Prophylactic low-molecular-weight heparin administration protected against severe acute pancreatitis partially by VEGF/Flt-1 signaling in a rat model

Abstract

Background:

The present study was aimed to explore the effects and the underlying mechanism of prophylactic low-molecular-weight heparin (LMWH) treatment on taurocholate-induced severe acute pancreatitis (SAP) in a rat model.

Methods:

Rat SAP model was induced by injection of 4% sodium taurocholate into the pancreatic duct. LMWH was applied half an hour before the induction of pancreatitis at the dose of 200 IU/kg subcutaneous injection. The rats were euthanized at 1 h, 6 h, and 12 h after taurocholate-induced SAP. The inflammatory and oxidative response markers were assessed. And the vascular endothelial growth factor (VEGF) and Fms-related tyrosine kinase 1 (Flt-1) expression were evaluated by immunohistochemistry (IHC) and western blot methods.

Results:

The expression of inflammatory and oxidative response markers increased after induction of SAP. IHC and western blot results showed the VEGF and Flt-1 expression were increased in SAP group. Prophylactic LMWH administration reduced the inflammatory and oxidative response markers expression and decreased the expression of VEGF and Flt-1.

Conclusions:

This study suggested that prophylactic LMWH treatment mitigated the severity of pancreatitis in rat SAP model by anti-inflammation and oxidative response. The underlying mechanism may result from downregulating VEGF/Flt-1 signaling of LMWH in SAP rat model.

Keywords

Low-molecular-weight heparin inflammation severe acute pancreatitis VEGF Flt-1

Introduction

Severe acute pancreatitis (SAP) is a common clinical problem with complicated etiology, rapid disease progression, and poor prognosis.¹ The incidence of SAP is still rising over time and SAP mortality rate is up to 20–30%.^2,3 SAP is characterized by local tissue edema, hemorrhage, necrosis, and inflammatory cell infiltration of the pancreas and accompanied by elevated level of pancreatic enzyme.⁴ However, the pathogenesis mechanism of SAP is still not fully understood.

Vascular endothelial growth factor (VEGF), as a heparin-binding protein, belonging to the platelet-derived growth factor supergene family, which has a wide range of pharmacological effects, is a well-known regulator in angiogenesis and microvascular permeability by binding to the VEGF receptors. The physiological and pathological roles of VEGF including regulation of endothelial cell proliferation, migration, secretion, and vascular permeability are mainly mediated by three receptors: VEGFR1 (Fms-related tyrosine kinase 1 (Flt-1)), VEGFR2 (kinase insert domain receptor (KDR)/Flk-1), and VEGFR3 (Flt-4).^5,6 Accumulating studies reported that VEGF plays a key role in regulating inflammation in clinical settings including rheumatoid arthritis,⁷ atherosclerosis,⁸ inflammatory bowel disease,⁹ as well as tumor formation.¹⁰ Moreover, the bioactivities of VEGF could regard as a heparin-binding growth factor and affected by heparin-like chemicals.¹¹ And several studies have confirmed that VEGF plays an effect in the inflammatory response mainly through combined with Flt-1.¹² VEGF can also activate neutrophils through Flt-1 and promote neutrophils chemotaxis.¹³

Low-molecular-weight heparin (LMWH) is widely used as an effective anticoagulant to prevent deep vein thrombosis, unstable angina pectoris, cerebral infarction, ischemic stroke, chronic pulmonary heart disease, cardiovascular surgery, disseminated intravascular coagulation, and other diseases.^14,15 It was also reported that LMWH had many other effects including inhibiting tumor metastasis, reducing proteinuria, and anti-inflammatory.^16
–18 However, the effects of LMWH on SAP have been controversial.¹⁹ A meta-analysis study showed LMWH could improve the prognosis of SAP, reduce hospital stay, incidences of multiple organ failure and operation rate.¹⁹ Researches showed that LMWH could enhance the effect of conventional treatment for SAP and could markedly decrease the mortality of SAP.²⁰ On the other hand, it was reported that LMWH could not significantly improve the operation rate and mortality in severe pancreatitis.²¹ As for the mechanism of SAP and the role of LMWH in SAP, it remains largely unknown. The aim of the present study is to evaluate the effect and mechanism of LMWH in rat SAP.

