Rat pancreatic acinar cell lines (AR42J) were purchased from Tongpai (Shanghai) Biotechnology Co., Ltd. and maintained in DMEM (Thermo Fisher Scientific, Inc.) containing 10% FBS (26), 1% penicillin and 1% streptomycin (Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. To mimic AP in vitro, AR42J cells were treated with LPS (10 µg/ml; Beijing Solarbio Science & Technology Co., Ltd.) and caerulein (100 nM; Beijing Solarbio Science & Technology Co., Ltd.) for 3 h, as previously described (27).

AR42J cells (3×10⁵ cells/well) were transfected with pcDNA3.1 (negative control; NC; 1 µg/µl) or pcDNA3.1-TUG1 [1 µg/µl, TUG1 overexpression (OE)] using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. pcDNA3.1 and pcDNA3.1-TUG1 were purchased from Shanghai GenePharma Co., Ltd. After 48 h of transfection, the transfected cells were used for subsequent analysis.

Briefly, AR42J cells were cotreated with LPS (10 µg/ml) and caerulein (100 nM) at 37°C for 3 h. Then, AR42J cells (3×10⁵ cells/well) or AR42J cells cotreated with LPS and caerulein were centrifuged at 300 × g for 15 min, 2,000 × g for 15 min, and 10,000 × g for 30 min at room temperature to collect the supernatant. The samples were filtered with a 0.22-µm filter and collected to isolate exosomes via ultracentrifugation at 4°C (120,000 × g for 70 min). For collection of the pellet, the samples were centrifuged at 10,000 × g for 1 h at 4°C. Then, the pellet was resuspended in PBS for further analysis. Meanwhile, the particle sizes of exosomes were investigated by Nanoparticle Tracking Analysis (NTA), the structure of exosomes was observed by transmission electron microscopy (TEM) and the exosome markers were detected by western blotting. Exosomes isolated from AR42J cells were the control-exo group; exosomes isolated from AR42J cells cotreated with LPS and caerulein were the AP-exo group.

Emodin was purchased from Beijing Solarbio Science & Technology Co., Ltd. IL-2 (cat. no. SRP3242) and anti-CD3 (cat. no. SAB4700040)/CD28 (cat. no. SAB4700739) were purchased from Sigma-Aldrich; Merck KGaA. Caerulein was obtained from MedChemExpress. Meanwhile, ARJ21 cells were treated with 20 and 40 µM emodin, according to previous references (28,29).

The exosome pellet was incubated for 5 min and subsequently immersed in 2% phosphotungstic acid solution for 1 min. The pellet was fixed using 2.5% glutaraldehyde (pH 7.2) at 4°C overnight. Then, 100 µl suspension was placed on a parafilm sheet and a copper grid coated with carbon was placed onto the drop for 10 sec and then removed. The grid was then rinsed 10 times with MiliQ H₂O (1 min each) at room temperature. Subsequently, the grid was laid on a drop of uranyl acetate (pH 7.0; cat. no. 2624; SPI-CHEM) at room temperature for 10 min. After rinsing with Milli-Q H₂O and methylcellulose uranyl (pH 4.0), the grid was incubated at room temperature for 10 min on a drop of methylcellulose uranyl (pH 4.0, cat. no. M-6385; Sigma-Aldrich; Merck KGaA). Finally, the grid was dried for 10 min at room temperature and observed using a transmission electron microscope (JEOL, Ltd.).

The viability of AR42J cells (5×10³/well) was determined in each group using the CCK-8 assay (Beyotime Institute of Biotechnology). In brief, ARJ21 cells were plated (5×10³ cells/well) into 96-well plates and treated for 0, 24, 48 or 72 h at 37°C. Subsequently, cells were incubated with 10 µl CCK-8 reagents for 2 h at 37°C. The optical density values were measured at 450 nm using a microplate reader to assess cell viability.

The early + late apoptosis of AR42J cells was detected by flow cytometry. In brief, AR42J cells (1×10⁴ per well) were trypsinized, washed with PBS and resuspended in binding buffer. Subsequently, the cells were stained with 5 µl Annexin V-FITC and PI (BD Biosciences) in the dark at 37°C for 30 min. Flow cytometry (FACScan™; BD Biosciences) was applied to investigate the apoptotic rate using FlowJo (version 10.6.2; BD Biosciences).

