rIL-22 Alleviates Severe Acute Pancreatitis and Secondary Multiple Organ Injury Induced by Caerulein in Mice.

Introduction
Acute pancreatitis (AP) is one of the most common inflammatory diseases, with a steadily increasing incidence over the past decade [1, 2]. While the majority of patients experience mild acute pancreatitis (MAP) that resolves spontaneously within a week, approximately 20% develop severe acute pancreatitis (SAP) [3]. SAP is a life-threatening inflammatory disorder characterized by pancreatic or peripancreatic tissue necrosis, systemic inflammatory response syndrome (SIRS), multi-organ dysfunction syndrome (MODS), resulting in a considerable mortality rate of 20–40% despite advancements in critical care [4, 5]. The pathogenesis of SAP involves premature activation of pancreatic enzymes, autodigestion, and a cascade of inflammatory mediators that drive both local pancreatic injury and distant organs failure [6]. Notably, patients with multiple organ failure (mainly involving the lungs, liver, kidneys and colon) often require prolonged intensive care and invasive interventions to manage local and systemic complications [7]. Given that current treatment strategies remain largely supportive and no effective therapeutic agent has been identified to mitigate SAP progression, there is an urgent and unmet clinical need to develop a drug capable of simultaneously protecting multiple target organs from damage in SAP.
IL-22, a member of the IL-10 cytokine family, is primarily secreted by T cells, with a core biological role in tissue repair, barrier integrity maintenance and epithelial protection through epithelial cell-specific receptor activation [8, 9]. In various disease models, IL-22 has been shown to mitigate injury in isolated organs such as the kidneys [10–13], liver [14, 15], lungs [16], and colon [17] through shared mechanisms, including anti-inflammatory [18, 19], anti-apoptotic [20] and pro-regenerative effects. However, these beneficial findings derive exclusively from single-organ or single-disease models. It remains unclear whether IL-22 can retain its coordinated protective function in a systemic crisis like SAP, where multi-organ injury is triggered from a single origin. Recently, several studies have suggested the protection of IL-22 in AP. IL-22 has been shown to alleviate pancreatic [21–23] or intestinal injury in MAP. Studies has extended to the role of IL‑22 in SAP, providing evidence that IL-22 can alleviate pancreatic injury and improve gut microbiota dysbiosis [5, 24]. However, the current studies, including MAP and SAP, have failed to demonstrate whether IL-22 functions as a systemic multi-organ protectant or to integrate its multifaceted protective mechanisms involving cell death pathways, such as apoptosis and autophagy. Specifically, it remains unproven whether IL-22 can simultaneously mitigate injury to the key distant organs, including the lungs, liver, kidneys and colon in SAP. The failure of these organs directly determines mortality. Therefore, the therapeutic value of IL-22 in SAP, especially its ability to improve the fatal process of MODS, is still inconclusive.
This study aims to elucidate the therapeutic effect of rIL-22 on CAE-induced SAP mice, focusing on its regulatory role in pathology, inflammation, apoptosis, and autophagy across multiple organs. By defining therapeutic potential of rIL-22, our work may provide a foundation for developing targeted immunomodulatory strategies of treating SAP and its associated subsequent multiorgan injury.

Materials and methods

Animals
Male Balb/c mice (20–25 g, 6–8 weeks old) were purchased from Ji’nan Pengyue laboratory Animal Breeding CO., Ltd. (JiNan, China). All the mice were housed at a constant temperature of 25℃ and a relative humidity of 55% on a 12-h light/dark cycle and allowed free access to laboratory diet and water. All the animal experiments were approved by the local ethical committee at Shandong Provincial Hospital Affiliated to Shandong First Medical University.

Mouse Model For SAP and Treatment
The mice were randomly assigned to 3 groups: normal saline group (NS group), SAP group (SAP group), and rIL-22 treatment group (SAP + rIL-22 group). Before the experiment, all mice were fasted for 12 h and free to drink water. Mice in the SAP and SAP + rIL-22 groups were injected intraperitoneally (ip) CAE (MedChemExpress (Monmouth Junction, NJ, USA), which was dissolved in 0.9% sterile saline, administered at 100 µg/kg body weight every hour for 12 injections [25]. Lipopolysaccharide 10 mg/kg was added to the last CAE injection. The NS group received physiological saline injections. rIL-22 (MedChemExpress (Monmouth Junction, NJ, USA) (30 µg/Kg, 4 times) was administered (ip) to mice in the SAP + rIL-22 groups (Fig. 1A). The mice were sacrificed 24 h after the first CAE injection. Blood, pancreas, liver, lungs, kidneys and colon were harvested.

