NQDI-1 protects against acinar cell necrosis in three experimental mouse models of acute pancreatitis

Abstract

NQDI-1, an inhibitor of ASK1, has been reported to have protective effects in several experimental human disease models. However, the role of NQDI-1 in acute pancreatitis (AP) has not been reported. In this study, we found that NQDI-1 could attenuate histological damage of pancreatic tissue as well as the levels of serum amylase and lipase in a mouse model of AP induced by caerulein. Moreover, the pro- duction of reactive oxygen species (ROS) and the expression of necrosis-related proteins (RIP3 and p- MLKL) were also reduced after NQDI-1 administration. Correspondingly, we elucidated the effect of NQDI-1 in vitro and found that NQDI-1 protected against pancreatic acinar cells necrosis via decreasing the ROS production and RIP3 and p-MLKL expression. In addition, we identified the protective effect of NQDI-1 on AP through two other mouse models induced by L-arginine and pancreatic duct ligation. Taken together, these findings showed that NQDI-1 could reduce the acinar cells necrosis and alleviate the severity of AP, which may afford a new therapeutic target on pancreatic necrosis in AP clinically.

1. Introduction

Acute pancreatitis (AP) is an inflammatory disease caused by premature activation of the zymogen, which leads to self-digestion of the pancreas [1]. Most cases of AP are mild and generally require only conservative treatment. Whereas, several patients may develop into severe acute pancreatitis (SAP) with necrosis of the local or surrounding tissues of the pancreas, and distant organ dysfunction, such as acute respiratory distress syndrome (ARDS),
acute kidney injury (AKI), and shock [2,3]. The mortality of SAP can be as high as 40%e70% [4]. Current researches suggest that necrosis of pancreatic acinar cells is one of the important mechanisms for the development of AP [5e7], and the necrosis of pancreatic acinar cells is closely related to the severity and prognosis of AP [8]. However, in clinical practice, there are no clear drugs to attenuate necrosis of AP.

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the stress-induced mitogen-activated protein kinase family and can be activated by a variety of stressors such as oxidative stress, endo- plasmic reticulum stress, calcium influx, inflammatory factors, and lipopolysaccharide (LPS) [9]. Activation of ASK1 could regulate cell death, cytokine secretion, immune response and cell differentiation [10e13]. A large number of studies have shown that ASK1 is involved in many human diseases, including cardiovascular dis- eases, nervous system diseases, infections, diabetes and cancer
[9,14,15]. Studies have shown that in the sodium taurocholate- induced rat AP model, the expression of ASK1 is significantly higher than that in normal group [16,17], indicating that ASK1 may also have an important regulatory role in AP. NQDI-1 is a novel selective inhibitor of ASK1, first identified by Volynets et al. [18]. Until now, researchers have found that NQDI-1 treatment has protective effect on renal function [19] and brain damage in animal models of ischemia-reperfusion injury [20,21]. Wu et al. found that the administration of emodin and curcumin could reduce the severity of SAP and decrease the release of IL-6 and TNFa in rat by down-regulating ASK1 [16,17]. However, whether NQDI-1 has protective effect on AP has not been reported. In this study, we investigated its effects in vivo and in vitro experiments.

2. Materials and methods

2.1. Animals

Male ICR mice weighting 25e30g were purchased from the QingLongshan Animal Breeding Field, Jiangning Nanjing. Before experiment, all mice were housed in specific pathogen-free (SPF) facilities, fed with standard rodent chow and water, kept in a controlled temperature (25 ◦C ± 2 ◦C), and maintained under a cycle lighting (12h light/12 h dark). The Principles of Laboratory An- imal Care (NIH publication no.85Y23, revised 1996) was followed, and all experimental protocols were approved by the experimental animal ethics committee of Jinling Hospital.

2.2. Induction of acute pancreatitis in mice

Three AP animal models were used to verify the protective effect of NQDI-1. First, a mild AP model was induced by intraperitoneal (i.p.) injection of caerulein (50 mg/kg, interval of 1 h, 10 times) (NJPeptide, Inc., Nanjing, China) [22]. The second model was an L- arginine-induced mouse SAP model [23]. Mice were induced by i. p injection of 8% L-arginine (L-Arg) (3.3 g/kg, 3 times, 1h interval) (Sigma Aldrich, St. Louis, MO, USA). The third model was induced by pancreatic duct ligation (PDL). Detailed methods are as follows: After intraperitoneal injection of sodium pentobarbital (50 mg/kg), 1e2 cm longitudinal incisions were made in the abdomen of the mice to expose the abdominal cavity and the duodenum was turned over to expose its distal side. At 1 cm above the duodenal papilla blunt dissection the tissue around the pancreatic duct and ligated with a silk thread. Complete obstruction of the pancreatic duct to simulate a model of biliary pancreatitis caused by cholelithiasis. After ligation, the abdominal cavity of mice was layered and closed. All the mice were placed on a 37 ◦C constant temperature heating table for 90 min [24].

