The Synthetic Triterpenoid RTA dh404 (CDDO-dhTFEA) Ameliorates Acute Pancreatitis

Abstract

Objectives

Nuclear factor-erythroid-2-related factor (Nrf2) is a ubiquitous transcriptional factor that regulates expression of cellular antioxidant and detoxifying molecules. This study was undertaken to test the hypothesis that administration of Nrf2 activator (dh404) may attenuate acute pancreatitis.

Methods

Rats were treated with dh404 (1 mg/kg) 24 hours prior to induction of pancreatitis, and for 3 days thereafter. Pancreatitis was induced with L-arginine (600mg/100g) or Cerulein (40µg/kg). Pancreases were processed for histology and Malondialdehyde (MDA), while serum was analyzed for amylase. Islet extracted human pancreatic tissue from organ donors were used for in vitro studies. The tissues were incubated with dh404 at 0, 250, 500 nM for 30, 60 min, 12 and 24 hrs. Nrf2 nuclear translocation, expression of Nrf2’s target genes and inflammatory mediators were determined.

Results

The dh404-treated rat pancreases demonstrated significantly less infiltration of inflammatory cells, destruction of acinar architecture, perilobar edema, and necrosis. Serum amylase and pancreatic MDA in the dh404-treated rats were significantly lower. Dh404-treated human pancreatic tissue showed a significantly higher expression of antioxidants enzymes, lower expression of inflammatory mediators and greater viability against oxidative stress.

Conclusion

Administration of dh404 attenuates acute pancreatitis by lowering oxidative stress and reducing pro-inflammatory mediators.

Introduction

Acute pancreatitis (AP) is a complex inflammatory disorder characterized by tissue edema, acinar cell necrosis, hemorrhage, and fat vacuolization ¹. Although advances in critical care have substantially improved mortality, the incidence of AP and the related hospitalization costs continues to rise, and the associated morbidity remains high ². In its severe form, morbidity and mortality ranges from 15% to 40% ³. Presently, therapy is aimed at supportive care with analgesics and intravenous fluid replacement. Few countries, such as Germany, have incorporated anti-oxidant agents in the pancreatitis treatment regimen^4,5. However, the effect of anti-oxidant and anti-inflammatory agents has been thoroughly investigated in experimental models of pancreatitis^6–11.

The pathophysiology of AP has been extensively studied, however, the exact pathogenesis remains poorly understood. It is clear that AP is an inflammatory disorder, with the recruitment of leukocytes and inflammatory cytokines playing an important role in the severity of the disease^12,13. Oxidative stress and inflammation are inseparably interconnected. By attacking and denaturing functional and structural molecules by activating redox sensitive transcription factors (e.g. NF-κB) and signal transduction pathways, reactive oxygen species (ROS) cause cytotoxicity, tissue damage, and trigger inflammation. Although, oxidative stress does not seem to be the primary trigger for the onset of pancreatitis it plays a major role in the pathogenesis of AP by causing tissue damage and promoting the recruitment of inflammatory mediators ^14,15. Given the critical role of oxidative stress in the pathogenesis and progression of many disorders, considerable attention is currently focused on the potential efficacy of antioxidant or anti-inflammatory mediators.

Under normal condition, disruption of the redox homeostasis by endogenous or exogenous oxidants, triggers upregulation of endogenous cytoprotective proteins, antioxidants, and enzymes to prevent/limit tissue injury and dysfunction. This process is mediated by activation of the Nuclear factor-erythroid-2-related factor 2 (Nrf2), which regulates the activity and induction of various genes that encode numerous phase II detoxifying and antioxidant enzymes, and related proteins¹⁶. Regulation of cellular antioxidant and anti-inflammatory machinery by Nrf2 plays a central part in defense against oxidative stress. Under normal “conditions”, most of the Nrf2 produced in the cell is held and driven to proteosomal degradation by the repressor molecule Keap1. In the presence of oxidative stress, or a Nrf2 inducer, covalent modifications of cysteine residues in the Keap1 molecule disable its ability to bind Nrf2, thereby promoting Nrf2 translocation to the nucleus and transcriptional upregulation of its target genes^16–19. Sulfur-containing and phenolic compounds are two classes of dietary compounds that can act as Nrf2-keap1 pathway activators. Natural Nrf2 activators have been used for centuries as Chinese herbal medicines in the treatment of a number of inflammatory conditions ²⁰. The possibility that Nrf2 activators can provide protection against AP has not been examined clinically.