Total protein was isolated from cell lysates using RIPA buffer (Beyotime Institute of Biotechnology). The protein was quantified by the BCA protein kit (Thermo Fisher Scientific, Inc.). Subsequently, the proteins (30 µg/lane) were separated with SDS-PAGE gel (10%) and separated proteins were subsequently transferred to PVDF membranes, which were incubated with primary antibodies overnight at 4°C following blocking with 3% non-fat milk at room temperature for 1 h. The membranes were incubated with Goat Anti-Rabbit antibody (HRP-conjugated, 1:5,000; cat. no. ab7090; Abcam) at room temperature for 1 h, scanned by an Odyssey Imaging System (Thermo Fisher Scientific, Inc.) and analyzed with ImageJ software (version 1.8.0; National Institutes of Health). The primary antibodies used in the present study were as follows: Anti-CD63 (1:1,000; cat. no. ab134045; Abcam), anti-Calnexin (1:1,000; cat. no. ab133615; Abcam), anti-IL-10 (1:1,000; cat. no. ab133575; Abcam), anti-TGF-β (1:1,000; cat. no. ab215715; Abcam) and anti-β-actin (1:1,000; cat. no. ab8226; Abcam). All the antibodies used were purchased from Abcam.

Total RNA from cells or exosomes was isolated using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the exoRNeasy Midi kit (Qiagen, Inc.). The All-in-One™ First-Strand cDNA Synthesis kit (GeneCopoeia, Inc.) was used to transcribe total RNA into cDNA according to the manufacturer's protocol. RT-qPCR was performed using the SYBR™ Green Master Mix (Qiagen, Inc.). qPCR was performed in triplicate under the following protocol: 2 min at 94°C, followed by 35 cycles for 30 sec at 94°C and 45 sec at 55°C. The 2^−ΔΔCq method (30) was used to quantify the data and GAPDH was used for normalization. The primer sequences used were as follows: TUG1 forward (F), 5′-ATCTAATCAGTAAGCGGA-3′ and reverse (R), 5′-AAAGCAAGTCAAGACCTC-3′; IL-10 F, 5′-GAAAAATTGAACCACCCGGCA-3′ and R, 5′-TTCCAAGGAGTTGCTCCCGT-3′; TGF-β F, 5′-CTGCTGACCCCCACTGATAC-3′ and R, 5′-AGCCCTGTATTCCGTCTCCT-3′; and GADPH F, 5′-ACAGCAACAGGGTGGTGGAC-3′ and R, 5′-TTTGAGGGTGCAGCGAACTT-3′.

The hydrodynamic radius and concentration of exosomes were detected via NTA, as previously described (31). In brief, a total of ~0.3 ml supernatant was loaded into the sample chamber of an LM10 Nanosight unit (Nanosight, Ltd.) and three videos of either 30 or 60 sec were recorded of each sample. Data analysis was performed using NTA 2.1 software (Nanosight, Ltd.). The paths of unlabeled particles acting as point scatterers undergoing Brownian motion in a 0.25 ml chamber through which a 635-nm laser beam is passed were determined from a video recording, with the mean squared displacement determined for each possible particle. The diffusion coefficient and sphere-equivalent hydrodynamic radius were subsequently determined using the Stokes-Einstein equation.

The levels of IL-6 (cat. no. SEA079Ra), IL-1β (cat. no. SEA563Ra) and TNF-α (cat. no. SEA133Ra) in the serum of mice or the cell supernatants were detected using ELISA kits (Wuhan USCN Business Co., Ltd.). The cell supernatants were harvested by centrifugation (500 × g, 4°C, 10 min). Subsequently, cells were incubated with a secondary antibody (cat. no. BA1054; Wuhan Boster Biological Technology, Ltd.) at room temperature for 1 h. Finally, following incubation with hydrochloric acid at room temperature for 5 min, the absorbance was measured using a microplate reader.

A male Wistar rat (12-weeks-old, 200 g) was obtained from The Chinese Academy of Sciences. The rat was housed within a dedicated specific pathogen-free (SPF) facility (it was raised in standard cages with 12-h light/dark cycle, a constant temperature of 23±1°C and a humidity of 50–60%) and had free access to food and water. The protocols for care and use of laboratory animals were approved by the Zhejiang Chinese Medical University (Hangzhou, China). The rat was euthanized using CO₂ at a displacement rate of 30% of the chamber volume/min (CO₂ flow rate, 2.5 l/min). Then, peripheral blood mononuclear cells (PBMCs, 5 ml) were collected. PBMCs were collected as described in a previous study (32). In brief, CD4⁺T cells were isolated from PBMCs, and then CD4⁺T cells were treated with 10 ng/ml IL-2 and 2 µg/ml anti-CD3/CD28 for 72 h in order to induce the activation of Treg cells (33).

