Skip to main content
NCBI home page
Gastro Hep Advances logoLink to Gastro Hep Advances
. 2026 Jul 29;5(10):101076. doi: 10.1016/j.gastha.2026.101076

Rapamycin Attenuates Acute Pancreatitis-Induced Acinar Injury in Mice With Pancreatic Loss of Gα13

Hidayatullah G Munshi 1,2,∗∗, Anastasia E Metropulos 1, Khadijeh Karbalaei 3, Christina Spaulding 1, Nida Mubin 1, Yaning Xi 1, Apurba Majumder 1, Thao D Pham 1,2, Mario A Shields 3,4,∗
PMCID: PMC13524613  PMID: 42668491

Abstract

Background and Aims

While Gα13 transduces signals from G protein-coupled receptors to regulate inflammation, its role in pancreatitis remains poorly understood. This study aims to understand how Gα13 regulates pancreatitis-induced inflammation and acinar injury.

Methods

Expression of Gα13 in mouse and human pancreas tissue was examined by immunostaining. Experimental pancreatitis was induced by administering cerulein to mice with wild-type and conditional knockout of Gα13 in the pancreas. The impact of Gα13 loss on pancreatitis was assessed by histology, immunostains, and cytokine proteome array. The Gα13-deficient mice were cotreated with the mammalian target of rapamycin (mTOR) inhibitor rapamycin during cerulein treatment to determine the effects of mTOR inhibition on pancreatitis.

Results

We found reduced Gα13 expression in acute and chronic human pancreatitis samples. We show that induction of acute and chronic pancreatitis in mice with cerulein treatment reduces Gα13 expression. Moreover, we found worsening of acute and chronic pancreatitis-induced acinar injury in mice with pancreas-specific deletion of Gα13. While pancreatic deletion of Gα13 did not exacerbate acute pancreatitis-induced inflammation, we found that Gα13 loss mildly exacerbated chronic pancreatitis. Gα13 loss increased cerulein-induced mTOR signaling in the acute and chronic pancreatitis mouse models; however, the mTOR inhibitor rapamycin attenuated the effects of cerulein only in the acute pancreatitis model, whereas it exacerbated the effects of cerulein in the chronic pancreatitis model.

Conclusion

This work demonstrates that Gα13 loss promotes tissue damage in acute and chronic pancreatitis, which can be attenuated by the mTOR inhibitor rapamycin in acute pancreatitis.

Keywords: Gα13, Pancreatitis, Inflammation, Acinar Injury

Introduction

Gα proteins transduce signals from G protein-coupled receptors, the largest class of cell surface proteins that regulate varied biological processes.1 Since G protein-coupled receptors signal in part through Gα13,2 many studies have evaluated the role of Gα13 in physiological and pathologic processes.3, 4, 5, 6 For example, Gα13 regulates lysophosphatidic acid– and thrombin-mediated inflammation.7,8 Targeting Gα13 decreases thrombin-induced arachidonic acid expression in Chinese hamster ovary cells and sphingosine 1-phosphate (S1P)-induced inflammatory genes in astrocytes.9,10 However, many studies evaluating Gα13 in inflammation were conducted in vitro, and studies evaluating the effects of epithelial targeting of Gα13 in vivo using genetically engineered mouse (GEM) models have been limited.

We recently showed that epithelial targeting of Gα13 in vivo in the KrasG12D/p53R172H/Pdx1-Cre (KPC) GEM model of pancreatic cancer promotes tumor development.11 We showed that loss of Gα13 increases mammalian target of rapamycin (mTOR) signaling in the KPC GEM model, and that treatment with the mTOR inhibitor rapamycin can decrease the growth of KPC tumors with Gα13 loss. Our results with the GEM model contrast with the previously published cell line studies showing that Gα13 is pro-tumorigenic,12, 13, 14, 15 warranting employing GEM models to further understand the role of Gα13 in physiological and pathologic processes.

Importantly, Gα proteins can potentially regulate pancreatitis. Gα proteins in acinar cells mediate zymogen granule secretion downstream of cholecystokinin (CCK)-A receptors.16,17 Studies in isolated acinar cells have shown that the Gα13 protein functions downstream of CCK-A receptors to mediate amylase secretion.18,19 While previous studies have evaluated the effects of Gα13 loss in isolated acinar cells,18,19 the effects of targeting Gα13 on inflammation and pancreatitis have not been studied in vivo in mouse models. Here, we target Gα13 in the pancreas of a GEM model and evaluate the effects on pancreatitis-induced inflammation and acinar injury.

Materials and Methods

Animal Experiments

Study approval

All animal work and procedures were approved by the Northwestern University Institutional Animal Care and Use Committee. Also, all animal experiments were performed per relevant guidelines and regulations.

Conditional knockout

Mice with loss of Gα13 in the pancreas were generated by crossing Pdx1-Cre mice (Jackson Laboratory #014647) to mice expressing the floxed allele of Gna13 (kindly provided by Stefan Offermanns, Max Planck Institute),7 to generate CGα13fl/+ and CGα13 fl/fl mice. All mice were bred on a C57/BL6 background.

Induction of pancreatitis

Pancreatitis was induced in age- and litter-matched mice between 8- and 12-week-old mice of both genders. For acute pancreatitis, mice were starved of food overnight, followed by 8 hourly intraperitoneal injections of cerulein (50 μg/kg). Chronic pancreatitis was induced by intraperitoneal injections of cerulein (250 μg/kg) 2 times daily for 14 days.

Blocking mammalian target of rapamycin in mice

Mice were treated with vehicle control (10% polyethylene glycol 400, 10% Tween80) or rapamycin (MedChemExpress) 60 mg/kg via intraperitoneal injections. For acute pancreatitis studies, mice were pretreated overnight followed by an additional dose 1 h before cerulein dosing. Once daily dosing of rapamycin was administered during chronic pancreatitis.

