Mouse Model Of Severe Acute Pancreatitis Research Articles

HP-MSCs attenuate severe acute pancreatitis in mice via inhibiting NLRP3 inflammasome-mediated acinar cell pyroptosis.

Severe acute pancreatitis (SAP) is a serious gastrointestinal disease that is facilitated by pancreatic acinar cell death. The protective role of human placental mesenchymal stem cells (hP-MSCs) in SAP has been demonstrated in our previous studies. However, the underlying mechanisms of this therapy remain unclear. Herein, we investigated the regularity of acinar cell pyroptosis during SAP and investigated whether the protective effect of hP-MSCs was associated with the inhibition of acinar cell pyroptosis. A mouse model of SAP was established by the retrograde injection of sodium taurocholate (NaTC) solution in the pancreatic duct. For the hP-MSCs group, hP-MSCs were injected via the tail vein and were monitored in vivo. Transmission electron microscopy (TEM) was used to observe the pyroptosis-associated ultramorphology of acinar cells. Immunofluorescence and Western blotting were subsequently used to assess the localization and expression of pyroptosis-associated proteins in acinar cells. Systemic inflammation and local injury-associated parameters were evaluated. Acinar cell pyroptosis was observed during SAP, and the expression of pyroptosis-associated proteins initially increased, peaked at 24h, and subsequently showed a decreasing trend. hP-MSCs effectively attenuated systemic inflammation and local injury in the SAP model mice. Importantly, hP-MSCs decreased the expression of pyroptosis-associated proteins and the activity of the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome in acinar cells. Our study demonstrates the regularity and important role of acinar cell pyroptosis during SAP. hP-MSCs attenuate inflammation and inhibit acinar cell pyroptosis via suppressing NLRP3 inflammasome activation, thereby exerting a protective effect against SAP.

Isorhamnetin Alleviates Mitochondrial Injury in Severe Acute Pancreatitis via Modulation of KDM5B/HtrA2 Signaling Pathway.

Severe acute pancreatitis (SAP), a widespread inflammatory condition impacting the abdomen with a high mortality rate, poses challenges due to its unclear pathogenesis and the absence of effective treatment options. Isorhamnetin (ISO), a naturally occurring flavonoid, demonstrates robust antioxidant and anti-inflammatory properties intricately linked to the modulation of mitochondrial function. However, the specific protective impact of ISO on SAP remains to be fully elucidated. In this study, we demonstrated that ISO treatment significantly alleviated pancreatic damage and reduced serum lipase and amylase levels in the mouse model of SAP induced by sodium taurocholate (STC) or L-arginine. Utilizing an in vitro SAP cell model, we found that ISO co-administration markedly prevented STC-induced pancreatic acinar cell necrosis, primarily by inhibiting mitochondrial ROS generation, preserving ATP production, maintaining mitochondrial membrane potential, and preventing the oxidative damage and release of mitochondrial DNA. Mechanistically, our investigation identified that high-temperature requirement A2 (HtrA2) may play a central regulatory role in mediating the protective effect of ISO on mitochondrial dysfunction in STC-injured acinar cells. Furthermore, through an integrated approach involving bioinformatics analysis, molecular docking analysis, and experimental validation, we uncovered that ISO may directly impede the histone demethylation activity of KDM5B, leading to the restoration of pancreatic HtrA2 expression and thereby preserving mitochondrial function in pancreatic acinar cells following STC treatment. In conclusion, this study not only sheds new light on the intricate molecular complexities associated with mitochondrial dysfunction during the progression of SAP but also underscores the promising value of ISO as a natural therapeutic option for SAP.