Materials and methods

Animals

Nine weeks old male Sprague-Dawley rats weighing 240–300 g were obtained from the Experimental Animal Center of Xi’an Jiaotong University. The rats were kept in compliant laboratory condition with free access to chow and water. The rats were given ketamine (50 mg/kg) intraperitoneal injection to induce anesthetization. Sodium taurocholate (4% in saline, Sigma, St. Louis, Missouri, USA) was administered by retrograde injection to induce acute pancreatitis. A dull needle was inserted into pancreatic duct through duodena following midline incision. Sodium taurocholate (1 ml/kg) was injected slowly and the bile duct clipped to prevent reflux.²² Forty-five rats were used in this study and separated randomly into following three groups (n = 15 per group): (1) the sham group, in which animals underwent injection of saline into the pancreatic duct; (2) the SAP group, in which SAP was induced with sodium taurocholate injection; (3) the LMWH group, in which SAP was induced and LMWH (Sanofi-Aventis, Paris, France) was applied at the dose of 200 IU/kg s.c (subcutaneous injection) half an hour before the induction of pancreatitis. The rats were euthanized 1 h, 6 h, and 12 h after the surgical procedure (n = 5 per time point). Serum samples were obtained from rat blood by centrifugation at 2600 r/min for 15 min and stored at −20°C. The pancreatic tissues were collected and stored at −80°C. All animal studies were in compliance with protocols approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University.

Tissue specimens staining

The pancreas was quickly removed. Representative small specimens of pancreatic tissue with typical changes were fixed in 10% formalin for routine hematoxylin and eosin (H&E) histology examination. Immunohistochemistry (IHC) was performed on paraformaldehyde-fixed paraffin sections. The sections were dewaxed and dehydrated. After rehydration and washing in phosphate buffer saline (PBS), antigen retrieval, the sections were blocked for 10 min by using 3.0% hydrogen peroxide and then blocked for half an hour using 10% goat serum. The sections were incubated with relevant primary antibodies against VEGF (1:50, MA1-16629, ThermoFisher Scientific, Waltham, Massachusetts, USA) and Flt-1 (1:200, ab32152, Abcam, Cambridge, Massachusetts, USA) at 4°C overnight. After washing the slides in PBS for three times, the sections were detected using biotinylated secondary antibodies (Goldenbridge Biotechnology, Zhongshan, China) following the manufacturer’s instructions. The staining was visualized with diaminobenzidine and counterstained with hematoxylin, then dehydrated in alcohol and xylene and mounted onto glass. The staining area scores were obtained by randomly evaluating of five different fields.

Assessment of pancreatitis severity

The blood amylase activity was assessed by kinetic spectrophotometric assay.²³ Samples of pancreas were cut into pieces, washed with PBS, dried, and weighed (wet weight). Then, the samples were desiccated for 10 h at 90°C and weighed (dry weight). The pancreatic water content was assessed by determination of the wet weight to dry weight ratio.²⁴ The severity of pancreatitis was detected in 5-µm thickness H&E-stained pancreatic sections according to the following indicators: edema, acinar necrosis, inflammatory infiltrate, hemorrhage, fat necrosis, and perivascular inflammation. Pancreatic sections were scored from 0 to 3. The total score was calculated together.

Assessment of inflammatory markers and oxidative stress

Myeloperoxidase (MPO) activity was applied to evaluate the infiltration of leukocytes in the pancreas. The samples of the pancreas were homogenized in neutral potassium phosphate buffer in ice. The tumor necrosis factor-α (TNF-α) activity was determined with enzyme-linked immunosorbent assays (ELISA) kit according to the manufacturer’s instructions (Wuhan Boster Biological Technology, Ltd, Wuhan, China). The malondialdehyde (MDA) concentration in pancreatic tissue was detected by using thiobarbituric acid test. MPO was measured using MPO detection kit (kt302357, MSKbio Co. Ltd, Wuhan, China) following the manufacturer’s instructions.

Immunoblotting

Rat pancreatic tissue sample proteins were extracted using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Guangzhou, China). The bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, Illinois, USA) was applied to measure the concentration of the proteins according to the manufacturer’s instructions. Briefly, proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked using 5% nonfat milk/Tris-buffered saline Tween-20. Subsequently, the anti-VEGF (1:1000, MA1-16629, ThermoFisher Scientific), anti-Flt-1 (1:1000, ab32152, Abcam), and anti-β-actin (1:1000, #4970, Cell Signaling Technology, Danvers, Massachusetts, USA) primary antibodies were used to incubate the membranes at 4°C overnight. Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Bio-Rad, Hercules, California, USA) were employed and the bands were visualized by using the chemiluminescence detection system through the peroxidase reaction. The images were revealed with ChemiDoc XRS imaging system (Bio-Rad).