Serum amylase assay

Blood was collected from euthanized mice via cardiac puncture and allowed to clot at room temperature for 20–30 min. The coagulated blood was centrifuged at 2000g for 10 min at 4 °C, and the supernatant was collected and stored in aliquots at −80 °C until use. The amylase activity in serum was determined using the EnzChek kit (Thermo Fisher E33651). Diluted serum samples or α-amylase standard from Bacillus species (Sigma-Aldrich A6380) were incubated with starch conjugated with boron-dipyrromethene (BODIPY) dye substrate for 30 min at room temperature in the dark. Fluorescent readings were acquired using Bio-Tek Synergy HT and the Gen5 software with excitation and emission wavelengths at 485 ± 20 nm and 520 ± 20 nm, respectively.

Cytokine array

Tissue samples were finely ground and lysed with cold radioimmunoprecipitation lysis buffer containing protease and phosphatase inhibitors. Tissue lysates were centrifuged, and the supernatant was used to detect cytokine levels using the proteome profiler mouse cytokine array kit (R&D Systems, ARY020).

Human pancreatitis samples

Formalin-fixed paraffin-embedded tissue arrays containing human pancreatitis samples were obtained from US Biomax (PA691). The array panel has 68 cores with 6 mild/normal, 5 acute, and 57 chronic pancreatitis cases. We restricted our analysis to 18 of the 57 chronic samples that were indicated as nonneoplastic. The requirement for instructional review board approval was not required because this study only used deidentified human samples obtained from a commercial source, in accordance with the applicable regulations and institutional policies. The tissue array was stained according to the immunohistochemistry protocol below using the validated antibody (Human Protein Atlas https://www.proteinatlas.org/ENSG00000120063-GNA13/tissue/pancreas) for human Gα13 (Sigma-Aldrich HPA010087). The whole array was scanned using an Olympus VS120 slide scanner, and the analysis of positivity was done with QuPath software, which returned the percent positive areas of each core.

Immunohistochemistry

For immunostains, paraffin-embedded sections were deparaffinized and rehydrated. Antigen retrieval was performed by boiling for 10 minutes in sodium citrate (10 mM, pH 6.0) or tris-ethylenediaminetetraacetic acid (10 mM, pH 9.0) buffer using a pressure cooker. Endogenous peroxidase activity in tissue was quenched, and sections were blocked with a mixture of goat serum and bovine serum albumin. Tissue sections were incubated with antibodies: Gα13 (Sigma-Aldrich HPA010087) CK19 (Abcam #ab52625, 1:500), Amylase (Cell Signaling #3796, 1:2000), F4/80 (Cell Signaling #70076, 1:800), Ly6.2B (clone 7/4, BioRad # MCA771GT, 1:400), or p-Rps6 (Cell Signaling #4858, 1:1000) overnight at 4 °C. Antibody binding was detected using horse radish peroxidase-conjugated antirabbit secondary antibody and visualized using ImmPACT 3,3′-diaminobenzidine Peroxidase Substrate kit (VectorLabs, #SK-4105). For Ly6.2B, antibody binding was detected using the Vectastain Elite ABC-HRP Kit (VectorLabs, #PK6104) Photographs were taken on the Olympus VS120 or TissueGnostics slide scanner systems and analyzed by QuPath, ImageJ or Aperio Software (Leica Biosystems).

Immunofluorescence

Paraffin-embedded sections were deparaffinized and rehydrated. Antigen retrieval was performed by boiling for 10 minutes in Tris buffer (10 mM, pH 9.0) using a pressure cooker. In select experiments, freshly isolated tissue samples were snap frozen in optimal cutting temperature compound and then sections were fixed in cold acetone:methanol (1:1) for 10 min. Tissue sections were stained using the mouse-on-mouse (MOM) immunodetection kit (Vector Laboratories [BMK-2202]). Tissue sections were blocked with MOM-blocking reagent for 1 h at room temperature, followed by washing and incubation with MOM-diluent for 5 min. Tissue sections were then incubated with primary antibody for Gα13 (Santa Cruz Biotechnology sc-293424, 1:100) for 1 h in MOM diluent. For multiplex immunofluorescence stains, tissue sections were incubated with a mix of antibodies for CK19 (DSHB Troma III 1:100), insulin (Cell Signaling # 3014 1:15,000) p-Rps6 (Cell Signaling #4858, 1:500), Gα13 (Sigma-Aldrich HPA010087 1:400) overnight at 4 °C, and then with antibody for Amylase (Santa Cruz Biotechnology sc-46657, 1:500) for 1 h at room temperature. After washing, the sections were incubated with Alexa Fluor conjugated secondary antibodies (Thermo Fisher Alexa fluor goat antimouse 647 #A32728, goat antirabbit 488 #A23731 and goat antirat 555 #A21434 1:250) for 30 min in the dark at room temperature. The tissue sections were washed and incubated with 4′,6-diamidino-2-phenylindole (Thermo Fisher D1306) for 10 min. Slides were mounted with fluorescence mounting media and images acquired using the EVOS M5000 microscope (Thermo Fisher) or Olympus VS120 slide scanning microscope. Fluorescence intensities were analyzed with Image J by averaging mean intensity values across at least 10 fields per sample.

Western blot

Tissue samples were finely ground and lysed with cold radioimmunoprecipitation lysis buffer containing protease and phosphatase inhibitors. The lysates were then clarified by centrifugation at 10,000 rpm for 10 min at 4 °C, and the protein concentration was determined using Precision Red solution (Cytoskeleton, Inc., Denver, CO). Equal amounts of protein were separated with a 10% SDS-PAGE electrophoresis gel. The separated proteins were transferred to a nitrocellulose membrane using the semi-dry transfer system (Bio-Rad). After blocking for 1 hour at room temperature with 5% bovine serum albumin, the membranes were incubated overnight at 4 °C with primary antibodies. Primary antibodies used include Gα13 (Santa Cruz sc-293424), p-Rps6 (Cell Signaling #4858), and Hsp90 (Santa Cruz Biotechnology sc-7947). Horse radish peroxidase-conjugated rabbit (A6667) or mouse (A4416) secondary antibody (Millipore-Sigma St. Louis, MO) was used with SuperSignal West Pico PLUS (Thermo Fisher Scientific) for protein detection.

