Adipose-derived Stem Cells Alleviate Acute Pancreatitis by Inhibiting Ferroptosis and Oxidative Damage in Canine

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This study aims to investigated the potential therapeutic benefits of adipose-derived stem cells (ADSCs) and conditioned medium (CM) in managing canine AP and the role in ferroptosis regulation. Methods Sixteen dogs were randomly divided into control (CON), AP, ADSCs and CM group. The AP model were established by injecting sodium taurocholate (5%, 0.1 mL/kg) and trypsin (3500 U/kg) through the pancreaticobiliary duct. ADSCs (1×10 6 /kg) and CM (0.1 mL/kg) were injected intravenously at 6 h after surgery, and the roles on ferroptosis and oxidative stress were analyzed. In addition, the changing pattern of ferroptosis and oxidative stress were investigated by LPS-induced cellular inflammation model of AR42J in vitro. Results Our study showed that ferroptosis occurs in the pancreas during AP, as evidenced by significant iron accumulation, with suppressed glutathione peroxidase 4 (GPx4) expression and increased transferrin receptor-1 (TFR1) and ferritin heavy chain (FTH). ADSCs and ADSCs-CM treatment achieved pathological remission and effectively restored abnormal amylase (AMY), lipase (LIPA) levels. ADSCs-CM showed similar ferroptosis alleviating effects as ADSCs treatment, with reduced iron accumulation and increased GPx4 expression. Furthermore, ADSCs promote nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into the nucleus and initiate transcription of detoxification enzymes to protect the pancreas from oxidative damage. Conclusions Based on these findings, ADSCs protect the pancreas by inhibiting ferroptosis and oxidative stress via paracrine function, which could be a therapeutic target for AP. Canine adipose-derived stem cells conditioned medium acute pancreatitis ferroptosis oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Acute pancreatitis (AP) is a common inflammatory disease that affects dogs, cats, and other small animals. AP can cause symptoms such as fever, nausea, vomiting, abdominal pain, diarrhea, and shock [ 1 ] . Complications such as renal failure, acute lung injury, disseminated intravascular coagulopathy, and pancreatic exocrine insufficiency can occur if treatment is delayed [ 2 ] . The pathogenesis of AP is complex, the standard of care for managing AP is symptomatic and supportive. Steiner et al. reported that fuzapladib was safe to administer to dogs, fuzapladib therapy of AP resulted in a significant improvement in the modified clinical activity index [ 3 ] . It is critical to discover new therapeutic drugs to improve AP treatment in the clinic. Ferroptosis is a newly regulatory cell death characterized by the accumulation of reactive oxygen species (ROS) and iron-dependent lipid peroxidation. Intracellular lipid peroxide metabolism is impaired and abnormal metabolism occurs under the catalytic effect of iron ions, and when the cellular antioxidant capacity is weakened, lipid reactive oxygen species accumulate, causing intracellular redox imbalance and inducing cell death [ 4 – 5 ] . According to reports, ferroptosis occurring in a single cell has the potential to spread to neighboring cells in a fast propagating wave, causing extensive tissue damage [ 6 ] . Adipose-derived stem cells (ADSCs) are multipotent stem cells with a high capacity for self-renewal that are obtained from adipose tissue that have been used to treat malignant illnesses, gastrointestinal disorders, musculoskeletal disorders, and skin conditions in companion animals [ 7 – 8 ] . ADSCs conditioned medium (CM) contains abundant bioactive substances secreted by ADSCs. Studies have shown that CM exhibited pancreatic protective effects by reducing the severity of caerulein-induced AP in mice, as well as decreasing interleukin-6 (IL-6) and myeloperoxidase (MPO) activities [ 9 ] . Wang et al. revealed that Klotho overexpressing MSC-derived extracellular vesicles mitigate cerulein-triggered apoptosis and NF-κB activation in AR42J cells. Therefore, the study of stem cell function is crucial for exploring the pathogenesis and treatment of pancreatitis [ 10 ] . In this study, we established an animal model of canine AP to determine the possible therapeutic benefits of ADSCs for AP and to investigate their role in the regulation of ferroptosis. Methods Ethics statement The experiment was approved by the Animal Protection and Academic Standardization Committee of Heilongjiang Bayi Agricultural University (Project: Molecular mechanism of adipose stem cells regulating transcription factor activity to attenuate canine pancreatitis. Date: 2023-03-23. Number: DWKJXY2023049). The work has been reported in line with the ARRIVE guidelines 2.0. ADSCs culture and preparation of ADSCs-CM Umbilical falciform ligament tissue was obtained from dogs, the adipose tissues were minced, and ADSCs were isolated by digestion with collagenase. Cells were suspended with low-sugar DMEM (HyClone, USA) containing 10%FBS (Senbeijia, China), 2 mM L-glutamine, 100 µg/mL penicillin and streptomycin (Solarbio, China), cultured at 37°C in a 5% CO 2 incubator (ThermoFisher, USA). Lipogenic and osteogenic induced differentiation medium (Cyagen, USA) was used to characterize the differentiation capacity of ADSCs. Calcium nodules were labeled using 0.1 mg/mL Alizarin Red and lipid droplets were labeled using 0.5% Oil Red O. Part of passage 3–5 ADSCs were collected for transplantation, and part of the cells were cultured with FBS-free medium for 48 h, 3 kDa ultrafiltration units were used to concentrate 10 7 ADSCs into 1 mL ADSCs-CM. Animals Sixteen adult dogs (5.97 ± 1.49 kg) were provided by the Laboratory Animal Center of Heilongjiang Bayi Agricultural University (Daqing, China). The dogs were fed a standard dog diet (Ouchong Pet Products, China) and housed in separate rooms at an animal research facility that was kept at 23 ± 0.5°C and 55%±15% humidity. The lights in each room switched on and off between 7:00 am and 9:00 evening, and the windows in each room let in extra natural light. The dogs were randomly divided into 4 groups (CON, AP, ADSCs and CM group, n = 4/group). Fifteen minutes following the dexmedetomidine injection, the dogs received induction anesthesia with propofol and maintenance anesthesia with isoflurane. After shaving and sterilizing the abdomen, an incision was made in the mid-abdominal line in front of the umbilicus, and the duodenum was retracted outside the abdominal. The pancreaticobiliary duct is identified, the duodenum is slit, and a soft indwelling needle is placed into the larger papilla of the duodenum. The AP, ADSC and CM groups established AP model by retrogradely injecting sodium taurocholate (5%, 0.1 mL/kg) and trypsin (3500 U/kg) through the pancreaticobiliary duct, while the CON group received an injection of 0.9% NaCl. During the injection, the fingers are used to compress the major and minor duodenal papilla to allow the injected fluid to penetrate the pancreas. ADSCs (1×10 6 /kg) were injected intravenously into the ADSCs group at 6 h after surgery, ADSCs-CM (0.1 mL/kg) into the CM group, and 0.9% NaCl into the CON and AP group. Blood samples were collected through the cephalic vein at 24 hours postoperatively, Glasgow composite pain scale (GCPS) was scored according to Reid's description, and clinical indicators were assessed [ 11 ] . After anesthesia, the pancreas was retracted out of the abdomen through the original incision, tissues with significant inflammation were selected for sample collection from the tail of the pancreas, and the resection site was ligated with sutures. The dogs received standard analgesic, antiemetic, anti-inflammatory, and rehydration therapy after surgery. Histological examination Hematoxylin-eosin (HE) stained 4 µm sections of pancreatic tissues were used to measure the severity of acute pancreatitis. According to Van Laethem's scoring guidelines, the degree of edema, inflammatory infiltration, and necrosis was evaluated [ 12 ] . Transmission electron microscope Glutaraldehyde-fixed pancreatic tissue samples were dehydrated, embedded, sliced, and electron-stained before being examined under a transmission electron microscope to check for alterations in the cellular ultrastructural. Blood indicators assay Blood samples were drawn and tested for white blood cells (WBC) serum amylase (AMY), lipase (LIPA), total protein (TP), albumin (ALB), total cholesterol (TCHO), total bilirubin (TBIL), pH, partial pressure of carbon dioxide (PaCO 2 ), total carbon dioxide (TCO 2 ) and bicarbonate (HCO 3 − ), using automated hematology system, automated biochemistry analyzer, and blood gas analyzer (IDEXX, USA). Iron assay The serum and pancreatic iron contents were measured and calculated according to the instructions (Nanjing Jiancheng Bioengineering Institute, China). In brief, acidic and reducing chemicals are used to remove iron from transferrin-containing proteins and reduce the Fe 3+ to Fe 2+ , which are then detected colorimetrically by the level of ferrous ions that bind to bipyridine to form a pink complex. Oxidative activities assay Pancreatic glutathione (GSH), glutathione peroxidase (GPx), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) concentrations were measured using assay kits (Nanjing Jiancheng Bioengineering Institute, China) according to the instructions. Cell Culture Rat pancreatic acinar cells (AR42J) were provided by Wuhan Pricella Biotechnology Co., Ltd., which were cultured in Ham's F-12K medium with 20% FBS and 1% penicillin-streptomycin. Cells were kept in 37℃ with 5% CO 2 . AR42J were co-cultured with ADSCs in a transwell chamber and treated with LPS for 24 h. The CCK-8 kit was used to detect AR42J cells activity. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from pancreatic tissue using the TRIzol reagent (Invitrogen, USA) and cDNA was generated using the PrimeScript RT reagent Kit (TAKARA, Japan). qRT-PCR reaction was performed in a 20-µL reaction volume containing 10 µL TB Green Premix Ex Taq (TAKARA, Japan), 2 µL cDNA, 0.8 µL of each primer, 6.4 µL ddH 2 O, and set as 95 ℃ for 5 min; 40 cycles of 95 ℃ for 15 s, 60 ℃ for 30 s. The relevant gene expression levels were calculated according to the 2 −△△Ct relative quantification method. The primers used in the study is shown in Table 1 . Table 1 Primers used for qPCR Gene Primer sequence GPx4 Forward 5’-GGCAAGACGGACGTAAACTACACTC-3’ Reverse 5’-CGGCGGCGAACTCTTTGATCTC-3’ FTH Forward 5’-CCATCAACCGCCAGATCAACCTG-3’ Reverse 5’-GTTTCTCAGCATGTTCCCTCTCCTC-3’ FTL Forward 5’-TGCGGATCTGTCTCTTGCTTCAAC-3’ Reverse 5’-CAGGAAGATGGCTCCGAAGGTTG-3’ Nrf2 Forward 5’-ACTCTTGCCGTTCAGTCAGTCATTG-3’ Reverse 5’-CACCATGCTAGTCTCGACCAACTTG-3’ Keap1 Forward 5’-CCTGGACAGTGTGGAGTGTTATGAC-3’ Reverse 5’-GTTCTGCTGGTCGATCTGCTTCC-3’ NQO1 Forward 5’-TGCAGCTCGCTGTCGGTATAATTC-3’ Reverse 5’-GCAACCTTGTTTAGCCTCCTCTCTC-3’ HO-1 Forward 5’-CTGTATCGCTCCCGCATGAACTC-3’ Reverse 5’-TGGTCCTCAGTGTCCTTGCTCAG-3’ GST Forward 5’-CTGCTCACACTGCTGTACTGATACG-3’ Reverse 5’-CACCTTGCCCTGTTCCCTAAACTC-3’ β-actin Forward 5’-TCCTGACCCTGAAGTACCCCATTG-3’ Reverse 5’-GTTGTAGAAGGTGTGGTGCCAGATC-3’ GPx4, Glutathione peroxidase; FTH, Ferritin heavy chain; FTL, Ferritin light chain; Nrf2, Nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; HO-1, Heme oxygenase-1; GST, Glutathione S-transferase. GPx4, Glutathione peroxidase; FTH, Ferritin heavy chain; FTL, Ferritin light chain; Nrf2, Nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; HO-1, Heme oxygenase-1; GST, Glutathione S-transferase. Western Blot Protein was extracted from pancreatic tissues using Nuclear and Cytoplasmic Protein Extraction Kit, and the protein concentration was detected using a BCA Kit (Beyotime, China). Protein samples (30 µg) were separated via 12% SDS-PAGE gels and electroblotted onto PVDF membranes (Millipore, Germany). Subsequently, the membranes were blocked with 5% nonfat milk and then incubated with anti-GPx4 (ABclonal, USA), Transferrin receptor-1 (TFR1), FTH (Affinity, USA), Nrf2, Keap1, HO-1, NQO1 (Wanleibio, China), Lamin B and β-Actin (Proteintech, China) at 4℃ overnight. The membranes were then washed in TBST, incubated with HRP-conjugated anti-IgG for two hours, and detected on a Tanon 5200 System (Tanon, China) using an ECL reagent (Biosharp, China). The ImageJ program was used to quantify relative protein expression. Immunofluorescence Cells were fixed in 4% paraformaldehyde, washed with TBST. ADSCs were incubated with polyclonal antibodies CD34, CD44, CD90, and CD105 (Servicebio, China) at 4°C overnight to stain cell surface antigens, and AR42J cells were incubated with Nrf2, FTH, and GPx4 antibodies. After that, secondary antibodies