HDAC3 improves intestinal function of mice by regulating cGAS-Sting pathway of intestinal glial cells

It is found that HDAC3 may be a potential therapeutic target for intestinal related diseases. At present, the role and mechanism of HDAC3 in the pathogenesis of severe acute pancreatitis (SAP) have not been reported, which needs to be further explored. The SAP mouse model was established and the expression of HDAC3 was detected by immunohistochemistry. H&E staining showed the intestinal pathological state of SAP mice. The expression of HDAC3 was measured by real-time quantitative PCR (RT qPCR) and Western blot. Apoptosis kit was used to determine cell apoptosis rate. The level of inflammatory factors was detected by ELISA kits. The expressions of HDAC3, cGAS and Sting were significantly increased in SAP patients and SAP mice. Silencing HDAC3 promoted the proliferation and adhesion of intestinal glial cells and inhibited the inflammation and apoptosis of intestinal epithelial cells. In addition, silencing HDAC3 inhibited oxidative stress in intestinal epithelial cells. Furthermore, silencing HDAC3 inhibited the activation of cGAS-Sting pathway in intestinal glial cells. More importantly, silencing HDAC3 alleviates intestinal barrier function in SAP mice. HDAC3 inhibition improves acute pancreatitis in mice by regulating cGAS-Sting pathway of intestinal glial cells.

Disulfiram relieves severe acute pancreatitis by inhibiting GSDMD-dependent NETs formation.

Severe acute pancreatitis (SAP) is characterized by pancreatic and systemic inflammation and persistent organ failure. Neutrophil extracellular traps (NETs) are considered to play an important role in the development of SAP. We aimed to explore the effect of disulfiram (DSL), a newly developed anti-inflammatory drug, on NETs in SAP. A mouse model of SAP was induced by caerulein and lipopolysaccharide, and the mice weredivided into the normal control group, the DSL group, the SAP group, and the SAP treated with 50 mg/kg (50DSLSAP) and 100 mg/kg DSL (100DSLSAP) groups, respectively. The severity of SAP was evaluated based on the morphological and biochemical changes. Gasdermin D (GSDMD) expression was evaluated in vivo and in vitro to verify the effect of DSL. Additionally, the expressions of NETs were also evaluated in vivo and in vitro in SAP with and without DSL treatment to explore the possible mechanism of DSL on SAP. Pancreatic inflammatory injury increased in the SAP group, which was alleviated by DSL. GSDMD, a protein related to the formation of NETs, increased in SAP. Expressions of NETs were also promoted in the in vivoSAP model and by phorbol myristate acetate (PMA) in vitro. Moreover, DSL inhibited the expressions of GSDMD and NETs in vivo. The results were further confirmed in the in vitro experiment. NETs are highly associated with inflammatory injury in SAP. DSL inhibits NETs formation by downregulating GSDMD, which in turn relieves the inflammation of SAP. Our study may provide a possible therapeutic target for SAP.

Salidroside protects against caerulein with the LPS-induced severe acute pancreatitis through suppression of oxidative stress and inflammation in mice

• Salidroside attenuated caerulein with LPS-induced pancreatic injury in a mouse model of SAP. • Salidroside significantly ameliorated oxidative stress and inflammation responses in the pancreas of SAP mice. • Depletion of Nrf2 weakened the therapeutic effect of salidroside and reduced the anti-oxidative stress and anti-inflammatory effects in salidroside treated SAP mice. • Molecular mechanism studies showed that salidroside mediated anti-oxidant stress and anti-inflammatory responses in SAP mice partially through the Nrf2/NF-κB p65 pathway. Severe acute pancreatitis (SAP) is a fatal inflammation with no effective treatment. Salidroside, a monomer isolated from the traditional Chinese medicine Rhodiola, has been shown cytoprotective roles in many inflammation-related diseases. However, whether salidroside plays a protective role in an acute inflammation like SAP in mice is unknown. In this study, we aimed to investigate the molecular mechanism underlying the pancreatic protection of salidroside. The results showed that salidroside attenuated caerulein with the LPS-induced pancreatic injury in a mouse model of SAP. Also, we found that salidroside ameliorated oxidative stress and inflammation responses in SAP mice. Furthermore, SAP mice treated with salidroside were associated with higher phosphorylation levels of Nrf2 and less nuclear accumulation of NF-κB p65. We further found that depletion of Nrf2 weakened the protective effect of salidroside in SAP mice. Collectively, salidroside mediated anti-oxidant stress and anti-inflammation in SAP mice partially through the Nrf2/NF-κB p65 pathway.