The aim of the present study was to test the hypothesis that treatment with a potent, synthetic Nrf2 activator (dh404) may attenuate severity of acute pancreatitis in experimental animals through regulation of cellular antioxidant and anti-inflammatory machinery in pancreatic acinar tissues. To this end the effect of dh404 was determined in rats with AP induced by two standard pancreatitis induction agents (L-arginine, Cerulein).

Materials and Methods

In vivo Acute Pancreatitis

In vivo studies were performed in accordance with the Institutional Animal Care and Use Committee of University of California, Irvine. (Irvine, CA). Experimental agents used to produce AP were purchased from Sigma (St. Louis, MO) unless otherwise specified. Sprague Daley rats (200–300g) were purchased from Charles River (Wilmington, MA) and fed ad libitum on a standard diet with free access to water. They were maintained on a 12h light/dark cycle. RTA dh404 was provided by Reata Pharmaceuticals, Inc. (Irving, TX). The chemical name for RTA dh404 is CDDO-9,11-dihydro-trifluoroethyl amide (CDDO-dhTFEA) ²¹.

The optimal dose of dh404 in AP rodent model was not known. We conducted a series of preliminary experiments to find the optimal dose of dh404. An optimal dose of 1mg/kg dose was used in this study.

Experimental animals were given oral dh404 (1 mg/kg) dissolved in Sesame oil and administered via oral gavage 24 hours prior to initiating AP and daily thereafter until the animals were sacrificed. Control animals were gavaged with sesame oil alone.

L-arginine

20% L-arginine was dissolved in normal saline and filtered through a syringe filter with pH adjusted to 7.0. The solution was administered to non-fasted rats in two intra-peritoneal injections at a dose of 300mg/100g body weight, each injection separated by 1 hour ²². Animals received pain medications and regular food and water. Experimental animals continued to receive daily dH404 via gavage along with a standard diet. The rats were sacrificed at 24 or 72 hours after induction^23,24.

Cerulein

Cerulein is administered to non-fasted rats at a dose of 40µg/kg body weight, as 4 separate hourly subcutaneous injections, each injection containing 25% of the dose. All of the animals were sacrificed at 2 hours after the last injection²⁵.

Histology

Rat pancreases were fixed in 10% buffered formalin and embedded in paraffin blocks. The pancreas tissue was processed for hematoxylin-eosin (H&E) staining using standard techniques. Two pathologists blinded to the experiment evaluated 20 pancreatic slides (5 Cerulein, 5 Cerulein+dh404, 6 L-arginine, 6 L-arginine+dh404) and provided a score. AP was scored using a quantitative grading system as described by Schmidt et al ²⁶. Classification (Table 1) was based on the presence of edema, leukocyte infiltration, acinar cell necrosis, and hemorrhage.

Table 1.

Quantitative grading score for acute pancreatitis

Score	0	1	2	3
Interstitial edema	none	Interlobular	Lobule involved	Isolated island like acinar cells
Leukocyte infiltration	none	<20%	20–50%	>50%
Acinar cell necrosis	none	<5%	5–20%	>20%
Hemorrhage	none	1–2 points	3–5 points	>20%

Immunohistochemistry for cleaved caspase-3

Cleaved caspase-3 was used for histological identification of acinar cell apoptosis. The detection system was DAKO LSAB2 system-HRP kit using the manufactures protocol (DAKO, K0675, Carpinteria, CA). Cleaved caspase-3 positive acinar cells (%) were quantified on 400x magnification by two pathologists. Cleaved caspase-3 index was defined as the proportion of caspase positive cells per high powered field in a minimum of 3 high powered fields. The results corresponded to the average number of positive acinar cells per field^23,27.

