All human blood samples were obtained as discarded clinical samples at the First Clinical College of Harbin Medical University. Procedures involving human blood samples were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (Protocol Number: 2021IIT060). All subjects signed an informed consent form.

Thirty-two male Wistar rats (180–200 grams) were provided by the Animal Research Center of the First Affiliated Hospital of Harbin Medical University (Harbin, China). The animal care and experimental protocols were all approved by the Institutional Animal Care and Use Committee of Harbin Medical University. All animals were fasted for 8 h before modeling and were maintained under a 12-h light/dark cycle. A rat model of AP was established. First, the rats were anesthetized by administering a intraperitoneal injection of 10 mg/kg sodium pentobarbital (Sigma–Aldrich, MO, USA) dissolved in 300 µl of saline. Then, the abdomen was opened along the midline, and the distal pancreaticobiliary duct was ligated. The rat AP model was established through the retrograde injection of 300 μl of 3.5% sodium taurocholate (Sigma-Aldrich) into the pancreatic duct. Two hours after the rat AP model was established, the rats were allocated into the following four groups to explore the effect of EVs on macrophages in vivo: sham (sham operation), AP (AP model), AP+Ctrl-ev and AP+Cae-ev (n=4 rats per group). Rats in the AP+Ctrl-ev group received EVs from AR42J cells (ACs) treated with phosphate-buffered saline (PBS) and rats in the AP+Cae-ev groups received EVs from ACs treated caerulein. Afterward, 200 μg (4x10¹¹ particles) of EVs were intraperitoneally injected. Six hours later, we sacrificed the rats and harvested the tissue. We allocated rats into another four groups 2 hours after the rat AP model was established to confirm the role of miR-183-5p in promoting M1 macrophage polarization in vivo: NC-mimic, NC-inhibitor, miR-183-5p mimic and miR-183-5p inhibitor (n = 4 rats per group). The AP rats in the miR-183-5p mimic or inhibitor groups were treated with EVs from ACs that were pre-transfected with the miR-183-5p mimic or inhibitor, respectively. The AP rats were sacrificed at 6 hours after the EVs (4x10¹¹ particles) were intraperitoneally injected.

NR8383 macrophages (Shanghai Chinese Academy of Sciences Cell Bank, Shanghai, China) were cultured in DMEM-F12 (Thermo Fisher Scientific, MA, USA) supplemented with 20% fetal bovine serum (FBS, Gibco, USA) and a 1% penicillin–streptomycin solution (Procell, Wuhan, China). AR42J cells (ACs, Shanghai Chinese Academy of Sciences Cell Bank) were cultured in DMEM supplemented with a 1% penicillin–streptomycin solution and 10% FBS. Bone marrow-derived macrophages (BMDMs) were obtained from the femurs of wild-type Wistar rats. After 10 days of culturing in DMEM supplemented with a 1% penicillin–streptomycin solution, 10% FBS and 10 ng/ml recombinant rat macrophage colony-stimulating factor (M-CSF; PeproTech, NJ, USA), BMDMs were artificially differentiated into macrophages.

The ACs were first treated with PBS or caerulein (1x10^-8 mol/L, Sigma–Aldrich) or GW4869 (1×10^-5 mol/L, Sigma–Aldrich) combined with caerulein for 6 hours before co-culture with macrophages to study whether caerulein affected miR-183-5p loading into EVs and transfer to recipient macrophages. Specifically, ACs were seeded in the upper chamber of 6-well Transwell permeable support coculture systems with a 0.4 µm pore filter (Corning, New York, USA). NR8383 macrophages were seeded into the lower chamber of a 6-well plate and co-cultured with ACs in the upper chamber for 12 h, and DMEM/F12 was used as the culture medium. The expression of miR-183-5p in ACs and macrophages was measured. We cocultured these NR8383 macrophages with caerulein-treated ACs for 12 h to determine whether macrophages overexpressing miR-183-5p damaged ACs. The NR8383 macrophages overexpressing miR-183-5p were plated in the upper chamber of the coculture system, whereas the ACs were plated in the lower chambers of the 6-well plate, and DMEM/F12 was used as the culture medium.

The NR8383 cells and BMDMs were plated in a 6-well plate. When cells reached 50% confluence, they were transfected with 10 nM NC-mimic, miR-183-5p mimic, NC-inhibitor, miR-183-5p inhibitor, siFoxO1 or si-NC (RiboBio, Guangzhou, China). Lipofectamine 3000 (11668019, Thermo) was used for transfection experiments according to the product instructions.

