the pancreas [18,19].
The changes occurred in the pancreatic gland lead to the release of active pancreatic enzymes that stimulate an inflammatory response [8].

Inflammation
Inflammatory mediators play an important role in AP and MODS which is the primary cause of death.Many studies evaluate & confirm the role of inflammatory mediators in AP such as TNF-α, IL-1β, IL-6, IL-8, Platelet-activating factor (PAF), IL-10, The complement activation product (C5a), Intercellular adhesion molecule-

Oxidative stress
Oxidative stress plays an important role in the initiation of AP.Reactive oxygen species (ROS) are generated at an early stage of the disease [37].Levels of the superoxide radical &lipid peroxides increased while ascorbic acid levels decreased in the early phase of AP.Also, the relation between disease severity & presence of oxidative stress was confirmed

Animal models
Conduction of studies on AP on the human pancreas is very difficult for many reasons, such as disease heterogenicity and limited samples, so it is more practical to conduct studies on animals [57].Animal models were used in recent years mostly to help in clarifying the underlying mechanisms of AP and/or examine therapies.Pathophysiological mechanisms of interest as well as the disease phase of interest are the determinant factors of the model type.The most commonly used animals are rodents (rats and mice) due to many reasons, such as that they are relatively inexpensive, easy to handle, and accessible.However, one of the drawbacks of animal models is that late complications are not usually studied in acute cases.Also, the differences between human and animal pancreas should be recognized.Several animal models of acute pancreatitis are available.The most common models are briefly discussed below.

Cerulein
Exocrine pancreas damage can be induced by neural stimulation.Cholecystokinin stimulates the pancreas and also does its analog, cerulein The L-arginine model is characterized by its reproducibility and suitability for the early and late phases of acute pancreatitis testing.However, it has a disadvantage in that its longterm administration induces chronic pancreatitis [69].The mechanism by which L-arginine causes AP is not well-known but it was thought that inflammatory mediators [65] or reactive oxygen species [70] could play an important role in this process.The increase in amylase and lipase levels occurring in L-arginine induced model was further supported by histopathological changes, which showed accumulation of fluid around acini, vacuolization, and marked necrosis of acinar cells which was significantly greater than that observed in cerulein-induced pancreatitis.

L-lysine
L-lysine is an amino acid used with different doses to induce AP.It was used at a dose of 2 g/kg causing mitochondrial damage that was followed by the activation of NF-κB & trypsinogen [62].Another study used L-lysine at a dose (400 mg/100 g body weight) and showed similar results [71].

L-ornithine
L-ornithine was used for induction of AP in rats by i.p. injection at a dose of 3 g/kg.Examination of the pancreatic tissue showed that L-ornithine use leads to necrosis of acinar cells and massive interstitial edema [63, 72].

L-histidine
Histidine was used for induction of AP in rats by i.p. injection at 2 × 4 g/kg L-histidine free base.Examination of the pancreatic tissue showed that L-ornithine use leads to necrosis of acinar cells and massive interstitial edema [73].

Bile duct ligation
Ligation of the common bile duct results in leakage of bile back into the pancreatic duct with subsequent inflammation.This model has been used in larger animals, including possums [15].This model clarified the importance of pancreatic duct obstruction as the central initiator of gallstone acute pancreatitis.However, this model has disadvantages, such as high intraanimal variability and the inability of opossums to be bred in the laboratory [83, 84].

Diet-induced pancreatitis
A diet deficient in choline and supplemented with ethionine induces hemorrhagic pancreatitis resembling human disease [15].It can be used for studying pathophysiology for AP and potential experimental treatment.It is a simple, cheap, non-invasive, and highly reproducible method.However, it can be only used in female mice, requires careful monitoring, and is associated with high mortality [69].

