Abstract
Background:
Acute pancreatitis (AP) is an inflammatory pancreatic disorder, often illustrated by neuropathic, inflammatory, and/or visceral pain. Pain is a medical indicator of pancreatic inflammation and growing evidence evokes that it exerts a significant role in AP progression via the widespread neuronal network surrounding the pancreas.
Objectives:
evaluate the possible defensive action of duloxetine (DUX), a serotonin/norepinephrine reuptake inhibitor (SNRI), in L-arginine (L-Arg)- provoked AP and multiple organ injury (MOI), and to explore the potential mechanism of action of DUX-mediated modulation of neuroinflammatory pain in AP.
Methods:
AP was induced in rats by intraperitoneal injection of L-Arg (100 mg/100 g) and two different doses of DUX (10 and 30 mg/kg) was examined.
Results:
DUX improved the histopathological architecture of the pancreas, liver, lung, and kidney. Additionally, it restored the normal levels of serum pancreatic enzymes, liver enzymes, and kidney biomarkers that were significantly elevated following L-Arg administration. In addition, DUX improved tissue antioxidant state. Moreover, DUX decreased the levels of transient receptor potential cation channel subfamily V member 1 (TRPV1), proteinase-activated receptor-2 (PAR2), substance P (SP), neurokinin, trypsin, leukotriene B4 (LTB4), signal transducer and activator of transcription 3 (STAT3) and significantly reduced TNF-α expression in the pancreatic tissues.
Conclusion:
DUX displayed analgesic, anti-inflammatory, and antioxidant effects. Therefore, DUX is a promising therapeutic candidate for the management of AP-related neuroinflammatory pain. The proposed analgesic and anti-inflammatory mechanisms of DUX may be linked to the modulation of SP related NK signaling and PAR2/TRPV1 pathways pointing to a possible role for these pathways in the observed beneficial action of DUX.
1 Introduction
Acute pancreatitis (AP) is an inflammatory disorder. It often presents neuropathic, inflammatory, and/or visceral pain (). AP Acute pancreatitis (AP) can occur at any age, with a reported annual incidence ranging from 10 to 50 cases/100,000 individuals (). The pathogenesis of AP is a dynamic process that starts with activation of the host immune response, leading to the release of proinflammatory cytokines and chemokine (). The leading causes of AP are gallstones and alcohol consumption, with gallstones responsible for approximately 60%–80% of cases. Additional causes include endoscopic retrograde cholangiopancreatography, drug-induced pancreatitis, metabolic disorders such as hypertriglyceridemia and hypercalcemia, autoimmune diseases, and idiopathic origins ().
Previous studies have revealed that mechanical factors, including pancreatic duct obstruction and elevated ductal and parenchymal pressures, are associated with pain in AP; however, these mechanisms alone are insufficient to fully explain the complexity of pain perception and signaling in AP (; ). Subsequent studies have underscored the contributory role of neurogenic inflammation in the pathogenesis of AP-related pain (). Consequently, pain research in animal models of AP has increasingly shifted from a predominant mechanical paradigm toward an emphasis on neurogenic inflammatory mechanisms (; ).
Noxious stimuli of pancreatic tissues lead to the production and release of several mediators, like trypsin, leukotriene B4 (LTB4) and protons. Such molecules are capable of engage diverse pain-related receptors and ion channels expressed on peripheral nerve terminals, including members of the transient receptor potential channels (TRP) family, e.g.,: TRP vanilloid 1 (TRPV1) and protease activated receptor 2 (PAR2). Neuropeptides including substance P (SP) and calcitonin-gene-related peptide (CGRP) were released triggered by peripheral nerve terminals within the pancreas, causing chemokine discharge from pancreatic acinar cells, plasma extravasation from venules, and interstitial edema within arterioles. Collectively, these alterations comprise neurogenic inflammation ().
The ensuing inflammatory response increases spinal cord excitability, thereby intensifying nociceptive signals arising from peripheral tissues. Similarly, a reciprocal interaction between damaged pancreatic tissue and activated neurons forms a self-perpetuating “auto-amplification loop” that connects inflammation and pain throughout the progression of AP (; ). After this pathogenic cycle surpasses critical limits, it may trigger a sustained systemic inflammatory response syndrome, resulting in irreversible numerous organ injuries, and consequently a significant increase in mortality risk ().