Clostridium butyricum Protects Against Pancreatic and Intestinal Injury After Severe Acute Pancreatitis via Downregulation of MMP9.

Background: Evidence have shown that gut microbiota plays an important role in the development of severe acute pancreatitis (SAP). In addition, matrix metalloproteinase-9 (MMP9) plays an important role in intestinal injury in SAP. Thus, we aimed to determine whether gut microbiota could regulate the intestinal injury during SAP via modulating MMP9. Methods: In this study, the fecal samples of patients with SAP (n = 72) and healthy controls (n = 32) were analyzed by 16S rRNA gene sequencing. In addition, to investigate the association between gut microbiota and MMP9 in intestinal injury during SAP, we established MMP9 stable knockdown Caco2 and HT29 cells in vitro and generated a MMP9 knockout (MMP9−/−) mouse model of SAP in vivo. Results: We found that the abundance of Clostridium butyricum (C. butyricum) was significantly decreased in the SAP group. In addition, overexpression of MMP9 notably downregulated the expressions of tight junction proteins and upregulated the expressions of p-p38 and p-ERK in Caco2 and HT29 cells (p < 0.05). However, C. butyricum or butyrate treatment remarkably upregulated the expressions of tight junction proteins and downregulated the expressions of MMP9, p-p38 and p-ERK in MMP9-overexpressed Caco2 and HT29 cells (p < 0.05). Importantly, C. butyricum or butyrate could not affect the expressions of tight junction proteins, and MMP9, p-p38 and p-ERK proteins in MMP9-knockdown cells compared with MMP9-knockdown group. Consistently, C. butyricum or butyrate could not attenuate pancreatic and intestinal injury during SAP in MMP9−/− mice compared with the SAP group. Conclusion: Collectively, C. butyricum could protect against pancreatic and intestinal injury after SAP via downregulation of MMP9 in vitro and in vivo.

Colonic interleukin-22 protects intestinal mucosal barrier and microbiota abundance in severe acute pancreatitis.

Intestinal barrier dysfunction plays a critical role in the pathophysiology of many diseases including severe acute pancreatitis (SAP). Interleukin-22 (IL-22) is a critical regulator of intestinal epithelial homeostasis. However, the mechanism, origin site, and characteristics of IL-22 in the intestinal barrier dysfunction remains elusive. Studies were conducted in patients with SAP and SAP mice model. SAP mice model was induced by intraductal infusion of 5% taurocholic acid. The level and source of IL-22 were analyzed by flow cytometry. The effect of IL-22 in SAP-associated intestinal injury were examined through knockout of IL-22 (IL-22-/- ) or administration of recombinant IL-22 (rIL-22). IL-22 increased in the early phase of SAP but declined more quickly than that of proinflammatory cytokines, such as IL-6 and TNF-α. CD177+ neutrophils contributed to IL-22 expression in SAP. IL-22 was activated in the colon rather than the small intestine during SAP. Deletion of IL-22 worse the severity of colonic injury, whereas administration of rIL-22 reduced colonic injury. Mechanistically, IL-22 ameliorates the intestinal barrier dysfunction in SAP through decreasing colonic mucosal permeability, upregulation of E-cadherin and ZO-1 expression, activation of pSTAT3/Reg3 pathway and restoration of fecal microbiota abundance. This study revealing that early decreased colonic IL-22 aggravates intestinal mucosal barrier dysfunction and microbiota dysbiosis in SAP. Colonic IL-22 is likely a promising treating target in the early phase of SAP management. Research in context Evidence before this study Intestinal barrier dysfunction plays a critical role in the pathophysiology of severe acute pancreatitis (SAP). Interleukin-22 (IL-22) is a critical regulator of intestinal epithelial homeostasis. However, the mechanism, origin site and characteristics of IL-22 in the intestinal barrier dysfunction remains elusive. Added value of this study Firstly, we determined the dynamic expression profile of IL-22 in SAP and found that IL-22 was mostly activated in the pancreas and colon and decreased earlier than proinflammatory cytokines. CD177+ neutrophils contributed to IL-22 expression in SAP. Furthermore, we found that IL-22 ameliorates intestinal barrier dysfunction in SAP through decreasing colonic mucosal permeability, upregulation of E-cadherin and ZO-1 expression, activation of pSTAT3/Reg3 pathway and restoration of fecal microbiota abundance. Implications of all the available evidence This study highlights the role of colonic injury and colonic IL-22 in SAP. IL-22 is likely a promising treating target in the early phase of SAP management.