Intestinal Permeability
Intestinal permeability was assessed by measuring the absorption of fluorescein isothiocyanate (FITC) - dextran (Shanghai Yuanye Bio-Technology Co., Ltd., China) following orogastric gavage (0.4 g/kg, 30 mg/ml). The extent of FITC diffusion was observed by small-animal imaging (IVIS Spectrum CT). Mice were sacrificed 4 h after gavage, and the blood was harvested in 1.5 ml EP tubes and centrifuged at 4 °C, 3000 g for 10 min under the condition of avoiding light. Plasma was diluted in an equal volume of PBS and measured for FITC–dextran concentration with a fluorescence microplate reader (Tecan Spark) at an excitation wavelength of 485 nm and emission wavelength of 530 nm. Standard curves were obtained by diluting FITC–dextran in non-treated plasma diluted with PBS.

Biochemical Marker Assays
The blood samples were centrifuged (3,000 g; 10 min), after centrifugation, serum was aspirated into 1.5 ml centrifuge tubes and stored at −80 ℃ until analysis. All the plasma biochemical parameters (Amylase, Lipase, aspartate transaminase (AST), alanine transaminase (ALT), creatinine (CREA), blood urea nitrogen (BUN), tumor necrosis factor-α (TNF-α), IL-6, IL-18 and IL-1β) were detected by the clinical laboratory of Shandong Provincial Hospital (Jinan China) using an Architect-i2000 system (Abbott Laboratories, USA). The quantitative determination of biomarkers was considered positive according to the criteria set by the manufacturer.

Hematoxylin and Eosin (H&E) and Histological Analysis
Pancreas, liver, lungs, kidneys and colon biopsy tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into 5-µm-thick sections. After deparaffinization and hydration, the sections were incubated in hematoxylin for 2 min and eosin for 1 min. Histopathological changes in different organs were observed using VS200 Research Slide Scanner (Olympus). The pancreas score system focused on four aspects: edema, inflammation, necrosis and vacuolization [26]. A liver injury score was assigned based on cell swelling, necrosis, inflammation and congestion [27, 28]. Kidney injury was scored based on renal tubular epithelial cell edema, vacuolar degeneration, cell lysis, loss of brush border and cast formation [29]. Lung injury was scored using the Smith scale [30], which considers edema, inflammation, hemorrhage, atelectasis and hyaline membrane. Colon pathology scores were assessed by inflammatory cell infiltration and basal crypt destruction [31]. Different tissue damage scores were evaluated by histopathologist in a blind manner (Tables 1, 2, 3, 4 and 5).

Immunohistochemistry (IHC) Staining
Pancreas, liver, lungs, kidneys and colon biopsy tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into 5-µm-thick sections. After deparaffinization and hydration, the sections were incubated with citrate and blocked with 3% H2O2. The slides were confined with 5% goat serum and incubated with primary antibody (anti-IL-6: 1:100, #DF6087, Affinity Biosciences; anti- myeloperoxidase (MPO): 1:300, #22225-1-AP, Proteintech; anti-IL-1β: 1:100, #AF5103, Affinity Biosciences; anti-Bcl-xl: 1:200, #2764, Cell Signaling Technology; anti-Bax: 1:1000, #db14911, Diagbio; anti-cleaved caspase-3: 1:200, #25128-AP, Proteintech; anti-Beclin1: 1:50, #11306-1-AP, Proteintech; anti-P62: 1:250, # 18420-1-AP, Proteintech), overnight at 4 °C. After washing in PBS, the sections were incubated with appropriate biotinylated secondary antibodies, stained with diaminobenzidine (DAB).

TUNEL Staining
The TUNEL assay was conducted using the TUNEL detection kit (TUNEL, Beyotime) to assay cell apoptosis according to the instructions. Briefly, paraffin sections of pancreas, liver, lungs, kidneys and colon tissues were first dewaxed in xylene for 5–10 min, then replaced with fresh xylene, and dewaxed again for 5–10 min. The resulting sections were treated with anhydrous ethanol for 5 min, 90% ethanol for 2 min, 70% ethanol for 2 min, distilled water for 2 min, in proper order. The deparaffinized sections were successively incubated with 20 µg/mL DNase-free proteinase K (30 min, 20–37°C). Then, they were washed three times by 1× PBS. Subsequently, 50 µl the TUNEL reaction mixture (5 µl TdT enzyme solution and 45 µl labeling solution) was added, and the resulting mixture was incubated for 1 h at 37 degrees Celsius in a dark humidified environment. The slides were counterstained with 4’,6-Diamidino-2-phenylindole (DAPI) and visualized with the EVOS M7000 Imaging System (Thermo Fisher Scientific).