Pain control has attracted increasing interest for the management of AP (). It is the most challenging and characteristic symptom in acute and chronic pancreatitis. In a prospective study, approximately 77% of patients reported pain. Those experiencing pain had marked reductions in quality of life, and over 25% were on disability benefits (). Analgesics, opioids, and non-opioids are usually used to control pain in patients with AP but are associated with side effects and may even exacerbate AP (). Consequently, the development of novel medical interventions and routines for pain management in patients with AP is necessary.
Improved management relies on the deeper understanding of the mechanisms underlying neuroinflammatory visceral pain, a topic that has recently progressed with the establishment of robust animal models and reliable experimental approaches for assessing sustained pancreatic pain. L-Arginine (L-Arg)-induced AP is an animal model of severe necrotizing AP. This model is extremely reproducible, noninvasive, and generates dose-dependent acinar necrosis, making it ideal for investigating the pathogenesis of AP ().
Duloxetine (DUX), a powerful serotonin/norepinephrine reuptake inhibitor (SNRI), is used in the management of major depressive disorders, painful diabetic neuropathy, and urinary incontinence (). Numerous preclinical studies have shown that DUX diminishes pain in a variety of insistent, neuropathic, and inflammatory pain models (). Hence, we speculated that DUX might have an ameliorative effect against AP-associated pain. The existing work was constructed to evaluate the possible defensive action of DUX in L-Arg-provoked AP and multiple organ injury (MOI), and to explore the potential mechanism of action of DUX-mediated modulation of neuroinflammatory pain in AP.
2 Materials and methods
2.1 Animals
Thirty-eight adult male Sprague–Dawley rats (190–240 g) from the MERC, Faculty of Medicine, Mansoura University, Egypt. The animals were kept in standard cages under regulated environmental conditions (22 °C ± 2 °C, 50%–60% relative humidity, and a 12-h light/dark cycle) with unrestricted access to standard chow and water. Following a 1-week acclimatization, rats were randomly distributed among the experimental groups. All procedures were approved from the Research Ethics Committee of the Faculty of Pharmacy, Mansoura University (approval number: MU-ACUC [PH.PHD.22.10.3]) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
2.2 Drugs and chemicals
L-Arg was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). DUX was purchased from Lilly (Indianapolis, IN, USA). Both L-Arg and DUX were thawed in 0.9% saline and administered intraperitoneally (i.p.) to the rats. Reagents of the highest available purity were used throughout the study.
2.3 Investigational procedure
Following period of acclimatization, age-matched rats with comparable body weights were randomly assigned into five groups and treated according to the protocols listed in Table 1 and Figure 1. The number of animals per group was selected based on previous studies employing similar L-Arg-induced AP models. Additional animals were initially included to compensate for the anticipated mortality associated with this severe experimental model. The selected duloxetine doses (10 and 30 mg/kg) were chosen based on previous experimental studies demonstrating their efficacy and safety in rodent models and to evaluate potential dose-dependent protective effects (; ; ). 24 h after the final L-Arg boosting, rats were deeply anesthetized with thiopental sodium (40 mg/kg, i. p.). Blood samples (1.5 mL) were collected from the retro-orbital venous plexus for biochemical analyses. Animals were subsequently euthanized by cervical dislocation under deep anesthesia in accordance with the approved Animal Care and Use Committee (ACUC) protocol. Death was confirmed prior to tissue harvesting, after which pancreatic, left lateral liver lobe, left lung, and left kidney tissues were collected for histopathological and molecular analyses. Blood samples were withdrawn from the retro-orbital plexus, centrifuged at 5000 rpm for 15 min, and the resulted sera were subsequently preserved at −80 °C for subsequent serum biochemical analysis. Portion of the pancreas, left lateral liver lobe, left lung, and left kidney were fixed in 10% neutral buffered formalin for histopathological and immunohistochemical (IHC) examination. The remaining tissues (pancreas, median liver lobe, right lung, and right kidney) were homogenized in ice-cold phosphate buffer (0.01 M, pH 7.4) to obtain 10% w/v homogenates then centrifuged at 3000 rpm for 20 min at 4 °C, and the supernatants were collected and stored at −80 °C for subsequent assays.