Quantitative reverse transcription-polymerase chain reaction

RNA was isolated from tissue using the Rneasy kit (74,104, QIAGEN, Hilden, Germany). Complementary DNA was synthesized from 1–2 μg of RNA with random hexamers and M-MLV reverse transcriptase in a single 50 μL reaction. Quantitative PCR was performed using gene-specific TaqMan probes, TaqMan Universal PCR Master Mix, and the CFX Connect Real-Time PCR Detection System (Bio-Rad Hercules, CA). Probes used to detect Gna13 (Mm01250415_m1) using mouse Gapdh (Mm99999915_g1) or Rn18s (Mm03928990_g1) as endogenous controls, and their expression levels were used to calculate the relative expression using the comparative Ct method.20

Flow cytometry

At the experimental end point, mice were euthanized, and the pancreas was quickly removed and placed on ice in Hank balanced salt solution (HBSS) containing 1% fetal bovine serum (FBS) and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Tissue samples were finely minced with a scalpel and then centrifuged at 1200 rpm for 3 min at 4 °C. The tissue pellet was resuspended in HBSS digestion buffer containing 10 mM HEPES, 200 U/mL Collagenase 1A, 0.25 mg/mL soybean trypsin inhibitor, and 4U/mL of DNase I. Tissue samples were digested at 37 °C with shaking on a rocker platform and by mechanical dissociation by pipetting at 10-minute intervals for a total of 30 min. The reaction was stopped by adding 10 mL cold HBSS wash buffer (5% FBS, 10 mM HEPES, and 0.25 mg/mL soybean trypsin inhibitor); the dissociated tissue was then filtered through 70 μm nylon mesh. The samples were centrifuged at 1200 rpm at 4 °C and then suspended in ammonium-chloride-potassium red blood cell lysis buffer for 3 min at room temperature. The lysis reaction was quenched with 10 mL of cold HBSS wash buffer, centrifuged, then resuspended in 2 mL of flow buffer (PBS with 2% FBS). Cells were counted, and viability recorded using trypan blue. One x 106 cells per well were dispensed in a 96-well round-bottom plate and blocked with TruStain FcX (101,320, BioLegend, San Diego, CA) at 1:50 dilution for 10 min on ice. The Fc block was removed by centrifugation, and cell pellets were resuspended and incubated in fluorophore-conjugated antibodies and LIVE/DEAD fixable blue viability dye (Thermo Fisher Scientific, L34962, 1:1000). Antimouse antibodies against CD45 (clone 30-F11 1:100), CD11b (clone M1/70 1:200), Ly6G (clone 1A8 1:300), and F4/80 (cone BM8 1:200) from Biolegend were used to stain cells. After 30 min of incubation in the dark and on ice, cells were washed 3 times with ice-cold flow buffer then fixed in 4% paraformaldehyde for 5 min. Samples were acquired on a 3C.A1 LSR Fortessa 1 Analyzer, and data were analyzed with FlowJo (Becton Dickinson).

Statistics

The in vivo results were compared using one-way analysis of variance and 2-tailed t-test analysis. Error bars represent the standard error of the mean or standard deviation as specified in the figure legends. All statistical analyses were done using GraphPad Prism. A P value of less than 0.05 was considered significant.

Results

Reduced Gα13 Expression in Acute and Chronic Pancreatitis Samples

Initially, we evaluated Gα13 protein expression in human acute and chronic pancreatitis specimens. Compared to pancreas specimens with mild changes, Gα13 protein expression was reduced in specimens exhibiting acute and chronic pancreatitis (Figure 1A). To demonstrate that pancreatitis directly suppresses Gα13 protein expression, we treated mice with cerulein to induce acute or chronic pancreatitis. Mice were treated with 8 hourly intraperitoneal injections of the CCK analog cerulein to induce acute pancreatitis.21,22 For chronic pancreatitis, mice received intraperitoneal injections of cerulein twice daily for 14 days, and the pancreata were collected 1 day after the last cerulein injection. As expected, mice treated with cerulein to induce chronic pancreatitis demonstrated reduced amylase expression and increased CK19 expression (Figure 1C). Importantly, in agreement with the human data, we found reduced Gα13 protein expression in the acinar cells with induction of both acute and chronic pancreatitis (Figure 1B and C).

Figure 1.

Figure 1

Open in a new tab

Gα13 expression is reduced in human and mouse pancreatitis tissue. (A) Human pancreas tissue array was stained for Gα13 by immunohistochemistry. Gα13 percent positivity was compared between normal/mild, acute, and chronic pancreatitis samples (n = 7, 5, 18). One-way analysis of variance, ∗∗P < .01 Scale bar = 100 μm (upper panels), 20 μm (lower panels). (B and C) Wild-type C57BL6 mice were treated with 8 hourly injections of saline or cerulein (50 μg/kg) to induce acute pancreatitis (B) or treated with cerulein (250 μg/kg) twice daily for 14 days to induce chronic pancreatitis (C). Pancreas tissue was collected 1 day after treatment and stained for Gα13, amylase and CK19 by immunofluorescence. Fluorescence intensity for Gα13 and percent positive for amylase and CK19 were quantified between saline and cerulein for acute (n = 4, 4) and chronic (n = 4, 4) pancreatitis samples. t-test, mean ± SD, ∗P < .05 ∗∗P < .01; ns, nonsignificant. Scale bar = 100 μm. SD, standard deviation.

Pancreatic Deletion of Gα13 Exacerbates Acute Pancreatitis-Induced Injury to the Acinar Compartment

We next evaluated the effects of Gα13 loss on experimental pancreatitis in a GEM model. We crossed Pdx1-Cre mice to mice expressing the floxed allele of Gna13 to generate CGα13fl/+ and CGα13 fl/fl mice (referred to as CGfl/+ and CGfl/fl, respectively), which express reduced Gα13 levels in the pancreas (Figure 2A–C). Initially, we evaluated the effects of Gα13 loss on pancreas development. Gα13 loss did not affect the acinar, ductal, the islet compartments (Figure 2D), or the pancreas weights (Figure 2E). We then treated wild-type (CG+/+) and CGfl/fl mice with cerulein to induce acute pancreatitis.21,22 Blood was collected at 1 and 12 h after the last injection, and the pancreata were collected at 1, 12, and 48 h after the last injection. There was no significant difference in the body or pancreas weights between CG+/+ and CGfl/fl mice following cerulein treatment (Figure 2F and G). However, compared to the cerulein-treated CG+/+ mice, the cerulein-treated CGfl/fl mice exhibit increased serum amylase levels at 1 h, with serum amylase levels decreasing in both CG+/+ and CGfl/fl mice at 12 h (Figure 2H). Moreover, the CGfl/fl mice demonstrated more pronounced histologic changes, including increased edema (evaluated by peri-acinar spacing), following cerulein treatment (Figure 2I), indicating increased acute pancreatitis-induced injury to the acinar compartment.

