Comprehensive analysis of intestinal barrier function and microbial diversity changes in l-arginine-induced acute pancreatitis mice

Abstract

Introduction

While it has been established that intestinal health and microbial variations can influence pancreatic well-being, the extent of intestinal damage and the composition and diversity of the microbiota in animal models of l-arginine-induced acute pancreatitis (AP) remain unclear.

Methods

This study aims to evaluate the extent of intestinal damage and microbial changes in AP mouse models. The male C57BL/6 mice were randomly divided into two groups: the AP group received an intraperitoneal injection of 10% l-arginine, while the control group received an equivalent volume of saline. Histological analysis was used to assess morphological changes and damage in the pancreas and the duodenum, ileum, and jejunum. Fluorescent immunohistochemistry and immunohistochemical techniques were employed to evaluate the expression of myeloperoxidase (MPO) in the pancreas and Zonula occludens-1 (ZO-1) and Occludin in the small intestine. High-throughput sequencing was utilized to analyze the composition and diversity of the intestinal microbiota.

Results

l-arginine-induced AP in mice significantly increased serum lipase activity and decreased calcium levels, with an increase in pancreatic MPO expression. Additionally, we observed varying degrees of intestinal villi necrosis, edema, and inflammatory cell infiltration in the duodenum, jejunum, and ileum of the AP group mice. Notably, the expression of epithelial Occludin and ZO-1 was reduced in these regions. Compared to healthy mice, AP mice exhibited reduced bacterial richness and diversity, with changes in the intestinal microbiota including increased prevalence of Bacteroidetes and Proteobacteria, while the abundance of Firmicutes decreased.

Discussion

l-arginine-induced acute pancreatitis in mice resulted in significant alterations in the intestinal barrier and microbiota.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40001-025-03199-7.

Introduction

Pancreatitis, a severe digestive system disease, can be classified into chronic pancreatitis and acute pancreatitis (AP), the pathogenesis of which involves bile reflux and Pancreatic autodigestion leading to Pancreatic injury and an inflammatory response. Globally, the annual incidence of AP is approximately 33.74 cases per 100,000 person-years [23]. AP presents suddenly with prominent symptoms such as acute abdominal pain, nausea, vomiting, and shock. Although the majority of patients exhibit mild symptoms with a self-limiting course, the overall mortality rate still reaches 5% [20], with 10–20% of patients progressing to severe AP, where the mortality rate can range from 10 to 30% [13].

Intestinal microbiota homeostasis is a crucial indicator of gut health. Previous studies have generally struggled to link pancreatic pathology with changes in the gut microbiota, as there is no direct contact between the pancreas and intestinal microbes, and the pancreas itself is devoid of microbial presence. However, an increasing number of reports confirm a direct connection between pancreatitis and alterations in the gut microbiota, with several studies indicating that acute pancreatitis (AP) alters the intestinal microbiome [3, 4, 6, 24]. The dysregulation of the gut microbiota can induce AP, suggesting that maintaining healthy gut microbiota could have therapeutic implications. Therefore, exploring changes in the gut microbiota of AP using an experimental animal model is essential. We believe that the intraperitoneal injection of l-arginine to induce an AP model in mice offers advantages such as ease of operation, high reproducibility, and minimal animal stress, making it an excellent method to replicate human edematous acute pancreatitis. Although both clinical trials and animal models report a relationship between AP and gut health, the changes in gut microbiota and function in the l-arginine-induced mouse AP model remain unclear. Hence, this study aims to investigate the diversity, composition, and functional changes of the intestinal microbiota in the l-arginine-induced AP model.

Materials and methods

Animal models

Ten male C57BL/6 J mice, aged 6–8 weeks and weighing 20–22 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, Liaoning, China) (experimental animal license number: SCXK2023-0001). All studies were conducted following protocols approved by the Experimental Animal Ethics Committee of Shenyang Institute of Technology (No. SITLLBA2023013). The animals were housed under a 12-h light/dark cycle at temperatures ranging from 18 to 26 °C, with ad libitum access to water and food.