Statistical analysis

GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, California, USA) was used in this study. All data are shown as the mean ± standard deviation. One-way analysis of variance was used for comparing difference between multiple groups and comparison between two groups was performed by the student’s t-test. p Values of <0.05 were considered having statistical significance.

Results

LMWH alleviated the degree of histological damage in pancreas

To explore the protective effects of LMWH against pancreatitis, the rats were divided into three groups and the flowchart of experiment was designed (Figure 1(a)). The main steps of the surgery are shown in Figure 1(b): (1) exposure of the pancreas and surrounding organs following midline incision; (2) imbedding the dull needle into pancreatic duct though duodena; (3) imbedding the clip to hepatic duct to prevent reflux. To assess the pathological changes, pancreas samples were collected at 1 h, 6 h, and 12 h after surgery. No significant changes were observed at all the time points in the sham group. Tissue congestion and mild swelling could be observed after 1 h in the SAP group. The degree of pancreatic edema aggravated over time. It appeared expanded necrosis and severe edema in the SAP group rat pancreatic tissue. Pancreatic edema degree in the LMWH group is lower than that in the SAP group at all the same time point (Figure 2(a)). Under microscopy, swollen pancreatic tissues, necrosis acinar, loosen, and disorganized glandular lobules could be observed in the SAP group. The injury scores and water content in the SAP group were significantly higher than that in the sham group at all the time points. Compared with the SAP group, LMWH treatment resulted in a significant alleviation in the pancreatic injury score and water content (Figure 2(b) and (c)).

Figure 1.

Study design and experimental flowchart. (a) Schematic illustration of experimental design. (b) Surgical procedure diagram to induce acute pancreatitis in rats: (b-1) exposure of the pancreas and surrounding organs following midline incision; (b-2) imbedding the dull needle into pancreatic duct though duodena; (b-3) imbedding the clip to biliary duct to prevent reflux. LMWH: low-molecular-weight heparin; TC: sodium taurocholate; SAP: severe acute pancreatitis.

Figure 2.

LMWH alleviated pancreatic injury in rat acute pancreatitis model. (a) Representative H&E-stained sections of pancreas from three different groups. Scale bar = 100 μm. (b) Injury scores of pancreas tissues in three groups were quantified and analyzed. *p < 0.05. (c) Water content of pancreas tissues in three groups was quantified. *p < 0.05. The differences between multiple groups were analyzed by one-way ANOVA analysis and student’s t-test was used for comparing the difference between two groups. H&E: hematoxylin and eosin; LMWH: low-molecular-weight heparin; SAP: severe acute pancreatitis; NS: no significance; ANOVA: analysis of variance.

LMWH reduced inflammatory and oxidative response

Next, we measured the expression of amylase, TNF-α, interleukin 6 (IL-6) in serum and MPO, MDA in pancreatic tissue. Compared with the sham group, the serum amylase content in the SAP group was higher at all time points, consistent with the pathological changes in pancreatitis. While the amylase in the LMWH group was lower than that in the SAP group (Figure 3(a)). ELISA assay showed the expression of TNF-α and IL-6 in the SAP group was also higher than that in the sham group at all time points. While LMWH treatment resulted in a reduction of these parameters compared with the SAP group (Figure 3(b) and (c)). Moreover, the MPO and MDA levels in pancreatic tissues were significantly elevated in the SAP group. LMWH treatment led to a remarkable reduction of MPO and MDA in comparison with the SAP group (Figure 3(d) and (e)).

Figure 3.

LMWH reduced the pro-inflammatory markers expression and oxidative stress response in rat acute pancreatitis model. The serum expression level of amylase (a), TNF-α (b), and IL-6 (c) were analyzed by ELISA assay. *p < 0.05. The tissue expression level of MPO (d) and MDA (e) were analyzed by ELISA assay. *p < 0.05. The differences between multiple groups were analyzed by one-way ANOVA analysis and student’s t-test was used for comparing the difference between two groups. ELISA: enzyme-linked immunosorbent assay; LMWH: low-molecular-weight heparin; SAP: severe acute pancreatitis; NS: no significance; TNF-α: tumor necrosis factor-α; IL-6: interleukin 6; ANOVA: analysis of variance; MDA: malondialdehyde; MPO: myeloperoxidase.