A Severe Acute Pancreatitis Mouse Model Transited from Mild Symptoms Induced by a "Two-Hit" Strategy with L-Arginine.

To develop a severe acute pancreatitis (SAP) model transited from mild symptoms, we investigated a “two-hit” strategy with L-arginine in mice. The mice were intraperitoneally injected with ice-cold L-arginine (4 g/kg) twice at an interval of 1 h on the first day and subjected to the repeated operation 72 h afterwards. The results showed the “two-hit” strategy resulted in the destructive damage and extensive necrosis of acinar cells in the pancreas compared with the “one-hit” model. Meanwhile, excessive levels of pro-inflammatory mediators, namely IL-6 and TNF-α, were released in the serum. Remarkably, additional deleterious effects on multiple organs were observed, including high intestinal permeability, kidney injury, and severe acute lung injury. Therefore, we confirmed that the SAP animal model triggered by a “two-hit” strategy with L-arginine was successfully established, providing a solid foundation for a deeper understanding of SAP initiation and therapy research to prevent worsening of the disease.

Activation of TLR4 induces severe acute pancreatitis-associated spleen injury via ROS-disrupted mitophagy pathway

Severe acute pancreatitis (SAP) is complicated by systemic inflammatory response syndrome and multiple organ dysfunction, the disease will eventually result in death in almost half of the case. The spleen, as the largest immune organ adjacent to the pancreas, is prone to damage in SAP, thereby aggravating the damage of other organs and increasing mortality. However, to date, the research on the mechanism and treatment of spleen injury caused by SAP is still in its infancy. Herein, we investigated the mechanism of spleen injury, and explored the application potential of tuftsin for relieving spleen damage in SAP mice. Firstly, SAP mice model was constructed via the retrograde infusion of 3.5 % sodium taurocholate into the biliopancreatic duct. Then, we proved that the up-regulation of Toll-like receptor 4 (TLR4) in spleen would lead to the accumulation of reactive oxygen species (ROS) and mitochondrial dysfunction under SAP conditions. The splenic ROS and mitochondrial dysfunction could be improved by N-acetylcysteine (NAC) treatment or knocking out TLR4 in SAP mice. Meanwhile, we found that NAC treatment could also improve the autophagy of spleen tissue, suggesting that splenic ROS may affect impaired autophagy, causing the accumulation of damaged mitochondria, aggravating spleen damage. Furthermore, we verified the mechanism of spleen injury is caused by splenic ROS affecting PI3K/p-AKT/mTOR pathway-mediated autophagy. In addition, we detected the spleen injury caused by SAP could decrease the concentration of tuftsin in the serum of mice. Whereas, exogenous supplementation of tuftsin ameliorated the pathological damage, ROS accumulation, impaired autophagy, inflammation expression and apoptosis in damaged spleen. In summary, we verified the new mechanism of SAP-caused spleen damage that TLR4-induced ROS provoked mitophagy impairment and mitochondrial dysfunction in spleen via PI3K/p-AKT mTOR signaling, and the application potential of tuftsin in treating spleen injury, which might expand novel ideas and methods for the treatment of pancreatitis.

Emodin Alleviates Intestinal Barrier Dysfunction by Inhibiting Apoptosis and Regulating the Immune Response in Severe Acute Pancreatitis.