2.3. Drug administration and sample collection

In the three AP mouse models, we administrated NQDI-1 after the model was prepared in order to observe the therapeutic effect on AP. In the Caerulein induced model, NQDI-1 (20 mg/kg) (MCE, Inc., Newjersey, USA) was administered 1h after the first injection of caerulein intraperitoneally. Mice were sacrificed at 12h after the first injection of caerulein. In the L-Arg induced model, NQDI-1 (20 mg/kg) was injected intraperitoneally at 1h, 24h and 48h af- ter the first injection of L-Arg. All the mice were sacrificed at 72h after the first administration of L-Arg. In the PDL induced model, NQDI-1 (20 mg/kg) was injected intraperitoneally at 1h and 24h after the operation. Mice were all sacrificed at 48h after pancreatic duct ligation.All animals were anesthetized with intraperitoneal administration of sodium pentobarbital (50 mg/kg) before sacrificed.Orbital venous blood samples were obtained for amylase and lipase measurements. Pancreatic and pulmonary tissues were harvested immediately and a part of the tissues were fixed for histological analysis, and the remaining tissues were stored at —80 ◦C for further investigations.

2.4. Histological examination

The pancreatic and pulmonary tissues were fixed with 4% paraformaldehyde, sliced by paraffin-embedded microtome. Paraffin tissue was treated with dewaxing, dehydration and stained with hematoxylin and eosin (H&E), followed by histological ex- amination. The histopathological scoring analysis of the pancreas and lung were evaluated by two independent pathologists with unknown group information according to previously described methods [25,26].

2.5. Serum amylase and lipase assay

Orbital venous blood samples were obtained for amylase (Bio- Sino Bio－Technology & Science Inc., Beijing, China) and lipase (Nanjing Jiancheng Corp., Nanjing, China) measurements according to the manufacturer’s instructions.

2.6. Immunofluorescence (IF)

ROS fluorescent probe-Dihydroethidium (DHE, Wuhan Serv- icebio Technology Co. Ltd., Wuhan, China) was employed to detect the content of ROS in pancreatic tissue. The fresh tissues of the pancreas were embedded in optimal cutting temperature (OCT) compound, and samples were cut into 7 mm sections. Tissues were incubated in the dark with DHE solution for 30 min at 37 ◦C. Slides were placed in the PBS (pH ¼ 7.4) and sloshed 3 times, each time for 5 min. Then tissues were incubated by 4’,6-Diamidino-2- Phenylindole, Dihydrochloride (DAPI, Wuhan Servicebio Technol- ogy Co. Ltd., Wuhan, China) solution at room temperature for 10 min and sloshed again. Finally, the slides were observed under the fluorescence microscope.

2.7. Immunohistochemistry (IHC)

In brief, for immunohistochemical staining of receptor- interacting protein 3 (RIP3) and phospho-mixed lineage kinase domain like pseudokinase (p-MLKL), the tissue slices were boiled in EDTA antigen repairing buffer. After natural cooling, slices were incubated to block endogenous peroxidase activity with 3% hydrogen peroxide solution at room temperature for 15 min. Slices were incubated overnight at 4 ◦C in a humid chamber with the
antibody against RIP3 (1:200 dilution, Santa Cruz, CA, USA) and p- MLKL (1:500 dilution, Abcam, Cambridge, UK). Then the tissue slices were incubated by biotinylated secondary antibody (1:200 dilution) for 1h. Finally, slices were counterstained with hematoxylin.

2.8. Cell culture and cell viability assay

The mouse pancreatic acinar 266-6 cell line was purchased from ATCC (Manassas, VA, USA) and cultured in DMEM medium sup- plemented with 10% FBS(Gibco, ThermoFisher Scientific), 100U/ml penicillin and 100 mg/ml streptomycin at 37 ◦C in a humidified at- mosphere of 5% CO2 in air. To induce pancreatitis in vitro, the 266- 6 cells were stimulated with 5 mM Cholecystokinin octapeptide (CCK, MCE, Inc., New jersey, USA). Six hours later, cells were collected for the following Western blot assay, DCFH-DA staining and flow cytometry analysis. Cell-Counting Kit-8 (CCK8) and cytotoxicity LDH assay kit (Dojindo Molecular Technologies, Inc., Tokyo, Japan) were used to assay cell viability. All procedures were done in accordance with the manufacturer’s instructions.