LMWH impacted VEGF/Flt-1 signaling

As known to us, VEGF signaling is an important mediator of inflammation. Activation of VEGF signaling will lead to pro-inflammatory cytokines including TNF-α and IL-6 release. We wondered whether VEGF/Flt-1 was involved in the acute pancreatitis pathological process. We explored whether the protective effect of LMWH treatment is associated with VEGF signaling by IHC and western blot after 12 h induction of SAP. IHC results showed that the VEGF and Flt-1 rarely expressed in sham group rat pancreas tissues. The IHC results showed that the expression of VEGF and Flt-1 were lower in the sham group than that in the SAP group. LMWH pretreatment decreased the VEGF and Flt-1 expression in taurocholate-induced rat SAP (Figure 4(a) and (b)). The western blot results also consistently supported the findings that LMWH administration decreased the VEGF and Flt-1 expression in taurocholate-induced rat SAP (Figure 5(a) and (b)).

Figure 4.

The expression of VEGF and Flt-1 was detected by IHC staining. (a) Representative IHC staining images of 12 h after operative process pancreas tissues in three different groups were shown. Scale bar = 100 μm. (b) IHC staining scores of 12 h after operative process pancreas tissues in three groups were quantified and analyzed. *p < 0.05. Student’s t-test was used for comparing the difference between two groups. IHC: immunohistochemical; LMWH: low-molecular-weight heparin; SAP: severe acute pancreatitis; VEGF: vascular endothelial growth factor; Flt-1: Fms-related tyrosine kinase 1.

Figure 5.

The expression of VEGF and Flt-1 expression were detected by immunoblotting. (a) Representative western blotting images of 12 h after operative process pancreas tissues in three different groups were shown. (b) Relative expression of VEGF and Flt-1 protein of 12 h after operative process pancreas tissues in three different groups detected by western blotting. *p < 0.05. Student’s t-test was used for comparing the difference between two groups. LMWH: low-molecular-weight heparin; SAP: severe acute pancreatitis; VEGF: vascular endothelial growth factor; Flt-1: Fms-related tyrosine kinase 1.

Discussion

SAP is commonly characterized by acute inflammation of the pancreas and increased expression level of serum amylase and lipase. The increased incidence and death rate make SAP one of the most devastating and insurmountable diseases.³ Furthermore, SAP requires intensive care and sometimes surgery is necessary for treating the complications induced by SAP.²⁵ Although the risks, measurements of severity, management of SAP, and its complications have evolved rapidly over the past decade, SAP is still a complicated disease and its pathogenesis mechanism remains unclear.²⁶ It urges us to search for new and better management strategies. Several experimental animal studies were already performed in the prevention of acute pancreatitis. It was reported that Myrtus communis leaf extract could exert protective effect acute pancreatitis.²⁷ Furthermore, urocortin 2 attenuated severity of an experimental pancreatitis model.²⁸ Although studies have assessed the value of antibiotics treatment for acute pancreatitis, the outcomes were controversial.^29
–31.A recent study was also carried out to identify risk factors of acute pancreatitis and help to prepare preventive recommendations for lifestyle elements.³²

Emerging evidence suggests that VEGF plays an important role in the host inflammatory response in many diseases.^33,34 Several studies have also confirmed that VEGF plays an effect in inflammatory response mainly by binding to Flt-1.¹³ VEGF can also activate neutrophils through Flt-1 and promote neutrophils chemotaxis.³⁴ More and more evidence showed that VEGF can participate in inflammatory response, especially in vascular endothelial cells.³⁵ However, the mechanism of VEGF system involved in inflammation and its role in the inflammatory process are still not fully understood. There are few studies on the effect of VEGF and Flt-1 on SAP. LMWH has been widely used as an effective anticoagulant in clinical practice.³⁶ Besides, it was reported that LMWH also has the anti-inflammatory effect.¹⁸