The intestinal barrier injury caused by severe acute pancreatitis (SAP) can induce enterogenous infection, further aggravating the inflammatory reactions and immune responses. This study aimed to test the hypothesis that emodin protects the intestinal function and is involved in the immune response in SAP. The network pharmacology was established using the Swiss target prediction and pathway enrichment analysis. The SAP mice model was induced by cerulein (50 μg/kg) and lipopolysaccharide (10 mg/kg) hyperstimulation. The pharmacological effect of emodin in treating SAP was evaluated at mRNA and protein levels by various methods. The network analysis provided the connectivity between the targets of emodin and the intestinal barrier-associated proteins and predicted the BAX/Bcl-2/caspase 3 signaling pathway. Emodin alleviated the pathological damages to the pancreas and intestine and reduced the high concentrations of serum amylase and cytokines in vivo. Emodin increased the expression of intestinal barrier-related proteins and reversed the changes in the apoptosis-related proteins in the intestine. Simultaneously, emodin regulated the ratio of T helper type 1 (TH1), TH2, TH17, γδ T cells, and interferon γ/interleukin 17 producing γδ T cells. These findings partly verified the mechanism underlying the regulation of the intestinal barrier and immune response by emodin.

MiR155 Disrupts the Intestinal Barrier by Inducing Intestinal Inflammation and Altering the Intestinal Microecology in Severe Acute Pancreatitis.

Intestinal dysfunction is a common complication of acute pancreatitis. MiR155 may be involved in the occurrence and development of intestinal dysfunction mediated by acute pancreatitis, but the specific mechanism is not clear. To investigate the effect of miR155 on severe acute pancreatitis (SAP)-associated intestinal dysfunction and its possible mechanism in a mice model. In this study, SAP mice model was induced by intraperitoneal injection of caerulein and LPS in combination. Adeno-associated virus (AAV) was given by tail vein injection before the SAP model. The pancreatic and intestinal histopathology changes were analyzed. Cecal tissue was collected for 16S rRNA Gene Sequencing. Intestinal barrier proteins ZO-1 and E-cad were measured by Immunohistochemistry Staining and Western Blot, respectively. Intestinal tissue miR155 and inflammatory factors TNF-α, IL-1β, and IL-6 were detected by Q-PCR. The expression levels of protein associated with TNF-α and TLR4/MYD88 pathway in the intestinal were detected. In miR155 overexpression SAP group, the levels of tissue inflammatory factor were significantly increased, intestinal barrier proteins were significantly decreased, and the injury of intestinal was aggravated. Bacterial 16S rRNA sequencing was performed, showing miR155 promotes gut microbiota dysbiosis. The levels of TNF-α, TLR4, and MYD88 in the intestinal were detected, suggesting that miR155 may regulate gut microbiota and activate the TLR4/MYD88 pathway, thereby affecting the release of inflammatory mediators and regulating SAP-related intestinal injury. After application of miR155-sponge, imbalance of intestinal flora and destruction of intestinal barrier-related proteins have been alleviated. The release of inflammatory mediators decreased, and the histopathology injury of intestinal was improved obviously. MiR155 may play an important role in SAP-associated intestinal dysfunction. MiR155 can significantly alter the intestinal microecology, aggravated intestinal inflammation through TLR4/MYD88 pathway, and disrupts the intestinal barrier in SAP mice.

Free Fatty Acid Increases the Expression of NLRP3-Caspase1 in Adipose Tissue Macrophages in Obese Severe Acute Pancreatitis.

Obesity is an important risk factor for severe acute pancreatitis. The necrosis of epididymal adipose tissue occurs in severe acute pancreatitis. Adipose tissue macrophages play an important role in metabolic related inflammation. Therefore, we explored the potential mechanisms between adipose tissue macrophages and obesity-related severe acute pancreatitis. Severe acute pancreatitis mice model was induced by caerulein with lipopolysaccharide. The severity of severe acute pancreatitis was evaluated according to the morphological, general, and biochemical change. We assessed the injury of epididymal white adipose tissue, pancreas, and adipose tissue macrophages in obese mice and lean mice with severe acute pancreatitis. Outcomes of caerulein-induced severe acute pancreatitis were studied in lean and obese mice with or without lipase inhibitor orlistat. Fat necrosis and pancreatic injury increased in the SAP groups. High levels of serum free fatty acid and triglyceride were increased significantly in the SAP group. The NLRP3-caspase1 inflammasome signal pathway in adipose tissue macrophages markedly enhanced in the SAP groups compared with control group. Free fatty acid can trigger macrophages inflammation through NLRP3-caspase1. Lipase inhibited by orlistat remarkably decreased in adipose tissue necrosis, and the levels of serum lipase, amylase, and pancreatic tissue damage decreased in the orlistat group compared with the SAP group. The NLRP3-caspase1 inflammasome pathway in adipose tissue macrophages markedly decreased in the orlistat groups compared with SAP group. The levels of serum free fatty acid and triglyceride were decreased significantly in the orlistat group. Inflammation increases in adipose tissue macrophages of obese mice with severe acute pancreatitis. Free fatty acid generated via adipocyte lipolysis worsens inflammation in adipose tissue macrophages and the outcome of severe acute pancreatitis in obese mice through the NLRP3-caspase1 inflammasome pathway.