Forty-eight hours before EV extraction from the cell culture supernatant, the FBS in the AC culture medium was replaced with EV-depleted FBS (System Biosciences, California, USA). Caerulein, a decapeptide molecule extracted from the skin of the Hyla caerulea frog, is a cholecystokinin analog that acts on pancreatic acinar cells and causes the secretion of a large amount of digestive enzymes and pancreatic juice, resulting in AP. After ACs were treated with PBS or caerulein (1x10^-8 mol/L) for 6 hours, the medium was refreshed, and the cells were cultured for another 48 h. EVs were purified by differential ultracentrifugation. In short, the samples were centrifuged at 300×g and 2000×g for 20 min each and then at 10,000×g for 30 min, and the collected supernatant was centrifuged at 100,000×g for 70 min. The precipitates were washed with PBS, centrifuged at 100,000 × g for 70 min and resuspended in 100 μl of PBS. The process of EV extraction from ACs with miR-183-5p overexpression and knockdown is described below. After the transfection of miR-183-5p mimic and inhibitor into ACs using the aforementioned transfection method, the culture was continued for 48 hours, and EVs were extracted from the cell supernatant by ultracentrifugation. Three milliliters of venous blood were collected from 15 pairs of patients with AP and healthy controls to extract EVs from sera. Serum was separated by centrifugation at 3,000 rpm for 10 min at 4°C to remove blood cells and debris. Finally, a 1 ml serum supernatant sample was diluted with 5 ml of PBS, and EVs were purified by differential ultracentrifugation.

All EV samples subjected to TEM were diluted 10-fold with PBS and then passed through 200-mesh nickel grids to remove any potential residual organelle debris. After being stained with 4% phosphotungstic acid for 3 min at room temperature, the samples were placed on filter paper and air-dried. EV morphology and size were imaged with a transmission electron microscope (HT7700, Hitachi, Japan).

NTA was performed with a nanoparticle size potentiometer (Zetasizer Ultra, United Kingdom). Briefly, 1 ml of sterile PBS-diluted EVs was dropped into the machine, and the diffusion rate of the particles in the solution was converted to the particle size of EVs.

ACs were stained with PKH67 (MIDI67, Sigma–Aldrich) in the dark at room temperature, and the unbound dye was removed by washing with PBS. EVs were separated by ultracentrifugation using the abovementioned method and added to BMDMs. After 24 h of culture, EV uptake by BMDMs was observed under a microscope. AP rats were sacrificed 6 h after treatment with PKH67-labeled EVs, and the distribution of the EVs in various organs was observed using frozen sections.

EVs were isolated from ACs treated with or without caerulein, and miRNAs were sequenced using a second-generation high-throughput sequencer (Illumina, CA, USA). The final data were compared with the reference genome using the BWA software to obtain genomic expression profiles.

To determine whether FoxO1 is a direct target of miR-183-5p, luciferase reporter constructs (3′UTR-NC, 3′UTR-FoxO1, and 3′UTR-FoxO1-mutant), miRNA (miRNA-NC or miR-183-5p) and Renilla luciferase (GeneChem, Shanghai, China) were transfected into 293T cells using Lipofectamine 3000 (Thermo Fisher Scientific). Cells were harvested at 48 h after transfection, and luciferase activity was determined using a dual-luciferase assay system (Promega, WI, USA) following a standard procedure.

Total RNA was isolated using an RNA extraction kit (Axygen, CA, USA). MiRNA and mRNA were transcribed into cDNA by a reverse transcription kit (Toyobo, Japan). The cDNA template was analyzed by qRT-PCR using SYBR Green assays (Roche, Germany). GAPDH was used as an internal reference for mRNA, and U6 was used as an internal reference for miRNA. PCR was performed using FastStart Universal SYBR Green Master Mix and a 7500 Real-Time PCR System (Basel, Switzerland). The absolute quantification of miR-183-5p in samples is described below. Briefly, a standard curve of the logarithmic values of miR-183-5p concentrations and the CT value was prepared by stepwise dilution of the miR-183-5p standard RNA (RiboBio, Guangzhou, China). By measuring the CT value of the sample, the absolute level of miR-183-5p in the sample was calculated. The primer sequences are shown in Supplemental Table 1 .