Serum Amylase

Blood was obtained from anesthetized rats via cardiac puncture just prior to euthanasia. Amylase level was determined to indicate the severity of pancreatitis using the Phadebas Amylase test (Magle AB, Lund, Sweden) ²³.

Malondialdehyde (MDA) content

MDA was measured by thiobarbituric acid colorimetric method using MDA assay kit (Cayman Chemical Company, Ann Arbor, MI). The dose of H₂O₂ were based on previously published works^23,24,28.

Inflammatory mediator production from rodent splenocytes

Spleens from normal anesthetized adult rats were harvest and cut into tiny morsels with a standard 15 blade scalpel. The spleens were then placed in an Erythrocyte lyses buffer (1.5 M NH₄CL, 100mM KHCO₃, 100 mM EDTA-2Na adjusted to a ph of 7.2) ²⁹. Whole splenocytes (1×10⁶ /ml), treated with endotoxin lipopolysaccharide 1µg/ml (LPS, Sigma), were cultured for 24 hours with or without dh404 500nM. Dh404 was dissolved in vehicle Dimethyl Sulphoxide Hybri-MAX (DMSO) prior to use. The supernatant was then placed on a standard rat cytokine kit (Ray Biotech, Norcross, GA). The cytokines IFN-γ, IL-1α, IL-1β, IL-4, IL-6, IL-10, and TNF-α were analyzed as well as growth factors, chemokines, and inflammatory mediators: Agrin, CD86, CINC-1, Fas-Ligand, Fractalkine, GM-CSF, ICAM-1, Leptin, and L-selectin using the manufacture protocol. Analysis was carried out using ImageQuant TL 7.0 (GE healthcare Life Sciences, Pittsburg, PA)^23,30.

In vitro human pancreatic tissue studies

In vitro studies were performed on human pancreatic tissue obtained from NIH/JDRF sponsored integrated islet distribution program or from collaborators. They were obtained after removal of islet cells. They were treated with dh404 at preconditioned concentrations dissolved in DMSO (Sigma, St. Louis, MO) and cultured in Roswell Park Memorial Institute (RPMI-1640) solution containing additionally 10% Newborn calf Serum and Penicillin 100U/ml/Streptomycin 50 µg/ml (Corning, Manassas, VA).

Nrf2 translocation in human non-endocrine cells

Translocation of Nrf2 into the nucleus of human pancreatic acini was confirmed with immunohistochemistry. Dh404 was dissolved in vehicle (DMSO) prior to use. Human pancreatic tissue was treated with dh404 500nM for 30 or 60 minutes. After washing the remaining pancreatic pellet was placed in 10% formalin and transferred into an embedding medium for frozen section specimens (OCT, Tissue Tek, Sakura, Torrance, CA) and cut into 5 µm sections onto slides. The treated and untreated samples were stained with Nrf2 monoclonal antibody (1:100) (ABcam, Cambridge, MA). For acinar cell specific Nrf2 translocation, the above protocol was modified by adding a double stain, antihuman amylase polyclonal antibody originated from rabbit (1:400) (Abcam Cambridge, MA). Samples were then incubated with goat anti-mouse (Alexa Fluor 488 goat anti-mouse IgG, 1:200 dilution) and goat anti-rabbit (Alexa Fluor 568 goat anti-rabbit IgG, 1:200 dilution) antibodies (Cell Signaling, Danvers, MA). 4′,6-diamidino-2-phenylindole (DAPI) was applied to stain cell nuclei. Images were taken using confocal microscopy (Zeiss LSM510, Jena, Germany) ³¹.

Protein Extraction and Western Blot

Human tissue was cultured with or without dh404. After a culture period of 0 or 30 minutes the tissue was homogenized (Power Gen 125, Fischer Scientific, Pittsburg, PA) with Lysis Buffer (RIPA, Thermo Scientific, Piscataway, NJ). The cytoplasm was removed and stored and the nuclear pellet was lysed again with RIPA buffer and sonicated. Nuclear protein aliquots (45 µg) were mixed with sample buffer (Bio-Rad laboratories, Richmond, CA) boiled for 5 minutes and separated via 4–12% BIS TRIS (2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol) (Life Technologies, Carlsbad, CA). After transfer to nitrocellulose membranes (Biorad) the membranes were blocked with PBS/0.1%Tween containing 2% dry milk for 90 minutes at room temperature. The membranes were then probed with mouse monoclonal antibody to Nrf2 (1:500) (Santa Cruz Biotech, Dallas, TX) followed by rabbit anti-mouse IgG (1:3000) (Cell Signaling, Danvers, MA).