with fluorescent labels were incubated, the nuclei were stained with DAPI. Images were captured using a fluorescence microscope (Leica, Germany) or confocal laser scanning biomicroscope (Olympus, Tokyo), then use Fiji software to adjust the threshold and remove the background from the images. Statistical Analysis Normal distribution tests were performed using GraphPad Prism 8.0 (GraphPad Software, USA), and the data was analyzed using one-way ANOVA with Turkey's post hoc test for statistical significance across groups. Results were provided as mean ± SD, with P < 0.05 indicating significance. Three replications of each experiment were performed. Results Characteristics of ADSCs Canine ADSCs had fibroblast-like spindle shape and adhered to the wall ( Fig. 1 A ) . After 14 days of lipogenic induction, Oil Red O staining revealed orange-red lipid droplets in the cytoplasm ( Fig. 1 B ) , and Alizarin Red staining revealed red calcium nodules after 21 days of osteogenic induction ( Fig. 1 C ) , demonstrating that ADSCs possess the capacity to differentiate into adipocytes and osteoblasts, respectively. Immunofluorescence analysis revealed that ADSCs express the stem cell-specific surface antigens CD44, CD90, and CD105, while not express CD34 (Fig. 1 D). Sodium taurocholate and trypsin induced canine AP SNAP cPL was effective in detecting pancreatitis, yielding positive results in the AP, ADSCs, and CM groups, indicating that sodium taurocholate and trypsin resembled AP. The dogs in these three groups exhibited considerable symptoms of pain, and the GCPS was higher than in the CON group, however, no differences were observed among the three groups. Systolic Blood Pressure (SBP) was significantly elevated in the AP and ADSCs groups, while diastolic blood pressure (DBP) was significantly higher in the AP group and significantly lower in the CM group compared to the AP group. No differences were found in temperature (T), respiratory rate (RR), heart rate (HR), or mean arterial pressure (MAP) among the four groups ( Table 2 ) . Table 2 Primers used for qPCR CON AP ADSCs CM GCPS 7.67 ± 2.89 17.00 ± 3.61* 16.33 ± 2.08* 17.33 ± 1.53* T (℃) 37.70 ± 0.78 38.67 ± 0.21 38.60 ± 0.30 38.27 ± 0.93 RR (Times/min) 25.33 ± 6.11 30.00 ± 6.00 28.00 ± 5.29 28.00 ± 6.93 HR (Times/min) 82.00 ± 15.10 87.67 ± 7.23 89.00 ± 9.54 89.33 ± 6.11 SBP (mmHg) 110.00 ± 9.17 152.00 ± 8.89* 146.00 ± 12.12* 136.00 ± 14.00 DBP (mmHg) 69.67 ± 4.73 98.67 ± 6.11* 85.67 ± 8.33 74.67 ± 5.77 # MAP (mmHg) 83.00 ± 4.36 104.33 ± 15.89 105.00 ± 5.20 92.67 ± 6.35 * P < 0.05 vs. CON; # P < 0.05 vs. AP. HE staining showed that pancreatic acinar cells in the CON group exhibited homogeneous interstitial stroma and clear structure, whereas the pancreas in the AP group displayed interstitial edema, massive inflammatory cell infiltration, multiple hemorrhages, widespread nuclear disintegration, and necrosis of the acinar cells ( Fig. 2 A ) . Transmission electron microscopy revealed that AP induced cell nuclear membrane wrinkling, chromatin edge aggregation, endoplasmic reticulum expansion, blurred structure, reduced rough endoplasmic reticulum, and mitochondrial cristae disorder ( Fig. 2 B ) . Serum biochemical tests revealed that the injection a mixed solution into the pancreaticobiliary duct increased plasma AMY and LIPA levels significantly ( Fig. 2 C-D ) . These findings indicate that sodium taurocholate and trypsin induce pancreatic dysfunction in dogs. Ferroptosis induced by canine AP We examined the protein expression levels of GPx4, TFR1, and FTH in order to investigate sodium taurocholate and trypsin induced ferroptosis in the canine pancreas. Notably, the expression of TFR1 and FTH was significantly elevated in the AP group compared to the CON group, while GPx4 expression was significantly reduced ( Fig. 2 E-H ) . Furthermore, the results revealed that the expression levels of GPx4, TFR1, and FTH mRNA were consistent with the protein alterations using qRT-PCR analysis ( Fig. 2 I-K ) . The iron content in pancreas of the AP group was significantly higher than that of the CON group ( Fig. 3 A ) . AP inhibited the activities of GSH, GPx and T-AOC in the pancreas, while simultaneously increasing MDA production ( Fig. 3 B-E ) . Further examination of the Kepa1/Nrf2 pathway showed that the levels of Keap1, HO-1, and NQO1 proteins were significantly lower in the AP group compared to the CON group, Nuclear Nrf2 protein was significantly increased in the AP group, whereas Total Nrf2 protein did not change significantly ( Fig. 3 F-K ) . The mRNA expression changes exhibited similar trends ( Fig. 3 L-O ) . These findings suggest that trypsin and sodium taurocholate-induced pancreatitis promote ferroptosis. ADSCs and ADSCs-CM treatment relieves pancreatic dysfunction To determine the effect of ADSCs on ferroptosis in AP, transmission electron microscopy and histological examination were performed. After transplantation of ADSCs, transmission electron microscopy showed that ADSCs and CM treatment improved the ultrastructural disorder of pancreatic cells ( Fig. 4 A ) . Additionally, these treatments significantly reduced pancreatic hemorrhage, inflammatory infiltration, edema, and vacuole formation, providing similar therapeutic benefits for AP ( Fig. 4 B ) . Correspondingly, the scores of edema and necrosis were significantly lower compared to the AP group ( Fig. 4 C-E ) . Biochemical parameters analyses revealed that ADSCs and CM treatment significantly inhibited AMY and LIPA secretion ( Fig. 4 F-G ) . AP significantly reduced TP and ALB levels, and plasma ALB levels were significantly raised by ADSC therapy ( Fig. 4 H-I ) . Meanwhile, TBIL levels were noticeably elevated in the AP and ADSCs groups, and CM treatment markedly reversed the trend of elevated TBIL ( Fig. 4 J ) . The AP group had the highest levels of TCHO, WBC, and PaCO 2 ; these levels were decreased by ADSCs and CM treatment, but there were no appreciable differences ( Fig. 4 K-M ) . Blood TCO 2 levels in the AP group slightly increased, which was significantly reduced by treatment with ADSCs and CM ( Fig. 4 N ) . There were no appreciable differences in pH and HCO 3 − levels between the four groups ( Fig. 4 O-P ) . These findings suggest that ADSCs-CM alleviate pancreatic dysfunction and lessen the body’s reaction to AP. ADSCs transplantation reduced pancreatic ferroptosis in AP According to the results of the iron content assay, significantly elevated plasma and pancreatic iron levels in the AP group served as a marker for the onset of ferroptosis, while ADSCs and CM treatments significantly downregulated pancreatic iron levels and had a lowering effect on plasma iron contents ( Fig. 4 Q-R ) . Results from the expression of ferroptosis and Keap1-Nrf2 pathway proteins revealed that AP induced significant upregulation of Nuclear Nrf2 and TFR1 levels, whereas downregulation of Keap1, GPx4, HO-1, and NQO1 protein levels. Nuclear Nrf2, TFR1, GPx4, HO-1, and NQO1 protein expression increased after treatment with ADSCs or CM, whereas Keap1 protein expression decreased ( Fig. 5 A-G ) . The mRNA expression was also examined. The pancreatic inflammatory response decreased the expression of GPx, Keap1, NQO1, and HO-1 mRNA while increasing the expression of FTH and FTL mRNA. In ADSCs or CM treatment caused upregulation of GPx, Nrf2, Keap1, GST mRNA expression, while FTH mRNA expression was downregulated ( Fig. 5 H-O ) . These findings suggest that ADSCs, through paracrine function, alleviate ferroptosis and pancreatic injury. ADSCs attenuate LPS-induced AR42J ferroptosis in vitro To elucidate the mechanism by which ADSCs alleviate ferroptosis, we established an AR42J inflammatory model in vitro using LPS at 0-200 mg/L. The results revealed that LPS at various concentrations inhibited AR42J cell activity and showed dose-dependent behavior. The IC 50 of LPS was calculated to be 42.53mg/L, and a concentration of 10mg/L was selected for the following experiments ( Fig. 6 A ) . Alterations in ferroptosis-related proteins and genes in the AR42J model were in line with the in vivo experiments. LPS treatment induced ferroptosis and down-regulated the expression of Nrf2 and its downstream in AR42J. Co-culturing ADSCs with AR42J reduced LPS-induced ferroptosis and lessened its inhibitory effect on the Keap1/Nrf2 pathway ( Fig. 6 B-P ) . In addition, we labeled Nrf2 with specific antibodies, and used Mito-tracker and DAPI label mitochondria and nucleus. The expression of Nrf2 protein in the nucleus was found to be significantly upregulated by ADSC treatment and promote its nuclear translocation under a laser confocal microscope, but there was no significant difference in the expression of Nrf2 in the nucleus of the LPS group ( Fig. 7 A ) . According to the results of the FTH and GPx4 immunofluorescence staining, LPS decreased the fluorescence intensity in AR42J, while ADSC treatment upregulated the expression levels of FTH and GPx4 ( Fig. 7 B-E ) . Discussion AP is a common veterinary clinical acute abdominal disease that is frequently accompanied by systemic inflammatory response syndrome, sepsis, and even multiple-organ dysfunction syndrome. It is a potentially fatal disease with a high morbidity and mortality [ 13 ] . Ferroptosis is a non-apoptotic type of programmed cell death in the morphological characteristics of the cells, which include mitochondrial shrinkage, higher mitochondria membrane density, and disappearance of mitochondrial cristae [ 14 ] . Increasing evidence suggested that ferroptosis-regulated cell death may be associated with the onset and complications of AP [ 6 , 15 – 17 ] . In this study, we found that sodium taurocholate and trypsin caused AP and ferroptosis, as evidenced by pancreatic histopathology, changes in plasma enzyme activities, GPx4 deletion and increased iron levels. As the study of ferroptosis progressed, researchers began to pay more attention to the role of ferroptosis and the associated regulatory roles. Ferroptosis brings together iron, selenium, amino acids, lipids, and redox reactions into a network and maintains homeostasis by participating in various pathways [ 18 ] . Abnormal distribution and levels of iron in the body can interfere with normal physiological processes. Fe 3+ binds to transferrin and forms a complex via TFR1, and the cell takes the complex via endocytosis. Then, Fe 3+ is reduced to Fe 2+ stored in the unstable iron pool and ferritin. Excess Fe 2+ is oxidized to Fe 3+ by ferritin. Ferritin is a heteropolymer composed of FTH and FTL, FTH is in charge of iron atom holding, and FTL may be involved in electron transfer [ 19 ] . Deposition of ferritin can result in pancreatitis and oxidative stress. Inhibiting ferroptosis has been proven in studies to reduce reactive oxidative stress and inflammation during AP, as well as improve multiple organ failure caused by severe AP. This finding emphasizes the tight association between ferroptosis and AP [ 13 , 20 ] . Ferroptosis causes lipid peroxidation and ROS production, both of which are linked to the pathogenesis of AP. The pancreas is not only a digestive organ, but it is also one of the major organs for iron storage, which suggests that the pancreas is more vulnerable to iron metabolism abnormalities [ 6 , 13 ] . Animal studies have shown that a high iron diet or conditional knockout of GPx4 in the pancreas promoted experimental pancreatitis in mice induced by the administration of cerulein or L-arginine. In contrast, ferroptosis inhibitors reversed this damage, implying a pathogenic role for ferroptosis in experimental pancreatitis [ 21 ] . The results of this study showed significant iron accumulation in the pancreas and plasma, with suppressed GPx4 expression and increased TFR1 and FTH, indicating the presence of ferroptosis during AP. Ferroptosis plays an important role in the development and progression of various diseases, thus, regulating the ferroptosis pathway offers potential therapeutic approaches for the treatment of diseases such as neurological diseases, myocardial infarction, ischemia/reperfusion injury and cancer. As an oxidative cell death mode, ferroptosis is caused by an imbalance in the production and degradation of intracellular lipid ROS [ 18 , 22 – 25 ] . According to Jia-Hong’s study, due to abnormalities in the iron-dependent Fenton counter, excess free iron causes a sustained accumulation of ROS and ultimately activates ferroptosis and necrosis sequentially [ 26 ] . ROS are also important in the pathogenesis of AP, as injured cells and activated immune cells produce large amounts of oxygen free radicals. ROS can cause cellular damage and initiate the inflammatory process, resulting in pancreatic edema and