2.9. Measurement of ROS generation in 266-6 cells

266-6 cells were seeded in 24-well plates overnight followed by treatment with CCK and NQDI-1 (1, 5, 10 mM) for 6h The cells were incubated with 2′,7′-Dichlorofluorescin diacetate (DCFH-DA, Sigma Aldrich, St. Louis, MO, USA) in the dark at 37 ◦C for 30 min. Cells were washed twice with PBS and detached with trypsin/EDTA. Cellular fluorescence was analyzed by flow cytometry.

2.11. Protein extraction and western blotting

The proteins were extracted in RIPA (Beyotime Biotechnology, Beijing, China) containing a cocktail of protease inhibitors (1:100 dilution, Roche, Shanghai, China) and 1%PMSF (1:1000 dilution, Beyotime Biotechnology, Beijing, China). The protein concentra- tions were measured using a BCA kit (Thermo Fisher Scientific, MA, USA) and subjected to 10% SDS-polyacrylamide gelelectrophoresis (PAGE), and the proteins were transferred to a PVDF membrane,blocked with 5% skim milk at room temperature for 2 h, and then incubated overnight at 4 ◦C with primary antibodies against RIP3
(1:1000 dilution), p-MLKL (1:1000 dilution) and GAPDH (1:2000 dilution, Santa Cruz, CA, USA) in blocking buffer. Membranes were washed with TBST (3*10 min) the next day and incubated with a secondary goat anti-mouse or goat anti-rabbit IgG horseradish peroxidase (HRP) antibody (1:10000 dilution, Abcam, Cambridge, UK) diluted in 5% (w/v) dry nonfat milk in TBST for 1 h at room temperature. Finally, membranes were washed with TBST (3*10 min), developed using the ECL detection system (Santa Cruz Biotechnology), quickly dried, and exposed to ECL film. Image in- tensity was analyzed with ImageJ software.

2.12. Statistical analysis

Statistical analysis was performed by GraphPad Prism 6 soft- ware (GraphPad, San Diego, CA, USA), and data were presented as the mean ± standard deviation (SD). The data represent the mean of at least three independent experiments. Statistical difference between the two groups was analyzed by the t-test, and the one- way ANOVA test was used to evaluate the difference more than two groups. P < 0.05 was considered statistically significant (two- tailed).

3. Results

3.1. NQDI-1 alleviated the severity of AP induced by caerulein in mice

To investigate the effects of NQDI-1 administration on AP induced by caerulein we evaluated the histopathological damage of the pancreas and the levels of serum amylase and lipase. As shown in Fig. 1AeE, the pancreatic tissue morphology of the control group was approximately normal. Caerulein stimulation could induce acute pancreatic injury in mice, which was characterized by edema, inflammatory cells infiltration and acinar cells necrosis. After NQDI- 1 administration, the histological damage of pancreatic tissue was significantly alleviated, with reduced edema, decreased infiltration of inflammatory cell and less necrosis of pancreatic acinar cells. In addition, the levels of serum amylase and lipase were also signifi- cantly decreased in Cae + NQDI-1 group (Fig. 1F and G).

3.2. NQDI-1 reduced the production of ROS and alleviated necrosis in pancrea of mice with caerulein induced-AP

The accumulation of ROS and its mediated necrosis pathway has been proposed to play a critical role in the necrosis of pancreatic acinar cells [27]. To clarify the potential mechanism of NQDI-1 protecting AP, we further examined the production of ROS and the expression levels of necrosis-related proteins (RIP3 and p- MLKL) in the pancreatic tissues. As shown in Fig. 2A and B, DHE staining illustrated a remarkable increase in ROS after AP onset, while NQDI-1 treatment could reduce the level of ROS significantly. Furthermore, immunohistochemistry staining was used to detect the expressions of RIP3 and p-MLKL in pancreatic tissues. We observed that the expressions of RIP3 and p-MLKL in pancreatic acinar cells were prominently increased after AP onset and decreased with NQDI-1 administration (Fig. 2CeF), which indicated that NQDI-1 could attenuate AP through necrosis pathway.