The study experiments were performed in accordance with relevant guidance and regulation. All experimental methods were performed in accordance with the ARRIVE guidelines (https://arriveguidelines.org), guidelines of the Chinese Institution of Laboratory Animal Sciences. The mice were randomly divided into two groups: a control group (CON) and an AP model group (AP). The AP model group received an intraperitoneal injection of 10% l-arginine at 4 g/kg, followed by a second injection after 1 h [7]. l-arginine (NO. 74-79-3 Purity ≥ 98%) were bought from Dalian Meilun Biotechnology Co., Ltd. The control group was given an equivalent volume of saline. 72 h after the l-arginine injection, the mice were euthanized, and two uncontaminated fecal samples from each mouse were collected with a sterile swab, stirred into the corresponding sample preservation solution, and sent to Suzhou Taihe Biotechnology Co., Ltd. (Zhejiang Province, China) for fecal microbiota analysis. Serum was collected for lipase and calcium ion monitoring. Mice from each group were anesthetized with isoflurane and sacrificed by cervical dislocation, parts of the pancreas and small intestine (duodenum, jejunum, and ileum) were fixed in 10% neutral buffered formalin.

Serological testing

Serum samples were collected and analyzed for calcium ion (C004-1-1) content and lipase (A054-2-1) activity using kits provided by Nanjing Jiancheng Bioengineering Institute.

Histological examination

Pancreatic and small intestinal tissues were fixed in 10% neutral buffered formalin for 48 h. Following fixation, the tissues were embedded in paraffin, sectioned, and processed through deparaffinization, hematoxylin and eosin staining, and dehydration before being cover-slipped. The pathological characteristics of each group’s pancreatic tissues were observed under a microscope, and images were captured for analysis.

Immunofluorescence

Paraffin-embedded samples were deparaffinized in xylene and rehydrated in ethanol. After washing three times in PBS (5 min each), the sections were incubated in EDTA buffer with autofluorescence quenching agent A for-antigen retrieval. The slides were then blocked with bovine serum albumin (BSA). They were incubated with MPO antibody (1:1000) at 4 °C overnight, followed by incubation with a fluorescently labeled secondary antibody (1:200) at room temperature for 50 min. DAPI was used for counterstaining. The slides were then treated with autofluorescence quenching agent B for 5 min at room temperature in the dark. Images were observed under a fluorescence microscope. Antibodies were purchased from Wuhan Servicebio Technology Co., Ltd.

Immunohistochemistry for detection of occludin and ZO-1 expression in small intestine tissues

Sections were routinely deparaffinized in xylene and digested with Proteinase K at 37 °C for 10 min, followed by PBS washes. Primary antibodies were added and incubated overnight at 4 °C in a refrigerator. Secondary antibodies were incubated for 30 min at room temperature in a humidified chamber. DAB staining was performed for 1 min followed by PBS washing. Staining time under the microscope was controlled, and PBS replaced the primary antibody as a negative control. Immunohistochemistry Results Interpretation: Positive staining for Occludin and Zonula occludens-1 (ZO-1) was defined as the presence of yellow or brownish-yellow staining in the cell membrane and cytoplasm. Each section was evaluated under 200 × magnification. Antibodies were purchased from Wuhan Servicebio Technology Co., Ltd.

16S rRNA gene sequencing

Total DNA was extracted using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Bacterial genomic DNA samples from all samples were stored at − 20 °C until further analysis. The V4 region of the bacterial 16S rRNA gene was amplified using the forward primer 357 F 5′-ACTCCTACGGGAGGCAGCAG-3′ and reverse primer 806R 5′-GGACTACHVGGGTWTCTAAT-3′. Sequencing was performed on the NovaSeq 6000 system (Illumina, USA).