Human pancreatic tissue cultured for 24 hours with or without dh404 was processed similarly to above. Membranes were probed with mouse monoclonal antibody to heme oxygenase (HO-1) (1:1000), super oxide dismutase (SOD) (1:5000), Catalase (1:1000), and glutamate-cysteine ligase-catalytic subunit (GCLC) (1:1000) (Cell Signaling, Danvers, MA) followed by rabbit anti-mouse IgG (1:3000). The membranes were developed with an enhanced chemoluminescence detection kit (Bio-Rad) and exposed to X-ray film (Kodak, Rochester, NY). To evaluate the results of the western blot, the relative intensities of individual bands were determined with ImageQuant TL 7.0 (GE healthcare Life Sciences, Pittsburg, PA).

Human pancreatic cell viability assay

Human pancreatic tissue was treated with or without dh404 (0–500nM) for 12 hours followed by 6 hour incubation with Hydrogen Peroxide (200 µM, Hydrox Laboratories, Elgin, IL). Human non-endocrine pancreatic cell viability was determined with previously reported methods^31,32.

Briefly, pancreatic tissue was washed with PBS and the remaining pellet (50 ul) aliquots were suspended in 1 ml prewarmed (37°C) Accutase (Innovative Cell Technologies, San Diego, CA). Digestion was stopped with 1 mL cold newborn calf serum (NCS, HyClone labs, Logan, UT). Washed Cells were transferred into filtered FACS tubes (BD Falcon, Franklin Lakes, NJ) to remove undigested tissue. Cells were resuspended in PBS and stained with tetramethylrhodamine ethyl ester perchlorate (TMRE, Life technologies for 30 minutes at 37°C. This was followed by 7-Aminoactinomycin D (7-AAD, Life technologies) stain on ice immediately prior to the FACS analysis. TMRE stains mitochondrial membrane potential and 7-AAD stains DNA binding. The percentage of 7-AAD⁺ cells was recorded for dead cells, and further analysis for TMRE⁺ (viable cells) and TMRE⁻ (apoptotic cells) was performed after their exclusion (gating out). 50,000 cells from each sample were assessed by flow cytometry (Accuri C6 cytometers, Ann Arbor, MI, BD Bioscience). Software (Accuri C6 software, San Jose, CA) software was used for analysis.

Cytokine and chemokine production from human pancreatic tissue

Human pancreatic tissue was cultured with or without dh404 for 24 hours and supernatant was collected for cytokine and chemokines assay. Analysis was performed using a human cytokine antibody array kit (Ray Biotech, Norcross, GA) using the manufacture protocol. ImageQuant TL 7.0 (GE healthcare Life Sciences, Pittsburg, PA) was used for analysis³⁰. Analysis of cytokines from dh404 treated acini were compared to the non-treated tissue and expressed as a percent of the control.

Statistical Analysis

Student's t test or one-way analysis of variance (ANOVA) was used in statistical analysis of the data using Excel for Windows software (Microsoft, Redmond, WA). P values equal to or less than 0.05 were considered significant. Data are expressed as mean ± SD.