inflammatory cell aggregation. In various animal models of AP, antioxidant treatment has been shown to reduce acinar cell necrosis and mitigate the severity of pancreatic tissue injury [ 6 , 27 ] . In this study, the pancreas demonstrated a significant oxidative stress response, as evidenced by an increase in MDA content and decrease in antioxidant enzyme activities. Keap1, HO-1, and NQO1 protein expression were reduced, while Nrf2 nuclear translocation and transcriptional activity were increased. Nrf2 is an essential transcription factor that regulates oxidative homeostasis and modulates GSH, iron, and lipid metabolism, as well as mitochondrial function, by activating the expression of various target genes required for iron death regulation [ 28 ] . Importantly, Nrf2 transcription regulates almost all ferroptosis genes, including GSH regulation, NADPH regeneration, and iron regulation [ 29 ] . Keap1 interacts with Nrf2 to regulate the expression of downstream antioxidant proteins and detoxification enzymes, reducing the damage caused by oxidative stress to cells and tissues [ 30 ] . The cysteine residues of the Keap1 protein change conformation and fail to remain bound to Nrf2, allowing Nrf2 to dissociate into the nucleus and bind to the ARE [ 31 ] . It was found that activation of Nrf2 in the nucleus ameliorated pancreatic injury caused by AP and the Keap1-Nrf2 pathway is important in the development of AP [ 32 ] . In addition, the Nrf2 pathway has been shown to inhibit ferritin formation by regulating HO-1, and Nrf2 knockdown enhances acute lung injury by promoting ferroptosis [ 28 ] . The current study demonstrates the regulatory effects of ADSCs and ADSCs-CM on oxidative stress and Nrf2, which alleviate pancreatic injury by inhibiting Keap1 expression, promoting Nrf2 translocation into the nucleus and triggering transcription of its downstream ROS detoxification enzymes, such as HO-1, NQO1, GPx, and GST. Several studies have found that mesenchymal stromal cells (MSCs) have significant therapeutic effects in animal models of AP, implying that MESs protect the pancreas through a variety of mechanisms [ 33 ] . First, MSCs down-regulate pro-inflammatory factors and up-regulate anti-inflammatory factors to attenuate the level of inflammatory response in the pancreas, and suppress the immune response [ 34 ] . Second, MSCs alleviated AP via antioxidant activity, which included increasing antioxidant enzyme activities and reducing MDA production [ 35 ] . Third, MSCs inhibit apoptosis in PACs while promoting angiogenesis in pancreatic tissues [ 36 ] . Furthermore, MSCs repair damaged pancreatic tissues via the effects of secreted VEGF, ANG-1, and HGF, reducing local/systemic inflammatory responses and effectively alleviating AP [ 37 ] . However, the specific underlying mechanisms of MSCs for AP treatment are still unknown, and clinical applications of MSCs for AP treatment has been rarely reported. ADSCs have a paracrine function of secreted cytokines, chemokines, growth factors, proteases, and extracellular vesicles, and growing evidence suggests that paracrine activity is more important for ADSCs' beneficial effects on tissue repairing than colonization and differentiation [ 38 – 39 ] . Qiang et al. demonstrated that ADSCs reduced the unfolded protein response induced by cerulein and lipopolysaccharide via TSG-6 auto-secretion and the inflammatory response by inhibiting NF-B expression, whereas ADSCs with TSG-6 gene knockdown lost the regulation of the unfolded protein response [ 40 ] . In addition, Zhao et al. reported that baicalein-pretreated MSCs-derived exosomes promoted liver function recovery in acute liver injury mice and activated the Keap1-Nrf2 pathway via P62, inhibiting ROS production and lipid peroxide-induced iron sagging [ 30 ] . We studied the protective effects of ADSCs and ADSCs-CM on the pancreas and discovered that both reduced pancreatic damage while efficiently restoring aberrant AMYL, LIPA, ALB, and TBIL levels. Inspiringly, cell-free ADSCs-CM treatment exhibited similar alleviation of oxidative damage and ferroptosis as transplantation of ADSCs, this is accomplished by reducing iron accumulation, promoting more Nrf2 translocation into the nucleus and initiating transcription of its downstream ROS-detoxifying enzymes to alleviate pancreatic injury. Conclusions In summarize, this study demonstrates that ferroptosis occurs during AP, characterized by iron accumulation and inhibition of GPx4 expression. ADSCs transplantation attenuate pancreatic injury, reduce iron accumulation, and mitigate oxidative stress by modulating Nrf2 nuclear translocation. Furthermore, cell-free ADSCs-CM treatment demonstrated therapeutic effects comparable to ADSCs, implying that ADSCs may protect the pancreas via paracrine mechanisms. The results of this study provide insights on the mechanisms of pancreatic ferroptosis and tissue protection in ADSCs following AP, laying the fundamental for future research into targeted therapies for AP. Abbreviations AP, Acute pancreatitis ADSCs, Adipose-derived stem cells CM, Conditioned medium CON, Control GPx4, Glutathione peroxidase 4 TFR1, Transferrin receptor-1 FTH, Ferritin heavy chain AMY, Amylase LIPA, Lipase Nrf2, Nuclear factor erythroid 2-related factor 2 ROS, Reactive oxygen species IL-6, Interleukin-6 MPO, Myeloperoxidase HE, Hematoxylin-eosin WBC, White blood cells TP, Total protein ALB, Albumin TCHO, Total cholesterol TBIL, Total bilirubin PaCO 2 , Partial pressure of carbon dioxide TCO 2 , Total carbon dioxide HCO 3 - , Bicarbonate GSH, Glutathione T-AOC, Glutathione peroxidase MDA, Malondialdehyde AR42J, Rat pancreatic acinar cells qRT-PCR, Quantitative real-time PCR FTL, Ferritin light chain Keap1, Kelch-like ECH-associated protein 1 HO-1, Heme oxygenase-1 GST, Glutathione S-transferase TFR1, Transferrin receptor-1 SBP, Systolic Blood Pressure DBP, Diastolic blood pressure T, Temperature RR, Respiratory rate HR, Heart rate MAP, Mean arterial pressure MSCs, Mesenchymal stromal cells Declarations Consent for publication Not applicable. Availability of data and materials The data sets used during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the Provincial Natural Science Foundation of Heilongjiang (LH2023C086), the Heilongjiang Postdoctoral Science Foundation (LBH-Z19088), the Research Fund (Clinical Diagnosis and Treatment of Pet) for Young College Teachers in Ruipeng Commonweal Foundation (RPJJ2020009), and the Personnel Foundation in Heilongjiang Bayi Agricultural University (XYB201915). Author’s Contributions YSG: Project administration; Writing-original draft; Data curation; Funding acquisition. MZC: Investigation; Formal analysis. MLL: Data curation; Investigation. ZW: Investigation; Formal analysis. RXD: Investigation; ZYW: Investigation; ESX: Writing-review & editing; Supervision. JSZ: Writing-review & editing. Acknowledgements The authors declare that they have not use AI-generated work in this manuscript. References Fabrès, Virginie, Dossin, et al. Development and validation of a novel clinical scoring system for short‐term prediction of death in dogs with acute pancreatitis[J]. Journal of Veterinary Internal Medicine, 2019, 33(2): 499-507. Garg, Pramod K, Singh, et al. Organ failure due to systemic injury in acute pancreatitis[J]. Gastroenterology, 2019, 156(7): 2008-2023. Steiner, Joerg M, Lainesse, et al. Fuzapladib in a randomized controlled multicenter masked study in dogs with presumptive acute onset pancreatitis[J]. Journal of Veterinary Internal Medicine, 2023, 37(6): 2084-2092. Xie, Y, Hou, et al. Ferroptosis: process and function[J]. Cell Death & Differentiation, 2016, 23(3): 369-379. Li, Jie, Cao, et al. Ferroptosis: past, present and future[J]. Cell Death & Disease, 2020, 11(2): 88. Li, Hongyao, Lin, et al. Ferroptosis and its emerging roles in acute pancreatitis[J]. Chinese Medical Journal, 2022, 135(17): 2026-2034. Bacakova, Lucie, Zarubova, et al. Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells–a review[J]. Biotechnology Advances, 2018, 36(4): 1111-1126. Kang, Min-Hee, Park, et al. Challenges of stem cell therapies in companion animal practice[J]. Journal of Veterinary Science, 2020, 21(3): e42. Abdolmohammadi, Kamal, Mahmoudi, et al. Effect of hypoxia preconditioned adipose-derived mesenchymal stem cell conditioned medium on cerulein-induced acute pancreatitis in mice[J]. Advanced Pharmaceutical Bulletin, 2020, 10(2): 297. Wang, Na, Ma, et al. Secreted klotho from exosomes alleviates inflammation and apoptosis in acute pancreatitis[J]. American Journal of Translational Research, 2019, 11(6): 3375. Reid, J, Nolan, et al. Development of the short-form Glasgow Composite Measure Pain Scale (CMPS-SF) and derivation of an analgesic intervention score[J]. Animal Welfare, 2007, 16(S1): 97-104. Van Laethem, Jean-Luc, Marchant, et al. Interleukin 10 prevents necrosis in murine experimental acute pancreatitis[J]. Gastroenterology, 1995, 108(6): 1917-1922. Lee, Peter J, Papachristou, et al. New insights into acute pancreatitis[J]. Nature Reviews Gastroenterology & Hepatology, 2019, 16(8): 479-496. Stockwell, Brent R, Angeli, et al. Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease[J]. Cell, 2017, 171(2): 273-285. Shan, Yuqiang, Li, et al. Ginsenoside Rg3 ameliorates acute pancreatitis by activating the NRF2/HO-1-mediated ferroptosis pathway[J]. International Journal of Molecular Medicine, 2022, 50(1): 89. Liu, Ke, Liu, et al. Trypsin-mediated sensitization to ferroptosis increases the severity of pancreatitis in mice[J]. Cellular and Molecular Gastroenterology and Hepatology, 2022, 13(2): 483-500. Fan, Rong, Sui, et al. Wedelolactone alleviates acute pancreatitis and associated lung injury via GPX4 mediated suppression of pyroptosis and ferroptosis[J]. Free Radical Biology and Medicine, 2021, 173: 29-40. Jiang, Xuejun, Stockwell, et al. Ferroptosis: mechanisms, biology and role in disease[J]. Nature Reviews Molecular Cell Biology, 2021, 22(4): 266-282. Wang, Dong, Zhang, et al. Mesenchymal stromal cell treatment attenuates repetitive mild traumatic brain injury-induced persistent cognitive deficits via suppressing ferroptosis[J]. Journal of Neuroinflammation, 2022, 19(1): 185. Liu, Yang, Wang, et al. The circadian clock protects against ferroptosis-induced sterile inflammation[J]. Biochemical and Biophysical Research Communications, 2020, 525(3): 620-625. Dai, Enyong, Han, et al. Ferroptotic damage promotes pancreatic tumorigenesis through a TMEM173/STING-dependent DNA sensor pathway[J]. Nature Communications, 2020, 11(1): 6339. Masaldan, Shashank, Bush, et al. Striking while the iron is hot: Iron metabolism and ferroptosis in neurodegeneration[J]. Free Radical Biology and Medicine, 2019, 133: 221-233. Li, Wenjun, Feng, et al. Ferroptotic cell death and TLR4/Trif signaling initiate neutrophil recruitment after heart transplantation[J]. The Journal of Clinical Investigation, 2019, 129(6): 2293-2304. Belavgeni, Alexia, Meyer, et al. Ferroptosis and necroptosis in the kidney[J]. Cell Chemical Biology, 2020, 27(4): 448-462. Weiland, Abigail, Wang, et al. Ferroptosis and its role in diverse brain diseases[J]. Molecular Neurobiology, 2019, 56: 4880-4893. Chu, Jia-Hong, Li, et al. Mercuric chloride induces sequential activation of ferroptosis and necroptosis in chicken embryo kidney cells by triggering ferritinophagy[J]. Free Radical Biology and Medicine, 2022, 188: 35-44. Sundar, Vaishnavi, Senthil Kumar, et al. Current trends in pharmacological approaches for treatment and management of acute pancreatitis–a review[J]. Journal of Pharmacy and Pharmacology, 2020, 72(6): 761-775. Dong, Hui, Qiang, et al. Nrf2 inhibits ferroptosis and protects against acute lung injury due to intestinal ischemia reperfusion via regulating SLC7A11 and HO-1[J]. Aging (albany Ny), 2020, 12(13): 12943. Yuan, Hanshu, Pratte, et al. Ferroptosis and its potential as a therapeutic target[J]. Biochemical Pharmacology, 2021, 186: 114486. Zhao, Shuxian, Huang, et al. Exosomes derived from baicalin‐pretreated mesenchymal stem cells alleviate hepatocyte ferroptosis after acute liver injury via the Keap1‐NRF2 pathway[J]. Oxidative Medicine and Cellular Longevity, 2022, 2022(1): 8287227. Suzuki, Takafumi, Yamamoto, et al. Stress-sensing mechanisms and the physiological roles of the Keap1–Nrf2 system during cellular stress[J]. Journal of Biological Chemistry, 2017, 292(41): 16817-16824. Liang, Xiaoqiang, Hu, et al. Dihydrokaempferol (DHK) ameliorates severe acute pancreatitis (SAP) via Keap1/Nrf2 pathway[J]. Life Sciences, 2020, 261: 118340. Ma, Zhilong, Zhou, et al. Mesenchymal stromal cell therapy for pancreatitis: Progress and challenges[J]. Medicinal Research Reviews, 2021, 