Figure 2.

Figure 2

Open in a new tab

Pancreatic deletion of Gα13 exacerbates acute pancreatitis-induced injury to the acinar compartment. (A) Pdx1-Cre mice were crossed to mice expressing the floxed allele of Gna13 to generate CGα13fl/+ and CGα13 fl/fl mice (referred to as CGfl/+ and CGfl/fl). Alleles of Pdx1-Cre (C) and Gα13fl (G). Western blot showing Gα13 protein levels in the pancreas of mice with the indicated genotypes, using Hsp90 as an endogenous control, with quantification of the Western blot bands by Image J. (B) Pancreas from control CG+/+ mice and CGfl/fl mice were immunostained for Gα13, using DAPI to stain nuclei. Scale bar = 50 μm. (C) Expression of Gna13 mRNA was determined by qRT-PCR, using Gapdh as an endogenous control. t-test, mean ± SD, ∗∗∗∗P < .0001. (D) Pancreas from control CG+/+ mice and CGfl/fl mice were stain with hematoxylin and eosin (top row) and immunostained for CK19, Insulin, and amylase, using DAPI to stain nuclei. Scale bar = 200 μm (low power) 50 μm (high power). (E) Relative weights of the pancreas from control CG+/+ mice and CGfl/fl mice were evaluated at 3 and 6 months of age (CG+/+ n = 8, 7; CGfl/fl n = 6, 10). One-way analysis of variance, mean ± SD; ns, nonsignificant. (F-H) Mice were treated with 8 hourly injections of saline or cerulein (50 μg/kg) per the acute pancreatitis protocol. The body weights were monitored over 48 h after the last injection (F) and the relative pancreas weights were determined at 48 h (G) (saline n = 4, 5; cerulein n = 4, 5). Serum amylase was quantified 1 h and 12 h after the last injection (H). Saline n = 5, 5 (1h, 12h); cerulein CG+/+ n = 4, 3 (1 h, 12 h); cerulein CGfl/fl n = 4, 4 (1h, 12h). One-way analysis of variance, mean ± SD, ∗P < .05, ∗∗∗∗P < .0001. (I) Representative hematoxylin and eosin stains of the pancreas from saline- and cerulein-treated mice of indicated genotypes 1 h, 12 h, and 48 h after the last injection. The extent of pancreas edema following induction of pancreatitis was quantified by ImageJ. t-test, mean ± SD, ∗P < .05. Scale bar = 100 μm. DAPI, 4′,6-diamidino-2-phenylindole; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; SD, standard deviation.

However, when we examined the effects on macrophage and neutrophil infiltration, we did not see a difference in the number of infiltrating macrophage and neutrophil between the CG+/+ and the CGfl/fl mice 24 h after the last cerulein injection (Figure 3A and B). Similarly, when we evaluated the effects of Gα13 loss on cytokine levels following cerulein treatment of CG+/+ and CGfl/fl mice, we found variable effects on the cytokines present on our cytokine array. While the protein levels of Ccl2, Ccl5, Il1a, and Il1b protein levels were increased in the pancreas of cerulein-treated CGfl/fl mice compared to the pancreas of cerulein-treated CG+/+ control mice (Figure 3C), the protein levels of Cxcl1 and Il1ra were decreased in the pancreas of cerulein-treated CGfl/fl mice compared to the pancreas of cerulein-treated CG+/+ control mice (Figure 3C).

Figure 3.

Figure 3

Open in a new tab

Pancreatic deletion of Gα13 does not exacerbate acute pancreatitis-induced inflammation. (A and B) Pancreas from saline- and cerulein-treated mice of indicated genotypes 1 day after the last injection were processed for macrophage (F4/80) and neutrophil (Ly6G, 7/4) infiltration by flow cytometry or IHC. Flow cytometric analysis of F4/80+ macrophages and Ly6G+ neutrophils after gating for CD45+, CD11b+ live cells in the pancreas of mice with acute pancreatitis (saline, CG+/+, CGfl/fl; n = 4, 3, 4) or IHC (F480-saline (4, 3), cerulein (4, 4), 7/4-saline (3, 3), cerulein (4, 4). t-test, mean ± SD; ns, nonsignificant. Scale bar = 100 μm. (C) Analysis of cytokine protein levels in the pancreatic tissue of mice 12 h after the last injection of cerulein. 100 μg of protein from pancreatic tissue lysates from three mice per treatment group (300 μg total) were combined, and the relative signal intensity was quantified with ImageJ. SD, standard deviation.

Pancreatic Deletion of Gα13 Mildly Exacerbates Chronic Pancreatitis

In parallel studies, we evaluated the effects of Gα13 loss on chronic pancreatitis.21,22 Chronic pancreatitis was induced by intraperitoneal injections of cerulein twice daily for 14 days, and the pancreata were collected 1 or 14 days after the last cerulein injection (Figure 4A). While mice treated with cerulein lost body weight, which they subsequently recovered, there was no significant difference in the body weights between the CG+/+ and the CGfl/fl mice (Figure 4B). However, there was a significant difference in pancreas weights between CG+/+ and CGfl/fl mice 1 day but not 14 days after the last cerulein injection (Figure 4C). There was decreased amylase staining in the 1-day samples in cerulein-treated CGfl/fl mice compared to the control cerulein-treated CG+/+ mice (Figure 4D and E).

Figure 4.