Results

Treatment with oral dh404 significantly attenuated pancreatitis

Administration of both L-arginine and Cerulein in rats caused severe acute pancreatitis. Treatment with oral dh404 in the experimental pancreatitis induced by L-arginine resulted in significant (p <0.001) reductions in infiltrating inflammatory cells, acinar architectural damage, edema, and necrosis (Figure 1A, Figure 1C, and Table 2). Moreover, dh404 significantly reduced the damage in Cerulein induced acute pancreatitis (Figure 1B, Figure 1C, and Table 2, p <0.05). Although, L-arginine has a mechanism of action that remains unclear there is evidence to support that L-arginine exerts its effects by producing oxidative stress and accumulation of nitric oxide in vivo (21, 22). Dh404 was extremely effective in protecting the treated rats against L-arginine induced AP – likely from the reduction in oxidative stress. Cerulein is a CCK analogue that promotes the intrapancreatic secretion of enzymes resulting in an edematous pancreatitis in experimental animals (47). Dh404 was effective in protecting the pancreas against damage from Cerulein however not to the extent of its protection against the L-arginine model. This is believed to be due to dh404’s inability to prevent the Cerulein from increasing pancreatic secretions. However, the dh404 likely had a role in blocking a secondary response to the increased pancreatic secretions. As was seen graphically in figure 1C a reduced level of inflammatory mediators and acinar destruction were seen in the dh404 treated animals however the level of edema was identical.

Figure 1.

A: L-arginine induced acute pancreatitis in a rodent model. Photomicrograph of an H&E stained section of pancreas after 72 hours of induction. Dh404 significantly reduced infiltration of inflammatory mediators within the acinar cells, destruction of normal acinar architecture, perilobular edema, necrosis of cells and surrounding fat in a rodent model of pancreatitis induced by L-arginine.

B: Cerulein induced acute pancreatitis in a rodent model. Photomicrograph of an H&E stained section of pancreas after 6 hours of induction. Dh404 significantly reduced the infiltration of acute inflammatory mediators within the acinar cells, and the presence of perilobar and intralobular edema.

C: Quantitative pancreatitis Score: interstitial edema, leukocyte infiltration, acinar cell destruction, and average in total pancreatitis score. Histological pancreas slides were evaluated by two blinded pathologists and given a score based on severity from 0–3 based on three criteria: Edema, infiltration, and acinar cell destruction. The total score was also calculated. Based on quantitative scores for all three criteria there was a significantly higher score for L-arginine compared to L-arginine + dh404. Likewise, the score was significantly higher for Cerulein compared to Cerulein+ dh404. There was very little variation between the histology slides scored (see Table 2). Standard deviation was low or non-existent.

Table 2.

L-arginine or Cerulein induced acute pancreatitis

Groups (n=5–6)	Intersitial Edema	Leukocyte infiltration	Acinar cell Destruction	hemorrhage	Total	P value
Control	0	0	0	0	0
L-arginine	3± 0	4±0	4±0	0	11	P<0.001
L-arginine + dh404	1±1	0	0	0	1±1
Cerulein	2±0	3±0	1±1	0	6±1	P<0.05
Cerulein + dh404	2±0	2±0	0	0	4

During our preliminary experiments to determine the best effective dose of dh404, we found significant worsening of the lesion in animals treated at doses between 5–20 mg/kg but significant improvement at 0.5–1 mg/kg. Therefore we selected 1mg/kg dose in this study. The adverse effects seen in the small experimental group (5 rats) that were treated with the supra-maximal doses included an increase in acute kidney injury seen on histology post necropsy and a slight but not statistically significant increase in mortality. Due to the small sample size of animals treated with this dose further analysis was not performed. The mechanism for adverse effect of higher doses of dh404 is currently unclear and requires further investigation.

Treatment with oral dh404 significantly reduced the concentration of cleaved caspase-3 positive cells

Cleaved caspase-3 was used to evaluate apoptotic cells in the pancreas. Compared to the control group, dh404 treated rats showed significantly lower cleaved caspase-3 positive cells in pancreatic sections (p <0.05) (Figure 2). The data indicated that the prevention of pancreatic acinar cells apoptosis contributed to better pancreatitis scores in the dh404 treated rats.

Figure 2. A: Cleaved caspase-3 IHC in L-arginine induced acute pancreatitis.

Representative photomicrograph of pancreas histology stained with cleaved caspase-3 antibody (arrow). In dh404treated rat pancreas, Cleaved caspase-3 staining revealed significantly lower apoptotic cells when compared with the L-arginine group. The slides were reviewed by two blinded pathologists who score the slides based on standardized criteria. Data are representative of three independent experiments.