TABLE 1
| Treatment protocol | Groups |
|---|---|
| Rats were injected with 0.9% normal saline (i.p.) for 10 days | Group I (Control) (n = 7) |
| Rats were administered DUX 30 mg/kg (i.p.) for 10 days | Group II (DUX Control) (n = 7) |
| Rats were injected with L-Arg dissolved in 0.9% saline (100 mg/100 g, i.p.) twice to create AP, with an interval of 1 h () | Group III (L-Arg) (n = 10) |
| Rats received DUX 10 mg/kg (i.p.) for 10 days prior to injection of 100 mg/100 g L-Arg twice with an interval of 1 h | Group IV (DUX10+L-Arg) (n = 7) |
| Rats received DUX 30 mg/kg (i.p.) for 10 days prior to injection of 100 mg/100 g L-Arg twice with an interval of 1 h | Group V (DUX30+L-Arg) (n = 7) |
Experimental protocol.
FIGURE 1
2.4 Spectrophotometric measurements
2.4.1 Assessment of serum biochemical markers reflecting pancreatic, hepatic, renal, and pulmonary function
Serum amylase, lipase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (SCr), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), and total protein (TP) levels were assessed spectrophotometrically using available kits. The absorbance was recorded using a spectrophotometer (Labomed, Los Angeles, USA) (Table 2).
TABLE 2
| Kit manufacturers | Techniques | Kits |
|---|---|---|
| MDSS, Germany | Spectrophotometry | Amylase and lipase kits |
| Agappe Hills, India | Spectrophotometry | Alanine aminotransferase (ALT) kit Aspartate aminotransferase (AST) kit |
| SPINREACT, S.A./S.A.U Ctra.Santa Coloma, Spain | Spectrophotometry | Serum creatinine (SCr) kit Blood urea nitrogen (BUN) kit |
| Sedmed Diagnostics, India | Spectrophotometry | Lactate dehydrogenase (LDH) kit |
| Biodiagnostic, Egypt | Spectrophotometry | Total protein kit |
| Biodiagnostic, Egypt | Spectrophotometry | Malondialdehyde (MDA) kit |
| Biodiagnostic, Egypt | Spectrophotometry | Total antioxidant capacity (TAC) kit |
| Cat. No. OKEH03613, Aviva system biology, USA | ELISA | Transient receptor potential cation channel subfamily V member 1 (TRPV1) kit |
| Cat. No. NBP3-06968, biotechne®, USA | ELISA | Proteinase-activated receptor-2 (PAR2) kit |
| Cat. No. E-EL-0067, Elabscience®, USA | ELISA | Substance P (SP) kit |
| Cat. No. ABIN772124, antibodies-online.com, USA | ELISA | Neurokinin (NK) kit |
| Cat. No. ABIN6970901, antibodies-online.com, USA | ELISA | Trypsin kit |
| Cat. No. E-EL-0061, Elabscience®, USA | ELISA | Leukotriene B4 (LTB4) kit |
| Cat. No. ER0163, Fine Test®, China | ELISA | Signal transducer and activator of transcription 3 (STAT3) kit |
| Cat. No.GB11188, Service bio, 21 Olympia Avenue, Woburn, USA | IHC | Tumor necrosis factor-α (TNF-α) antibodies |
Spectrophotometric, ELISA, And IHC kits.
2.4.2 Evaluation of the oxidative and antioxidant status in tissues
Malondialdehyde (MDA), lipid peroxidation end-product, and total antioxidant capacity (TAC) were assessed using spectrophotometric available kits (Table 2).
2.5 Enzyme-linked immunosorbent assays (ELISA)
The pancreatic content of transient receptor potential cation channel subfamily V member 1 (TRPV1), proteinase-activated receptor-2 (PAR2), substance P (SP), neurokinin (NK), trypsin, leukotriene B4 (LTB4), and signal transducer and activator of transcription 3 (STAT3) were determined using commercially available ELISA kits (Table 2).
2.6 Histopathological examination
2.6.1 Haematoxylin and Eosin (H&E) staining
Formalin-preserved sections of the pancreatic, hepatic, pulmonary, and renal tissues were placed in paraffin blocks. Thin sections (5 μm) were sliced and stained with H&E to evaluate any histopathological alterations. Histopathological abrasions were scored based on the criteria established in (; ; ).
2.6.2 Immunohistochemical (IHC) analysis
Pancreatic TNF-α expression was evaluated via IHC analysis using the Avidin–Biotin Complex (ABC) technique as previously described (). Fiji ImageJ software (version 1.51r; NIH, Maryland, USA) was utilized for quantification of % of positively stained areas. This approach generated three distinct digital channels corresponding to H&E, DAB, and a residual image. For quantitative assessment, five randomly selected microscopic fields (each measuring 200 × 200 µm) were analyzed per slide.