BALB/c mice (n=18, male, 28–35 g; age, 6–8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were maintained under SPF conditions. All procedures performed in this study involving animals were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (34). The animal experiments were approved by the Animal Care and Use Committee of The First Affiliated Hospital of Zhejiang Chinese Medical University (approval no. 20210201003B). To mimic AP in vivo, mice were fasted for 12 h, and a mouse model of AP was constructed according to a previous method (35). Briefly, mice in the AP group were injected intraperitoneally with 50 µg/kg caerulein 12 times (0.2 ml/mice, each time interval was 1 h). In addition, mice in the control group were injected with saline with the same procedure. Meanwhile, before the second injection and after the eighth injection of caerulein, emodin (40 mg/kg) was injected intraperitoneally into mice (AP + emodin group). The mice were sacrificed 48 h after the last injection of caerulein, and the tissues and serum were then collected. All mice were euthanized using CO₂ at a displacement rate of 30% of the chamber volume/min (CO₂ flow rate, 2.5 l/min).

To evaluate the effects of emodin in AP mice, inflammatory factor (IL-1β, IL-6 and TNF-α) contents in serum of mice were detected with the ELISA kits, as mentioned above.

H&E staining was used to observe pancreatic injury and inflammation as previously described (36). Paraffin-embedded tissues were cut to 4 µm thickness, deparaffinized, rehydrated and stained with H&E using standard procedures. The slides were then mounted with BioMount mounting media and observed under a light microscope (Olympus Corporation).

A total of 2×10⁶ PBMCs/ml were seeded in 6-well cell culture plates. The cells were treated with 50 ng/ml Phorbol-12-myristate-13-acetate (Beijing Solarbio Science & Technology Co., Ltd.), Ionomycin (2 µg/ml; Beijing Solarbio Science & Technology Co., Ltd.) and Brefelin (2 µg/ml; Beijing Solarbio Science & Technology Co., Ltd.). These reagents were incubated with the cells at 37°C for 4 h. The CD4⁺T cell population was isolated from the cell cultures by immunomagnetic selection (MidiMACS™ Separator; Thermo Fisher Scientific, Inc.).

To further induce the differentiation of Treg cells, CD4⁺T cells (5×10³/well) were treated with anti-CD3 (cat. no. SAB4700040)/anti-CD28 (cat. no. SAB4700739; 2 µg/ml; Sigma-Aldrich; Merck KGaA) and IL-2 (10 ng/ml; Sigma-Aldrich; Merck KGaA) at 37°C for 72 h. The cell suspension was collected and the cells were incubated with anti-CD4 (labeled with FITC; cat. no. 11-0040-82; Thermo Fisher Scientific, Inc.) and anti-CD25 (labeled with PE; cat. no. MA1-90766; Thermo Fisher Scientific, Inc.) for 30 min. Subsequently, the cells were centrifuged at 3,000 × g at 4°C for 10 min, washed and fixed with 4% paraformaldehyde for 20 min at 4°C. The samples were resuspended and incubated with anti-FOXP3 antibody (labeled with APC; cat. no. 17-5773-82; Thermo Fisher Scientific, Inc.) for 30 min at 4°C. Finally, the cells were centrifuged at 3,000 × g at 4°C for 10 min, washed, fixed with 4% paraformaldehyde at 4°C for 10 min and the ratio of CD4⁺/CD25⁺/FOXP3⁺ T cells was measured by FACS (FACSLyric™; BD Biosciences). FlowJo (version 10.6.2; BD Biosciences) was used to analyze the data.

All data are expressed as the mean ± SEM. The CCK-8 assay was performed five times. Western blotting, ELISA, RT-qPCR and flow cytometry assays were performed in triplicate. One-way ANOVA followed by the Tukey's post hoc test were used for comparisons between ≥3 groups. P<0.05 was considered to indicate a statistically significant difference.

To mimic AP in vitro, AR42J cells were cotreated with caerulein and LPS. The levels of IL-1β and TNF-α in the supernatants of AR42J cells were significantly increased following treatment of the cells with caerulein and LPS (Fig. 1A and B). The data suggested that the in vitro model of AP was successfully established.