Treatment with oral dh404 significantly decreased serum amylase and pancreatic MDA content

Although, a number of other conditions can result in elevated amylase levels, in patients with clinical AP, the sensitivity and specificity of serum amylase is over 90%. Administration of oral dh404 significantly lowered the serum amylase level. A lower amylase level in the dh404-treated rats is consistent with the observed histological improvement in rats with L-arginine induced pancreatitis.

MDA is an indicator of lipid peroxidation and cellular damage under oxidative stress. Dh404 treated rats showed a lower pancreatic MDA level after 24 hours of L-arginine induced pancreatitis (p<0.05) (Figure 3). Likewise the lower MDA level is indicative of the efficacy of dh404 in attenuating oxidative stress in this model.

Figure 3. Serum Amylase and pancreatic MDA level.

Serum amylase levels and pancreatic MDA content were obtained after 24 hours induction with L-arginine. Compared to the L-arginine alone rats, the dh404 treated rats resulted in a significant decrease in serum amylase levels and pancreatic MDA concentration (p <0.05). Data are representative of at least three independent experiments.

Dh404 significantly lowered production of inflammatory mediators in LPS-treated rodent splenocytes

To confirm dh404’s ability to reduce inflammatory mediators, whole splenocytes from normal rats were treated with LPS alone or with dh404 + LPS 1µg/ml in vitro for 24 hours. LPS stimulated splenocytes were examined for the intended purpose of mimicking an acute gram negative infection, such as one that occurs commonly with severe pancreatitis in clinical settings. Dh404 significantly decreased the production of many inflammatory mediators from rodent splenocytes treated with LPS (Figure 4)

Figure 4. Inflammatory mediator levels from LPS stimulated rat splenocytes treated with dh404.

Rat splenocytes were isolated from normal rats and stimulated with LPS. They were cultured for 24 hours with or without dh404 in vitro. Dh404 significantly decreased the production of inflammatory mediators except Il-1β from LPS stimulated rat splenocytes. Data are representative of six independent experiments (* indicates a statistically significant difference, p<0.05).

Nrf2 translocation to nucleus in human acinar cells

To confirm the ability of dh404 to activate Nrf2 pathway in pancreatic tissues, human pancreatic tissues from cadaveric donors were used. The image analysis by con-focal microscope revealed Nrf2 translocation to the nucleus in a time dependent manner. Compared to 30 minute incubation with 500 nm dh404, pancreatic cells after 1 hour incubation revealed more Nrf2 antibody stain uptake in the nucleus (Figure 5A). The nuclear translocation of Nrf2 was represented by a teal color which revealed a merging of the nuclear stain in blue and the nrf2 antibody stain in green. The confocal data was also supplemented with nuclear Nrf2 protein expression (Figure 5B). Nuclear Nrf2 protein expression is almost doubled after incubation with 500 nM dh404 for 30 minutes. Double staining with amylase further confirmed Nrf2 translocation, specifically in the nucleus of human acinar cells (Figure 5C).

Figure 5.

A: Translocation of Nrf2 in human pancreatic cells. To investigate Nrf2 translocation from cytoplasm to nucleus, human pancreatic cells were treated with dh404 500nm for 0.5 or 1 hours. The treated and untreated samples were stained with Nrf2 antibody and DAPI. Nrf2 translocation to nuclear is represented in the merged slides as a teal color (arrow), representing the combination of the green nrf2 antibody stain and the blue nuclear stain. Data are representative of three independent experiments.

B: Western blot assay in nuclear Nrf2 expression. Human pancreatic tissue was treated with dh404 (500 nM) for 0.5 hours. Enhanced Nrf2 expression in nuclear fraction was observed. Data are representative of three independent experiments.

C: Translocation of Nrf2 in human acinar cells. After treatment with dh404 for 1 hour, the double staining of Nrf2 and amylase antibodies demonstrated acinar cell specific Nrf2 translocation to nucleus. An Arrow reveals an area where the images have merged, indicating Nrf2 translocation to the nucleus in human acinar cells. Pictures were taken with confocal microscopy with 63x magnification. Data are representative of three independent experiments.