As for the protective effect of LMWH in acute pancreatitis, researchers mainly focus on its anticoagulation currently, but the anti-inflammatory effect of LMWH in SAP and its influence on VEGF and VEGFR are rarely reported. The sequestration of inflammatory cell and alleviation of pro-inflammation cytokine cascade were widely thought to be vital in managing pancreatitis.^37,38 MPO activity is a marker of leukocyte inflammation.³⁹ TNF-α and IL-6 are dominating pro-inflammatory cytokines in pancreatitis.⁴⁰ The present study was aimed to evaluate the protective effect and the underlying mechanism of prophylactic LMWH treatment on taurocholate-induced SAP in a rat model. It was found LMWH treatment resulted in reduction of the serum amylase activities at 1 h, 6 h, and 12 h and alleviation in pancreatic injury score and water content in rat SAP model. This phenomenon was an evidence to support protective effect of LMWH treatment in a rat SAP model. We further found LMWH treatment significantly decreased the inflammatory markers expression in a rat pancreatitis model. These results demonstrated that LMWH treatment exerted a protective effect by suppressing inflammation. Our results also showed LMWH treatment reduced MDA content and suggested a protective role of LMWH against oxidative stress. Importantly, the elevated expression of VEGF and Flt-1 were found after induction of SAP. The expression of VEGF and Flt-1 were consistently decreased in LMWH pretreated rat SAP. These results demonstrated that LMWH treatment exerted a protective effect by suppressing inflammation and downregulating VEGF/Flt-1 signaling. Thus, it provided a theoretical basis for the clinical application of LMWH.

These results were in line with the alleviator role of LMWH treatment in inflammation and also demonstrated protective effect of LMWH in SAP. Here, it was worthy to mention that the commonly adopted dose of heparin in other literature was 100 IU/kg or 150 IU/kg and was used as a treatment for SAP.^41
–43 While LMWH was applied at the dose of 200 IU/kg s.c half an hour before the induction of pancreatitis in our study. In other words, LMWH was used prophylactically in our study. So, the usage is different. We also found that no bleeding or other complications happened after injection of 200 IU/kg LMWH in these animals. After reviewing other literatures, we invesitaged whether LMWH could be as a prevention strategy to SAP.^43,44 LMWH was applied half an hour before the induction of pancreatitis in our study. What’s more, we wanted to look into the effects of LMWH in different stages of SAP. According to our experience and other experiment results,^24,45 we then thought an inflammatory reaction of pancreatic tissue occurred 1 h after the induction of pancreatitis and could be regarded as the initial stage of SAP; 6 h after the induction of pancreatitis could be considered as the development stage of SAP; 12 h after the induction of pancreatitis was the most severe. Moreover, 6 h and 12 h were the generally accepted time points for performing experiments of SAP. Thus, 1 h, 6 h, and 12 h time points were selected for this study. And this is a normal course of this disease. Although the value of some parameters at 6 h and 12 h in SAP and LMWH groups was close, the results showed pancreatic injury scores, water content, amylase, TNF-α, and IL-6 existed differences between 6 h and 12 h in SAP and LMWH groups. While the oxidative markers including MPO and MDA did not change as above indicators. This is properly that inflammatory markers and oxidative stress markers did not change at the same time. Trying to perform experiments at another time point during 1–6 h might be helpful to address this question. There were some limitations in this study. First, whether LMWH could inhibit the binding of VEGF to Flt-1 leading to the downregulation of VEGF and Flt-1. Second, two major VEGF receptors expressed in pancreatic tissues. VEGFR1 also called Flt-1 and VEGFR2 also called KDR. And KDR is generally thought to be the classical signaling receptor for VEGF, however, Flt-1 not only possesses higher affinity for VEGF but also mediates ligand access to KDR. We can’t determine whether VEGFR2 plays a role in acute pancreatitis in rats. At last, the rational and precise LMWH administration time and dose in rat SAP treatment are required to be further investigated.

Footnotes

Author contributions

S Li, S Zhang, and R Li contributed equally to this work and should be considered as co-first authors.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Key Research and Development Project of Shaan’Xi Province (2017SF-035) and the National Natural Science Foundation of China (NSFC, No. 81902449).

ORCID iDs

R Li

M Xu

References

Hines

Pandol

. Management of severe acute pancreatitis. BMJ 2019; 367: l6227.

Trikudanathan

Wolbrink

DRJ

van Santvoort

, et al. Current concepts in severe acute and necrotizing pancreatitis: an evidence-based approach. Gastroenterology 2019; 156: 1994–2007.

Peery

Crockett

Murphy

, et al. Burden and cost of gastrointestinal, liver, and pancreatic diseases in the United States: update 2018. Gastroenterology 2019; 156: 254–272.