Bioinformatics and statistical analysis

The sequencing reaction was performed by Suzhou Taihe Biotechnology Co., Ltd. (Zhejiang). Sequence quality control and filtering were conducted using Trimmomatic (Version 0.35), cutadapt (version 1.16), and mothur (Version 1.33.3). Operational taxonomic units (OTUs) were clustered using UPARSE software (usearch Version V8.1.1756, http://drive5.com/uparse/), with representative sequences annotated against the Silva 128 database for species information. Statistical analysis of community structure was conducted at various taxonomic levels including phylum, class, order, family, genus, and species, based on taxonomic information. Visualization was performed using ggplot2 (version 3.3.3) in R (version 3.6.3). Alpha diversity analyses, including species richness estimates (Chao, Ace) and diversity indices (Shannon, Simpson), and beta diversity analyses (based on OTU using Bray–Curtis and Jaccard, based on species information using Bray–Curtis, and based on phylogenetic distance using Weighted and Unweighted Unifrac) were conducted using the vegan package in R (version 3.6.3). Further statistical and visual analyses such as rarefaction curves, abundance rank curves, VENN diagrams, PCA, community structure bar plots, heatmaps, PCoA, and NMDS were performed using R (version 3.6.3).

We employed the Wilcoxon rank-sum test to determine whether there were differences in intra-group (α) diversity among different treatments. Multiple group comparisons were conducted using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test.

Results

Serum lipase activity and calcium ion content

During acute pancreatitis, pancreatic cells sustain severe damage, secreting large amounts of enzymes such as lipase (LPS), digestive enzymes, and proteases. These enzymes break down fats into a large quantity of fatty acids, which react with calcium ions (Ca²⁺) in the body, leading to a reduction in blood calcium levels and consequently, hypocalcemia associated with acute pancreatitis. As shown in Fig. 1, compared to the control group, the AP group mice exhibited a highly significant increase in lipase activity (P < 0.01) (Fig. 1A) and a highly significant decrease in serum calcium ion content (P < 0.01) (Fig. 1B).

Fig. 1.

Results of Serum Lipase Activity and Calcium Ion Content in Mice. A The activity of LPS in serum was determined. B The concentration of Ca²⁺ in serum was examined. **P < 0.01, compared with control group. AP: acute pancreatitis; LPS: Lipase. Data are expressed as the means ± SD, n = 5 in each group

Pathological observations of mouse pancreas and MPO expression results

Upon dissection, compared to the control group, we observed significantly aggravated damage in the pancreatic tissues, characterized by swelling and evident edema, with some necrosis around the pancreas (Fig. 2A). Microscopic examination revealed interlobular edema, diffuse acinar cell vacuolar degeneration, and extensive inflammatory cell infiltration and acinar cell necrosis at the lobular margins (Fig. 2B). Additionally, through fluorescent immunotechniques, we noted markedly higher expression of MPO in the pancreas of the AP group mice compared to the control group (Fig. 2C).

Fig. 2.

Pathological Observations of Pancreatic Tissue in Mice and MPO Expression Results. A Necropsy showed severe pancreatic edema in AP mice. B The pathological changes of pancreatic tissues in mice were observed under a light microscope using HE staining (H&E staining, scale bar = 200 μm). and C Immunofluorescence staining of MPO in pancreatic tissues was observed under a microscope (× 400). DAPI was used to stain the nucleus (blue). AP, acute pancreatitis group; Control, control group

Pathological observations of mouse small intestine tissues and expression of occludin and ZO-1

In the normal group, the small intestine mucosal structure was essentially normal with intact and orderly villi and no infiltration of inflammatory tissue. In the AP group mice, the duodenum, jejunum, and ileum exhibited significant villous necrosis, edema, and inflammatory infiltration (Fig. 3A). In the normal group, Occludin and ZO-1 showed pronounced positive expression in the intestinal epithelial tissues, appearing brown and brownish. The positive expression of Occludin in the epithelial tissue of the model group was reduced compared to the normal group (Supplementary Table 1). Concurrently, a notable positive expression of Occludin was detected in the intestine, suggesting an inward movement of this protein (Fig. 3B). Additionally, ZO-1 exhibited positive expression in the epithelial tissues of the model group (Fig. 3C), although this was decreased relative to the normal group (Supplementary Table 2).

Fig. 3.