2.7 Statistical analysis
Statistical analysis was performed using GraphPad Prism version 8.0.1 (San Diego, CA, USA). Data normality was assessed with the Shapiro–Wilk test. Normally distributed data were analyzed using one-way ANOVA followed by Tukey–Kramer post hoc test for multiple comparisons and are expressed as mean ± SEM. Non-parametric scoring data are presented as median ± IQR and were analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test. A P value <0.05 was considered statistically significant.
3 Results
Current data revealed insignificant changes in any measured parameters concerning the normal control and DUX control groups. Mortality was observed exclusively in the L-arg group (40%), whereas no deaths occurred in the normal control, DUX control, or DUX-treated groups during the study period. Nevertheless, the study was not designed to evaluate survival as a primary outcome.
3.1 DUX deceased the elevated serum amylase, lipase, and LDH levels induced by L-Arg
A noteworthy elevation in the levels of serum amylase, lipase, and LDH was detected in the L-Arg-treated group when matched the control group. DUX (10 mg/kg) treatment caused a substantial lowering in amylase, lipase, and LDH levels, comparative to the L-Arg group. On the other hand, their levels were remained meaningfully dissimilar to the control group. Conversely, DUX (30 mg/kg) administration in rats triggered a marked reduction in the amylase, lipase, and LDH levels in serum compared to L-Arg group, but there was still a significant difference in serum lipase and LDH levels between the DUX 30 and control groups. In addition, DUX 30 significantly lowered serum levels of amylase, lipase, and LDH compared with DUX 10 (Figure 2).
FIGURE 2
3.2 DUX decreased the elevated serum liver enzymes and total protein (TP) levels induced by L-Arg
As shown in Figure 3, a noteworthy increase in the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels in serum, with a reduction in TP levels, was observed in L-Arg-administered rats compared to the control group. Nevertheless, rats treated with DUX 10 or DUX 30 showed dose dependent amelioration in serum ALT, AST, and TP levels relative to the L-Arg-injected group. However, a substantial dissimilarity among DUX 10, DUX 30 and the control group was noticed. In addition, pretreatment with DUX 30 ameliorated serum ALT and TP levels comparative to DUX 10-treated animals.
FIGURE 3
3.3 DUX mitigated the elevated kidney function biomarkers induced by L-Arg
As shown in Figure 4, L-Arg triggered a noteworthy rise in serum creatinine (SCr) and blood urea nitrogen (BUN) levels comparative to control rats. In contrast, DUX10 administration led to noteworthy lessening in SCr and BUN relative to L-Arg group, but still significantly higher than control rats. DUX 30 pretreatment significantly diminished SCr and BUN relative to L-Arg group, but still significantly higher than the control group. Moreover, DUX 30 significantly decreased the serum level of BUN compared to DX10.
FIGURE 4
3.4 DUX attenuated the L-Arg induced oxidative stress and improved the tissue anti-oxidant status
In L-Arg-injected animals, A noteworthy upsurge in malondialdehyde (MDA) levels in pancreatic, hepatic, pulmonary, and kidney tissues with a marked decrease in total antioxidant capacity (TAC) levels in the previously mentioned tissues compared to the control group was noticed. As matched to the L-Arg-treated group, DUX 10 group exhibited significant decrease in MDA content in the previous tissues in comparison with the L-Arg group and significantly elevated TAC| content in the previously mentioned tissues.
Both markers in all measured tissues were significantly higher than the levels in the control group. Conversely, oral ingestion of DUX 30 led to a noteworthy lessening in MDA content in the previously mentioned tissues and drastically augmented TAC content compared to the L-Arg group, but both markers in all measured tissues were still significantly higher than in the control group. Interestingly, DUX 30 significantly decreased MDA content in the pancreas, liver, lungs, and kidneys compared with DUX 10. In the same manner, DUX 30 significantly increased the level of TAC in the pancreas, liver, lungs, and kidneys compared to DUX 10 (Table 3).