Samples were lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors, and the protein concentration was measured by the BCA assay (Beyotime, Shanghai, China). Protein samples were separated on 10% SDS-PAGE gels, and the separated proteins were transferred onto nitrocellulose membranes, which were blocked with 5% milk and then incubated overnight with the following antibodies: anti-Alix (1:1,000, ab275377, Abcam, UK), anti-TSG101 (1:1,000, ab125011, Abcam), anti-CD63 (1:1,000, A5271, ABclonal), anti-FoxO1 (1:1,000, C29H4, Cell Signaling Technology), anti-P-FoxO1 (1:1,000, 9461, Cell Signaling Technology), β-actin (1:2,000, BA2305, Boster), anti-iNOS (1:1,000, BA0362, Boster), anti-Arg-1 (1:1,000, 93668, Cell Signaling Technology), anti-P65 (1:2,000, 8242S, Cell Signaling Technology), anti-P-P65 (1:1,000, 93H1, Cell Signaling Technology) and anti-TNF-α (1:1,000, ab205587, Abcam). After incubation for 1 h with the corresponding fluorescent secondary antibody.

After pancreatic tissue samples were embedded using a cryostat, the tissue was sliced at 10 μm thickness and placed on a glass slide. HE-stained paraffin sections were subjected to xylene dewaxing, hydration in an ethanol gradient, HE staining, dehydration in an alcohol gradient and xylene, and mounting. Tissue damage was observed under the microscope. We applied the scoring system defined by Kusshe et al. (13), and the final score for each group was calculated.

The IF protocol has been previously described (14). Briefly, the pancreatic tissues were blocked with 5% donkey serum containing 0.1% Triton X-100 for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: CD86 (1:200, BU63, Novus, CO, USA), iNOS (1:100, GB11119, Servicebio) and CD68 (1:200, KP1, Novus). Subsequently, the slides were washed with PBS (0.01 M, pH 7.4) and incubated with the corresponding fluorophore-conjugated secondary antibodies, namely, goat anti-rabbit IgG (1:100, A27039, Invitrogen) and goat anti-mouse IgG (1:100, ab6785, Abcam), at 37°C for 1 h. 4′,6-Diamidino-2-phenylindole (DAPI, Abcam) was used for nuclear staining. When cells were stained, BMDMs overexpressing and inhibiting miR-183-5p were blocked with 5% serum and permeabilized with 0.3% Triton X-100, then incubated with the FoxO1 antibody (1:100, C29H4, Cell Signaling Technology) overnight at 4°C. Finally, BMDMs were incubated with appropriate secondary antibodies and DAPI. After staining, BMDMs were examined using a fluorescence microscope.

Amylase, lipase, creatinine and urea nitrogen levels in the blood samples and myeloperoxidase levels in lung tissue were measured using standard diagnostic kits (Jiancheng Biotech, Nanjing, China). Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in the pancreatic tissue samples and peripheral blood were measured using standard diagnostic kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Single-cell suspensions of NR8383 cells and BMDMs were prepared as previously described. BMDMs were incubated first with CD16/CD32 (Thermo Fisher Scientific) and then with FITC-CD68 (MA5-28262, Thermo Fisher Scientific) and PERCPEF710-CD11b/c (46-0110-82, Thermo Fisher Scientific). The percentage of double-positive cells indicates the purity of macrophages. To identify macrophage polarization, cells were first incubated with CD16/CD32, followed by PE-CD86 (12-0860-83, Thermo Fisher Scientific) and CD163 (PA5-78961, Thermo Fisher Scientific). Finally, the cells were incubated in the dark for 1 h with APC (A-10931, Thermo Fisher Scientific). Apoptosis was determined by an Annexin V-FITC/PI double staining cell apoptosis detection kit (Sevenbio, Beijing, China) following the manufacturer’s instructions. Each sample was analyzed by flow cytometry and FlowJo v.10 software.

Macrophages were seeded into a 6-well plate (20 × 10⁵ cells per well). Fluorescein isothiocyanate (FITC)-OVA (100 μg/ml, Solarbio, Beijing, China) was added to the 6-well plate, which was incubated for 6 h at 37°C in the dark and washed 3 times with 0.01 M PBS, pH 7.2. Then, the fluorescence intensity of each sample was measured on a flow cytometer.

All data were processed using GraphPad Prism 7.0. Data are presented as the mean ± standard deviation. Paired data in two groups were compared using a t-test. One-way ANOVA followed by the Bonferroni correction was performed to compare count data in more than two groups. A P-value of <0.05 was considered to be statistically significant.