Pathological Observations of Small Intestinal Tissue in Mice and Expression of Occludin and ZO-1. A The pathological changes of small intestine tissues in mice were observed under a light microscope (× 200) using HE staining. B Comparison of occludin protein expression in small intestine between groups (× 200). C Comparison of ZO-1 protein expression in small intestine between groups (× 200). Yellow star—Inflammatory infiltration; yellow triangle—Fibrous necrosis; yellow diamond—edema

Changes in intestinal microbiotarichness and diversity of the gut microbiota

We observed the fecal color of the two groups of mice; compared to the healthy group, the feces of the AP group were yellowish, more closely resembling the color of the rodent chow (Fig. 4A), and were also softer, indicating a decline in digestive function. Following Illumina sequencing analysis, the Venn diagram (Fig. 4B) illustrated the similarity and overlap in OTU numbers between the two groups. The Rank-abundance curve demonstrated that the richness and evenness of the gut microbiota in the AP group were significantly lower than those in the control group (Fig. 4C). Alpha diversity analyses of the fecal microbiota in both groups showed that the ACE index (Fig. 5A) and Chao index (Fig. 5B) were significantly lower in the AP group, indicating a reduction in microbial richness. Similarly, the Shannon and Simpson indices showed that the diversity of microbiota in the AP group was lower than that in the control group (Fig. 5C and D). Beta diversity analysis using PCoA revealed a distinct separation in the community composition between the two groups, with PC1 and PC2 explaining a total variance of 38.49% and 34.01%, respectively (Fig. 5E). PCA and NMDS analyses yielded similar results (Fig. 5F and G). Anosim analysis (Fig. 5H) resulted in an R value of 0.5996, greater than 0 with a P-value of < 0.01, indicating significant differences between the AP and control groups.

Fig. 4.

The appearance of the two groups of mice feces after modeling was completed (A), Venn diagram (B) and OTU-level ranked abundance curves (C)

Fig. 5.

OTU-level alpha diversity indices, such as observed species (A), Chao (B), Shannon diversity index (C) and Simpson index (D) richness of each group, and beta diversity analysis visualized via principal coordinate analysis (PCoA) (E), nonmetric multidimensional scaling (NMDS) (F), principal component analysis (PCA) (G) and analysis of similarities (ANOSIM) (H)

Changes in intestinal microbiota

Based on species information, we analyzed the differences between the AP group and the control group, comparing the richness from phylum to species. We observed differences between the AP group and the control group (Fig. 6). At the phylum level, the dominant microbial groups included Bacteroidetes, Firmicutes, and Proteobacteria. In the control group, Bacteroidetes accounted for 53.25%, Firmicutes for 42.83%, and Proteobacteria for 1.8%, while in the AP group, Bacteroidetes constituted 57.11%, Firmicutes 37.30%, and Proteobacteria 3.60% (Fig. 6A). The results indicated that the AP group had an increased abundance of Bacteroidetes and Proteobacteria, with a decrease in Firmicutes. At the family level, the primary families included Lactobacillaceae, Lachnospiraceae, Bacteroidaceae, and Rikenellaceae (Fig. 6B). Compared to the control group, the AP group showed a significant increase in the abundance of Lactobacillaceae, while Lachnospiraceae, Ruminococcaceae, and Veillonellaceae experienced significant declines (Fig. 6D, E). At the genus level, the predominant genera were Lactobacillus, Escherichia, Bacteroides, and Alistipes (Fig. 6C). Compared to the control group, the AP group demonstrated a significant increase in the abundance of Lactobacillus and Escherichia, while the abundance of Bacteroides and Alistipes significantly decreased (Fig. 6D). Additionally, we used LEfSe to detect differences in the microbial spectra between the two groups. Firmicutes.c was present in both groups; however, the AP group was primarily characterized by Enterobacteriaceae.g and Lactobacillaceae, while the control group was dominated by Lachnospiraceae.g and Ruminococcaceae.g. Moreover, the AP group predominantly featured Proteobacteria.c and Actinobacteria.c, whereas the control group was characterized by Bacteroidetes.c and Deferribacteres.c (Fig. 6F).

Fig. 6.