41(4): 2474-2488. Kim, Hyun-Wook, Song, et al. Canine adipose tissue-derived mesenchymal stem cells ameliorate severe acute pancreatitis by regulating T cells in rats[J]. Journal of Veterinary Science, 2016, 17(4): 539. Gori, E, Lippi, et al. Acute pancreatitis and acute kidney injury in dogs[J]. The Veterinary Journal, 2019, 245: 77-81. Kawakubo, Kazumichi, Ohnishi, et al. Effect of fetal membrane-derived mesenchymal stem cell transplantation in rats with acute and chronic pancreatitis[J]. Pancreas, 2016, 45(5): 707-713. He, Zhigang, Hua, et al. Intravenous hMSCs ameliorate acute pancreatitis in mice via secretion of tumor necrosis factor-α stimulated gene/protein 6[J]. Scientific Reports, 2016, 6(1): 38438. Kumar, Praveen, Kandoi, et al. The mesenchymal stem cell secretome: A new paradigm towards cell-free therapeutic mode in regenerative medicine[J]. Cytokine & Growth Factor Reviews, 2019, 46: 1-9. Yu, Bo, Zhang, et al. Exosomes derived from mesenchymal stem cells[J]. International Journal of Molecular Sciences, 2014, 15(3): 4142-4157. Song, Woo-Jin, Li, et al. TSG-6 secreted by human adipose tissue-derived mesenchymal stem cells ameliorates DSS-induced colitis by inducing M2 macrophage polarization in mice[J]. Scientific Reports, 2017, 7(1): 5187. Supplementary Files ARRIVEguidelines2.0.pdf Westernblotsimages.pdf Cite Share Download PDF Status: Published Journal Publication published 07 Jul, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted Editorial decision: Major Revision 27 Mar, 2025 Reviewers agreed at journal 25 Mar, 2025 Reviewers invited by journal 25 Mar, 2025 Editor assigned by journal 20 Mar, 2025 First submitted to journal 19 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6218655","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433685222,"identity":"103da1eb-3797-4996-a9ae-04b04270ac9c","order_by":0,"name":"Yansong Ge","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYLCCDwYScvzMzIcfEK2DcUaFhbFkO1uaAdFamHnOVCRuOM+jIEGUcoPzxy8+4G2TMDY+zMNgwFBjE01Yy42cYgPJNgk5s8O8Bx4wHEvLbSCshSdNwhBoi9lhvgQDxobDRGg5fyb9R2KbROLmZh4DCeK0HEg/xnDgjETiBmZitUjeyGGWbKiQMJY4DAzkBGL8wnf++MPPfwzq5Pj7Dx9+8KHGhrAWhQM8SBGYQEg5CMg3sD8gRt0oGAWjYBSMZAAANvVCBSmhlDwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4270-2716","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Yansong","middleName":"","lastName":"Ge","suffix":""},{"id":433685223,"identity":"a172d756-8c2f-466b-afa2-8ed652d52f20","order_by":1,"name":"Mingzhen Chen","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mingzhen","middleName":"","lastName":"Chen","suffix":""},{"id":433685224,"identity":"d44cb2c3-4951-4ba1-b423-0d8088f032e2","order_by":2,"name":"Meilin Li","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Meilin","middleName":"","lastName":"Li","suffix":""},{"id":433685225,"identity":"260eb99c-dda5-484e-bae2-74006ccfe44d","order_by":3,"name":"Zheng Wang","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Wang","suffix":""},{"id":433685226,"identity":"99331a94-5799-46ec-adb9-2c59e413baea","order_by":4,"name":"Ruxin Ding","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ruxin","middleName":"","lastName":"Ding","suffix":""},{"id":433685227,"identity":"8a9c5522-491a-4dcf-adb5-074d8eca259e","order_by":5,"name":"Zhiying Wan","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhiying","middleName":"","lastName":"Wan","suffix":""},{"id":433685228,"identity":"9cbfc945-6790-4953-b4d4-a7b76226d6ff","order_by":6,"name":"Enshuang Xu","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Enshuang","middleName":"","lastName":"Xu","suffix":""},{"id":433685229,"identity":"8e741541-ad99-495e-be23-72c86376c1ff","order_by":7,"name":"Jiasan Zheng","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiasan","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2025-03-13 09:42:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6218655/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6218655/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-025-04466-4","type":"published","date":"2025-07-07T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80050277,"identity":"fbe5a89d-5298-4456-8456-326319f5193b","added_by":"auto","created_at":"2025-04-07 10:19:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4108600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of ADSCs and immunofluorescence. \u003c/strong\u003e(A) In vitro cultured canine ADSCs. Scale bar = 50 μm. (B) ADSCs lipogenic differentiation and Oil Red O staining. Scale bar = 50 μm. (C) ADSCs osteogenic differentiation and Alizarin Red staining. Scale bar = 100 μm. (D) Immunofluorescence of ADSCs, CD34, CD44, CD90, and CD105 were incubated with Alexa Fluor 594 (red), and the nuclei were incubated with DAPI (blue). Scale bar = 200 μm.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6218655/v1/84c535def456059600c7854a.jpg"},{"id":80050274,"identity":"cb8b36bc-0e22-47e9-a765-864c19afdb60","added_by":"auto","created_at":"2025-04-07 10:19:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3119398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSodium taurocholate and trypsin caused AP and ferroptosis. \u003c/strong\u003e(A) HE staining. Scale bar = 200 μm and 50 μm. (B) Transmission electron microscope. Scale bar = 2 μm. (C-D) Levels of plasma AMY and LIPA. (E-H) Western blot analysis of GPx4, TFR1, and FTH as evidence of iron death during AP, and optical densitometry was used to determine the relative expressions. (I-K) Analysis of the relative expression of GPx, FTH, and FTL mRNA. n = 3 each group with similar results. The data are presented as means ± SD (* \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6218655/v1/0346bffd9869b71e3d4203cb.jpg"},{"id":80050267,"identity":"746f591c-3632-4a78-aeb7-8c79dc67b4e7","added_by":"auto","created_at":"2025-04-07 10:19:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1287802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment for iron content and antioxidant capacity.\u003c/strong\u003e (A) Levels of pancreatic iron. (B-E) GSH, GPx, T-AOC activities and MDA level in plasma were measured using antioxidant kits. (F-K) Analysis of the relative expression of Total Nrf2, Nuclear Nrf2, Keap1, HO-1, and NQO1 proteins. (L-O) Analysis of the relative expression of Nrf2, Keap1, HO-1, and NQO1 mRNA. The data are presented as means ± SD (* \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6218655/v1/d08c3b2ced407c494b0a2636.jpg"},{"id":80051615,"identity":"05561add-51ec-4fef-a28c-e206ef5c71cf","added_by":"auto","created_at":"2025-04-07 10:27:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3450615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eADSCs and ADSCs-CM treatment relieves pancreatic dysfunction.\u003c/strong\u003e (A) HE staining. Scale bar = 50 μm. (B) Transmission electron microscope. Scale bar = 2 μm. (C-E) Van Laethem's scoring guidelines were used to assess edema, inflammatory infiltration, and necrosis in pancreatic tissues. (E-K) AMY, LIPA, TP, ALB, TCHO, and TBIL were detected using an automated biochemistry analyzer. (L) WBC were detected using an automated hematology system. (M-R) PaCO\u003csub\u003e2\u003c/sub\u003e, TCO\u003csub\u003e2\u003c/sub\u003e, pH, and HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e were detected using a blood gas analyzer. (Q-R) Levels of plasma and pancreatic iron. The data are presented as means ± SD (* \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, vs. CON group, \u003csup\u003e#\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, vs. AP group).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6218655/v1/cd69e850d241125b93d72d3e.jpg"},{"id":80050269,"identity":"ba4b2148-8bfb-4cb8-8231-551b0f242810","added_by":"auto","created_at":"2025-04-07 10:19:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1410396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eADSCs treatment reduced pancreatic ferroptosis in AP. \u003c/strong\u003e(A-G) Analysis of the relative expression of Nuclear Nrf2, TFR1, Keap1, GPx4, HO-1, and NQO1 proteins. (H-O) Analysis of the relative expression of GPx, FTH, FTL, Nrf2, Keap1, NQO1, HO-1, and GST mRNA. The data are presented as means ± SD (* \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, vs. CON group, \u003csup\u003e#\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, vs. AP group, \u003csup\u003e▲\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, vs. AP group).\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6218655/v1/6a9bf056756b15a5ec4fb0cb.jpg"},{"id":80051613,"identity":"057a3762-18b0-4ddd-b112-9bbbca1a4862","added_by":"auto","created_at":"2025-04-07 10:27:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1387798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eADSCs alleviated LPS induced ferroptosis in AR42J cells.\u003c/strong\u003e (A) CCK-8 assay was performed in AR42J treated with LPS. (B-H) Analysis of the relative expression of Nuclear Nrf2, FTH, Keap1, GPx4, HO-1, and NQO1 proteins. (I-P) Analysis of the relative expression of GPx, FTH, FTL, Nrf2, Keap1, NQO1, HO-1, and GST mRNA. The data are presented as means ± SD (* \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, vs. CON group, \u003csup\u003e#\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, vs. AP group).\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6218655/v1/c8c06afb62b069f5093c6809.jpg"},{"id":80050282,"identity":"df3a2d53-3df5-4af7-82b7-50d34318d0d1","added_by":"auto","created_at":"2025-04-07 10:19:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2668053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eADSCs attenuate ferroptosis in AR42J cells by promoting nuclear translocation of Nrf2. \u003c/strong\u003e(A) Stained AR42J cells with Mito-Tracker Red and Nrf2 specific antibody (Green) were observed by confocal microscopy. Scale bar = 10 μm. (B-E) The protein expressions of GPx4 and FTH were detected by immunoblotting. Scale bar = 200 μm.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6218655/v1/614e123083f5167138ec34ad.jpg"},{"id":86699344,"identity":"709e458c-e564-4c30-b88e-f37c4e0fff06","added_by":"auto","created_at":"2025-07-14 16:08:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18523780,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6218655/v1/0d5a0588-2cbc-4554-a1e6-31abd58f177c.pdf"},{"id":80050283,"identity":"83cabc60-cd35-4a1f-a192-db9ff1c120e4","added_by":"auto","created_at":"2025-04-07 10:19:19","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":126689,"visible":true,"origin":"","legend":"","description":"","filename":"ARRIVEguidelines2.0.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6218655/v1/6779b69d13f5c74beec41591.pdf"},{"id":80051619,"identity":"aba3af1a-6f8c-46ff-8ca2-f6d150c3910f","added_by":"auto","created_at":"2025-04-07 10:27:19","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":416244,"visible":true,"origin":"","legend":"","description":"","filename":"Westernblotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6218655/v1/bb98845bc8970b77100885b3.pdf"}],"financialInterests":"","formattedTitle":"Adipose-derived Stem Cells Alleviate Acute Pancreatitis by Inhibiting Ferroptosis and Oxidative Damage in Canine","fulltext":[{"header":"Background","content":"\u003cp\u003eAcute pancreatitis (AP) is a common inflammatory disease that affects dogs, cats, and other small animals. AP can cause symptoms such as fever, nausea, vomiting, abdominal pain, diarrhea, and shock\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Complications such as renal failure, acute lung injury, disseminated intravascular coagulopathy, and pancreatic exocrine insufficiency can occur if treatment is delayed\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The pathogenesis of AP is complex, the standard of care for managing AP is symptomatic and supportive. Steiner et al. reported that fuzapladib was safe to administer to dogs, fuzapladib therapy of AP resulted in a significant improvement in the modified clinical activity index\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. It is critical to discover new therapeutic drugs to improve AP treatment in the clinic.