TABLE 3
| Organ | Control | DUX | L-Arg | L-Arg +DUX 10 | L-Arg +DUX 30 |
|---|---|---|---|---|---|
| Malondialdehyde (MDA) (nmol/g tissue) | |||||
| Pancreas | 330.80 ± 7.64 | 363.50 ± 9.82 | 1908.0 ± 52.42a | 1138.0 ±35.35a b | 650.7 ± 4.54a b c |
| Liver | 250.00 ± 20.74 | 312.20 ± 4.36 | 1314.0 ± 68.92a | 736.5 ±7.17a b | 486.2 ± 5.95a b c |
| Kidney | 218.30 ± 2.18 | 251.10 ± 4.36 | 793.3 ± 28.64a | 508.7 ±2.52a b | 342.8 ± 10.08a b c |
| Lung | 153.90 ± 7.64 | 197.00 ± 3.27 | 633.2 ±7.87a | 449.8 ±7.87a b | 272.9 ± 8.26a b c |
| Total antioxidant capacity (TAC) (mM/L) | |||||
| Pancreas | 0.63 ± 0.01 | 0.612 ± 0.004 | 0.12 ± 0.02a | 0.33 ± 0.001a b | 0.497 ± 0.020a b c |
| Liver | 0.51 ± 0.01 | 0.517 ± 0.002 | 0.21 ± 0.01a | 0.34 ± 0.005ab | 0.467 ± 0.001a b c |
| Kidney | 0.55 ± 0.01 | 0.519 ± 0.003 | 0.25 ± 0.01a | 0.40 ± 0.004ab | 0.479 ± 0.002a b c |
| Lung | 0.58 ± 0.01 | 0.579 ± 0.013 | 0.27 ± 0.01a | 0.38 ± 0.01a b | 0.530 ± 0.010a b c |
Effect of duloxetine (DUX) on oxidant/antioxidant level in different tissues in L-Arginine (L-Arg) injected rats.
Data are presented as mean ± SEM (n = 6 for L-Arg group; n = 10 for all other groups).
abc vs. the control, L-Arg, and L-Arg + DUX10 groups, respectively, as determined by one-way ANOVA, followed by Tukey-Kramer multiple comparison post hoc tests (P < 0.05).
3.5 DUX ameliorated the histopathological alterations in the pancreas, liver, pulmonary, and renal tissues induced by L-Arg
3.5.1 Pancreatic tissues
The control group (Figures 5A,a) and DUX group (Figures 5B,b) maintained normal pancreatic architecture, with intact exocrine acini and islets of Langerhans. In contrast, the L-Arg group (Figures 5C,c), exhibited disruption of exocrine acinar structure, vascular congestion, cellular damage, and infiltration of the inflammatory cells. The DUX10 group (Figures 5D,d) displayed moderate architectural disruption, congested blood vessels, and cellular injury. Notably, the DUX30 + L-Arg group (Figure 4E,e) demonstrated only minimal disturbance of acinar architecture, with no detectable cellular damage or vascular congestion. Semi-quantitative analysis of pancreatic edema, acinar necrosis, hemorrhage, and inflammation (Figures 5F–I respectively) revealed significant increases in the L-Arg group compared to controls. Treatment with DUX10 and DUX30 substantially reduced these pathological scores relative to the L-Arg group, although differences remained when compared with the normal control group.
FIGURE 5
3.5.2 Hepatic tissues
Liver sections from the normal control group (Figures 6A,a) and the DUX control group (Figures 6B,b) demonstrated a normal hepatic architecture characterized by polyhedral hepatocytes arranged in cords separated by blood sinusoids containing Kupffer cells surrounding the central vein. In the L-Arg-treated group (Figures 6C,c), marked sinusoidal congestion, infiltration of the inflammatory cells, and increased the Kupffer cell activity were observed. The DUX 10 group (Figures 6D,d) exhibited moderate sinusoidal congestion with elevated Kupffer cell activity and minimal inflammatory infiltration. In contrast, the DUX 30 group (Figures 6E,e) showed only mild sinusoidal congestion without inflammatory cell infiltration. Moreover, L-Arg administration significantly increased portal inflammation and hepatic centrilobular necrosis compared with the control group. Treatment with DUX at 10 and 30 mg noticeably reduced portal inflammation scores relative to the L-Arg group (Figure 6F–H).