Figure 4

Open in a new tab

Effect of chronic pancreatitis in mice with pancreatic deletion of Gα13. (A) Schema for the chronic pancreatitis protocol, in which the mice were treated with twice daily injections of saline or cerulein (250 μg/kg) for 14 days. (B and C) Body weights were monitored during the induction of chronic pancreatitis and for 14 days after the last injection (saline n = 8, 3; cerulein n = 11, 6). The relative pancreas weights were determined at 1 day and 14 days after the last injection (d1 cerulein n = 10, 7; d14-cerulein n = 9, 4). t-test, mean ± SD, ∗P < .05; ns, nonsignificant. (D) Representative hematoxylin and eosin stains of the pancreas 1 day after the last cerulein injection on the chronic pancreatitis protocol. Scale bar = 100 μm. (E) Representative stains for amylase and CK19 of the pancreas 1 day after the last cerulein injection. Scale bar = 100 μm. Relative quantification of amylase (n = 3, 5, 3) and CK19 (n = 3, 8, 4) in the treated mice. t-test, mean ± SD, ∗P < .05. SD, standard deviation.

We also observed increased pancreatic infiltration by macrophages but not neutrophils in the cerulein-treated CGfl/fl mice compared to the cerulein-treated CG+/+ mice (Figure 5A and B). In addition, there was no difference in the extent of fibrosis, as determined by trichrome staining (Figure 5C). Moreover, when we assessed the effects of Gα13 loss on cytokine expression 1 day after the last cerulein injection on the chronic pancreatitis protocol, we found variable effects on the cytokine expression. While there was increased Ccl5 expression in the pancreas of cerulein-treated CGfl/fl mice compared to the pancreas of cerulein-treated CG+/+ control mice (Figure 5D), there was decreased expression of Cxcl1 in the pancreas of cerulein-treated CGfl/fl mice compared to the pancreas of cerulein-treated CG+/+ control mice. Overall, these results indicate that loss of Gα13 mildly exacerbates chronic pancreatitis.

Figure 5.

Figure 5

Open in a new tab

Pancreatic deletion of Gα13 mildly exacerbates chronic pancreatitis. (A and B) Pancreas from saline- and cerulein-treated mice of indicated genotypes 1 day after the last injection on the chronic pancreatitis protocol were processed for macrophage (F4/80) and neutrophil (Ly6G, 7/4) analysis by flow cytometry or IHC. Flow cytometric analysis of F4/80+ macrophages and Ly6G+ neutrophils after gating for CD45+, CD11b+ live cells in the pancreas of mice with chronic pancreatitis. Saline, CG+/+, CGfl/fl; n = 4, 4, 3. IHC: F480; saline n = 1, 2; cerulein 4, 3, 7/4 saline n = 5, 2; cerulein 6, 6. t-test, mean ± SD, ∗P < .05; ns, nonsignificant. Scale bar = 100 μm. (C) Pancreatic specimens were stained for fibrosis using trichrome staining (cerulein 4, 4). Scale bar = 100 μm. (D) Analysis of cytokine protein levels in the pancreatic tissue of mice 1 day after cerulein treatment on the chronic pancreatitis protocol. 100 μg of protein from pancreatic tissue lysates from three different mice of each treatment group (300 μg total) were combined, and the relative intensity of the signal was quantified with ImageJ. SD, standard deviation.

Rapamycin Exacerbates Chronic Pancreatitis-Induced Injury to the Acinar Compartment in Mice With Gα13 Loss

Since we found that loss of Gα13 increases mTOR signaling in the KPC GEM model,11 we next evaluated the effects of targeting mTOR using rapamycin on cerulein-induced histologic changes in the CGfl/fl mice treated with cerulein. Initially, we evaluated Rps6 phosphorylation, a downstream target of mTOR, in pancreatic specimens from mice treated on the chronic pancreatitis protocol. Compared to the control cerulein-treated CG+/+ mice, cerulein-treated CGfl/fl mice demonstrate increased Rps6 phosphorylation in acinar cells (Figure 6A). The CGfl/fl mice were then treated with rapamycin daily during cerulein treatment, which was given twice daily for 2 weeks. As expected, treatment with rapamycin decreased Rps6 phosphorylation (Figure 6B). We found that rapamycin decreased amylase and CK19 levels in our model while not impacting the levels of macrophages and neutrophil in our model (Figure 6C–E). These results indicate that rapamycin exacerbates cerulein-induced injury to the acinar compartment mice with Gα13 loss.

Figure 6.

Figure 6

Open in a new tab

Rapamycin exacerbates chronic pancreatitis-induced injury to the acinar compartment in mice with Gα13 loss. (A) Representative immunostain and quantification of p-Rps6 (co-IF amylase/DAPI) in chronic pancreatitis tissue sections from saline- or cerulein-treated CG +/+ or CG fl/fl mice. Saline, cerulein CG+/+, cerulein CGfl/fl (n = 4, 8, 5). t-test, mean ± SD, ∗∗P < .01. Scale bar = 100 μm. (B) Schema showing cotreatment of Rapamycin and cerulein in the chronic pancreatitis protocol. Representative immunostain and quantification of p-Rps6 (co-IF amylase/DAPI) in pancreatic tissue from cerulein-treated CG fl/fl mice treated with vehicle or Rapamycin. Vehicle, rapamycin (n = 5, 5). t-test, mean ± SD, ∗∗∗∗P < .0001. Scale bar = 100 μm. (C) Representative hematoxylin and eosin, Amylase, and CK19 immunostains of pancreas tissue from cerulein-treated CG fl/fl mice treated with vehicle or rapamycin. Relative quantification of Amylase and CK19 stains; vehicle, rapamycin (n = 5, 5). t-test, mean ± SD, ∗P < .05, ∗∗∗P < .001. Scale bar = 100 μm. (D and E) Representative F4/80+ macrophage and 7/4+ neutrophil immunostains of pancreas tissue from saline or cerulein-treated CG+/+ and CGfl/fl mice treated with vehicle or rapamycin. Relative quantification of percentage F4/80 macrophage (n = 3, 5, 5, 5, 5) and 7/4 neutrophil (n = 3, 6, 5, 4, 5) stains in vehicle, rapamycin treated samples. t-test, mean ± SD, ∗P < .05, ∗∗P < .01, ∗∗∗P < .001; ns, nonsignificant. Scale bar = 100 μm. (F and G) The body weights were monitored during cotreatment with cerulein and rapamycin and for 14 days after the last treatment (saline n = 7, vehicle + cerulein n = 7, 5, rapamycin + cerulein n = 6, 6). The relative pancreas weights were determined at 14 days after the last injection (vehicle + cerulein n = 7, 5, rapamycin + cerulein n = 6, 6). (H) Representative hematoxylin and eosin stains of the pancreas from cerulein-treated mice and cotreated with vehicle control or rapamycin of indicated genotypes 1 day and 14 days after the last injection. Scale bars = 100 μm. DAPI, 4′,6-diamidino-2-phenylindole; SD, standard deviation.