We intraperitoneally injected PKH67-labeled EVs from ACs into AP rats to evaluate whether EVs reached the pancreatic tissue and aggravated pancreatitis. IF staining showed that PKH67-labeled EVs reached the pancreatic tissue, as well as the lungs, liver, and kidneys, which are vulnerable to the attack of pancreatitis ( Figure 1A ). Subsequently, we extracted EVs from ACs treated with PBS or caerulein and injected them into AP rats to confirm the effect of AC-derived EVs on pancreatitis. Caerulein-treated AC-derived EVs did not increase MPO (neutrophil marker) levels in the pancreas ( Supplemental Figure 1A ). Interestingly, EVs from caerulein-treated ACs aggravated pancreatic tissue damage ( Figure 1B ) and increased CD68-positive macrophages infiltration ( Figure 1C ). Subsequently, we measured the levels of lipase and amylase in the blood and inflammatory cytokines in the pancreas, and the results showed that caerulein-treated AC-derived EVs increased lipase, amylase, IL-6 and TNF-α levels ( Figure 1D ). Additionally, caerulein-treated AC-derived EVs also increased the levels of IL-6 and TNF-α in peripheral blood ( Supplemental Figure 1B ). Based on these results, EVs from caerulein-treated ACs aggravate AP.

We next evaluated the characteristics of EVs isolated and purified from ACs. First, the size and morphology of EVs secreted from ACs treated with PBS or caerulein were determined by TEM, and the results revealed discoid vesicles between 50 and 150 nm in diameter ( Figure 2A ). NTA showed that the mean particle size of EVs purified from ACs treated with or without caerulein was 83.34 ± 32.17 nm and 75.65 ± 28.5 nm, respectively ( Figure 2B ). The EV protein markers Alix, TSG101 and CD63 were also used for western blotting to identify EVs ( Figure 2C ). The extracted material was EVs. Subsequently, we incubated PKH67-stained EVs with BMDMs to ascertain whether EVs were internalized by macrophages. The fluorescence analysis showed that AC-derived EVs were internalized by BMDMs, and this phenomenon was more obvious for EVs derived from caerulein-treated ACs ( Figure 2D ). Next, we investigated whether AC-derived EVs impact macrophages by extracting monocytes from the rat bone marrow and transforming them into macrophages by treatment with M-CSF. Flow cytometry showed that 78.8% of the macrophage population was positive for both the macrophage markers CD68 and CD11B/C ( Supplemental Figure 2A ). After treating macrophages with PBS or caerulein-treated AC-derived EVs for 12 h, the proportion of macrophages treated with EVs from caerulein-treated ACs that were positive for CD86 (an M1 macrophage marker) was significantly increased ( Figure 2E ). Western blot analysis showed a significant increase in the expression of inducible nitric oxide synthase (iNOS) (an M1 macrophage marker) in BMDMs treated with EVs from caerulein-treated ACs ( Figure 2F ). qRT-PCR showed the significant upregulation of the expression of inflammatory cytokines, including IL-1β, IL-6 and TNF-α, in BMDMs treated with EVs from caerulein-treated ACs ( Figure 2G ). Thus, EVs derived from caerulein-treated ACs promote M1 macrophage polarization.

We analyzed the differentially expressed miRNAs in EVs from ACs treated with PBS or caerulein by high-throughput sequencing to explore whether EV miRNAs derived from AC affected AP. Compared with EVs from ACs treated with PBS, those from caerulein-treated ACs contained 30 (22 upregulated and 8 downregulated) differentially expressed miRNAs ( Figures 3A, B ). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation and Gene Ontology (GO) analyses were performed on the upregulated miRNAs to identify the functions of these differentially expressed miRNAs in EVs. GO analysis showed that the upregulated miRNAs mainly regulate the binding of intracellular proteins ( Figure 3C ). The KEGG annotation analysis showed that the upregulated miRNAs may play a role in inflammation by regulating leukocytes. In addition, this analysis predicted the potential involvement of the FoxO signaling pathways ( Figure 3D ). Sequencing results showed that the top 8 significantly upregulated miRNAs were detected in EVs from caerulein-treated ACs. The expression of miR-183-5p in EVs derived from ACs treated with PBS (Ctrl-ev) and caerulein (Cae-ev) was verified by qRT-PCR, and miR-183-5p was upregulated in Cae-ev, which again suggested that the sequencing was accurate ( Figure 3E ). Subsequently, we overexpressed the top 5 differentially expressed miRNAs in macrophages ( Supplemental Figure 3A ), and iNOS and arginase 1 (Arg-1) expression levels were verified by qRT-PCR. The results showed a significant increase in iNOS expression in macrophages overexpressing miR-183-5p ( Figure 3F ). Thus, miR-183-5p may induce M1 macrophage polarization.