FIGURE 6
3.5.3 Lung tissues
Lung sections from the normal control group (Figures 7A,a) and the DUX control group (Figures 7B,b) displayed normal pulmonary architecture, with well-defined alveoli separated by thin interalveolar septa along with normal bronchioles and bronchial arterioles. The L-Arg-injected group (Figures 7C,c) showed marked distortion of lung architecture, characterized by thickened interalveolar septa, reduced alveolar spaces, inflammatory cell infiltration within the septa and around bronchi, and congested, thickened pulmonary arterioles. In the DUX 10 group (Figures 7D,d), moderate thickening of the interalveolar septa with smaller alveoli, inflammatory cell infiltration, and congested pulmonary arterioles were observed. Meanwhile, the DUX 30 group (Figures 7E,e) demonstrated nearly normal pulmonary septa with only minimal thickening of the pulmonary arteriolar wall. Histopathological scoring revealed a significant increase in lung tissue damage in the L-Arg group compared with the normal control. Treatment with DUX (10 and 30 mg) reduced the damage score relative to the L-Arg group; however, both groups still showed significant differences compared with the normal group (Figure 7F).
FIGURE 7
3.5.4 Kidney tissues
H&E-stained kidney sections from the control (Figures 8A,a) and DUX control groups (Figures 8B,b) displayed normal renal architecture, with appropriately sized glomeruli within Bowman’s space and intact proximal and distal tubules. Figure 8C, C revealed that injection of L-Arg disrupted the renal architecture, a glomerular shrinkage, expanded Bowman’s space, tubular epithelial cells degeneration and inflammatory cell infiltration were noticed.
FIGURE 8
In the DUX10 group (Figures 8D,d), moderate architectural distortion was observed, including glomerular shrinkage, expanded Bowman’s space, accumulation of the inflammatory cells, and tubular epithelial degeneration. The DUX30 group (Figures 8E,e) revealed only minimal collapsed glomeruli with a widened Bowman’s space and mild tubular epithelial changes. Tubular necrosis scoring indicated a significant increase in the L-Arg group compared to controls, whereas treatment with DUX10 and DUX30 significantly reduced necrosis scores relative to the L-Arg-treated group (Figure 8F).
3.6 Effect of DUX on pancreatic protein levels of trypsin, proteinase-activated receptor-2 (PAR2), transient receptor potential cation channel subfamily V member 1 (TRPV1), substance P, neurokinin, leukotriene B4 (LTB4), and signal transducer and activator of transcription 3 (STAT3)
Figure 9 demonstrated that L-Arg inoculation triggered a considerable elevation in the pancreatic trypsin, PAR2, TRPV1, SP, neurokinin, LTB4, and STAT3 levels compared to control animals. Management with DUX 10 led to a marked decrease in previous markers compared to the L-Arg group. DUX 30 handling considerably lessened pancreatic levels of measured parameters when matched to L-Arg rats. Furthermore, it was noted that DUX 30 significantly decreased levels of trypsin, PAR2, TRPV1, SP, LTB4, and STAT3 compared with DUX 10.
FIGURE 9
3.7 DUX suppressed the elevated tumor necrosis factor-α (TNF-α) immune-expression in pancreatic tissues induced by L-Arg
Immunohistochemical staining for TNF-α in rat pancreatic tissue sections revealed no detectable immunoreactivity in the control group (Figures 9A,a). In contrast, the L-Arg group (Figures 10B,b) exhibited strong cytoplasmic expression in the exocrine acinar cells and moderate staining in the islets of Langerhans. The DUX 10 group (Figures 10C,c) demonstrated moderate cytoplasmic TNF-α expression in both exocrine acinar cells and the islets of Langerhans. Meanwhile, the DUX 30 group (Figures 9D,d) showed only mild cytoplasmic immunoreactivity in these pancreatic structures.
FIGURE 10
4 Discussion
Acute pancreatitis (AP) is characterized by complex neuroimmune crosstalk that amplifies inflammatory cascades and nociceptive transmission. Pain in AP comprises both nociceptive and neuropathic components driven by neurogenic inflammation, ion channel activation, and cytokine-mediated central sensitization (; ; ). The present study demonstrates that DUX confers marked protection against L-arginine–induced AP, attenuating pancreatic injury, systemic organ injury, and pain-related behavioral alterations through coordinated modulation of inflammatory, neurogenic, and oxidative pathways.
Biochemically and histologically, the L-Arg model reliably reproduced severe AP, evidenced by marked elevations in serum amylase and lipase, destruction of acinar architecture, inflammatory infiltration, vascular congestion, and multi-organ injury reflected by increased ALT, AST, creatinine, BUN, and LDH levels (; ; ). These findings confirm the robustness and translational relevance of this model for evaluating therapeutic interventions targeting both pancreatic and systemic sequelae.
