Introduction

Acute pancreatitis (AP) is a severe inflammatory condition primarily affecting the pancreas, often leading to systemic inflammation and multiple organ dysfunction, necessitating frequent hospitalization1. AP ranges from 13 to 45 cases per 100,000 persons per year. The annual incidence of AP has increased with an average annual percent change of 3.07%, showing significant geographical variation with highest rates reported in Western countries. AP is associated with significant morbidity and mortality, with hospitalization costs exceeding $30,000 per person in the United States. While overall mortality is approximately 1%, this rate can increase dramatically to 30–40% among hospitalized patients with organ failure or pancreatic necrosis2,3. The etiology of AP includes various factors such as excessive alcohol consumption, gallstones, hyperlipidemia, physical trauma, viral infections, sepsis, shock, and adverse drug reactions4.

During the early stages of AP, the excessive production of reactive oxygen species (ROS) by pancreatic acinar cells and activated immune cells significantly exacerbates pancreatic tissue damage5. This oxidative stress is reflected in elevated levels of biomarkers such as malondialdehyde (MDA) and decreased levels of glutathione (GSH), which are critical indicators of lipid peroxidation and antioxidant capacity, respectively6.

Damaged pancreatic cells release damage-associated molecular patterns like high-mobility group box 1 (HMGB1), which acts as a potent pro-inflammatory cytokine. HMGB1 interacts with receptors such as toll-like receptor 4 (TLR4), triggering the activation of nuclear factor kappa B (NF-κB). NF-κB is a key transcription factor that, upon activation, translocates to the nucleus and stimulates the expression of various inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)7,8. The upregulation of these cytokines leads to a cascade of inflammatory responses, contributing to the severity and progression of AP7. TNF-α and IL-1β further amplify the inflammatory response by recruiting additional immune cells to the site of injury, exacerbating tissue damage and promoting a cycle of ongoing inflammation9.

Addressing the pathophysiological mechanisms of AP requires therapeutic strategies that focus on both suppressing the extensive inflammatory response and enhancing cytoprotective signaling6. Resveratrol (RES), a natural polyphenolic compound with 3,5,4'-trihydroxystilbene structure, exhibits potent antioxidant properties through direct free radical scavenging and enhancement of endogenous antioxidant systems10. β‐carotene (βC), a lipid-soluble tetraterpene precursor of vitamin A, demonstrates significant membrane-protective effects through its conjugated double bond system that effectively neutralizes reactive oxygen species11. RES has demonstrated significant therapeutic efficacy in experimental pancreatic injury models through NF-κB pathway modulation12, while βC has shown protective effects against oxidative damage in pancreatic tissue and has been successfully used in post-ERCP pancreatitis prevention13. Despite these established therapeutic properties, the potential effects of RES and βC in the context of L-arginine-induced AP remain underexplored, particularly regarding their influence on inflammatory pathways and oxidative stress markers.

Therefore, the aim of our study was to evaluate the effects of RES and βC on L-arginine-induced AP in rats. We focused on several key parameters: levels of oxidative stress markers (MDA and GSH) in pancreatic tissues, immunohistochemical staining with anti-insulin antibody to assess β + Langerhans islets, relative mRNA transcript levels of inflammatory genes (NF-κB, TNF-α, and IL-1β), and relative mRNA transcript levels of apoptotic genes (Bax and Caspase-3) and the anti-apoptotic gene (Bcl-2) in pancreatic tissues. Through these analyses, we aimed to elucidate the mechanisms by which RES and βC confer their effects against AP.

Materials and methods

Animals

A total of 48 male Sprague Dawley rats, aged 2 months and weighing 250 g, were obtained from Atatürk University Medical Experimental Application and Research Center. The rats were acclimatized to the laboratory conditions for one day before the commencement of the experiment and housed under controlled temperature and light conditions, with ad libitum access to food and water. The study strictly adhered to the ARRIVE guidelines and was conducted following the national regulations for the ethical use and care of laboratory animals. This study protocol was approved by Atatürk University Animal Experiments Local Ethics Committee (Decision No: 2023/13). All procedures performed in studies involving animals were in accordance with the ethical standards of the institution.

Drugs and chemical reagents

Chemicals utilized in this study included resveratrol, β‐carotene, L-arginine were purchased from Sigma-Aldrich (USA). All chemicals utilized in the experiment were sourced from the highest-quality commercially available options.