We chose the well-known EV inhibitor GW4869 to inhibit the secretion of EVs and better determine the function of EVs. The inhibitory effect of GW4869 on EVs secreted by ACs was confirmed by western blotting. The amount of EVs released by GW4869-treated ACs was significantly reduced, and the levels of TSG101 and Alix in lysates of EVs released by GW4869-treated ACs were also reduced ( Supplemental Figure 4A ). We explored whether EV miR-183-5p from ACs reached macrophages and promoted M1 macrophage polarization by treating ACs with PBS, caerulein, or GW4869 combined with caerulein for 6 hours and co-cultured them with NR8383 macrophages. Cells in the two chambers were separated by a 0.4 μm Transwell filter. The caerulein-treated ACs released more miR-183-5p and the release of miR-183-5p was significantly inhibited by GW4869. Therefore, ACs stimulated with caerulein secrete miR-183-5p-rich EVs that are taken up by macrophages, and EVs play a major role in the process of transporting miR-183-5p ( Figure 4A ). We added the extracted EVs to NR8383 macrophages and incubated them for 12 hours to further assess the effect of EVs on macrophages. qRT-PCR showed higher miR-183-5p expression in Cae-ev than in Ctrl-ev, and its expression was significantly increased in Cae-ev-treated NR8383 macrophages, further confirming that EVs play a key role in transporting miR-183-5p to macrophages ( Figure 4B ). We transfected an miR-183-5p mimic and miR-183-5p inhibitor into NR8383 macrophages and BMDMs to investigate whether AC-derived EV miR-183-5p mediated M1 macrophage polarization. The mRNA expression levels of IL-6 and TNF-α increased significantly in NR8383 macrophages overexpressing miR-183-5p (mimic group) but decreased in the miR-183-5p inhibitor group ( Figure 4C ). Western blot analysis showed that iNOS expression was significantly upregulated in NR8383 macrophages overexpressing miR-183-5p and suppressed in the miR-183-5p inhibitor group ( Figure 4D ). The expression of Arg-1 was not significantly changed in NR8383 macrophages with miR-183-5p overexpression and knockdown ( Supplemental Figure 4B ). Flow cytometry showed that the overexpression of miR-183-5p in NR8383 cells significantly increased the proportion of CD86-positive cells ( Figure 4E ). Similar results were confirmed in isolated BMDMs ( Figures 4F–H ). In addition, OVA-FITC staining showed that miR-183-5p overexpression reduced macrophage-mediated phagocytosis ( Figure 4I ). Therefore, miR-183-5p plays a role in promoting M1 polarization and reducing macrophage phagocytosis.

The downstream target genes of miR-183-5p were predicted using online analysis tools, including TargetScan (http://www.targetscan.org), miRWalk (http://mirwalk.umm.uni-heidelberg.de) and miRDB (http://www.mirdb.org). FoxO1 is one of the 118 genes predicted by all three tools, and there is evidence that it is closely related to macrophage polarization ( Figure 5A ). TargetScan revealed that the 3’UTR of FoxO1 contains a complementary binding site for miR-183-5p ( Figure 5B ). We exposed macrophages to EVs from ACs treated with PBS or caerulein to verify that EV miR-183-5p regulated macrophage polarization by targeting FoxO1 in macrophages. Western blot analysis showed that EVs derived from caerulein-treated ACs decreased the levels of FoxO1 and P-FoxO1 ( Figure 5C ). Subsequently, we confirmed the relationship between miR-183-5p and FoxO1 and P-FoxO1 levels by overexpressing or silencing miR-183-5p in NR8383 macrophages and BMDMs, respectively. Western blot analysis showed decreased FoxO1 and P-FoxO1 levels in cells overexpressing miR-183-5p, but their levels were significantly increased by the miR-183-5p inhibitor ( Figure 5D ). The results of immunofluorescence staining also confirmed that FoxO1 levels were decreased in BMDMs overexpressing miR-183-5p, but significantly increased in BMDMs with miR-183-5p knockdown ( Supplemental Figure 5A ). Moreover, the transfection of macrophages with the miR-183-5p inhibitor and siFoxO1 ameliorated the inhibitory effect of miR-183-5p and increased iNOS expression ( Figure 5E ). To confirm this hypothesis, we used dual-luciferase reporter gene assays. Compared with the 3’UTR-FoxO1-mutant group, the 3’UTR-FoxO1 group showed significantly reduced luciferase activity, indicating that miR-183-5p directly targets FoxO1 ( Figure 5F ). In summary, miR-183-5p can directly target FoxO1 and promote M1 macrophage polarization.