\u003c/p\u003e \u003cp\u003eFerroptosis is a newly regulatory cell death characterized by the accumulation of reactive oxygen species (ROS) and iron-dependent lipid peroxidation. Intracellular lipid peroxide metabolism is impaired and abnormal metabolism occurs under the catalytic effect of iron ions, and when the cellular antioxidant capacity is weakened, lipid reactive oxygen species accumulate, causing intracellular redox imbalance and inducing cell death\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. According to reports, ferroptosis occurring in a single cell has the potential to spread to neighboring cells in a fast propagating wave, causing extensive tissue damage\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdipose-derived stem cells (ADSCs) are multipotent stem cells with a high capacity for self-renewal that are obtained from adipose tissue that have been used to treat malignant illnesses, gastrointestinal disorders, musculoskeletal disorders, and skin conditions in companion animals\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. ADSCs conditioned medium (CM) contains abundant bioactive substances secreted by ADSCs. Studies have shown that CM exhibited pancreatic protective effects by reducing the severity of caerulein-induced AP in mice, as well as decreasing interleukin-6 (IL-6) and myeloperoxidase (MPO) activities\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Wang et al. revealed that Klotho overexpressing MSC-derived extracellular vesicles mitigate cerulein-triggered apoptosis and NF-κB activation in AR42J cells. Therefore, the study of stem cell function is crucial for exploring the pathogenesis and treatment of pancreatitis\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. In this study, we established an animal model of canine AP to determine the possible therapeutic benefits of ADSCs for AP and to investigate their role in the regulation of ferroptosis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e The experiment was approved by the Animal Protection and Academic Standardization Committee of Heilongjiang Bayi Agricultural University (Project: Molecular mechanism of adipose stem cells regulating transcription factor activity to attenuate canine pancreatitis. Date: 2023-03-23. Number: DWKJXY2023049). The work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eADSCs culture and preparation of ADSCs-CM\u003c/h3\u003e\n\u003cp\u003eUmbilical falciform ligament tissue was obtained from dogs, the adipose tissues were minced, and ADSCs were isolated by digestion with collagenase. Cells were suspended with low-sugar DMEM (HyClone, USA) containing 10%FBS (Senbeijia, China), 2 mM L-glutamine, 100 \u0026micro;g/mL penicillin and streptomycin (Solarbio, China), cultured at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator (ThermoFisher, USA). Lipogenic and osteogenic induced differentiation medium (Cyagen, USA) was used to characterize the differentiation capacity of ADSCs. Calcium nodules were labeled using 0.1 mg/mL Alizarin Red and lipid droplets were labeled using 0.5% Oil Red O. Part of passage 3\u0026ndash;5 ADSCs were collected for transplantation, and part of the cells were cultured with FBS-free medium for 48 h, 3 kDa ultrafiltration units were used to concentrate 10\u003csup\u003e7\u003c/sup\u003e ADSCs into 1 mL ADSCs-CM.\u003c/p\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eSixteen adult dogs (5.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49 kg) were provided by the Laboratory Animal Center of Heilongjiang Bayi Agricultural University (Daqing, China). The dogs were fed a standard dog diet (Ouchong Pet Products, China) and housed in separate rooms at an animal research facility that was kept at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C and 55%\u0026plusmn;15% humidity. The lights in each room switched on and off between 7:00 am and 9:00 evening, and the windows in each room let in extra natural light.\u003c/p\u003e \u003cp\u003eThe dogs were randomly divided into 4 groups (CON, AP, ADSCs and CM group, n\u0026thinsp;=\u0026thinsp;4/group). Fifteen minutes following the dexmedetomidine injection, the dogs received induction anesthesia with propofol and maintenance anesthesia with isoflurane. After shaving and sterilizing the abdomen, an incision was made in the mid-abdominal line in front of the umbilicus, and the duodenum was retracted outside the abdominal. The pancreaticobiliary duct is identified, the duodenum is slit, and a soft indwelling needle is placed into the larger papilla of the duodenum. The AP, ADSC and CM groups established AP model by retrogradely injecting sodium taurocholate (5%, 0.1 mL/kg) and trypsin (3500 U/kg) through the pancreaticobiliary duct, while the CON group received an injection of 0.9% NaCl. During the injection, the fingers are used to compress the major and minor duodenal papilla to allow the injected fluid to penetrate the pancreas. ADSCs (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg) were injected intravenously into the ADSCs group at 6 h after surgery, ADSCs-CM (0.1 mL/kg) into the CM group, and 0.9% NaCl into the CON and AP group. Blood samples were collected through the cephalic vein at 24 hours postoperatively, Glasgow composite pain scale (GCPS) was scored according to Reid's description, and clinical indicators were assessed\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. After anesthesia, the pancreas was retracted out of the abdomen through the original incision, tissues with significant inflammation were selected for sample collection from the tail of the pancreas, and the resection site was ligated with sutures. The dogs received standard analgesic, antiemetic, anti-inflammatory, and rehydration therapy after surgery.\u003c/p\u003e\n\u003ch3\u003eHistological examination\u003c/h3\u003e\n\u003cp\u003eHematoxylin-eosin (HE) stained 4 \u0026micro;m sections of pancreatic tissues were used to measure the severity of acute pancreatitis. According to Van Laethem's scoring guidelines, the degree of edema, inflammatory infiltration, and necrosis was evaluated\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eTransmission electron microscope\u003c/h3\u003e\n\u003cp\u003eGlutaraldehyde-fixed pancreatic tissue samples were dehydrated, embedded, sliced, and electron-stained before being examined under a transmission electron microscope to check for alterations in the cellular ultrastructural.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBlood indicators assay\u003c/h2\u003e \u003cp\u003eBlood samples were drawn and tested for white blood cells (WBC) serum amylase (AMY), lipase (LIPA), total protein (TP), albumin (ALB), total cholesterol (TCHO), total bilirubin (TBIL), pH, partial pressure of carbon dioxide (PaCO\u003csub\u003e2\u003c/sub\u003e), total carbon dioxide (TCO\u003csub\u003e2\u003c/sub\u003e) and bicarbonate (HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), using automated hematology system, automated biochemistry analyzer, and blood gas analyzer (IDEXX, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIron assay\u003c/h3\u003e\n\u003cp\u003eThe serum and pancreatic iron contents were measured and calculated according to the instructions (Nanjing Jiancheng Bioengineering Institute, China). In brief, acidic and reducing chemicals are used to remove iron from transferrin-containing proteins and reduce the Fe\u003csup\u003e3+\u003c/sup\u003e to Fe\u003csup\u003e2+\u003c/sup\u003e, which are then detected colorimetrically by the level of ferrous ions that bind to bipyridine to form a pink complex.\u003c/p\u003e\n\u003ch3\u003eOxidative activities assay\u003c/h3\u003e\n\u003cp\u003ePancreatic glutathione (GSH), glutathione peroxidase (GPx), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) concentrations were measured using assay kits (Nanjing Jiancheng Bioengineering Institute, China) according to the instructions.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eRat pancreatic acinar cells (AR42J) were provided by Wuhan Pricella Biotechnology Co., Ltd., which were cultured in Ham's F-12K medium with 20% FBS and 1% penicillin-streptomycin. Cells were kept in 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. AR42J were co-cultured with ADSCs in a transwell chamber and treated with LPS for 24 h. The CCK-8 kit was used to detect AR42J cells activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from pancreatic tissue using the TRIzol reagent (Invitrogen, USA) and cDNA was generated using the PrimeScript RT reagent Kit (TAKARA, Japan). qRT-PCR reaction was performed in a 20-\u0026micro;L reaction volume containing 10 \u0026micro;L TB Green Premix Ex Taq (TAKARA, Japan), 2 \u0026micro;L cDNA, 0.8 \u0026micro;L of each primer, 6.4 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO, and set as 95 ℃ for 5 min; 40 cycles of 95 ℃ for 15 s, 60 ℃ for 30 s. The relevant gene expression levels were calculated according to the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e relative quantification method. The primers used in the study is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used for qPCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer sequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGPx4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward 5\u0026rsquo;-GGCAAGACGGACGTAAACTACACTC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo;-CGGCGGCGAACTCTTTGATCTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFTH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward 5\u0026rsquo;-CCATCAACCGCCAGATCAACCTG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo;-GTTTCTCAGCATGTTCCCTCTCCTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFTL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward 5\u0026rsquo;-TGCGGATCTGTCTCTTGCTTCAAC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo;-CAGGAAGATGGCTCCGAAGGTTG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNrf2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward 5\u0026rsquo;-ACTCTTGCCGTTCAGTCAGTCATTG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo;-CACCATGCTAGTCTCGACCAACTTG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKeap1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward 5\u0026rsquo;-CCTGGACAGTGTGGAGTGTTATGAC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo;-GTTCTGCTGGTCGATCTGCTTCC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNQO1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward 5\u0026rsquo;-TGCAGCTCGCTGTCGGTATAATTC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo;-GCAACCTTGTTTAGCCTCCTCTCTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHO-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward 5\u0026rsquo;-CTGTATCGCTCCCGCATGAACTC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo;-TGGTCCTCAGTGTCCTTGCTCAG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGST\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward 5\u0026rsquo;-CTGCTCACACTGCTGTACTGATACG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo;-CACCTTGCCCTGTTCCCTAAACTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward 5\u0026rsquo;-TCCTGACCCTGAAGTACCCCATTG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo;-GTTGTAGAAGGTGTGGTGCCAGATC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eGPx4, Glutathione peroxidase; FTH, Ferritin heavy chain; FTL, Ferritin light chain; Nrf2, Nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; HO-1, Heme oxygenase-1; GST, Glutathione S-transferase.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGPx4, Glutathione peroxidase; FTH, Ferritin heavy chain; FTL, Ferritin light chain; Nrf2, Nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; HO-1, Heme oxygenase-1; GST, Glutathione S-transferase.