Proteinase-activated receptor-2 (PAR2), transient receptor potential cation channel subfamily V member 1 (TRPV1) levels were further determined by ELISA represent tissue protein expression and should not be interpreted as direct measures of receptor activation. Mechanistically, pancreatic pain is largely driven by neurogenic inflammation originating from T5–L2 dorsal root ganglia afferents innervating the pancreas (; ). Activation of TRPV1 channels enhances Ca2+ influx and stimulates SP release, which binds NK1R and amplifies inflammatory signaling within pancreatic tissue (; ; ; ). Upregulation of SP related NK1R signaling has been documented in experimental and clinical AP (; ). In the present study, L-Arg significantly increased SP, NK, and TRPV1 expression, confirming activation of this neuroinflammatory axis. DUX markedly suppressed these mediators, indicating concomitant downregulation of the SP-related NK1R and TRPV1 pathways indicating disruption of the SP related NK1R–TRPV1 feed-forward loop that perpetuates pancreatic inflammation and pain. Protease-driven signaling represents an additional amplification pathway. Trypsin released during acinar injury activates PAR2 on nociceptive neurons, which is known to transactivates TRPV1 and enhances neuronal excitability (; ; ). Elevated pancreatic trypsin and PAR2 levels in AP animals confirm engagement of this cascade. Importantly, DUX significantly reduced both trypsin and PAR2 expression, suggesting modulation of protease-dependent nociceptive sensitization.
Previous studies have demonstrated that experimental acute pancreatitis may be associated with behavioral manifestations of pain, including mechanical hypersensitivity and thermal hyperalgesia. In the present study, however, behavioral nociceptive assessments were not conducted. Therefore, our findings are limited to biochemical and molecular alterations in pain-related pathways, including SP-, PAR2-, and TRPV1-associated signaling, and should not be interpreted as direct evidence of analgesic activity (; ). Furthermore, while these mediators are biochemically linked, our current expression data are correlative; hence, the precise sequential hierarchy of the trypsin/PAR2/TRPV1/SP axis remains a proposed mechanism rather than a definitively proven pathway in this specific setting. Future studies incorporating pathway-specific inhibitors or gene-knockdown models, alongside behavioral endpoints, are needed to establish the causal relationships and functional significance of these molecular changes.
Leukotriene B4 (LTB4) further sustains TRPV1 activation by sensitizing pancreatic sensory neurons (; ). Increased LTB4 levels observed in the AP group provide additional evidence of lipid mediator–driven neurogenic inflammation. The reduction of LTB4 by DUX indicates broader suppression of inflammatory lipid signaling contributing to nociceptive amplification.
Beyond peripheral mechanisms, transcriptional regulation via STAT3 plays a critical role in sustaining neuroinflammation and chronic pain states (; ). Increased STAT3 expression following L-Arg administration suggests activation of downstream inflammatory gene programs that reinforce central sensitization. DUX significantly attenuated STAT3 expression, highlighting its capacity to interfere with the overall expression of transcriptional machinery that links immune activation to persistent pain signaling.
Tumor necrosis factor-α (TNF-α), a master pro-inflammatory cytokine elevated in severe AP, contributes to neutrophil recruitment, endothelial activation, and tissue injury (). Importantly, TNF-α also facilitates neuropathic pain by disrupting the perineural barrier and promoting axonal sensitization (). The marked increase in TNF-α observed in AP animals underscores its dual inflammatory and nociceptive role. DUX significantly reduced TNF-α levels, thereby attenuating both inflammatory progression and pain amplification.
Oxidative stress constitutes a pivotal link between tissue injury and nociceptive sensitization. Excess reactive oxygen species (ROS) enhance TRPV1 activity, disrupt mitochondrial function, and propagate inflammatory cascades (). Consistent with previous evidence demonstrating antioxidant effects of DUX via modulation of Ca2+ influx and ROS production (), our results show reduced MDA levels and restoration of total antioxidant capacity across pancreatic and remote tissues. This antioxidant action likely synergizes with anti-inflammatory and neurogenic modulation to confer organ protection and analgesia.
Pharmacologically, DUX acts as a serotonin and norepinephrine reuptake inhibitor, enhancing descending inhibitory pathways within the spinal cord (; ). Increased synaptic 5-HT and NE strengthen endogenous analgesic circuits and counterbalance ascending nociceptive transmission. Thus, DUX integrates central neuromodulation with peripheral anti-inflammatory and antioxidative effects, resulting in comprehensive attenuation of AP-associated pain.