Portelli

Jones

. Severe acute pancreatitis: pathogenesis, diagnosis and surgical management. Hepatobiliary Pancreat Dis Int 2017; 16: 155–159.

Apte

Chen

Ferrara

. VEGF in signaling and disease: beyond discovery and development. Cell 2019; 176: 1248–1264.

Melincovici

Bosca

Susman

, et al. Vascular endothelial growth factor (VEGF)—key factor in normal and pathological angiogenesis. Rom J Morphol Embryol 2018; 59: 455–467.

Chen

Wang

Xia

, et al. Therapeutic potential of mesenchymal cell-derived miRNA-150-5p-expressing exosomes in rheumatoid arthritis mediated by the modulation of MMP14 and VEGF. J Immunol 2018; 201: 2472–2482.

Zhu

Xie

Liang

, et al. Bilayered nanoparticles with sequential release of VEGF gene and paclitaxel for restenosis inhibition in atherosclerosis. ACS Appl Mater Interf 2017; 9: 27522–27532.

D’Alessio

Correale

Tacconi

, et al. VEGF-C-dependent stimulation of lymphatic function ameliorates experimental inflammatory bowel disease. J Clin Invest 2014; 124: 3863–3878.

10.

Siveen

Prabhu

Krishnankutty

, et al. Vascular endothelial growth factor (VEGF) signaling in tumour vascularization: potential and challenges. Curr Vasc Pharmacol 2017; 15: 339–351.

11.

Koch

Tugues

, et al. Signal transduction by vascular endothelial growth factor receptors. Biochem J 2011; 437: 169–183.

12.

Mohr

Haudek-Prinz

Slany

, et al. Proteome profiling in IL-1β and VEGF-activated human umbilical vein endothelial cells delineates the interlink between inflammation and angiogenesis. PloS One 2017; 12: e0179065.

13.

Shibuya

. VEGF-VEGFR system as a target for suppressing inflammation and other diseases. Endocr Metab Immune Disord Drug Targets 2015; 15: 135–144.

14.

Hirsh

Warkentin

Shaughnessy

, et al. Heparin and low-molecular-weight heparin: mechanisms of action, pharmacokinetics, dosing, monitoring, efficacy, and safety. Chest 2001; 119: 64S–94 S.

15.

Garcia

Lyman

, et al. Direct oral anticoagulant (DOAC) versus low-molecular-weight heparin (LMWH) for treatment of cancer associated thrombosis (CAT): a systematic review and meta-analysis. Thromb Res 2019; 173: 158163.

16.

, et al. LMWH and its derivatives represent new rational for cancer therapy: construction strategies and combination therapy. Drug Discov Today 2019; 24: 2096–2104.

17.

Liu

Qiao

Liu

, et al. Low molecular weight heparin improves proteinuria in rats with L-NAME induced preeclampsia by decreasing the expression of nephrin, but not podocin. Hypertens Pregnancy 2015; 34: 24–35.

18.

Luan

Naranpurev

. Treatment of low molecular weight heparin inhibits systemic inflammation and prevents endotoxin-induced acute lung injury in rats. Inflammation 2014; 37: 924–932.

19.

Qiu

Tang

, et al. The efficacy of low molecular weight heparin in severe acute pancreatitis: a systematic review and meta-analysis of randomized controlled trials. J Dig Dis 2019; 20: 512–522.

20.

Qiu

, et al. Low molecular weight heparin in the treatment of severe acute pancreatitis: a multiple centre prospective clinical study. Asian J Surg 2009; 32: 89–94.

21.

Tozlu

Kayar

Ince

, et al. Low molecular weight heparin treatment of acute moderate and severe pancreatitis: a randomized, controlled, open-label study. Turk J Gastroenterol 2019; 30: 81–87.

22.

Lichtenstein

Milani

Jr Fernezlian

, et al. Acute lung injury in two experimental models of acute pancreatitis: infusion of saline or sodium taurocholate into the pancreatic duct. Crit Care Med 2000; 28: 1497–1502.

23.

Chen

Cai

Shen

, et al. Netrin-1 protects against L-arginine-induced acute pancreatitis in mice. PloS One 2012; 7: e46201.

24.

Hackert

Werner

Gebhard

, et al. Effects of heparin in experimental models of acute pancreatitis and post-ERCP pancreatitis. Surgery 2004; 135: 131–138.

25.