Diagnosis
Diagnosis of AP depends on many criteria, like laboratory tests, imagining techniques, and physical examination [85,86].AP can be diagnosed when two of three conditions are met, two of them are abdominal pain & increase of serum amylase and/or lipase level at least three times the upper limit of normal level which is between 100-300 U/L for amylase and 50 -160 U/l for lipase.The third condition is imaging techniques of the abdomen after 72 h from the symptoms start mainly by contrast-enhanced computed tomography to show findings like gland edema and peripancreatic fat stranding [1].Imagining techniques include Magnetic Resonance Imaging [86].There are some challenges to these criteria, for example in alcoholics and patients suffering from high levels of triglycerides, the amylase levels can be normal.Another challenge is that there are other cases in which the amylase and lipase levels are increased, like obstruction and abdominal aortic aneurysm in the case of amylase and acute intestinal pathologies, cholecystitis, peptic ulcer disease, and biliary obstruction in case of lipase While it is very important to early diagnose for better management, it is difficult to identify MODS cases early.Many predictors of MODS exist including pancreatic injury markers and inflammatory response markers & clinical features [52].

Management of acute pancreatitis
Incompatibility between the rapid onset of the AP attacks and the slow rate of hospitalization makes the management of AP challenging.It is important to identify the risk factors early during the first 24 h after hospital admission [88].
Management of AP mainly aims to the reduction of complications by focusing on preserving organ perfusion.Thus, management includes analgesics & antiemetic administration, oxygen administration, fluid restoration & continuous patient evaluation to detect any organ dysfunction or complication [8,89].Loss of a large volume of fluid increase the incidence rate of renal failure & associated mortality.To restore intravascular fluid volume rapidly the patient may administer fluids at a rate of 300 to 500 ml/h which may lead to electrolyte imbalance [90].Balanced electrolyte solutions (9% saline or Ringer"s lactate) should be given rapidly then frequent assessment of the patient"s volume status should be done by assessing heart rate, blood pressure, and urine output [11].
Control of pain is very important as patients with pain tend to have a high respiratory rate from hypoxic "drive", which can lead finally to the reduction of lung function.Furthermore, it can increase the risk of deep venous thrombosis.The therapy of choice, in this case, is Narcotic analgesics which are administered via an epidural catheter.As AP results in rapid loss of body weight, fat, and protein, so nutritional support is an essential step in patient care [11].Early introduction of a solid, low-fat diet in patients with mild or moderately severe pancreatitis is supported by evidence & is preferable for those patients who can tolerate an oral diet [91,92].If patients with mild-moderate AP do not tolerate oral food within approximately 3-5 days, the enteral tube feeding should be used [93] (total parenteral nutrition) plays a role in seriously ill patients with acute pancreatitis Otherwise, patients with severe disease who are not expected to eat for a week or more should be considered for (preferably enteral) nutritional support at an early stage [94].It is useful to use prophylactic antibiotics in severe acute pancreatitis as approximately 80 percent of deaths from AP result from infectious complications but should be of broad spectrum, and should be used for a short period (5-7 days) [95].Moreover surgical, endoscopic, or radiologic drainage procedures may be required for patients with infected necrosis [96].Management of AP resulting from gallstone, endoscopic intervention, and endoscopic intervention is recommended for removal of the stone(s) which has success rates over 90% [11].MODS that may result need close monitoring and may require management in the intensive care unit, accordingly as a minimum in this case peripheral venous access, a central venous line, and a urinary catheter are needed.Also, management of acute respiratory complications may require intubation and mechanical ventilation.Acute renal failure can occur & it will be diagnosed depending on the presence of one of the following: (i) increase in serum creatinine > 0.5 mg/dL (44  VB12 prevents AP in mice models, so it can be a promising treatment option for AP.clinical studies can be launched to test this beneficial effect on patients [116].Diosgenin derivatives D (Drug D) are derivatives of Diosgenin (a kind of natural steroidal sapogenin) which has excellent anti-inflammatory properties and prevents AP through mitochondrial protection and PI3Kγ/Akt inhibition.Diosgenin derivatives D was explored for its beneficial effect on L-arginine-induced AP & its effect was demonstrated through mediating Gasdermin D (GSDMD) which causes holes in the acinar cells &release of inflammatory factors after accumulation in the Endoplasmic reticulum.Based on the fact that the inactivation of GSDMD significantly reduces necrosis and systemic inflammation in AP, drug D is expected to be a potentially effective therapeutic strategy for the development of new drugs [117].A novel self-nanomicellizing formulation of EMP with phytochemical was examined for its effect on AP & it was found to suppress the effects of oxidative stress and inhibit proinflammatory cytokines [118].Disulfiram was shown to inhibit NF-κB activation in acini & reduce expression of TLR4, so it was concluded that disulfiram ameliorates the severity of AP in mice [119].