HO-1, SOD, Catalase, and GCLC expressions were upregulated in human pancreatic tissue treated with dh404 in vitro for 24 hours

To confirm the over expression of Nrf2 target enzymes, western blot was performed using antibodies against HO-1, GCLC, Catalase, and SOD 24 hours after incubation in media containing dh404. Western blot revealed a significant increase in Nrf2 target genes, Catalase, HO-1, SOD and GCLC (Figure 6). Enhanced antioxidant production in pancreatic tissue treated with dh404 strengthens our hypothesis that dh404 is an Nrf2 pathway activator and mediates oxidative stress through regulation of anti-oxidative enzymes.

Figure 6. Expression of HO-1, SOD, GCLC, and Catalase in human pancreatic tissue.

To confirm higher expression of antioxidants induced by dh404, they were treated with dh404 250–1000nM for 24 hrs. The Western blot assay demonstrated an increase in the expression of anti-oxidative enzymes in the dh404 treated human non-endocrine pancreatic tissue. The blots pictured represent a single western blot experiment which was repeated four independent times‥ The graphs depict the average protein expressions of the four independent experiments for HO-1, SOD, GCLC, and Catalase with significance (p <0.05) is indicated by a * in the graph.

Significant dh404 concentration-dependent reduction in oxidative stress was observed in human pancreatic tissue

Since dh404 was effective in significantly reducing oxidative stress in our animal model of AP, the effect of dh404 was examined on human pancreatic tissue in vitro. Human pancreatic tissue was cultured for 24 hours in media containing 0, 250 and 500 nM dh404 with or without H2O2 (200µM). As expected the addition of H2O2 resulted in a significant rise in MDA level which was reduced in a dh404 concentration dependent manner (Figure 7A). These findings clearly demonstrate dh404 is able to increase the anti-oxidant capacity in human pancreatic tissue.

Figure 7.

A: MDA level of human pancreatic tissue in vitro. Human pancreatic tissue was cultured for 24 hours in media containing 0, 250 and 500 nM dh404 with or without H2O2 (200µM). The addition of H2O2 resulted in a significant rise in MDA level which was reduced in a dh404 concentration dependent manner.

B: Viability assay of human pancreatic cells. To test cytoprotective effects of dh404, viability assay in human pancreatic cells was performed using flow cytometry after H2O2 treatment. After dissociation of pancreatic cell, single-cell suspensions were stained with 7-AAD and TMRE. The percentage of TMRE^- cells were calculated for apoptotic cells after the exclusion (gating out) of 7-AAD⁺ cells. The graph reveals a protective effect of dh404 (250nM) against oxidative stress. (* and * * indicate a statistically significant difference, p<0.05 and <0.001, respectively)

Dh404 has cytoprotective effects against oxidative stress in vitro

Having demonstrated the upregulation of antioxidant enzymes and attenuation of oxidative stress in dh404 treated human pancreatic tissue, we explored its cytoprotective effects against oxidative stress on human pancreatic cells. To this end we employed flow cytometry using markers of apoptosis (TMRE) and cell death (7AAD). H2O2 significantly increased cell apoptosis which was significantly reduced by dh404 treatment (Figure 7B).

Taken together, our results demonstrate the efficacy of dh404 in promoting Nrf2 activation, upregulation of endogenous antioxidant enzymes and enhancing cell viability by attenuating oxidative stress and apoptosis.

Significant decreases in production of cytokines and chemokines were seen in dh404-treated human pancreatic tissue

To explore the effect of dh404 on production of inflammatory mediators in pancreatic acinar cells, the cytokine and chemokine were measured in the supernatant of cultured human pancreatic tissue treated with or without dh404 (500 nM) for 24 hours (Figure 8). The level (% of untreated group) of cytokines and chemokines in dh404-treated tissues was significantly lower than in the untreated tissues. A significant reduction in the key inflammatory cytokines for pancreatitis such as IL-1β, Il-4, and IFN-γ were observed. Dh404 significantly reduced production of cytokines and chemokines in human pancreatic tissue pointing to its anti-inflammatory properties.