Notably, CGfl/fl mice cotreated with rapamycin and cerulein regained body and pancreas weights to a similar extent as CGfl/fl mice treated with vehicle and cerulein once the cerulein treatment was stopped (Figure 6F and G). Histologic examination at Day 14 of recovery following cerulein treatment found no apparent difference in the pancreas between vehicle and rapamycin-treated CG+/+ and CGfl/fl mice (Figure 6H).

Rapamycin Attenuates Acute Pancreatitis-Induced Injury to the Acinar Compartment in Mice With Gα13 Loss

Finally, we evaluated the effects of rapamycin on cerulein-induced changes in the CGfl/fl mice treated with cerulein on the acute pancreatitis protocol. Compared to the control cerulein-treated CG+/+ mice, cerulein-treated CGfl/fl mice demonstrate increased Rps6 phosphorylation in acinar cells (Figure 7A). The CGfl/fl mice were then pretreated with rapamycin 16 h and 1 h before 8 hourly injections of cerulein. Blood was collected 1 h after the last cerulein injection, and the pancreata were collected 1 h and 12 h after the last injection. As expected, rapamycin decreased Rps6 phosphorylation (Figure 7B). Rapamycin suppressed the increased serum amylase levels and pancreatic edema seen in the cerulein-treated CGfl/fl mice (Figure 7C and D). Rapamycin had no effect on macrophages and neutrophils in the cerulein-treated CGfl/fl mice (Figure 7E). Notably, cotreatment with rapamycin did not affect the body weights of cerulein-treated mice during the recovery period (Figure 7F). There was preservation of the pancreas weights and no apparent difference in the pancreas between vehicle and rapamycin-treated CG+/+ and CGfl/fl mice at day 7 of recovery from acute pancreatitis (Figure 7G and H). These results demonstrate that rapamycin attenuates acute pancreatitis-induced injury to the acinar compartment in mice with Gα13 loss.

Figure 7.

Figure 7

Open in a new tab

Rapamycin attenuates acute pancreatitis-induced injury to the acinar compartment in mice with Gα13 loss. (A) Representative immunostain and quantification of p-Rps6 (co-IF amylase/DAPI) in acute pancreatitis tissue section from saline- or cerulein-treated CG +/+ or CG fl/fl mice (n = 4, 8, 5). t-test, mean ± SD. Scale bar = 100 μm. (B) Schema showing cotreatment of rapamycin and cerulein in the acute pancreatitis protocol. Representative immunostain and quantification of p-Rps6 (co-IF amylase/DAPI) in pancreatic tissue from cerulein-treated CG fl/fl mice treated with vehicle or rapamycin. Vehicle, rapamycin (n = 5, 5). t-test, mean ± SD, ∗∗∗∗P < .0001. Scale bar = 100 μm. (C) Serum amylase quantification from cerulein-treated CG fl/fl mice treated with vehicle or rapamycin (n = 4, 5, 6). Analysis of variance, mean ± SD, ∗∗P < .01, ∗∗∗∗P < .0001. (D) Representative hematoxylin and eosin stains of the pancreas from cerulein-treated CG fl/fl mice cotreated with vehicle or rapamycin. The extent of pancreas edema was quantified by ImageJ. t-test, mean ± SD, ∗P < .05. Scale bar = 100 μm. (E) Representative F4/80+ macrophage and 7/4+ neutrophil immunostains of pancreas tissue from cerulein-treated CG fl/fl mice treated with vehicle or rapamycin. Relative quantification of percentage F4/80 macrophage (n = 3, 4, 4, 5, 5) and 7/4 neutrophil (n = 2, 4, 4, 5, 6) stains in saline, vehicle, rapamycin treated samples. Scale bar = 100 μm. (F and G) The body weights were monitored for 7 days after the last treatment (vehicle + cerulein n = 4, 6, rapamycin + cerulein n = 4, 9). The relative pancreas weights were determined at 7 days after the last injection. (H) Representative hematoxylin and eosin stains of the pancreas from cerulein-treated mice and cotreated with vehicle control or rapamycin of indicated genotypes 1 day and 7 days after the last injection. Scale bars = 100 μm. DAPI, 4′,6-diamidino-2-phenylindole; SD, standard deviation.

Discussion

Our results demonstrate that Gα13 restrains pancreatitis-induced acinar cell injury, thereby maintaining pancreas homeostasis. Our data agree with the published studies showing that Gα13 restrains osteoclast-driven bone resorption in GEM models, thereby maintaining bone homeostasis.23,24 Loss of Gα13 in osteoclasts in GEM models increases NFATc1 signaling to cause bone resorption. Importantly, overexpression of Gα13 blocks bone resorption in a TNFα transgene-expressing mouse model of autoimmune arthritis and a lipopolysaccharides-injection mouse model of osteolysis.23 Also, mice with myeloid-specific deletion of Gα13 demonstrate that Gα13 restrains osteoclast function, thereby maintaining bone homeostasis.24

Importantly, while mTOR signaling functions in tissue repair and homeostasis,25,26 depending on the timing of activation, it can mediate either tissue injury or repair.27, 28, 29, 30 We found that rapamycin protects against acute pancreatitis-induced injury in mice with Gα13 loss but not against chronic pancreatitis-induced injury. Others have also shown that rapamycin ameliorates acute pancreatitis injury31, 32, 33 but worsens chronic pancreatitis-induced fibrotic changes.34 We have also found that rapamycin exacerbates chronic pancreatitis injury in mice with Gα13 loss. Our data suggest that mTOR signaling functions as a protective mechanism in chronic pancreatitis, limiting further pancreatic injury in mice with Gα13 loss.