5 Conclusion
Collectively, our findings demonstrate that protective effects of DUX in experimental AP are associated with modulation of markers linked to SP/NK1R signaling, PAR2/TRPV1 activation, LTB4 sensitization, STAT3-activity, TNF-α liberation, and oxidative stress. As far as we know, this is the first study to demonstrate concurrent alteration in these interconnected pathways following DUX in experimental AP, supporting its possible repurposing as a multimodal therapeutic strategy targeting management of both pancreatic inflammation and pain; however, further mechanistic studies are required to establish causal pathway involvement (Figure 11).
FIGURE 11
6 Limitation
Despite the promising findings of the present study, several limitations should be acknowledged. First, the study was conducted using an acute L-Arg-induced AP model, which may not fully recapitulate the complexity of human AP. Second, the mechanistic conclusions are based primarily on alterations in pathway-related biomarkers and protein expression levels. Although changes in SP, PAR2, TRPV1, STAT3, TNF-α, and oxidative stress markers were observed, receptor activation and downstream signaling were not directly assessed, and no receptor antagonists or pathway-specific inhibitors were employed to establish causality. Furthermore, NK1R expression was not directly quantified, and PAR2/TRPV1 measurements reflected tissue protein expression rather than functional receptor activity. In addition, complementary techniques such as Western blotting, immunofluorescence, or gene expression analyses were not performed. In addition to, although additional animals were included to compensate for the anticipated mortality associated with the L-Arg model, a formal a priori power analysis was not conducted. Therefore, future studies incorporating larger sample sizes, mechanistic interventions, and comprehensive molecular analyses are warranted to further validate and extend the present findings. Also, an additional limitation is the absence of behavioral pain assessments. Although several nociception-related biomarkers, including SP, PAR2, and TRPV1, were evaluated, validated behavioral measures of pain sensitivity, such as von Frey, thermal nociception, or abdominal withdrawal tests, were not performed. Therefore, the present findings provide indirect evidence regarding pain-related mechanisms and do not establish a direct analgesic effect of duloxetine. Finally, an important limitation is that DUX was administered before the induction of acute pancreatitis. Consequently, the present experimental design evaluated the preventive (prophylactic) rather than the therapeutic effects of DUX. Therefore, the observed protective effects cannot be directly extrapolated to the treatment of established AP in clinical settings. Future studies employing post-induction treatment protocols are necessary to determine the therapeutic potential of duloxetine after disease onset.
Statements
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
The animal study was approved by Research Ethics Committee of the Faculty of Pharmacy, Mansoura University University (approval number: MU-ACUC [PH.PHD.22.10.3]). The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
KA: Formal Analysis, Funding acquisition, Investigation, Methodology, Software, Writing – original draft, Writing – review and editing. AE: Conceptualization, Project administration, Supervision, Writing – original draft, Writing – review and editing. MN: Conceptualization, Data curation, Formal Analysis, Project administration, Supervision, Validation, Writing – original draft, Writing – review and editing. MZ: Investigation, Methodology, Software, Supervision, Writing – original draft, Writing – review and editing.
Funding
The author(s) declared that financial support was not received for this work and/or its publication.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Summary
Keywords
acute pancreatitis, duloxetine, neuroinflammatory pain, PAR2, TRPV1
Citation
Almutary KH, Elsheakh AR, Nader MA and Zaghloul MS (2026) Duloxetine ameliorates acute pancreatitis-associated inflammation and modulates pain related neuroinflammatory pathways via modulation of SP-related neurokinin signaling and PAR2/TRPV1 pathways. Front. Toxicol. 8:1891784. doi: 10.3389/ftox.2026.1891784
Received
26 May 2026
Revised
13 July 2026
Accepted
14 July 2026
Published
07 August 2026
Volume
8 - 2026
Edited by
Yupei Li, Sichuan University, China
Reviewed by
Maedeh Ghasemi, Isfahan University of Medical Sciences, Iran
Jagtar Singh, National Institute of Pharmaceutical Education and Research, India
Updates
Copyright
© 2026 Almutary, Elsheakh, Nader and Zaghloul.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Marwa S. Zaghloul, dr_marwasalah@mans.edu.eg, marwazaghloul@mansnu.edu.eg
ORCID: Marwa S. Zaghloul, orcid.org/0000-0002-7082-481X;
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