Conclusion
AP is a common disorder of the pancreas and could range from mild to potentially lifethreatening cases.It has an increasing incidence rate and is associated with many complications that cause a high burden to the healthcare system.Animal models appear to be appropriate for the study of acute pancreatitis.The exact underlying pathophysiological mechanisms are not fully clear, but several studies on experimental models of acute pancreatitis have revealed many of those mechanisms that could be potential therapeutic targets.This is supported by the evidence that many processes are involved in animal models and humans, such as a strong inflammatory response.It was confirmed by elevated levels of many cytokines, including TNF-α and IL-1β.Other elevated factors include TLR4, NF-κB, and HMGB1.Autophagy and oxidative stress also play an important role in AP pathogenesis.In conclusion, there is an urgent need for the development of new therapeutic agents, and more research is required to reveal many unclear gaps.
[38].Levels of malondialdehyde (MDA) also increased in AP[34] & levels of glutathione were reduced and depleted during the development of AP[39].Antioxidant use in cerulein-induced pancreatitis reduced pancreatic tissue damage, and also hampered the extrapancreatic complications, thus improving the outcome of the disease[37].It was found in a study that used antioxidant in an AP animal model that acinar cell injury and edema is reduced after treatment [39].3.1.3.AutophagyA process in which damaged proteins and organelles are transferred to lysosomes for digestion and degradation.Normally autophagy prevents cancer development.But if cancer already exists autophagy usually supports the cancer cell growth & survival [40].Autophagy can be stimulated in other diseases as autophagy is stimulated by multiple factors, like nutrient deprivation and stress, as a cell survival mechanism.However, deregulation of the autophagic process can lead to harmful effects in AP [41].LC3 is autophagic activity marker [42, 43], and is significantly increased in AP [44] to activate autophagy in AP rat models [41].HMGB1 regulates autophagy [45], which was demonstrated to be activated in AP [41], as evidenced by the elevated levels of autophagic genes such as Beclin1 and microtubuleassociated protein 1A/1B-light chain 3 (LC3-II) [41, 43].LC3& berlin-1 are autophagy proteins, their levels were elevated in AP while reduction of their levels is associated with improvement in AP [46].Beclin1 is an autophagic gene that induces autophagy through the formation of a complex with Atg14, Vps34/ class 3 phosphatidylinositol 3-kinase (PI3k), and Vps15 [47].afterstimulation by HMGB1 [48].4. Pathophysiology AP can cause MODS including progressive renal and liver failure [49] which is considered the primary cause of morbidity and mortality [50, 51].Pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α,) play a significant role in the pathogenesis of AP and systemic complications [52].AP pathophysiology can be explained as stages.Firstly, activation of pancreatic trypsin in acinar cells and activation of many cytokines & inflammatory mediators occurs.Activated trypsin activates other enzymes including phospholipase and elastase.The active enzymes and cytokines then digest cellular membranes and cause edema, proteolysis, interstitial hemorrhage, vascular damage, and cell necrosis [53].During AP progression, Cytokines continue to be released from the necrotic acinar cells & the immune cells to cause more pancreatic inflammation, which leads to the systemic inflammatory response.Moreover, it can cause distant organ damage including acute respiratory distress syndrome, or MODS [53, 54].MODS are associated with high levels of cytokine and C-reactive protein in the circulation.The endothelial barrier damage in various organs may be involved in the pathophysiology of MODS resulting from AP.It is also accompanied by increased permeability which allows the transfer of blood constituents to various tissues, including the pancreas, lungs, kidneys, colon, spleen, and intestine [55].4.1.AP severity Pancreatitis severity can be categorized according to the revised Atlanta classification, as mild, moderate, or severe.Mild AP is characterized by the absence of organ failure, local complications, or systemic complications.Moderately severe AP is characterized by the presence of transient organ failure (<2 days) and local complications, while severe acute pancreatitis is characterized by the presence of persistent organ failure (>2 days) [56].Table2shows the difference between the revised Atlanta classification and determinant-based classifications of AP.