Notably, we have found that Gα13 loss has minimal to no effects on pancreatitis-induced inflammation. Consistent with this observation, we show that the epithelial targeting of Gα13 in our GEM model has variable effects on several inflammatory cytokines following induction of acute and chronic pancreatitis, with Ccl5 increasing in both acute and chronic pancreatitis, but Cxcl1 and Il1ra decreasing in both acute and chronic pancreatitis. Others have also evaluated the role of Gα13 in regulating cytokines, chemokines, and other inflammatory mediators; however, these studies were conducted primarily using in vitro models. For example, Gα13 mediated the increase in IL-6 and COX-2 expression in astrocytes downstream of S1P receptor 3.9 Gα13 also mediated the increase in NLR family pyrin domain containing 3, interleukin-1 beta, and interleukin-18 in bone marrow-derived monocytes/macrophages following sphingosine-1-phosphate treatment.35 Thus, the differences between our findings and the in vitro data are likely due to the more complex in vivo microenvironment in the GEM models.

Conclusion

Overall, we show that Gα13 loss exacerbates pancreatitis-induced tissue damage, which can be attenuated by the mTOR inhibitor rapamycin in the context of acute pancreatitis. Thus, we demonstrate a protective role for Gα13 in maintaining pancreas homeostasis following acute and chronic pancreatitis-induced acinar cell injury.

Acknowledgments

Authors’ Contributions

Hidayatullah G. Munshi: Cowrote the initial and revised manuscript-guided and designed experiments, interpreted results, and secured funding. Anastasia E. Metropulos: Data acquisition for histology experiments. Khadijeh Karbalaei: Data acquisition for histology experiments and reviewed and edited manuscript. Christina Spaulding: Data acquisition for animal and histology and flow cytometry experiments. Nida Mubin: Data acquisition for animal experiments. Yaning Xi: Data acquisition for cytokine analysis. Apurba Majumder: Animal experiments. Thao D. Pham: Experimental designs and interpretation, and reviewed and edited manuscript. Mario A. Shields: Conceived the project, guided and designed experiments, performed the main experiments, cowrote the initial and revised manuscript, data visualization, interpreted the results, and secured funding.

Footnotes

Conflicts of Interest: The authors disclose no conflicts.

Funding: This work was supported by the American Pancreatic Association/APA Foundation 2020 Young Investigator Pancreatitis Grant (to Mario A. Shields) and the NIH/NCI grant R01CA283925 (to Mario A. Shields).

Ethical Statement: All animal work and procedures were approved by the Northwestern University Institutional Animal Care and Use Committee. The requirement for instructional review board approval was not required because this study uses deidentified human tissue samples obtained from a commercial source.

Data Transparency Statement: Materials and data related to this manuscript are available upon request.

Reporting Guidelines: The Guide for the Care and Use of Laboratory Animals.

Material associated with this article can be found, in the online version, at https://doi.org/10.1016/j.gastha.2026.101076.

Contributor Information

Hidayatullah G. Munshi, Email: h-munshi@northwestern.edu.

Mario A. Shields, Email: mario.shields@stonybrook.edu.

Supplementary Materials

Supplementary Materials
mmc1.pdf (2.4MB, pdf)
Extended PDF
mmc2.pdf (52.9MB, pdf)