The separation efficiency of exosomes was examined by TEM. Exosomes demonstrated disc-shaped crescent-shaped and double-layered membrane structure (Fig. 2A). In addition, the expression levels of CD63 were notably higher in the AP-exo group than those noted in the control-exo, while western blotting demonstrated the absence of calnexin expression in the control-exo and AP-exo groups (Fig. 2B). In addition, rounded particles of ~80–100 nm in diameter were noted following treatment of the cells with LPS and caerulein (Fig. 2C). The data suggested that exosomes were successfully separated from AR42J cells. Furthermore, the expression levels of TUG1 in AR42J cells and exosomes derived from AR42J cells were significantly increased following treatment of the cells with caerulein and LPS (Fig. 2D and E). In summary, the data indicated that lncRNA TUG1 expression was upregulated in caerulein and LPS pre-treated AR42J cells and exosomes derived from AR42J cells cotreated with caerulein and LPS.

The effects of emodin on AR42J cell viability were assessed using the CCK-8 assay. The viability of AR42J cells was significantly inhibited by caerulein and LPS, while the effects of caerulein and LPS were reversed following treatment of the cells with 20 or 40 µM emodin (Fig. 3A). In addition, the apoptotic rate of AR42J cells cotreated with caerulein and LPS was significantly inhibited following treatment of the cells with 20 or 40 µM emodin (Fig. 3B). Moreover, ELISA demonstrated that the levels of IL-1β and TNF-α in supernatants derived from AR42J cells were significantly increased following treatment of the cells with caerulein and LPS, while application of 20 or 40 µM emodin significantly reversed this effect (Fig. 3C and D). Furthermore, caerulein and LPS significantly increased the levels of TUG1 in AR42J cells or exosomes derived from AR42J cells, while their effects were significantly reversed by emodin treatment of the cells (20 or 40 µM; Fig. 3E and F). Since AR42J cells were more sensitive to 40 µM emodin, this concentration was selected for subsequent analysis. Taken together, the data indicated that emodin inhibited the expression levels of cellular and exosomal TUG1 in AR42J cells cotreated with caerulein and LPS.

To investigate the effects of TUG1 on emodin-treated AR42J cells, cells were transfected with a TUG1 OE plasmid. The expression levels of TUG1 were upregulated following transfection of AR42J cells with TUG1 OE (Fig. 4A). Moreover, overexpression of TUG1 caused a significant reduction in the emodin-induced increase of AR42J cell viability following treatment with caerulein and LPS (Fig. 4B). The inhibitory effect of emodin on the induction of apoptosis in AR42J cells cotreated with caerulein and LPS was significantly reduced following overexpression of TUG1 (Fig. 4C). In conclusion, TUG1 overexpression reversed the effects of emodin on the proliferation of AR42J cells cotreated with caerulein and LPS by inducing cell apoptosis.

In order to confirm whether emodin mediates Treg cell differentiation via regulation of exosomal TUG1, RT-qPCR was performed. The expression levels of TUG1 in CD4⁺T cells cotreated with anti-CD3/anti-CD28 and IL-2 were significantly increased by AP-exo (exosomes derived from AR42J cells cotreated with LPS and caerulein), while the effects of AP-exo were reversed by emodin treatment (Fig. 5A). In addition, the ratio of CD4⁺/CD25⁺/FOXP3⁺ T cells in CD4⁺ T cells was significantly increased following their combined treatment with anti-CD3/anti-CD28 and IL-2, while this effect was reversed by AP-exo treatment (Fig. 5B and C). Moreover, emodin significantly inhibited the effects of AP-exo on Treg cell differentiation (Fig. 5B and C). The expression levels of IL-10 and TGF-β in CD4⁺T cells were notably increased by combined treatment of anti-CD3/anti-CD28 and IL-2, while these effects were partially reversed by AP-exo treatment (Fig. 5D-F). However, emodin restored the effects of anti-CD3/anti-CD28 and IL-2 (Fig. 5D-F). In summary, emodin may promote the differentiation of Treg cells by inhibition of exosomal lncRNA TUG1 expression.

To further confirm the function of emodin in AP, an in vivo model of AP was established. As indicated in Fig. 6A, notable injury of the pancreas tissue and inflammatory infiltration were observed in AP mice, while emodin markedly reversed this phenomenon. In addition, AP-induced upregulation of IL-6, IL-1β and TNF-α was significantly inhibited in the presence of emodin (Fig. 6B). Overall, emodin significantly inhibited the progression of AP in vivo.