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003eProtein was extracted from pancreatic tissues using Nuclear and Cytoplasmic Protein Extraction Kit, and the protein concentration was detected using a BCA Kit (Beyotime, China). Protein samples (30 \u0026micro;g) were separated via 12% SDS-PAGE gels and electroblotted onto PVDF membranes (Millipore, Germany). Subsequently, the membranes were blocked with 5% nonfat milk and then incubated with anti-GPx4 (ABclonal, USA), Transferrin receptor-1 (TFR1), FTH (Affinity, USA), Nrf2, Keap1, HO-1, NQO1 (Wanleibio, China), Lamin B and β-Actin (Proteintech, China) at 4℃ overnight. The membranes were then washed in TBST, incubated with HRP-conjugated anti-IgG for two hours, and detected on a Tanon 5200 System (Tanon, China) using an ECL reagent (Biosharp, China). The ImageJ program was used to quantify relative protein expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eCells were fixed in 4% paraformaldehyde, washed with TBST. ADSCs were incubated with polyclonal antibodies CD34, CD44, CD90, and CD105 (Servicebio, China) at 4\u0026deg;C overnight to stain cell surface antigens, and AR42J cells were incubated with Nrf2, FTH, and GPx4 antibodies. After that, secondary antibodies with fluorescent labels were incubated, the nuclei were stained with DAPI. Images were captured using a fluorescence microscope (Leica, Germany) or confocal laser scanning biomicroscope (Olympus, Tokyo), then use Fiji software to adjust the threshold and remove the background from the images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eNormal distribution tests were performed using GraphPad Prism 8.0 (GraphPad Software, USA), and the data was analyzed using one-way ANOVA with Turkey's post hoc test for statistical significance across groups. Results were provided as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating significance. Three replications of each experiment were performed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCharacteristics of ADSCs\u003c/h2\u003e \u003cp\u003eCanine ADSCs had fibroblast-like spindle shape and adhered to the wall \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. After 14 days of lipogenic induction, Oil Red O staining revealed orange-red lipid droplets in the cytoplasm \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, and Alizarin Red staining revealed red calcium nodules after 21 days of osteogenic induction \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, demonstrating that ADSCs possess the capacity to differentiate into adipocytes and osteoblasts, respectively. Immunofluorescence analysis revealed that ADSCs express the stem cell-specific surface antigens CD44, CD90, and CD105, while not express CD34 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSodium taurocholate and trypsin induced canine AP\u003c/h2\u003e \u003cp\u003eSNAP cPL was effective in detecting pancreatitis, yielding positive results in the AP, ADSCs, and CM groups, indicating that sodium taurocholate and trypsin resembled AP. The dogs in these three groups exhibited considerable symptoms of pain, and the GCPS was higher than in the CON group, however, no differences were observed among the three groups. Systolic Blood Pressure (SBP) was significantly elevated in the AP and ADSCs groups, while diastolic blood pressure (DBP) was significantly higher in the AP group and significantly lower in the CM group compared to the AP group. No differences were found in temperature (T), respiratory rate (RR), heart rate (HR), or mean arterial pressure (MAP) among the four groups \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used for qPCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eADSCs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGCPS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT (℃)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e37.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e38.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e38.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e38.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRR (Times/min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.33\u0026thinsp;\u0026plusmn;\u0026thinsp;6.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e30.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e28.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e28.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHR (Times/min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e82.00\u0026thinsp;\u0026plusmn;\u0026thinsp;15.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e87.67\u0026thinsp;\u0026plusmn;\u0026thinsp;7.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e89.00\u0026thinsp;\u0026plusmn;\u0026thinsp;9.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e89.33\u0026thinsp;\u0026plusmn;\u0026thinsp;6.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSBP (mmHg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e110.00\u0026thinsp;\u0026plusmn;\u0026thinsp;9.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e152.00\u0026thinsp;\u0026plusmn;\u0026thinsp;8.89*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e146.00\u0026thinsp;\u0026plusmn;\u0026thinsp;12.12*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e136.00\u0026thinsp;\u0026plusmn;\u0026thinsp;14.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDBP (mmHg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e69.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e98.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.11*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e85.67\u0026thinsp;\u0026plusmn;\u0026thinsp;8.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e74.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.77\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMAP (mmHg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e83.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e104.33\u0026thinsp;\u0026plusmn;\u0026thinsp;15.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e105.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e92.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. CON; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. AP.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHE staining showed that pancreatic acinar cells in the CON group exhibited homogeneous interstitial stroma and clear structure, whereas the pancreas in the AP group displayed interstitial edema, massive inflammatory cell infiltration, multiple hemorrhages, widespread nuclear disintegration, and necrosis of the acinar cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Transmission electron microscopy revealed that AP induced cell nuclear membrane wrinkling, chromatin edge aggregation, endoplasmic reticulum expansion, blurred structure, reduced rough endoplasmic reticulum, and mitochondrial cristae disorder \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Serum biochemical tests revealed that the injection a mixed solution into the pancreaticobiliary duct increased plasma AMY and LIPA levels significantly \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e. These findings indicate that sodium taurocholate and trypsin induce pancreatic dysfunction in dogs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFerroptosis induced by canine AP\u003c/h2\u003e \u003cp\u003eWe examined the protein expression levels of GPx4, TFR1, and FTH in order to investigate sodium taurocholate and trypsin induced ferroptosis in the canine pancreas. Notably, the expression of TFR1 and FTH was significantly elevated in the AP group compared to the CON group, while GPx4 expression was significantly reduced \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-H\u003cb\u003e)\u003c/b\u003e. Furthermore, the results revealed that the expression levels of GPx4, TFR1, and FTH mRNA were consistent with the protein alterations using qRT-PCR analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-K\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe iron content in pancreas of the AP group was significantly higher than that of the CON group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. AP inhibited the activities of GSH, GPx and T-AOC in the pancreas, while simultaneously increasing MDA production \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-E\u003cb\u003e)\u003c/b\u003e. Further examination of the Kepa1/Nrf2 pathway showed that the levels of Keap1, HO-1, and NQO1 proteins were significantly lower in the AP group compared to the CON group, Nuclear Nrf2 protein was significantly increased in the AP group, whereas Total Nrf2 protein did not change significantly \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-K\u003cb\u003e)\u003c/b\u003e. The mRNA expression changes exhibited similar trends \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL-O\u003cb\u003e)\u003c/b\u003e. These findings suggest that trypsin and sodium taurocholate-induced pancreatitis promote ferroptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eADSCs and ADSCs-CM treatment relieves pancreatic dysfunction\u003c/h2\u003e \u003cp\u003eTo determine the effect of ADSCs on ferroptosis in AP, transmission electron microscopy and histological examination were performed. After transplantation of ADSCs, transmission electron microscopy showed that ADSCs and CM treatment improved the ultrastructural disorder of pancreatic cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Additionally, these treatments significantly reduced pancreatic hemorrhage, inflammatory infiltration, edema, and vacuole formation, providing similar therapeutic benefits for AP \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Correspondingly, the scores of edema and necrosis were significantly lower compared to the AP group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBiochemical parameters analyses revealed that ADSCs and CM treatment significantly inhibited AMY and LIPA secretion \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-G\u003cb\u003e)\u003c/b\u003e. AP significantly reduced TP and ALB levels, and plasma ALB levels were significantly raised by ADSC therapy \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I\u003cb\u003e)\u003c/b\u003e. Meanwhile, TBIL levels were noticeably elevated in the AP and ADSCs groups, and CM treatment markedly reversed the trend of elevated TBIL \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e. The AP group had the highest levels of TCHO, WBC, and PaCO\u003csub\u003e2\u003c/sub\u003e; these levels were decreased by ADSCs and CM treatment, but there were no appreciable differences \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK-M\u003cb\u003e)\u003c/b\u003e. Blood TCO\u003csub\u003e2\u003c/sub\u003e levels in the AP group slightly increased, which was significantly reduced by treatment with ADSCs and CM \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN\u003cb\u003e)\u003c/b\u003e. There were no appreciable differences in pH and HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e levels between the four groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO-P\u003cb\u003e)\u003c/b\u003e. These findings suggest that ADSCs-CM alleviate pancreatic dysfunction and lessen the body\u0026rsquo;s reaction to AP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eADSCs transplantation reduced pancreatic ferroptosis in AP\u003c/h2\u003e \u003cp\u003eAccording to the results of the iron content assay, significantly elevated plasma and pancreatic iron levels in the AP group served as a marker for the onset of ferroptosis, while ADSCs and CM treatments significantly downregulated pancreatic iron levels and had a lowering effect on plasma iron contents \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eQ-R\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eResults from the expression of ferroptosis and Keap1-Nrf2 pathway proteins revealed that AP induced significant upregulation of Nuclear Nrf2 and TFR1 levels, whereas downregulation of Keap1, GPx4, HO-1, and NQO1 protein levels. Nuclear Nrf2, TFR1, GPx4, HO-1, and NQO1 protein expression increased after treatment with ADSCs or CM, whereas Keap1 protein expression decreased \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-G\u003cb\u003e)\u003c/b\u003e. The mRNA expression was also examined. The pancreatic inflammatory response decreased the expression of GPx, Keap1, NQO1, and HO-1 mRNA while increasing the expression of FTH and FTL mRNA. In ADSCs or CM treatment caused upregulation of GPx, Nrf2, Keap1, GST mRNA expression, while FTH mRNA expression was downregulated \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH-O\u003cb\u003e)\u003c/b\u003e. These findings suggest that ADSCs, through paracrine function, alleviate ferroptosis and pancreatic injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eADSCs attenuate LPS-induced AR42J ferroptosis in vitro\u003c/h2\u003e \u003cp\u003eTo elucidate the mechanism by which ADSCs alleviate ferroptosis, we established an AR42J inflammatory model in vitro using LPS at 0-200 mg/L. The results revealed that LPS at various concentrations inhibited AR42J cell activity and showed dose-dependent behavior. The IC\u003csub\u003e50\u003c/sub\u003e of LPS was calculated to be 42.53mg/L, and a concentration of 10mg/L was selected for the following experiments \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Alterations in ferroptosis-related proteins and genes in the AR42J model were in line with the in vivo experiments. LPS treatment induced ferroptosis and down-regulated the expression of Nrf2 and its downstream in AR42J. Co-culturing ADSCs with AR42J reduced LPS-induced ferroptosis and lessened its inhibitory effect on the Keap1/Nrf2 pathway \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-P\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, we labeled Nrf2 with specific antibodies, and used Mito-tracker and DAPI label mitochondria and nucleus. The expression of Nrf2 protein in the nucleus was found to be significantly upregulated by ADSC treatment and promote its nuclear translocation under a laser confocal microscope, but there was no significant difference in the expression of Nrf2 in the nucleus of the LPS group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. According to the results of the FTH and GPx4 immunofluorescence staining, LPS decreased the fluorescence intensity in AR42J, while ADSC treatment upregulated the expression levels of FTH and GPx4 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-E\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAP is a common veterinary clinical acute abdominal disease that is frequently accompanied by systemic inflammatory response syndrome, sepsis, and even multiple-organ dysfunction syndrome. It is a potentially fatal disease with a high morbidity and mortality\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Ferroptosis is a non-apoptotic type of programmed cell death in the morphological characteristics of the cells, which include mitochondrial shrinkage, higher mitochondria membrane density, and disappearance of mitochondrial cristae\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Increasing evidence suggested that ferroptosis-regulated cell death may be associated with the onset and complications of AP\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. In this study, we found that sodium taurocholate and trypsin caused AP and ferroptosis, as evidenced by pancreatic histopathology, changes in plasma enzyme activities, GPx4 deletion and increased iron levels.