Cell culture and Treatment
Rat pancreatic acinar cells (AR42J) were maintained in Ham’s F‑12 K (Gibco) basal medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Human non-small cell lung cancer cells (A549) were cultured in Ham’s F‑12 K (Gibco) basal medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Human colon carcinoma cell lines (Caco-2) were maintained in Minimum Essential Medium (MEM, Gibco) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were maintained at 37 °C and 5% CO2. Autophagy activator, Rapamycin (MCE, HY-10219) was dissolved in Dimethyl sulphoxide (DMSO) at a stock concentration of 1mM and stored at − 20 °C. The above-mentioned cells were incubated with Rapamycin concentrations of 100 nM or the DMSO control. After 2 h, rIL-22 was added dissolved in dH2O (100nM). After further incubation at 37˚C for 24 h, the cells were collected for Western Blot. Apoptosis activator MDK83190 (MCE, HY-18633) was dissolved in DMSO at a stock concentration of 1mM. The above-mentioned cells were incubated with MDK83190 concentrations of 100 µM or the DMSO control. After 2 h, rIL-22 was added dissolved in dH2O (100nM). After further incubation at 37˚C for 24 h, the cells were collected for Western Blot.

Western Blot Analysis
The proteins of tissues or cells were extracted with protein lysate buffer (#AG21502, ACCURATE BIOTECHNOLOGY(HUNAN)CO.,LTD, ChangSha, China) containing protease inhibitor mix PMSF (#GC10477, Glpbio (Montclair, CA, USA). After determining the protein concentration by a bicinchoninic acid (BCA) protein assay kit (Solarbio, Beijing, China), the proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA), which were blocked in 5% nonfat milk for 1 h at room temperature and then incubated with the indicated primary antibodies against Claudin-1 (1:1000, abcam, #ab307692, UK), Occludin (1:1000, abcam, #ab216327, UK), IL-6 (1:500, Affinity Biosciences, #DF6087, China), MPO (1:1000, Proteintech, #22225-1-AP, China), IL-1β (1:500, Affinity Biosciences, #AF5103, China), Bcl-xl (1:1000, Cell Signaling Technology, #2764, USA), Bax: (1:1000, Diagbio, #db14911, China), cleaved caspase-3 (1:200, Proteintech, #25128-AP, China), LC3B (1:1000, ABclonal, #A19665, China),
Beclin1 (1:1000, Proteintech, #11306-1-AP, China), P62 (1:1000, Proteintech, #18420-1-AP, China) and GAPDH (1:1000, Abcam, ab8245, UK) overnight at 4 °C. The next day, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (Millipore, USA) with an Amersham Imager 680 (GE Healthcare, USA), and quantified with Image-Pro Plus 6.0 software. GAPDH served as an internal control.

Statistical Analysis
All statistical analyses were performed with GraphPad Prism 8.0 software (GraphPad Software, La Jolla, CA, USA). Data are presented as the means ± standard deviations (SDs). Differences were analyzed with Student’s t-test for the comparison of two groups and by one-way analysis of variance for the comparison of multiple groups. Experiments were repeated at least three independent times. For all analyses, the P-values reported were two-tailed, and P-values < 0.05 were considered statistically significant.