Table 2 .
Difference between revised Atlanta classification & determinant-based classifications of acute pancreatitis [

4 . 4 .
[58].Excessive doses of cerulein at repeated intervals result in AP [59], which is characterized by cytoplasmic vacuolization, acinar cell necrosis, edema formation, and inflammatory response [1].Cerulein can be administered subcutaneously, or intravenously.Acute pancreatitis caused by cerulein is mild [15], rapidly resolves, and is not accompanied by any mortality.Cerulein-induced pancreatitis models are characterized by low cost and high reproducibility [57, 60].Basic Amino Acids Intraperitoneal (i.p.) administration of many doses of certain amino acids causes AP in mice or rats, such as L-arginine [61], L-lysine [62], Lornithine [63], and L-histidine [64].4.4.1.L-arginine L-arginine-induced AP is currently the most commonly used amino acid-induced AP model in rats and mice.L-arginine was used to induce AP for the first time in 1984 as a single i.p. injection at 5 g/kg and led to necrosis of tissues in the rat pancreas, without affecting islets of Langerhans and other organs [65].After that, L-arginine was used with different doses & gave reproducible results [66], such as two doses of 4 g/kg each, at 1 h apart [67].When higher doses (7.5 g/kg) of L-arginine were used, it caused a lethal effect on animals, while the lower dose of 2.5 g/kg caused mild injury in the pancreas.Many studies used either single or double injections of L-arginine at different doses to induce AP in rats or mice [68].

[ 1 ]
. The abdominal pain and elevated levels of pancreatic enzymes in the serum are very important in diagnosis [8].Pancreatic enzyme assessment is a critical step used in the diagnosis with the preference for lipase over amylase [85] because it is slightly more specific and sensitive than amylase and has a longer half-life.Their levels rise to the peak early while and turn down over 3-4 days of the initial attack [8].Quick diagnosis is extremely essential in the reduction of the morbidity and mortality associated with AP [52].In case of suspicion of a biliary etiology, a trans-abdominal ultrasound (T-A US) must be done first to demonstrate gallstones and any other pathological change .andif it appears normal with strong suspicious of biliary cause Magnetic resonance cholangiopancreatography or the endoscopic US should be performed to investigate the causes of duct obstruction [8].The most common symptom of AP is acute abdominal pain and tenderness in the upper abdomen which is commonly occurred in another abdominal diseases.Abdominal pain occurs in nearly 95% of the patients.The pain is usually acute & reaches maximum intensity rapidly and it is usually generalized to the upper abdomen [8], but it could be more localized to the right upper quadrant, epigastric area, or, rarely, left upper quadrant [87].Moreover, nausea and vomiting occur in the majority of the patients [8].Fever also is an important sign in patients with acute pancreatitis.Most patients develop a fever at the start of the illness [11].

[35, 36].
[113]rotective effect in AP was demonstrated by reducing pancreatic edema and reducing the production of serum pancreatic enzymes and pro-inflammatory cytokines.In addition, BCA's beneficial effect on AP is partly mediated by improving intestinal homeostasis as evidenced by reduced serum amylase and lipase activities, and pancreatic edema[113].