\u003c/p\u003e \u003cp\u003eAs the study of ferroptosis progressed, researchers began to pay more attention to the role of ferroptosis and the associated regulatory roles. Ferroptosis brings together iron, selenium, amino acids, lipids, and redox reactions into a network and maintains homeostasis by participating in various pathways\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Abnormal distribution and levels of iron in the body can interfere with normal physiological processes. Fe\u003csup\u003e3+\u003c/sup\u003e binds to transferrin and forms a complex via TFR1, and the cell takes the complex via endocytosis. Then, Fe\u003csup\u003e3+\u003c/sup\u003e is reduced to Fe\u003csup\u003e2+\u003c/sup\u003e stored in the unstable iron pool and ferritin. Excess Fe\u003csup\u003e2+\u003c/sup\u003e is oxidized to Fe\u003csup\u003e3+\u003c/sup\u003e by ferritin. Ferritin is a heteropolymer composed of FTH and FTL, FTH is in charge of iron atom holding, and FTL may be involved in electron transfer\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Deposition of ferritin can result in pancreatitis and oxidative stress. Inhibiting ferroptosis has been proven in studies to reduce reactive oxidative stress and inflammation during AP, as well as improve multiple organ failure caused by severe AP. This finding emphasizes the tight association between ferroptosis and AP\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFerroptosis causes lipid peroxidation and ROS production, both of which are linked to the pathogenesis of AP. The pancreas is not only a digestive organ, but it is also one of the major organs for iron storage, which suggests that the pancreas is more vulnerable to iron metabolism abnormalities\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Animal studies have shown that a high iron diet or conditional knockout of GPx4 in the pancreas promoted experimental pancreatitis in mice induced by the administration of cerulein or L-arginine. In contrast, ferroptosis inhibitors reversed this damage, implying a pathogenic role for ferroptosis in experimental pancreatitis\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. The results of this study showed significant iron accumulation in the pancreas and plasma, with suppressed GPx4 expression and increased TFR1 and FTH, indicating the presence of ferroptosis during AP.\u003c/p\u003e \u003cp\u003eFerroptosis plays an important role in the development and progression of various diseases, thus, regulating the ferroptosis pathway offers potential therapeutic approaches for the treatment of diseases such as neurological diseases, myocardial infarction, ischemia/reperfusion injury and cancer. As an oxidative cell death mode, ferroptosis is caused by an imbalance in the production and degradation of intracellular lipid ROS\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. According to Jia-Hong\u0026rsquo;s study, due to abnormalities in the iron-dependent Fenton counter, excess free iron causes a sustained accumulation of ROS and ultimately activates ferroptosis and necrosis sequentially\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. ROS are also important in the pathogenesis of AP, as injured cells and activated immune cells produce large amounts of oxygen free radicals. ROS can cause cellular damage and initiate the inflammatory process, resulting in pancreatic edema and inflammatory cell aggregation. In various animal models of AP, antioxidant treatment has been shown to reduce acinar cell necrosis and mitigate the severity of pancreatic tissue injury\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. In this study, the pancreas demonstrated a significant oxidative stress response, as evidenced by an increase in MDA content and decrease in antioxidant enzyme activities. Keap1, HO-1, and NQO1 protein expression were reduced, while Nrf2 nuclear translocation and transcriptional activity were increased.\u003c/p\u003e \u003cp\u003eNrf2 is an essential transcription factor that regulates oxidative homeostasis and modulates GSH, iron, and lipid metabolism, as well as mitochondrial function, by activating the expression of various target genes required for iron death regulation\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Importantly, Nrf2 transcription regulates almost all ferroptosis genes, including GSH regulation, NADPH regeneration, and iron regulation\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Keap1 interacts with Nrf2 to regulate the expression of downstream antioxidant proteins and detoxification enzymes, reducing the damage caused by oxidative stress to cells and tissues\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. The cysteine residues of the Keap1 protein change conformation and fail to remain bound to Nrf2, allowing Nrf2 to dissociate into the nucleus and bind to the ARE\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. It was found that activation of Nrf2 in the nucleus ameliorated pancreatic injury caused by AP and the Keap1-Nrf2 pathway is important in the development of AP\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. In addition, the Nrf2 pathway has been shown to inhibit ferritin formation by regulating HO-1, and Nrf2 knockdown enhances acute lung injury by promoting ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. The current study demonstrates the regulatory effects of ADSCs and ADSCs-CM on oxidative stress and Nrf2, which alleviate pancreatic injury by inhibiting Keap1 expression, promoting Nrf2 translocation into the nucleus and triggering transcription of its downstream ROS detoxification enzymes, such as HO-1, NQO1, GPx, and GST.\u003c/p\u003e \u003cp\u003eSeveral studies have found that mesenchymal stromal cells (MSCs) have significant therapeutic effects in animal models of AP, implying that MESs protect the pancreas through a variety of mechanisms\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. First, MSCs down-regulate pro-inflammatory factors and up-regulate anti-inflammatory factors to attenuate the level of inflammatory response in the pancreas, and suppress the immune response\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Second, MSCs alleviated AP via antioxidant activity, which included increasing antioxidant enzyme activities and reducing MDA production\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Third, MSCs inhibit apoptosis in PACs while promoting angiogenesis in pancreatic tissues\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Furthermore, MSCs repair damaged pancreatic tissues via the effects of secreted VEGF, ANG-1, and HGF, reducing local/systemic inflammatory responses and effectively alleviating AP\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. However, the specific underlying mechanisms of MSCs for AP treatment are still unknown, and clinical applications of MSCs for AP treatment has been rarely reported.\u003c/p\u003e \u003cp\u003eADSCs have a paracrine function of secreted cytokines, chemokines, growth factors, proteases, and extracellular vesicles, and growing evidence suggests that paracrine activity is more important for ADSCs' beneficial effects on tissue repairing than colonization and differentiation\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Qiang et al. demonstrated that ADSCs reduced the unfolded protein response induced by cerulein and lipopolysaccharide via TSG-6 auto-secretion and the inflammatory response by inhibiting NF-B expression, whereas ADSCs with TSG-6 gene knockdown lost the regulation of the unfolded protein response\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. In addition, Zhao et al. reported that baicalein-pretreated MSCs-derived exosomes promoted liver function recovery in acute liver injury mice and activated the Keap1-Nrf2 pathway via P62, inhibiting ROS production and lipid peroxide-induced iron sagging\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. We studied the protective effects of ADSCs and ADSCs-CM on the pancreas and discovered that both reduced pancreatic damage while efficiently restoring aberrant AMYL, LIPA, ALB, and TBIL levels. Inspiringly, cell-free ADSCs-CM treatment exhibited similar alleviation of oxidative damage and ferroptosis as transplantation of ADSCs, this is accomplished by reducing iron accumulation, promoting more Nrf2 translocation into the nucleus and initiating transcription of its downstream ROS-detoxifying enzymes to alleviate pancreatic injury.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summarize, this study demonstrates that ferroptosis occurs during AP, characterized by iron accumulation and inhibition of GPx4 expression. ADSCs transplantation attenuate pancreatic injury, reduce iron accumulation, and mitigate oxidative stress by modulating Nrf2 nuclear translocation. Furthermore, cell-free ADSCs-CM treatment demonstrated therapeutic effects comparable to ADSCs, implying that ADSCs may protect the pancreas via paracrine mechanisms. The results of this study provide insights on the mechanisms of pancreatic ferroptosis and tissue protection in ADSCs following AP, laying the fundamental for future research into targeted therapies for AP.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAP, Acute pancreatitis\u003c/p\u003e\n\u003cp\u003eADSCs, Adipose-derived stem cells\u003c/p\u003e\n\u003cp\u003eCM, Conditioned medium\u003c/p\u003e\n\u003cp\u003eCON, Control\u003c/p\u003e\n\u003cp\u003eGPx4, Glutathione peroxidase 4\u003c/p\u003e\n\u003cp\u003eTFR1, Transferrin receptor-1\u003c/p\u003e\n\u003cp\u003eFTH, Ferritin heavy chain\u003c/p\u003e\n\u003cp\u003eAMY, Amylase\u003c/p\u003e\n\u003cp\u003eLIPA, Lipase\u003c/p\u003e\n\u003cp\u003eNrf2, Nuclear factor erythroid 2-related factor 2\u003c/p\u003e\n\u003cp\u003eROS, Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eIL-6, Interleukin-6\u003c/p\u003e\n\u003cp\u003eMPO, Myeloperoxidase\u003c/p\u003e\n\u003cp\u003eHE, Hematoxylin-eosin\u003c/p\u003e\n\u003cp\u003eWBC, White blood cells\u003c/p\u003e\n\u003cp\u003eTP, Total protein\u003c/p\u003e\n\u003cp\u003eALB, Albumin\u003c/p\u003e\n\u003cp\u003eTCHO, Total cholesterol\u003c/p\u003e\n\u003cp\u003eTBIL, Total bilirubin\u003c/p\u003e\n\u003cp\u003ePaCO\u003csub\u003e2\u003c/sub\u003e, Partial pressure of carbon dioxide\u003c/p\u003e\n\u003cp\u003eTCO\u003csub\u003e2\u003c/sub\u003e, Total carbon dioxide\u003c/p\u003e\n\u003cp\u003eHCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, Bicarbonate\u003c/p\u003e\n\u003cp\u003eGSH, Glutathione\u003c/p\u003e\n\u003cp\u003eT-AOC, Glutathione peroxidase\u003c/p\u003e\n\u003cp\u003eMDA, Malondialdehyde\u003c/p\u003e\n\u003cp\u003eAR42J, Rat pancreatic acinar cells\u003c/p\u003e\n\u003cp\u003eqRT-PCR, Quantitative real-time PCR\u003c/p\u003e\n\u003cp\u003eFTL, Ferritin light chain\u003c/p\u003e\n\u003cp\u003eKeap1, Kelch-like ECH-associated protein 1\u003c/p\u003e\n\u003cp\u003eHO-1, Heme oxygenase-1\u003c/p\u003e\n\u003cp\u003eGST, Glutathione S-transferase\u003c/p\u003e\n\u003cp\u003eTFR1, Transferrin receptor-1\u003c/p\u003e\n\u003cp\u003eSBP, Systolic Blood Pressure\u003c/p\u003e\n\u003cp\u003eDBP, Diastolic blood pressure\u003c/p\u003e\n\u003cp\u003eT, Temperature\u003c/p\u003e\n\u003cp\u003eRR, Respiratory rate\u003c/p\u003e\n\u003cp\u003eHR, Heart rate\u003c/p\u003e\n\u003cp\u003eMAP, Mean arterial pressure\u003c/p\u003e\n\u003cp\u003eMSCs, Mesenchymal stromal cells\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets used during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Provincial Natural Science Foundation of Heilongjiang (LH2023C086), the Heilongjiang Postdoctoral Science Foundation (LBH-Z19088), the Research Fund (Clinical Diagnosis and Treatment of Pet) for Young College Teachers in Ruipeng Commonweal Foundation (RPJJ2020009), and the Personnel Foundation in Heilongjiang Bayi Agricultural University (XYB201915).