Relative abundances of the gut microbiota in theAP and CON groups at the phylum level (A), family level (B) and genus level (C), heatmap cluster at genus levels (D), cladogram of the LDA scores showing the abundant genera in AP (blue) and CON (red) (E) and cladogram generated by LEfSe analysis showing the enriched taxa in AP (blue) and CON (red) (F). AP, acute pancreatitis group; Control, control group; LDA, linear discriminant analysis; LEfSe, linear discriminant analysis effect size

Discussion

The l-arginine-induced acute pancreatitis model is a highly reproducible model extensively used in research related to acute pancreatitis. Previous studies have indicated that the gut microbiota of patients with acute pancreatitis undergoes changes; however, the intestinal function and microbial changes in the l-arginine-induced AP model have not yet been studied, and it is unknown whether there are differences compared to patients with acute pancreatitis. For the first time, we explored the intestinal function and microbial changes in the l-arginine model, providing a reference for further research in this area.

The intraperitoneal injection method is one of the non-invasive methods, known for its ease of operation, high reproducibility, and minimal animal stress. In 1984, Mizunuma et al. successfully established the first l-arginine-induced rat model of acute pancreatitis (AP), utilizing an intraperitoneal injection of l-arginine at a dose of 500 mg/(100 g · bw), which caused significant damage to pancreatic cells [12]. Studies have found that a single intraperitoneal injection of 500 mg/(100 g · bw) l-arginine can cause 70–80% necrosis of pancreatic cells [16, 17]. Similarly, our study employed intraperitoneal injection of l-arginine, with HE staining revealing extensive necrosis in the pancreatic cells of AP mice and increased MPO expression. Delaney CP administered three intraperitoneal injections of l-arginine (250 mg/100 g) within 10 days to rats, resulting in up to 90% acinar destruction [5, 11].

In recent years, more studies have discovered that changes in the gut microbiota occur during the development of AP, potentially relating to the severity of the disease. Both in AP patients and animal models of AP, it has been reported that abnormal secretion of pancreatic enzymes and structural damage to the pancreas lead to abnormal pancreatic secretion. This disrupts the intestinal homeostasis and alters the gut microbiota [1, 18]. Compared to healthy controls, patients with AP have been found to have higher abundances of Bacteroidetes and Proteobacteria, while Firmicutes and Actinobacteria are less abundant [25]. Tan et al. observed significant changes in the microbial composition between AP patients and healthy controls, characterized by a decrease in the genus Lactobacillus and a noticeable increase in the genus Enterococcus [19]. Animal experimental evidence also indicates similar changes in the gut microbiota of AP [3]. Using 16S rRNA high-throughput sequencing analysis, studies found that the gut microbiota diversity in rats with acute necrotizing pancreatitis significantly decreased. At the phylum level, the abundance of Saccharibacteria and Tenericutes significantly decreased. At the genus level, the abundance of Shigella and Lactobacillus significantly increased, while the abundance of Gluconobacter, Prevotellaceae UCG-001, Lachnospiraceae UCG-001, Ruminococcus, and Ruminococcaceae UCG-008 significantly decreased. In our experiment, it was found that in the AP group, the phyla Bacteroidetes and Proteobacteria were more abundant, while the abundance of Firmicutes decreased. Zhang also used this method to test human feces and found that AP patients, compared to healthy individuals, had a higher abundance of Bacteroidetes and Proteobacteria, with a decreased abundance of Firmicutes and Actinobacteria [26]. These results indicate that our l-arginine-induced mouse model can better simulate the changes in the gut microbiota of AP patients, with typical changes including a significant increase in the abundance of Escherichia and dominance of the family Enterococcaceae.