Figure 8. Cytokine and chemokine production by human pancreatic tissue.

The human pancreatic tissue were treated with or without dh404 (500nM) for 24 hours. The cytokine and chemokine production in the supernatant was determined in each experimental group. Dh404 significantly decreased many inflammatory mediators related to acute pancreatitis. Data are representative of four independent experiments. (* indicates a statistically significant difference, p<0.05)

Discussion

Currently, treatment for acute pancreatitis is largely limited to supportive care. There is increasing interest in the development of more effective treatments for this disorder.

In this study, we evaluated the effects of Nrf2 activator, dh404, on human acinar cells, and on a rodent acute pancreatitis model induced by L-arginine or Cerulein. Our data demonstrated that the pharmacological activation of Nrf2 pathway in human acinar cells by dh404 resulted in a significant increase of antioxidant enzymes, and a substantial reduction of inflammatory mediators. Moreover, treatment with dh404 significantly improved acute pancreatitis in two different rodent acute pancreatitis models.

Previous studies have found impaired Nrf2 activity in animal models of various diseases including chronic kidney disease, diabetes, and inflammatory bowel disease^33–36. The protective effects of dh404 are directly related to its action as an inducer of the Nrf2-keap1 pathway^37,38. ROS activate Nrf2, by preventing its binding to Keap1 thereby enabling its migration to the nucleus where it binds to the antioxidant response element (ARE) in the promoter region of transcriptional upregulation of its target genes^17–19. The phase II genes are involved in the inactivation of reactive oxygen species (ROS), increased antioxidant capacity, and suppression of inflammation^18,39. While ROS act as critical signaling messengers in cellular redox reactions, sustained oxidative stress leads to cellular damage ³³. Cells protect themselves against oxidative stress by a combination of physical barriers, repair enzymes, and antioxidant mechanisms ³⁴. The antioxidant defense system is the major protective mechanism for cells to neutralize ROS; however, tissue damage ensues when production of ROS exceeds the antioxidant capacity³⁵. Expressions of cytoprotective compounds and proteins such as nicotinamide adenine dinucleotide phosphate (NADPH), NAD(P)H:Quinone oxidoreductase (NQO1), SOD, GCLC, glutathione peroxidase (GPx) and catalase are regulated by the Nrf2-Keap1 pathway ³⁵. These compounds catalyze a variety of chemical detoxification reactions, and are involved in the generation of several endogenous antioxidants ⁴⁰. Clinical pancreatitis is an inflammatory disorder initiated by the disruption of acinar cells and the leakage of pancreatic enzymes. This results in the development of oxygen free radicals, recruitment of inflammatory mediators and oxidation of lipids and proteins ⁴¹. Numerous clinical trials examining the effects of various antioxidants in patients with acute and recurrent chronic pancreatitis have yielded mixed results. In the 90’s, two regions in Germany began using Selenium in the treatment of patients with AP. In retrospective analysis investigators observed a significant reduction in mortality, morbidity and the number of necessary operations, following the implementation of Selenium therapy ⁵. In contrast, other studies have revealed either no benefit or equivocal results with inclusion of antioxidants in the treatment regimen for AP or CP^42–47.

The authors wish to point out that the natural antioxidant defense system consists of numerous antioxidant and detoxifying enzymes and substrates which work in concert to contain oxidative stress and prevent cytotoxicity and tissue damage. Thus the effects of activation of the endogenous antioxidant system can’t be replicated by administration of one or more exogenous antioxidant compounds. For this reason strategies aimed at eliciting endogenous antioxidant defense system by restoring Nrf2 activity are more effective in management of oxidative stress as shown in the present study.

In conclusion, administration of the synthetic Nrf2 activator, dh404, attenuated oxidative stress and inflammation by restoring expression of the key antioxidant enzymes and molecules, and by lowering production of inflammatory mediators in rats with experimental AP. This was accompanied by significant amelioration of pancreatic tissue damage and reduction in plasma amylase level and pancreatic MDA levels. Clinical trials are needed to determine the potential utility of Nrf2 activators in patients with AP.