Experimental design

The rats were randomly divided into six groups (n = 8 per group) as follows:

Control (C) group Rats in this group received 1 ml isotonic intraperitoneally (i.p.).

Resveratrol (RES) group Healthy rats in this group were given 20 mg/kg RES by gavage for 7 days14, with no changes to standard feeding conditions.

β-carotene (βC) group Rats in this group were administered 50 mg/kg βC by gavage for 7 days15.

Acute Pancreatitis (AP) group Experimental pancreatitis was induced in rats by administering 250 mg/100 g L-arginine i.p. twice daily with a 1-h interval16, with no changes to standard feeding conditions.

Acute Pancreatitis + Resveratrol (AP + RES) group One hour after the second L-arginine injection, rats in this group received 20 mg/kg RES by gavage at the same dose and time daily for 7 days. No changes to standard feeding conditions were made.

Acute Pancreatitis + β-carotene (AP + βC) group One hour after the second L-arginine injection, rats in this group received 50 mg/kg ββC by gavage at the same dose and time daily for 7 days. No changes to standard feeding conditions were made.

Blood and tissue sampling

The duration of the experiment was seven days. At the end of the experimental period, rats were fasted overnight and deeply anesthetized using a combination of intraperitoneal thiopental sodium (20 mg/kg) and inhalation anesthesia with 5% sevoflurane. The depth of anesthesia was confirmed by the absence of pedal withdrawal reflexes. Blood samples were collected via cardiac puncture under anesthesia. The animals were then euthanized by administering a high dose of thiopental sodium (50 mg/kg). Pancreatic tissues were immediately excised and processed under sterile conditions. Tissue samples were either snap-frozen in liquid nitrogen and stored at -80°C for biochemical analysis or fixed in 10% neutral buffered formalin for histopathological and immunohistochemical examination.

Histopathological methods

The tissues were processed through routine alcohol-xylol series and embedded in paraffin blocks. Sections of 5 µm thickness were taken on poly-L-lysine-coated slides, stained with hematoxylin–eosin, and examined for degenerative and necrotic changes in the endocrine and exocrine pancreas in 10 randomly selected areas17. The histopathological changes were scored based on the criteria presented in Table 1.

Table 1 Histopathological scoring.
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Evaluation of oxidative stress biomarkers

Pancreatic tissues were powdered using a homogenizer (Tissue Lyser II, Qiagen, Netherlands) under liquid nitrogen. For MDA level analysis, the tissues were diluted at a 1:10 ratio with 1.15% phosphate buffer and homogenized with stainless steel beads using the same device. The homogenates were centrifuged at 3500 RPM for 15 min, and the supernatants were used to determine MDA levels, an indicator of lipid peroxidation, following the method of Ohkawa et al.18. For GSH level analysis, the tissues were also diluted at a 1:10 ratio and homogenized, followed by centrifugation at 10,000 RPM for 20 min. The supernatants were analyzed for GSH levels using the method based on the reduction of Ellman’s reagent [5,5'-dithiobis (2-nitrobenzoic acid)]19.

Immunohistochemical methods

The tissue sections of 5 µm thickness were deparaffinized, rehydrated through xylol and alcohol series, and washed with PBS. Endogenous peroxidase activity was blocked by incubating the sections in 3% H2O2 for 10 min. Antigen retrieval was performed using an antigen retrieval solution in a microwave at 500 watts for 2 × 5 min. After washing with PBS, the tissues were incubated overnight at + 4 °C with anti-Insulin primary antibody (Sigma, Cat. no. I2018) at a 1/500 dilution. The secondary antibody, Large Volume Detection System: anti-Polyvalent, HRP (Thermofischer, Cat. no: TP-125-HL), was applied according to the manufacturer’s instructions. DAB (3,3′-Diaminobenzidine) was used as the chromogen. After counterstaining with Mayer’s hematoxylin, the sections were mounted with entellan and examined under a light microscope. The diameter and area of 10 randomly selected Langerhans islets with immunopositivity (diameter and area) were measured using Leica LAS EZ software20.