The normal intestinal mucosal physical barrier consists of the mucous layer, intestinal mucosal epithelial cells, and tight junctions between cells [8, 9], with continuous renewal of mucosal epithelial cells to maintain the integrity of the mucosal barrier. Research has shown that damage to the intestinal mucosal barrier is one of the main complications of acute pancreatitis (AP). A meta-analysis indicated that 59% of AP patients experience intestinal barrier injury [21], leading to increased intestinal mucosal permeability. This, in turn, causes bacterial translocation from the intestines, pancreatic tissue necrosis and infection, resulting in multiple organ dysfunction syndrome (MODS). This study employed immunohistochemistry to assess the expression of tight junction proteins Occludin and ZO-1. While no significant changes were observed, we noted a reduction in the expression of Occludin and ZO-1 in the small intestine of AP mice. Occludin is typically expressed on the surface of intestinal epithelial cells and glandular cells, as well as in the cytoplasm. Results indicated a reduction in the expression of Occludin and ZO-1 in AP mice, suggesting impairment of the intestinal barrier. Lipopolysaccharides (LPS) secreted by bacteria such as Escherichia coli inhibit the expression of ZO-1 and occludin in intestinal epithelial cells, leading to the accumulation of ZO-1 at cell junctions and altering its spatial distribution, thereby increasing intercellular permeability and causing damage to the epithelial barrier [2, 27]. Research by Pan demonstrated a significant reduction in the expression of the tight junction proteins ZO-1 and Occludin in a NaT-induced SAP rat model. Similar results were shown in caerulein-induced AP in BALB/c mice, where the expression of intestinal tight junction proteins ZO-1 and Occludin also decreased [14, 15]. In recent years, an increasing number of studies in animal models have observed that dysbiosis of the gut microbiota and the integrity of the intestinal barrier are closely related and also influence the severity of acute pancreatitis (AP). Xueyang Li found that increasing the content of the beneficial bacterium Lactobacillus can inhibit the growth of the pathogenic Escherichia coli-Shigella, thereby maintaining intestinal mucosal barrier function and ultimately mitigating the pathological changes of AP [8, 9]. Conversely, the intestinal barrier also affects changes in the gut microbiota; damage to the intestinal mucosal barrier is one of the main complications of AP. A meta-analysis showed that 59% of AP patients experience increased permeability due to intestinal mucosal barrier injury [22]. Increased intestinal permeability allows bacterial translocation into the bloodstream, and the endotoxins produced by these bacteria can also enter the bloodstream, leading to necrotizing Pancreatitis. In 68.8% of AP patients, bacterial DNA representing the gut microbiota can be detected in the blood, indicating that during AP, gut microbiota translocated to the blood through the damaged intestinal mucosal barrier [10]. Therefore, the interaction between the gut microbiota and the intestinal barrier is crucial to the pathogenesis and progression of AP.

In summary, our results demonstrate that l-arginine-induced AP in mice leads to significant pathological changes, with a decrease in the richness and diversity of intestinal bacteria. The primary changes include an increased abundance of Bacteroidetes and Proteobacteria, while the abundance of Firmicutes is reduced. Additionally, there is a decrease in the positive expression of Occludin and ZO-1 in the intestinal epithelium, indicating an impaired intestinal barrier. These findings support the utility of this model for preclinical drug development targeting AP. However, this study has certain limitations. First, the mechanistic link between gut microbiota dysbiosis and intestinal barrier dysfunction in AP requires further validation. Second, the long-term effects of l-arginine-induced acute pancreatitis on gut microbiota composition and metabolic characteristics have not been explored, and future studies should include multi-omics analysis to elucidate these dynamic changes. Additionally, while mouse models provide valuable insights, differences between mouse and human acute pancreatitis pathophysiology mean that caution is needed when directly extrapolating these findings to clinical settings.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, P.Z; methodology, K. I. P. and Y.L.; software, Z.C.; validation, Z.C. and X.W.; investigation, Y.L.; resources, P.Z.; data curation, Y.L.; writing—original draft preparation, P.Z.; funding acquisition, P.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Shenyang institute of technology major issue support fund projects, under grant no.DZ202404 and Joint Program of Science and Technology Program of Liaoning Province (Technical Research Program) (2024JH2/102600210).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The protocol for this study was approved by the Ethics Committee of Shenyang Institute of Technology (SITLLBA2023013, approval date: March 3, 2023). The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) and guidelines of the Chinese Institution of Laboratory Animal Sciences (https://cnilas.org/en/).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.