Johnson

Besselink

Carter

. Acute pancreatitis. BMJ 2014; 349: g4859.

26.

Tenner

Baillie

DeWitt

, et al. American College of Gastroenterology guideline: management of acute pancreatitis. Am J Gastroenterol 2013; 108: 1400–1415.

27.

Ozbeyli

Sen

Cilingir Kaya

, et al. Myrtus communis leaf extract protects against cerulein-induced acute pancreatitis in rats. J Food Biochem 2020; 44: e13130.

28.

Yuan

Hasdemir

Tan

, et al. Protective effects of urocortin 2 against caerulein-induced acute pancreatitis. PloS One 2019; 14: e0217065.

29.

Ding

Sun

Wen

, et al. Assessment of prophylactic antibiotics administration for acute pancreatitis: a meta-analysis of randomized controlled trials. Chin Med J 2020; 133: 212–220.

30.

Cagle

Chopra

. Antibiotic prophylaxis for severe acute pancreatitis. Am Fam Physician 2019; 99: 49.

31.

Mandal

Chaudhary

Shrestha

, et al. Efficacy of prophylactic use of ciprofloxacin and metronidazole in mild and moderately severe acute pancreatitis. JNMA J Nepal Med Assoc 2017; 56: 207–210.

32.

Koncz

Darvasi

Erdosi

, et al. LIFEStyle, Prevention and Risk of Acute PaNcreatitis (LIFESPAN): protocol of a multicentre and multinational observational case-control study. BMJ Open 2020; 10: e029660.

33.

Arjamaa

Aaltonen

Piippo

, et al. Hypoxia and inflammation in the release of VEGF and interleukins from human retinal pigment epithelial cells. Graefes Arch Clin Exp Ophthalmol 2017; 255: 1757–1762.

34.

Janela

Patel

Lau

, et al. A subset of type I conventional dendritic cells controls cutaneous bacterial infections through VEGFα-mediated recruitment of neutrophils. Immunity 2019; 50: 1069–1083.

35.

Mura

Binnie

Han

, et al. Functions of type II pneumocyte-derived vascular endothelial growth factor in alveolar structure, acute inflammation, and vascular permeability. Am J Pathol 2010; 176: 1725–1734.

36.

Rodger

Gris

de Vries

JIP

, et al. Low-molecular-weight heparin and recurrent placenta-mediated pregnancy complications: a meta-analysis of individual patient data from randomised controlled trials. Lancet 2016; 388: 2629–2641.

37.

Stigliano

Sternby

de Madaria

, et al. Early management of acute pancreatitis: a review of the best evidence. Dig Liver Dis 2017; 49: 585–594.

38.

Greenberg

Hsu

Bawazeer

, et al. Clinical practice guideline: management of acute pancreatitis. Can J Surg 2016; 59: 128–140.

39.

Batistel

Osorio

Tariq

, et al. Peripheral leukocyte and endometrium molecular biomarkers of inflammation and oxidative stress are altered in peripartal dairy cows supplemented with Zn, Mn, and Cu from amino acid complexes and Co from Co glucoheptonate. J Anim Sci Biotechnol 2017; 8: 33.

40.

Ercan

Ilbar Tartar

Solmaz

, et al. Examination of protective and therapeutic effects of ruscogenin on cerulein-induced experimental acute pancreatitis in rats. Ann Surg Treat Res 2019; 97: 271–281.

41.

Qiu

Huang

. Protective effect of low-molecular-weight heparin on pancreatic encephalopathy in severe acute pancreatic rats. Inflamm Res 2012; 61: 1203–1209.

42.

Qiu

Huang

. Effect of low molecular weight heparin on pancreatic micro-circulation in severe acute pancreatitis in a rodent model. Chin Med J 2007; 120: 2260–2263.

43.

Ceranowicz

Dembinski

Warzecha

, et al. Protective and therapeutic effect of heparin in acute pancreatitis. J Physiol Pharmacol 2008; 59(Suppl 4): 103–125.

44.

Warzecha

Dembinski

Ceranowicz

, et al. Heparin inhibits protective effect of ischemic preconditioning in ischemia/reperfusion-induced acute pancreatitis. J Physiol Pharmacol 2012; 63: 355–365.

45.

Wang

Zhao

Hong

, et al. Serum thyroid hormones levels are significantly decreased in pregnant rats with acute pancreatitis. Biochem Biophys Res Commun 2018; 505: 657–663.