Quantitative real-time polymerase chain reaction (qRT-PCR.) analyses

100 mg of pancreatic tissues were weighed and 1 mL of QIAzol Lysis Reagent (79,306; Qiagen) was added. Then, the tissues were homogenized for 1 min in a homogenizer (Tissue Lyser II, Qiagen, Netherlands) with the help of stainless steel balls, and then the homogenates were incubated at room temperature for 5 min. After the incubation period was completed, 200 µL of chloroform was added to the samples and incubated for 3 min after vortexing. The tubes were then centrifuged at 12,000 g for 15 min. The upper transparent phase was transferred to new sterile tubes and 500 µL of isopropanol was added. Samples incubated for 10 min were centrifuged at 12,000 g for 10 min. Then, isopropanol was removed and the pellet was washed with 1 mL of 75% ethanol. Centrifugation was performed at 7500 g for 5 min to remove ethanol. Ethanol was removed from the tubes and the pellet was dissolved with DNase/RNase free water. The concentration and RNA integrity of the obtained total RNA solution were measured on the NanoDrop (BIO-TEK INSTRUMENTS EPOCH, USA) device. The quality of RNAs was determined according to the 260/280 ratio. Accordingly, RNA samples with a 260/280 ratio of 2.0 were used in cDNA synthesis. cDNA synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad). All procedures were performed by strictly following the manufacturer’s instructions. For this purpose, a mixture was prepared by adding 5 μl 5 × iScript Reaction Mix, 1 μl iScript Reverse Transcriptase and 4 μl iScript Nuclease-free water to 10 μl total RNA. The mixture was then incubated at 25 °C for 5 min, 46 °C for 20 min and 95 °C for 1 min in the ROTOR-GENE Q (Qiagen, Germany) device to generate cDNAs. After the concentrations of cDNAs were measured on the NanoDrop (BİOTEK INSTRUMENTS EPOCH, USA) device, the qRT-PCR stage was started. For quality control of cDNAs, the 260/280 ratio was taken as reference. cDNAs with a 260/280 ratio of 1.8 were used. The synthesized cDNA was then subjected to qRT-PCR using the primers listed in Table 2 and iTaq Universal SYBR Green Supermix (Bio-Rad). The reactions were performed in a Rotor-Gene Q (Qiagen) machine according to the manufacturer’s instructions. Gene expression was normalized to β-Actin using the 2-ΔΔCT method21.

Table 2 List of primer sequences.
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Statistical analysis

Histopathological and immunohistochemical data were analyzed using IBM SPSS, version 20.0 (IBM, USA; URL: https://www.ibm.com/products/spss-statistics). The differences between groups for histopathological data were analyzed using the non-parametric Kruskal–Wallis test, and the groups causing the differences were identified using the Mann–Whitney U test (p < 0.05). Immunohistochemical data were analyzed using one-way ANOVA followed by post hoc Tukey’s test (p < 0.001).

Results

Histopathological findings

Histopathological evaluation revealed statistically significant differences among the groups (Table 3, p < 0.05). The AP group exhibited the most severe degenerative changes, while treatments with AP + RES and AP + βC mitigated these changes to varying extents.

Table 3 Comparison of endocrine and exocrine pancreas among the groups.
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As shown in Fig. 1, histopathological examinations showed that the pancreas of rats in the C, RES, and βC groups had normal histological appearances. In other treatment groups, various degrees of pathological findings were observed in both endocrine and exocrine pancreas. Severe degenerative-necrotic cells were found in the Langerhans islets of the endocrine pancreas in the AP and AP + RES groups, whereas these changes were mild in the AP + βC group. Degenerative-necrotic changes were also observed in the acinar cells of the exocrine pancreas. These changes were severe in the AP group, moderate in the AP + RES group, and mild in the AP + βC group (Fig. 1).

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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Histopathological examination of the pancreas in different groups: (A) Control group, (B) RES group, (C) βC group. Normal histological appearance. (D) AP group. Severe degenerative-necrotic changes in the cells of Langerhans islets (arrow) and acinar cells (arrowhead). (E) AP + RES group. Severe degenerative-necrotic changes in the cells of Langerhans islets (arrow) and moderate changes in acinar cells (arrowhead). (F) AP + βC group. Mild degenerative-necrotic changes in the cells of Langerhans islets (arrow) and acinar cells (arrowhead) (H&E staining, magnification ×40).

Oxidative stress markers in pancreatic tissues

The MDA and GSH levels in pancreatic tissues are presented in Fig. 2. The results indicate that lipid peroxidation occurred in the pancreatic tissues following acute pancreatitis (AP), as evidenced by increased MDA levels (p < 0.001) and decreased GSH stores (p < 0.001). On the other hand, treatments with RES and βC significantly reduced MDA levels (p < 0.01 for both) and increased GSH levels (p < 0.01 for both) compared to the AP group.