Results

rIL-22 Ameliorates CAE-induced Pathological Injury in the Pancreas and Multiple Organs
Mice in the SAP group exhibited evident intra-abdominal inflammation, along with visible pancreatic congestion (Fig. 1B). After rIL-22 treatment, abdominal inflammation and pancreatic congestion were significantly reduced, but pancreatic edema was still observed. H&E staining and histological analysis revealed inflammatory cell infiltration in the pancreas, liver, lungs, kidneys and colon and varying pathological damage in the pancreas, lungs and colon of CAE-treated mice (Fig. 1C). In the pancreas, distinctively pancreatic acinar edema, acinar cell necrosis, extensive inflammatory cell infiltration (arrowheads), and bleeding were observed in the SAP group. In the liver, SAP group showed hepatocyte swelling, a small amount of inflammatory cells infiltration, and congestion in central vein while compared with NS group (Fig. 1C). Similarly, the lungs of SAP mice exhibited alveolar wall swelling, alveolar interstitial thickening, alveolar deformation, hemorrhage, and significant lymphocyte infiltration (arrowheads) (Fig. 1C). In the kidneys, hemorrhage occurs within and between the glomeruli, partial renal tubular epithelial vacuole degeneration or edema, minimal lymphocyte infiltration (arrowheads) (Fig. 1C). In the colon, intestinal injury includes submucosal edema, reduction of goblet cells, crypt atrophy, infiltration of inflammatory cells and red blood cell effusion (arrowheads) (Fig. 1C). Notably, rIL-22 treatment alleviated pathological changes across all examined organs in SAP group (Fig. 1C). These findings suggested that rIL-22 mitigates pancreatic injury and protects against secondary multiple organs, especially the lungs and colon damage in SAP.

rIL-22 Alleviates CAE-induced Dysfunction in the Pancreas and Multiple Organs
To evaluate the functional impairment of various organs, we measured the serum levels of various biochemical markers including pancreatic injury indicators serum Amylase (Fig. 2A), Lipase (Fig. 2B), liver function indicators ALT (Fig. 2C) and AST (Fig. 2D), kidney function indexes CREA (Fig. 2E) and BUN (Fig. 2F) in serum samples collected from the mice in different groups. These biomarkers were significantly elevated in SAP group compared to the NS group, indicating severe multiple organs dysfunction. However, treatment with rIL-22 effectively reduced the levels of these biomarkers of SAP group. In addition, we also detected that the expression of IL-22RA1 in multiple organs increased after rIL-22 treatment (Fig. 2G), indicating that the effect of rIL-22 on multiple organs may be achieved through IL-22RA1. Furthermore, given the critical role of tight junction proteins including Occludin and Claudin1 in maintaining gut barrier function, we assessed their expression in colon tissues using Western blot and IHC. As shown in Fig. 2H and I, rIL-22 treatment significantly upregulated Occludin and Claudin-1 expression in the colon of SAP group. We found that SAP group exhibited elevated serum levels of FITC, which were mitigated by treatment with rIL-22 (Fig. 2J). It was observed that FITC fluorescence was more concentrated in the SAP group, while the fluorescence area significantly decreased after rIL-22 treatment via small-animal imaging (Fig. 2K), suggesting the potential role of rIL-22 in preserving gut barrier function. The above results reveal that rIL-22 improves CAE-induced dysfunction of pancreas, liver, lungs and kidney, and the intestinal permeability of colon.

rIL-22 Attenuates the CAE-induced Systemic Inflammation
To evaluate the systemic inflammatory response, we measured the serum inflammatory cytokines, including TNF-α (Fig. 3A), IL-6 (Fig. 3B), IL-1β (Fig. 3C), and IL-18 (Fig. 3D). Compared with NS group, SAP mice exhibited a significant increase in these cytokine levels, indicating a robust inflammatory response. However, rIL-22 treatment markedly reduced cytokine levels, suggesting its anti-inflammatory properties. To further investigate inflammation at the tissue level, we assessed the expression of IL-6, MPO, and IL-1β in multiple organs using IHC and Western blot. MPO, a neutrophil-specific protein, is commonly used to quantify neutrophil infiltration. As shown in Fig. 3E-H, compared to those in the NS group, the expression levels of IL-6, MPO and IL-1β were dramatically increased in SAP group. In contrast, rIL-22 treatment significantly reduced the expression of IL-6, MPO and IL-1β of SAP group. The above findings suggest that rIL-22 significantly decreased the systemic inflammatory responses of SAP mice.

rIL-22 Reduces CAE-induced Apoptosis in the Pancreas and Multiple Organs
To assess cell apoptosis in various organs, we performed IHC and Western blot on the pancreas, liver, kidneys, lungs, and colon. Results demonstrated that rIL-22 treatment successfully suppressed the expression of Bax and cleaved caspase-3 (Fig. 4A, C and E). Conversely, the expression of Bcl-xl was significantly upregulated in all examined organs following rIL-22 treatment compared with the SAP group (Fig. 4B). Consistent with IHC and Western blot, TUNEL assay revealed that significant numbers of apoptotic cells of pancreas and colon were observed in SAP group, whereas rIL-22 treatment markedly reduced apoptosis (Fig. 4D). Although the apoptotic cells in the liver, lungs and kidneys of the SAP group were not obvious, the expression of apoptotic proteins was significant, and rIL-22 significantly improved the expression of apoptotic proteins. Nevertheless, in general, rIL-22 confers a protective effect by inhibiting apoptosis in SAP-induced multiple organs damage.