3.3. NQDI-1 protected PACs and decreased the ROS generated by 266-6 cells

In order to verify the role of NQDI-1 on acinar cell injury in vitro, we first observed the damage of 266-6 cells, a pancreatic exocrine cell line, stimulated by CCK at different times. The degree of cell death was found to be time-dependent by the cell proliferation and toxicity analysis (Fig. 3A and B). Accordingly, 6h CCK-stimulated cells injury model was used to assess the in vitro protection of the drug. After administered different doses of NQDI-1 (1, 5, 10 mM), we found that 5 mM of NQDI-1 was the most protective (Fig. 3E and F). Similarly, we detected the production of ROS from 266-6 cells after AP induction, which was also time-dependent (Fig. 3C and D). As shown in Fig. 3G and H, 5 mM NQDI-1 could significantly sup- press the production of ROS in pancreatic acinar cells. Additionally, by western blot detected, we found that after cell injury the ex- pressions of RIP3 and p-MLKL protein increased pronouncedly, but it could be reduced by NQDI-1 (Fig. 3IeK).

3.4. NQDI-1 alleviated the histopathological damage of the pancreas and lungs in AP mice induced by L-Arg and PDL

To further explore the protective effect of NQDI-1 on acinar cell necrosis and pancreatic inflammatory injury, other two classic mouse AP models, induced by L-Arg and pancreatic duct ligation were employed respectively. In these two SAP models, marked acinar cell necrosis and severe pancreatic tissue damage were observed. As respect, HE staining of the pancreas suggested that NQDI-1 could alleviate the pathological damage of the pancreas in two SAP mouse models (Fig. 4A, C, 4G, 4I), which was consistent with the results of caerulein induced mild AP model. Similarly, serum amylase and lipase were also reduced after NQDI-1 admin- istration (Fig. 4E, F, 4K, 4L). In clinical practice, SAP can be complicated by multiple distant organ injuries, among which acute lung injury is the most common one. Therefore, we also performed histological analysis of pulmonary tissues. The results showed that NQDI-1 also improved pulmonary tissue damage after AP, demonstrated as less inflammatory cells infiltration, decreased alveolar thickness and reduced alveolar congestion (Fig. 4B, D, 4H, 4J). All the results indicated that NQDI-1 not only protected the pancreas, but also protected the distant organs through a systemic mechanism.

4. Discussion

In this present study, we demonstrated that NQDI-1, an inhibitor of ASK1, could alleviate acinar cell necrosis and ameliorate the severity of AP in mice probably by inhibiting ROS aggregation and down-regulating the necrosis-related proteins RIP3 and p-MLKL.Pancreatic acinar cell necrosis is associated with the infection of pancreatic tissue and systemic inflammatory response, affecting the progression and prognosis of AP [8]. Therefore, searching for effective targets and therapeutic drugs to reduce acinar cells ne- crosis has always been the focus of AP basic research. As a member of the stress-activated mitogen-activated protein kinase, many definitive studies showed that ASK1 may be a good target for reducing cell necrosis. Notably, novel ASK1 inhibitors have shown promise in Phase 2 clinical trials for nonalcoholic steatohepatitis, pulmonary arterial hypertension and diabetic kidney disease [28,29]. In this study, we clearly observed the protective effect of NQDI-1 on AP through three models of AP in mice, which was re- flected in the decrease of serum enzymes, the reduction of pancreatic tissue damage (including acinar cell necrosis), and the relief of damage from distant organs. All these results suggested the therapeutic potential of NQDI-1 for AP.

RIP3 and p-MLKL are two key proteins involved in necrosis [30e32]. Studies have shown that RIP3 or MLKL knockout mice have reduced necrosis of pancreatic tissue in caerulein induced AP model compared to wild type mice [6,33,34]. Hence, the expression levels of RIP3 and p-MLKL are often used to assess the extent of acinar cell necrosis. Studies have reported that mitochondria- derived ROS can autophosphorize RIP3, which in turn phosphory- lates MLKL to form RIP3-MLKL-containing necrosis death complex (necrosome). Then necrosome can target membranes and disrupt membrane integrity, leading to cell necrosis [30,31]. In addition, many studies have shown that the production of ROS is a very important regulatory factor in the pathogenesis of pancreatic acinar cells necrosis [7,27,35]. There are data showing that ROS may aggravate acinar cell damage, causing lesions to the cells mem- branes and cytoskeleton, impairing the functions of intracellular proteins, destroying DNA, inducing lipid peroxidation, down- regulating the levels of antioxidants [35]. In this study, we found that ROS production, RIP3 and p-MLKL protein expression were increased significantly after acinar cell injury both in vitro and in vivo experiments, and these phenomena could be effectively corrected by NQDI-1, suggesting that NQDI-1 had a good protective effect on pancreatic necrosis.

5. Conclusion

Overall, our study firstly demonstrates that the protective effect of NQDI-1 on acinar cell necrosis and pancreatic inflammatory injury. In clinic application, NQDI-1 maybe a promising therapeutic treatment for AP.