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYSG: Project administration; Writing-original draft; Data curation; Funding acquisition. MZC: Investigation; Formal analysis. MLL: Data curation; Investigation. ZW: Investigation; Formal analysis. RXD: Investigation; ZYW: Investigation; ESX: Writing-review \u0026amp; editing; Supervision. JSZ: Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not use AI-generated work in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFabr\u0026egrave;s, Virginie, Dossin, et al. Development and validation of a novel clinical scoring system for short‐term prediction of death in dogs with acute pancreatitis[J]. Journal of Veterinary Internal Medicine, 2019, 33(2): 499-507.\u003c/li\u003e\n\u003cli\u003eGarg, Pramod K, Singh, et al. Organ failure due to systemic injury in acute pancreatitis[J]. Gastroenterology, 2019, 156(7): 2008-2023.\u003c/li\u003e\n\u003cli\u003eSteiner, Joerg M, Lainesse, et al. Fuzapladib in a randomized controlled multicenter masked study in dogs with presumptive acute onset pancreatitis[J]. Journal of Veterinary Internal Medicine, 2023, 37(6): 2084-2092.\u003c/li\u003e\n\u003cli\u003eXie, Y, Hou, et al. Ferroptosis: process and function[J]. Cell Death \u0026amp; Differentiation, 2016, 23(3): 369-379.\u003c/li\u003e\n\u003cli\u003eLi, Jie, Cao, et al. Ferroptosis: past, present and future[J]. Cell Death \u0026amp; Disease, 2020, 11(2): 88.\u003c/li\u003e\n\u003cli\u003eLi, Hongyao, Lin, et al. Ferroptosis and its emerging roles in acute pancreatitis[J]. Chinese Medical Journal, 2022, 135(17): 2026-2034.\u003c/li\u003e\n\u003cli\u003eBacakova, Lucie, Zarubova, et al. Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells\u0026ndash;a review[J]. Biotechnology Advances, 2018, 36(4): 1111-1126.\u003c/li\u003e\n\u003cli\u003eKang, Min-Hee, Park, et al. Challenges of stem cell therapies in companion animal practice[J]. Journal of Veterinary Science, 2020, 21(3): e42.\u003c/li\u003e\n\u003cli\u003eAbdolmohammadi, Kamal, Mahmoudi, et al. Effect of hypoxia preconditioned adipose-derived mesenchymal stem cell conditioned medium on cerulein-induced acute pancreatitis in mice[J]. Advanced Pharmaceutical Bulletin, 2020, 10(2): 297.\u003c/li\u003e\n\u003cli\u003eWang, Na, Ma, et al. Secreted klotho from exosomes alleviates inflammation and apoptosis in acute pancreatitis[J]. American Journal of Translational Research, 2019, 11(6): 3375.\u003c/li\u003e\n\u003cli\u003eReid, J, Nolan, et al. Development of the short-form Glasgow Composite Measure Pain Scale (CMPS-SF) and derivation of an analgesic intervention score[J]. Animal Welfare, 2007, 16(S1): 97-104.\u003c/li\u003e\n\u003cli\u003eVan Laethem, Jean-Luc, Marchant, et al. Interleukin 10 prevents necrosis in murine experimental acute pancreatitis[J]. Gastroenterology, 1995, 108(6): 1917-1922.\u003c/li\u003e\n\u003cli\u003eLee, Peter J, Papachristou, et al. New insights into acute pancreatitis[J]. Nature Reviews Gastroenterology \u0026amp; Hepatology, 2019, 16(8): 479-496.\u003c/li\u003e\n\u003cli\u003eStockwell, Brent R, Angeli, et al. Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease[J]. Cell, 2017, 171(2): 273-285.\u003c/li\u003e\n\u003cli\u003eShan, Yuqiang, Li, et al. Ginsenoside Rg3 ameliorates acute pancreatitis by activating the NRF2/HO-1-mediated ferroptosis pathway[J]. International Journal of Molecular Medicine, 2022, 50(1): 89.\u003c/li\u003e\n\u003cli\u003eLiu, Ke, Liu, et al. Trypsin-mediated sensitization to ferroptosis increases the severity of pancreatitis in mice[J]. Cellular and Molecular Gastroenterology and Hepatology, 2022, 13(2): 483-500.\u003c/li\u003e\n\u003cli\u003eFan, Rong, Sui, et al. Wedelolactone alleviates acute pancreatitis and associated lung injury via GPX4 mediated suppression of pyroptosis and ferroptosis[J]. Free Radical Biology and Medicine, 2021, 173: 29-40.\u003c/li\u003e\n\u003cli\u003eJiang, Xuejun, Stockwell, et al. Ferroptosis: mechanisms, biology and role in disease[J]. Nature Reviews Molecular Cell Biology, 2021, 22(4): 266-282.\u003c/li\u003e\n\u003cli\u003eWang, Dong, Zhang, et al. Mesenchymal stromal cell treatment attenuates repetitive mild traumatic brain injury-induced persistent cognitive deficits via suppressing ferroptosis[J]. Journal of Neuroinflammation, 2022, 19(1): 185.\u003c/li\u003e\n\u003cli\u003eLiu, Yang, Wang, et al. The circadian clock protects against ferroptosis-induced sterile inflammation[J]. Biochemical and Biophysical Research Communications, 2020, 525(3): 620-625.\u003c/li\u003e\n\u003cli\u003eDai, Enyong, Han, et al. Ferroptotic damage promotes pancreatic tumorigenesis through a TMEM173/STING-dependent DNA sensor pathway[J]. Nature Communications, 2020, 11(1): 6339.\u003c/li\u003e\n\u003cli\u003eMasaldan, Shashank, Bush, et al. Striking while the iron is hot: Iron metabolism and ferroptosis in neurodegeneration[J]. Free Radical Biology and Medicine, 2019, 133: 221-233.\u003c/li\u003e\n\u003cli\u003eLi, Wenjun, Feng, et al. Ferroptotic cell death and TLR4/Trif signaling initiate neutrophil recruitment after heart transplantation[J]. The Journal of Clinical Investigation, 2019, 129(6): 2293-2304.\u003c/li\u003e\n\u003cli\u003eBelavgeni, Alexia, Meyer, et al. Ferroptosis and necroptosis in the kidney[J]. Cell Chemical Biology, 2020, 27(4): 448-462.\u003c/li\u003e\n\u003cli\u003eWeiland, Abigail, Wang, et al. Ferroptosis and its role in diverse brain diseases[J]. Molecular Neurobiology, 2019, 56: 4880-4893.\u003c/li\u003e\n\u003cli\u003eChu, Jia-Hong, Li, et al. Mercuric chloride induces sequential activation of ferroptosis and necroptosis in chicken embryo kidney cells by triggering ferritinophagy[J]. Free Radical Biology and Medicine, 2022, 188: 35-44.\u003c/li\u003e\n\u003cli\u003eSundar, Vaishnavi, Senthil Kumar, et al. Current trends in pharmacological approaches for treatment and management of acute pancreatitis\u0026ndash;a review[J]. Journal of Pharmacy and Pharmacology, 2020, 72(6): 761-775.\u003c/li\u003e\n\u003cli\u003eDong, Hui, Qiang, et al. Nrf2 inhibits ferroptosis and protects against acute lung injury due to intestinal ischemia reperfusion via regulating SLC7A11 and HO-1[J]. Aging (albany Ny), 2020, 12(13): 12943.\u003c/li\u003e\n\u003cli\u003eYuan, Hanshu, Pratte, et al. Ferroptosis and its potential as a therapeutic target[J]. Biochemical Pharmacology, 2021, 186: 114486.\u003c/li\u003e\n\u003cli\u003eZhao, Shuxian, Huang, et al. Exosomes derived from baicalin‐pretreated mesenchymal stem cells alleviate hepatocyte ferroptosis after acute liver injury via the Keap1‐NRF2 pathway[J]. Oxidative Medicine and Cellular Longevity, 2022, 2022(1): 8287227.\u003c/li\u003e\n\u003cli\u003eSuzuki, Takafumi, Yamamoto, et al. Stress-sensing mechanisms and the physiological roles of the Keap1\u0026ndash;Nrf2 system during cellular stress[J]. Journal of Biological Chemistry, 2017, 292(41): 16817-16824.\u003c/li\u003e\n\u003cli\u003eLiang, Xiaoqiang, Hu, et al. Dihydrokaempferol (DHK) ameliorates severe acute pancreatitis (SAP) via Keap1/Nrf2 pathway[J]. Life Sciences, 2020, 261: 118340.\u003c/li\u003e\n\u003cli\u003eMa, Zhilong, Zhou, et al. Mesenchymal stromal cell therapy for pancreatitis: Progress and challenges[J]. Medicinal Research Reviews, 2021, 41(4): 2474-2488.\u003c/li\u003e\n\u003cli\u003eKim, Hyun-Wook, Song, et al. Canine adipose tissue-derived mesenchymal stem cells ameliorate severe acute pancreatitis by regulating T cells in rats[J]. Journal of Veterinary Science, 2016, 17(4): 539.\u003c/li\u003e\n\u003cli\u003eGori, E, Lippi, et al. Acute pancreatitis and acute kidney injury in dogs[J]. The Veterinary Journal, 2019, 245: 77-81.\u003c/li\u003e\n\u003cli\u003eKawakubo, Kazumichi, Ohnishi, et al. Effect of fetal membrane-derived mesenchymal stem cell transplantation in rats with acute and chronic pancreatitis[J]. Pancreas, 2016, 45(5): 707-713.\u003c/li\u003e\n\u003cli\u003eHe, Zhigang, Hua, et al. Intravenous hMSCs ameliorate acute pancreatitis in mice via secretion of tumor necrosis factor-\u0026alpha; stimulated gene/protein 6[J]. Scientific Reports, 2016, 6(1): 38438.\u003c/li\u003e\n\u003cli\u003eKumar, Praveen, Kandoi, et al. The mesenchymal stem cell secretome: A new paradigm towards cell-free therapeutic mode in regenerative medicine[J]. Cytokine \u0026amp; Growth Factor Reviews, 2019, 46: 1-9.\u003c/li\u003e\n\u003cli\u003eYu, Bo, Zhang, et al. Exosomes derived from mesenchymal stem cells[J]. International Journal of Molecular Sciences, 2014, 15(3): 4142-4157.\u003c/li\u003e\n\u003cli\u003eSong, Woo-Jin, Li, et al. TSG-6 secreted by human adipose tissue-derived mesenchymal stem cells ameliorates DSS-induced colitis by inducing M2 macrophage polarization in mice[J]. Scientific Reports, 2017, 7(1): 5187.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Canine, adipose-derived stem cells, conditioned medium, acute pancreatitis, ferroptosis, oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-6218655/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6218655/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCanine acute pancreatitis (AP) is a common exocrine pancreatitis disease that can lead to systemic inflammatory response syndrome and multi-organ failure. This study aims to investigated the potential therapeutic benefits of adipose-derived stem cells (ADSCs) and conditioned medium (CM) in managing canine AP and the role in ferroptosis regulation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSixteen dogs were randomly divided into control (CON), AP, ADSCs and CM group. The AP model were established by injecting sodium taurocholate (5%, 0.1 mL/kg) and trypsin (3500 U/kg) through the pancreaticobiliary duct. ADSCs (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/kg) and CM (0.1 mL/kg) were injected intravenously at 6 h after surgery, and the roles on ferroptosis and oxidative stress were analyzed. In addition, the changing pattern of ferroptosis and oxidative stress were investigated by LPS-induced cellular inflammation model of AR42J in vitro.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur study showed that ferroptosis occurs in the pancreas during AP, as evidenced by significant iron accumulation, with suppressed glutathione peroxidase 4 (GPx4) expression and increased transferrin receptor-1 (TFR1) and ferritin heavy chain (FTH). ADSCs and ADSCs-CM treatment achieved pathological remission and effectively restored abnormal amylase (AMY), lipase (LIPA) levels. ADSCs-CM showed similar ferroptosis alleviating effects as ADSCs treatment, with reduced iron accumulation and increased GPx4 expression. Furthermore, ADSCs promote nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into the nucleus and initiate transcription of detoxification enzymes to protect the pancreas from oxidative damage.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eBased on these findings, ADSCs protect the pancreas by inhibiting ferroptosis and oxidative stress via paracrine function, which could be a therapeutic target for AP.\u003c/p\u003e","manuscriptTitle":"Adipose-derived Stem Cells Alleviate Acute Pancreatitis by Inhibiting Ferroptosis and Oxidative Damage in Canine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 10:19:14","doi":"10.21203/rs.3.rs-6218655/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-03-28T03:30:25+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-25T22:38:19+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-25T10:28:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T08:02:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2025-03-20T03:42:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"77f4ca46-7cc5-434a-9324-c181f25c81f2","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-14T16:00:35+00:00","versionOfRecord":{"articleIdentity":"rs-6218655","link":"https://doi.org/10.1186/s13287-025-04466-4","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-07-07 15:57:14","publishedOnDateReadable":"July 7th, 2025"},"versionCreatedAt":"2025-04-07 10:19:14","video":"","vorDoi":"10.1186/s13287-025-04466-4","vorDoiUrl":"https://doi.org/10.1186/s13287-025-04466-4","workflowStages":[]},"version":"v1","identity":"rs-6218655","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6218655","identity":"rs-6218655","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}