rIL-22 Alleviates CAE-Induced Autophagy in the Pancreas and Multiple Organs
As reported in literature, autophagy and apoptosis are closely related that often occur within the same cell. Moreover, it has shown that inflammatory cytokines raise autophagy levels in cells, potentially causing apoptosis and damage [32, 33]. While autophagy mainly serves as a survival mechanism in response to different stressors, its overactivation can lead to cell death [34–37]. Therefore, we detected the expression level of critical autophagic indicators using both IHC and Western blot in the pancreas liver, lungs, kidneys and colon tissues. As shown in Fig. 5A and B, the expression of Beclin1 was increased in SAP group compared with the NS group, while the expression of p62 was decreased. However, the expression of Beclin1 was reduced, as well as p62 levels were elevated in the SAP + rIL-22 group compared to those in the SAP group (Fig. 5A and B). Similarly, Western blot results indicated that the levels of Beclin1 and LC3BⅡ were upregulated, whereas p62 expression was downregulated in the SAP group (Fig. 5C). Notably, rIL-22 intervention reversed these changes, decreasing Beclin1 and LC3BⅡ expression while increasing p62 levels (Fig. 5C). These findings collectively suggest that rIL-22 plays a protective role in SAP mice by modulating autophagy-related proteins, thereby potentially alleviating the severity of SAP.

rIL-22 Reverses Apoptosis and Autophagy Induced by Stimuli Other Than Inflammation
To investigate whether the alleviating effect of rIL-22 on autophagy and apoptosis is a direct action, we stimulated Rat pancreatic acinar cells (AR42J), Human non-small cell lung cancer cells (A549) and Human colon carcinoma cell lines (Caco-2) with apoptosis activator (MDK83190) and autophagy activator (Rapamycin), and then treated them with rIL-22. As shown in Fig. 6A and D, the expression of Beclin1 and LC3B Ⅱ was increased in the Rapamycin group, while the expression of p62 was decreased compared with the DMSO group. However, the expression of Beclin1 and LC3B Ⅱ was reduced, and p62 levels were elevated in rIL-22 treatment group compared with the Rapamycin group. In the MDK83190 + rIL-22 group, rIL-22 successfully suppressed the expression of Bax and cleaved caspase-3 induced by MDK83190 (Fig. 6E and H). Conversely, the expression of Bcl-xl was significantly upregulated in all examined cells following rIL-22 treatment compared with the MDK83190 group (Fig. 6E and G). The above results reveal that rIL-22 could directly reverse cell apoptosis and autophagy induced by stimuli other than inflammation.