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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Levels of oxidative stress markers MDA and GSH in pancreatic tissues. Values are expressed as mean ± SD. Statistical significance: Control vs. others: *** p < 0.001; AP vs. others: ### p < 0.001.

Immunohistochemical findings

Immunohistochemical staining for anti-insulin revealed statistically significant differences in the diameter and area of β + Langerhans islets among the groups (Table 4, p < 0.001).

Table 4 Comparison of the diameter and area of β + Langerhans islets among the groups.
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Immunohistochemical analysis showed that the diameter and area of β + Langerhans islets were normal in the C, RES, and βC groups. In other treatment groups, these measurements varied significantly. The AP group had the smallest diameter and area of β + Langerhans islets, while the AP + RES group had larger diameters and areas, and the AP + βC group had the largest diameters and areas (Fig. 3).

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Diameter and area of β + Langerhans islets (arrowhead) in different groups (immunohistochemical staining with anti-insulin antibody). (A) Control group, (B) RES group, (C) βC group. Normal histological appearance of β + Langerhans islets. (D) AP group. Group with the smallest β + Langerhans islets. (E) AP + RES group. Group with larger β + Langerhans islets. (F) AP + βC group. Group with the largest β + Langerhans islets (Immunohistochemistry, magnification × 20).

Relative mRNA transcript levels of inflammatory genes in pancreatic tissues

To determine the inflammatory status in pancreatic tissues, the relative mRNA transcript levels of NF-κB, TNF-α, and IL-1β genes were analyzed, and the results are shown in Fig. 4. The data indicate that these genes were upregulated in the pancreatic tissues following AP (p < 0.001 for all). However, treatments with RES and βC suppressed the expression of NF-κB (p < 0.01 for both), TNF-α (p < 0.01 for both), and IL-1β (p < 0.01 for both), demonstrating an anti-inflammatory effect.

Fig. 4
Fig. 4The alternative text for this image may have been generated using AI.
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Relative mRNA transcript levels of inflammatory genes NF-κB, TNF-α, and IL-1β in pancreatic tissues. Statistical significance: Control vs. others: *** p < 0.001; AP vs. others: ### p < 0.001.

Relative mRNA transcript levels of apoptotic and anti-apoptotic genes in pancreatic tissues

The relative mRNA transcript levels of the apoptotic genes Bax and Caspase-3 and the anti-apoptotic gene Bcl-2 in pancreatic tissues are shown in Fig. 5. The results indicate that AP induced an increase in the expression of Bax (p < 0.001) and Caspase-3 (p < 0.001) and a decrease in Bcl-2 levels (p < 0.001). Conversely, RES and βC treatments exhibited anti-apoptotic effects by downregulating Bax (p < 0.01 for both) and Caspase-3 (p < 0.01 for both) and upregulating Bcl-2 (p < 0.01 for both) compared to the AP group.

Fig. 5
Fig. 5The alternative text for this image may have been generated using AI.
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Relative mRNA transcript levels of apoptotic genes Bax and Caspase-3, and anti-apoptotic gene Bcl-2 in pancreatic tissues. Statistical significance: Control vs. others: *** p < 0.001; AP vs. others: ### p < 0.001.

Discussion

In this study, we investigated the potential therapeutic properties of RES and βC against AP induced by L-arginine using a rat model. The utilization of L-arginine in the induction of AP serves as a straightforward and effective experimental model, enabling the examination of various histological and biochemical alterations that closely mimic AP in humans22,23.

Histopathological assessment of the AP group revealed significant degenerative changes in both endocrine and exocrine pancreas, characterized by necrosis, edema, and inflammatory infiltration. Treatment with RES and βC effectively reduced these alterations, with βC showing superior efficacy in preserving pancreatic tissue integrity24,25,26. Similarly, immunohistochemical analysis demonstrated that AP significantly reduced β + Langerhans islets size, while RES and βC treatments preserved islet structure and size, maintaining pancreatic endocrine function. These findings correspond with recent studies showing antioxidant-mediated preservation of β-cell viability under inflammatory conditions11,27.