EVs were extracted from ACs transfected with miR-183-5p mimic or inhibitor and injected into AP rats, which were sacrificed 6 h later, to verify the function of AC-derived EV miR-183-5p in vivo. First, we examined the efficiency of the overexpression and inhibition of miR-183-5p in ACs, EVs and pancreatic tissue. Transfection of the miR-183-5p mimic significantly increased the level of miR-183-5p in ACs and AC-derived EVs, while transfection of the miR-183-5p inhibitor produced the opposite result. We injected EVs with miR-183-5p overexpression or knockdown into AP rats, which effectively increased or decreased the level of miR-183-5p in pancreatic tissue ( Figure 6A ). Electron microscopy results showed that EVs from ACs that overexpressed miR-183-5p exacerbated nuclear membrane contraction, endoplasmic reticulum swelling, and disordered arrangement ( Figure 6B ). Most importantly, EVs from miR-183-5p inhibitor-treated ACs alleviated AP and decreased the proportion of macrophages co-expressing CD68 and iNOS, while EVs that overexpressed miR-183-5p aggravated AP and increased the proportion of macrophages co-expressing CD68 and iNOS ( Figures 6C, D ). However, AC-derived EVs with miR-183-5p overexpression and silencing did not directly damage ACs ( Supplemental Figure 6A ), and thus EVs damaged pancreatic tissue by inducing M1 macrophage polarization. In addition, EVs from miR-183-5p mimic-treated ACs even damaged the lung and kidney ( Supplemental Figure 6B ). The levels of MPO in lung tissue and blood creatinine and urea nitrogen levels further confirmed that miR-183-5p-enriched EVs may aggravate lung and kidney damage ( Figure 6E ). Subsequently, we measured lipase and amylase levels in the blood and inflammatory cytokine levels in the pancreas, and the results showed that EVs overexpressing miR-183-5p increased the levels of lipase, amylase, IL-6 and TNF-α ( Figure 6F ). These results indicate that miR-183-5p is delivered to pancreatic tissue through AC-derived EVs and aggravates AP by inducing M1 macrophage polarization.

We cocultured such macrophages with caerulein-treated ACs to prove whether macrophages overexpressing miR-183-5p damaged acinar cells. Cocultivation with miR-183-5p-overexpressing macrophages increased the ratio of P-P65/P65 and TNF-α levels in caerulein-treated ACs ( Figure 7A ). As a method to explore the degree of trypsinogen activation in acinar cells, we used the trypsin substrate BZiPAR to detect trypsin activation and the proportion of positive cells, which represented the degree of trypsin activation. The results showed that macrophages overexpressing miR-183-5p increased trypsinogen activation in caerulein-treated ACs ( Figure 7B ). Subsequently, flow cytometry was performed to further verify the damage to acinar cells, and the proportion of necrotic ACs was significantly increased after coculture with miR-183-5p-overexpressing macrophages ( Figure 7C ). Our results show that overexpressing miR-183-5p in macrophages induces trypsinogen activation and aggravates acinar cell damage.

We isolated and purified EVs present in blood samples from patients with AP (n=15) and normal individuals (n=15) to confirm the clinical significance of our findings. EVs were discoid vesicles between 50 and 150 nm in diameter, as determined using TEM ( Figure 8A ). NTA showed that the mean particle size of EVs purified from serum was 95.35 ± 32.13 nm ( Figure 8B ). The EV protein markers Alix, TSG101 and CD63 were also used to identify EVs by performing western blotting, which confirmed that the extracted material was EVs ( Figure 8C ). qRT-PCR results showed a significant increase in miR-183-5p levels in serum EVs from patients with AP compared with those of normal individuals ( Figure 8D ). Lower levels of miR-183-5p were detected in the blood of patients with mild to moderate pancreatitis than in patients with severe pancreatitis. The blood amylase levels represent the disease severity, and the results showed that the EV miR-183-5p in blood was positively correlated with the amylase level ( Figure 8E ).