Discussion
SAP is an inflammatory condition characterized by pancreatic edema, necrosis, and extensive immune cell infiltration [1]. Pancreatic acinar cell injury triggers the release of inflammatory mediators such as cytokines and chemokines, which recruit both innate (e.g., neutrophils, macrophages, NK cells) and adaptive (e.g., T and B cells) immune cells [38]. This cascade initiates an uncontrolled inflammatory response within the acinar cells, eventually leading to SIRS and MODS, which significantly contribute to the high mortality and unfavorable prognosis observed in SAP [39, 40]. The development of a therapeutic agent capable of concurrently mitigating multiple organs injury remains a critical unmet need in the management of SAP. IL-22 has been confirmed to have various protective effects, such as promoting liver regeneration [14, 15], alleviating lung injury [16], and ameliorating renal fibrosis [41, 42]. Still, these effects have not been proved in systemic multi-organ damage secondary to SAP. Our findings demonstrated that rIL-22 attenuated necrosis, hemorrhages, and inflammation of multiple organs. In addition, rIL-22 treatment decreased serum levels of amylase, lipase and inflammatory cytokines in SAP group. The expression of IL-22RA1 in multiple organs increased after rIL-22 treatment, indicating that the effect of rIL-22 on multiple organs may be achieved through IL-22RA1.
IL-22 has demonstrated tissue-protective effects by enhancing epithelial integrity, suppressing oxidative stress, promoting cellular regeneration, and enhancing anti-apoptosis genes in models of single organ injury [43–46]. Our previous studies have shown that IL-22 could mitigate CAE-induced MAP by activating AKT/mTOR pathway [22] and L-arginine‐induced SAP - associated lung injury by inhibiting inflammation through the STAT3 signaling pathway [47]. These results suggest rIL-22 as a potential therapeutic candidate for SAP and its subsequent multiple organ injury.
Our study confirms that rIL-22 alleviated the pathological damage of kidneys, liver and lungs tissue secondary to SAP, while reducing the expression of inflammatory cytokines of tissues. It is worth noting that in the TUNEL staining, rIL-22 did not show significant effects on liver, lungs and kidneys apoptosis, as the number of apoptotic cells in the SAP group was not obvious. This may be because the observation time was short (only 24 h) and the apoptosis of the liver, lungs and kidneys had not yet occurred on a large scale. Future studies could extend the observation time to 36–72 h.
Beyond parenchymal organ damage, SAP disrupts intestinal barrier integrity, increasing permeability and leading to bacterial translocation and endotoxin release [48]. Studies have shown that IL-22 can improve intestinal microflora imbalance and maintain the integrity of intestinal mucosal barrier in intestinal diseases [49–51]. In our study, we found that rIL-22 not only improved colonic mucosal damage, but also decreased the expression of colonic tight junction proteins, reduced intestinal permeability, and reduced inflammatory cytokines in intestinal injury secondary to SAP. This is a critical step in preventing the development of SIRS and MODS, given that SAP progression is closely linked to intestinal permeability. We have found that a recent study [24] indicates that IL-22 plays a crucial role in ameliorating the intestinal mucosal barrier dysfunction and microbiota dysbiosis during SAP. Our findings complement and extend this observation by demonstrating that therapeutically administered rIL-22 not only corroborates the protection of the intestinal barrier but also provides simultaneous protection to the pancreas, liver, lungs, and kidneys. The concurrent suppression of apoptosis and autophagy across these organs, coupled with the amelioration of systemic inflammation, suggests that the therapeutic value of rIL-22 on multiple organs in SAP, positioning it as a systemic therapeutic agent.
It was previously suggested that autophagy and apoptosis could happen at the same time, with autophagy being induced by certain apoptosis inducers [52]. So, we presume that in SAP, especially in the most severe stages, apoptosis and autophagy occur simultaneously. Our results confirm this idea, showing that the expression of apoptotic proteins and autophagic proteins increased in SAP mice across multiple organs, with rIL-22 treatment reducing the apoptotic and autophagic level. Although IL-22 has been shown to inhibit the autophagic pathway in MAP mice [53], our findings extended the current understanding of IL-22 beyond its known single-organ effects, a dimension less explored in prior IL-22 research. To investigate the alleviating effect of rIL-22 on autophagy and apoptosis is a direct action, we treated three types of cells with Rapamycin and MDK83190. Results revealed that rIL-22 could directly inhibit cell apoptosis and autophagy induced by stimuli other than inflammation. This suggests that IL-22 acts as a protective role against cellular damage by directly acting on multiple organs in SAP. The regulation of apoptosis and autophagy by rIL-22, as observed in our study, offers a mechanistic explanation for its therapeutic potential.
A key strength of this study is its comprehensive and systematic approach to evaluating the therapeutic effect of rIL-22. Unlike previous reports focusing on a single organ, we concurrently assessed pathology, function, inflammation, apoptosis, and autophagy across five major organs (pancreas, liver, lungs, kidneys, and colon) affected by SAP, providing a holistic view of its protective efficacy. Furthermore, our findings enhance the clinical translational potential of rIL-22, demonstrating that a single cytokine can concurrently mitigate a cascade of distant organ injuries, which is the primary challenge in SAP management. Despite the systemic evidence presented, our study has limitations. Firstly, the precise molecular mechanisms by which rIL-22 exerts its protective effects require further elucidation. Secondly, as with any pre-clinical study, the translatability of findings from a murine model to human SAP needs to be verified in other experimental models, and ultimately in clinical settings. Finally, our study focused on the acute phase of organ injury. The long-term therapeutic benefits and potential safety profile of rIL-22 administration beyond 24 h were not evaluated.
In summary, we suggest that IL-22 has the potential to improve SAP and secondary multiple organ injury, especially the lungs and the colon, by concurrently attenuating pathological damage, inflammation, apoptosis and autophagy. This study underscores the therapeutic versatility rIL-22 in systemic inflammatory conditions and fills a critical gap in managing SAP complications.