References

  • 1.Oldham WM, Hamm HE. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat Rev Mol Cell Biol. 2008;9(1):60–71. doi: 10.1038/nrm2299. [DOI] [PubMed] [Google Scholar]
  • 2.Kurose H. Galpha12 and Galpha13 as key regulatory mediator in signal transduction. Life Sci. 2003;74(2–3):155–161. doi: 10.1016/j.lfs.2003.09.003. [DOI] [PubMed] [Google Scholar]
  • 3.Sriram K, Salmeron CS, Wiley SZ, et al. GPCRs in pancreatic adenocarcinoma: contributors to tumor biology and novel therapeutic targets. Br J Pharmacol. 2020;177:2434–2455. doi: 10.1111/bph.15028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Syrovatkina V, Huang XY. Signaling mechanisms and physiological functions of G-protein Galpha13 in blood vessel formation, bone homeostasis, and cancer. Protein Sci. 2019;28(2):305–312. doi: 10.1002/pro.3531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Tutunea-Fatan E, Lee JC, Denker BM, et al. Heterotrimeric Galpha12/13 proteins in kidney injury and disease. Am J Physiol Renal Physiol. 2020;318:F660–F672. doi: 10.1152/ajprenal.00453.2019. [DOI] [PubMed] [Google Scholar]
  • 6.Guo P, Tai Y, Wang M, et al. Galpha(12) and Galpha(13): versatility in physiology and pathology. Front Cell Dev Biol. 2022;10:809425. doi: 10.3389/fcell.2022.809425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Moers A, Nieswandt B, Massberg S, et al. G13 is an essential mediator of platelet activation in hemostasis and thrombosis. Nat Med. 2003;9(11):1418–1422. doi: 10.1038/nm943. [DOI] [PubMed] [Google Scholar]
  • 8.Gong H, Shen B, Flevaris P, et al. protein subunit Galpha13 binds to integrin alphaIIbbeta3 and mediates integrin “outside-in” signaling. Science. 2010;327(5963):340–343. doi: 10.1126/science.1174779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Dusaban SS, Chun J, Rosen H, et al. Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes. J Neuroinflammation. 2017;14(1):111. doi: 10.1186/s12974-017-0882-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Mariggio S, Bavec A, Natale E, et al. Galpha13 mediates activation of the cytosolic phospholipase A2alpha through fine regulation of ERK phosphorylation. Cell Signal. 2006;18(12):2200–2208. doi: 10.1016/j.cellsig.2006.05.003. [DOI] [PubMed] [Google Scholar]
  • 11.Shields MA, Spaulding C, Metropulos AE, et al. Galpha13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin. Cell Rep. 2022;38(9):110441. doi: 10.1016/j.celrep.2022.110441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Xu N, Voyno-Yasenetskaya T, Gutkind JS. Potent transforming activity of the G13 alpha subunit defines a novel family of oncogenes. Biochem Biophys Res Commun. 1994;201(2):603–609. doi: 10.1006/bbrc.1994.1744. [DOI] [PubMed] [Google Scholar]
  • 13.Voyno-Yasenetskaya TA, Pace AM, Bourne HR. Mutant alpha subunits of G12 and G13 proteins induce neoplastic transformation of Rat-1 fibroblasts. Oncogene. 1994;9(9):2559–2565. [PubMed] [Google Scholar]
  • 14.Maziarz M, Federico A, Zhao J, et al. Naturally occurring hotspot cancer mutations in Galpha13 promote oncogenic signaling. J Biol Chem. 2020;295(49):16897–16904. doi: 10.1074/jbc.AC120.014698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Chow CR, Ebine K, Knab LM, et al. Cancer cell invasion in three-dimensional collagen is regulated differentially by Galpha13 protein and discoidin domain receptor 1-Par3 protein signaling. J Biol Chem. 2016;291(4):1605–1618. doi: 10.1074/jbc.M115.669606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Williams JA, Sans MD, Tashiro M, et al. Cholecystokinin activates a variety of intracellular signal transduction mechanisms in rodent pancreatic acinar cells. Pharmacol Toxicol. 2002;91(6):297–303. doi: 10.1034/j.1600-0773.2002.910606.x. [DOI] [PubMed] [Google Scholar]
  • 17.Williams JA. Intracellular signaling mechanisms activated by cholecystokinin-regulating synthesis and secretion of digestive enzymes in pancreatic acinar cells. Annu Rev Physiol. 2001;63:77–97. doi: 10.1146/annurev.physiol.63.1.77. [DOI] [PubMed] [Google Scholar]
  • 18.Sabbatini ME, Bi Y, Ji B, et al. CCK activates RhoA and Rac1 differentially through Galpha13 and Galphaq in mouse pancreatic acini. Am J Physiol Cell Physiol. 2010;298(3):C592–C601. doi: 10.1152/ajpcell.00448.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Kim M, Nozu F, Kusama K, et al. Cholecystokinin stimulates the recruitment of the src-rhoa-phosphoinositide 3-kinase pathway by Vav-2 downstream of G(alpha13) in pancreatic acini. Biochem Biophys Res Commun. 2006;339(1):271–276. doi: 10.1016/j.bbrc.2005.11.014. [DOI] [PubMed] [Google Scholar]
  • 20.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
  • 21.Ebine K, Chow CR, DeCant BT, et al. Slug inhibits pancreatic cancer initiation by blocking Kras-induced acinar-ductal metaplasia. Sci Rep. 2016;6:29133. doi: 10.1038/srep29133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Saluja AK, Saluja M, Printz H, et al. Experimental pancreatitis is mediated by low-affinity cholecystokinin receptors that inhibit digestive enzyme secretion. Proc Natl Acad Sci U S A. 1989;86(22):8968–8971. doi: 10.1073/pnas.86.22.8968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Wu M, Chen W, Lu Y, et al. Galpha13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3beta-NFATc1 signalling pathway. Nat Commun. 2017;8:13700. doi: 10.1038/ncomms13700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Nakano S, Inoue K, Xu C, et al. G-protein Galpha(13) functions as a cytoskeletal and mitochondrial regulator to restrain osteoclast function. Sci Rep. 2019;9(1):4236. doi: 10.1038/s41598-019-40974-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Wei X, Luo L, Chen J. Roles of mTOR signaling in tissue regeneration. Cells. 2019;8(9):1075. doi: 10.3390/cells8091075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149(2):274–293. doi: 10.1016/j.cell.2012.03.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Codeluppi S, Svensson CI, Hefferan MP, et al. The Rheb-mTOR pathway is upregulated in reactive astrocytes of the injured spinal cord. J Neurosci. 2009;29(4):1093–1104. doi: 10.1523/JNEUROSCI.4103-08.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Du K, Zheng S, Zhang Q, et al. Pten Deletion promotes regrowth of corticospinal tract axons 1 year after Spinal cord injury. J Neurosci. 2015;35(26):9754–9763. doi: 10.1523/JNEUROSCI.3637-14.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Guan Y, Zhang L, Li X, et al. Repression of mammalian target of rapamycin complex 1 inhibits intestinal regeneration in acute inflammatory bowel disease models. J Immunol. 2015;195(1):339–346. doi: 10.4049/jimmunol.1303356. [DOI] [PubMed] [Google Scholar]
  • 30.Nagahama Y, Shimoda M, Mao G, et al. Regnase-1 controls colon epithelial regeneration via regulation of mTOR and purine metabolism. Proc Natl Acad Sci. 2018;115(43):11036–11041. doi: 10.1073/pnas.1809575115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Mei Q, Zeng Y, Huang C, et al. Rapamycin alleviates hypertriglyceridemia-related acute pancreatitis via restoring autophagy flux and inhibiting endoplasmic reticulum stress. Inflammation. 2020;43(4):1510–1523. doi: 10.1007/s10753-020-01228-7. [DOI] [PubMed] [Google Scholar]
  • 32.Xu D, Xie R, Xu Z, et al. mTOR-Myc axis drives acinar-to-dendritic cell transition and the CD4(+) T cell immune response in acute pancreatitis. Cell Death Dis. 2020;11(6):416. doi: 10.1038/s41419-020-2517-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Sharma MK, Priyam K, Kumar P, et al. Effect of calorie-restriction and rapamycin on autophagy and the severity of caerulein-induced experimental acute pancreatitis in mice. Front Gastroenterol. 2022;1:977169. doi: 10.3389/fgstr.2022.977169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ozturk K, Tasci I, Yasar M, et al. Effects of rapamycin treatment on pancreatic fibrosis, cellular apoptosis and oxidative stress in experimental chronic pancreatitis model. Acta Gastro-Enterologica Belgica. 2015;78(1):3–7. [PubMed] [Google Scholar]
  • 35.Hou L, Zhang Z, Yang L, et al. NLRP3 inflammasome priming and activation in cholestatic liver injury via the sphingosine 1-phosphate/S1P receptor 2/Galpha((12/13))/MAPK signaling pathway. J Mol Med (Berl) 2021;99(2):273–288. doi: 10.1007/s00109-020-02032-4. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Materials
mmc1.pdf (2.4MB, pdf)
Extended PDF
mmc2.pdf (52.9MB, pdf)

Articles from Gastro Hep Advances are provided here courtesy of Elsevier

RESOURCES