Oxidative stress is a critical factor in the pathogenesis of various diseases, including AP. The excessive formation of ROS during AP leads to lipid peroxidation, protein oxidation, and DNA damage, exacerbating cellular injury28. The levels of MDA, a lipid peroxidation product, reflect the level of free radicals in tissues29. Glutathione deficiency and the involvement of oxygen free radicals have been linked to acute pancreatitis in humans30. In this study, we observed significantly elevated levels of MDA, a marker of lipid peroxidation, and decreased levels of GSH, a crucial antioxidant, in the AP group. These changes highlight the imbalance between ROS production and antioxidant defenses in AP and these findings are consistent with the literature31,32. Studies have shown that treatments with RES and βC significantly reduced oxidative stress and restored antioxidants levels, suggesting that these compounds enhance the antioxidant defense system and mitigate oxidative stress. It has been shown that RES, with its potent antioxidant properties, could protect the intestinal tissue from I/R injury and could have a beneficial effect in inhibiting the oxidative damage in the liver induced by the chronic administration of ethanol33,34.

Inflammation is a hallmark of acute pancreatitis and is driven by the activation of various inflammatory pathways35. NF-κB is a central molecule that regulates the expression of many pro-inflammatory cytokines and mediators. Its activation leads to the recruitment of immune cells, exacerbating tissue injury and inflammation36. Previous studies have confirmed that NF-κB activation during AP is capable of producing numerous types of cytokines such as TNF-α, IL-1β, IL-2, IL-6 and IL-18, and various chemokines such as IL-837,38,39. In this study, we found that, the relative mRNA transcript levels of NF-κB, TNF-α, and IL-1β were significantly upregulated in the L-Arginine induced AP group, reflecting an intense inflammatory response. Treatments with RES and βC significantly suppressed the expression of NF-κB, TNF-α, and IL-1β, demonstrating their potent anti-inflammatory effects. These findings are consistent with previous research showing the anti-inflammatory properties of RES. For instance, RES has been shown to decrease serum levels of TNF-α and IL-6 in severe acute pancreatitis (SAP) modeled rats, reduce mortality, and suppress the production, activation, and release of inflammatory mediators40. Additionally, data have shown that NF-κB levels in SAP rats increased after modeling, while RES inhibited NF-κB activity and decreased the expression of TNF-α and IL-8, indicating that RES modulates cytokine expression by inhibiting NF-κB and the inflammatory response41. Furthermore, studies have demonstrated that RES effectively reduces NF-κB activation in inflammatory conditions, upregulating anti-inflammatory cytokines like IL-10 while suppressing pro-inflammatory mediators42,43. Similarly, βC exhibits significant anti-inflammatory properties by modulating the TLR4 signaling pathway, enhancing barrier function, and reducing inflammatory agents44,45. These findings align with our results showing that both compounds effectively suppress NF-κB activation and downregulate pro-inflammatory cytokines, thereby alleviating pancreatic inflammation.

Apoptosis, or programmed cell death, is another critical aspect of acute pancreatitis46. In current study, the imbalance between pro-apoptotic and anti-apoptotic factors determines the fate of pancreatic cells during L-arginine induced AP. The relative mRNA transcript levels of the pro-apoptotic genes Bax and Caspase-3 were significantly increased in the AP group, while the anti-apoptotic gene Bcl-2 was decreased, indicating enhanced apoptotic activity. Treatment with RES and βC exhibited anti-apoptotic effects by downregulating Bax and Caspase-3 and upregulating Bcl-2 levels. This suggests that RES and βC can modulate apoptotic pathways to protect pancreatic cells from programmed cell death, thus preserving pancreatic function. These findings are consistent with previous studies demonstrating the anti-apoptotic properties of antioxidants in various models of oxidative stress and inflammation9,11,31,44,47,48. The interplay between oxidative stress, inflammation, and apoptosis in the context of acute pancreatitis creates a vicious cycle that exacerbates pancreatic injury. Concurrently, the imbalance between pro-apoptotic and anti-apoptotic signals results in increased apoptosis of pancreatic cells, further compromising pancreatic function49,50.

Conclusion

RES and βC demonstrate therapeutic effects against L-arginine-induced acute pancreatitis through three key mechanisms: inhibition of the NF-κB inflammatory pathway, reduction of oxidative stress, and modulation of apoptotic pathways. These effects are evidenced by decreased inflammatory markers (NF-κB, TNF-α, IL-1β), improved oxidative status (reduced MDA, increased GSH), and regulated apoptotic gene expression (decreased Bax/Caspase-3, increased Bcl-2). Consequently, RES and Βc hold potential as therapeutic agents for the treatment of acute pancreatitis induced by L-arginine.