Mechanism of gasdermin D on intestinal injury in severe acute pancreatitis by mediating pyroptosis

To investigate the function of gasdermin D (GSDMD) in intestinal damage of mice with severe acute pancreatitis (SAP). The healthy C57BL/6 mice were divided into four groups randomly, including normal saline (NS) group, small interfering RNA (siRNA)-NS group, SAP model group and siRNA-SAP group, with 6 mice in each group. The SAP mouse model was reproduced by intraperitoneal injection of caerulein 50 μg/kg combined with lipopolysaccharide (LPS) 10 mg/kg; the NS group was given the same amount of NS; in the siRNA-SAP group and siRNA-NS group, siRNA 50 mg/kg was injected through the tail vein three times before modeling or injection of NS. The blood of mice eyeball in each group was taken 12 hours after modeling, and serum interleukins (IL-1β, IL-18) levels were detected by enzyme linked immunosorbent assay (ELISA). The mice were sacrificed to observe the general changes in abdominal cavity, the pancreas and ileum tissues were taken to observe the pathological changes under a light microscope. The expression of long-chain non-coding RNA uc.173 (lnc uc.173) was detected by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical method was used to detect the expression of tight junction proteins zonula occluden-1 (ZO-1) and Occludin in intestinal mucosal epithelial cells. Western blotting was used to detect the GSDMD protein expression level in the intestinal tissue. The serum levels of IL-1β and IL-18 in the SAP model group were significantly higher than those in the NS group and the siRNA-NS group [IL-1β (ng/L): 146.66±1.40 vs. 44.48±5.76, 81.49±10.75, IL-18 (ng/L): 950.47±177.09 vs. 115.43±16.40, 84.84±21.90, all P < 0.05]; and the levels of IL-1β and IL-18 in the siRNA-SAP group were significantly lower than those in the SAP model group [IL-1β (ng/L): 116.26±15.54 vs. 146.66±1.40, IL-18 (ng/L): 689.96±126.08 vs. 950.47±177.09, both P < 0.05]. General observation showed that there were no obvious abnormalities in the abdominal cavity of the mice in the NS and siRNA-NS groups; the mice in the SAP model group and the siRNA-SAP group had different degrees of edema and congestion in the intestine; compared with the SAP model group, the abnormalities in the siRNA-SAP group was significantly reduced. Under light microscope, there were no obvious changes in the pancreas and intestinal mucosa in the NS group and the siRNA-NS group; the pancreatic tissue of the SAP model group and the siRNA-SAP group had different degrees of edema, inflammatory cell infiltration, and lobular structure damage, and the intestinal mucosa was damaged to a certain degree, and the villi were broken to varying degrees, but the damage in the siRNA-SAP group was lighter. The results of RT-PCR showed that the expression of lnc uc.173 in the intestinal tissues of the model SAP group was significantly lower than that of the NS group and the siRNA-NS group (2-ΔΔCt: 0.26±0.12 vs. 1.01±0.37, 0.67±0.32, both P < 0.05), while the expression of lnc uc.173 in the siRNA-SAP group was significantly higher than that in the SAP model group (2-ΔΔCt: 0.60±0.39 vs. 0.26±0.12, P < 0.05). Immunohistochemistry showed that ZO-1 and Occludin proteins in the NS group were distributed along the epithelial cells of the intestinal mucosa, showing a strong expression; ZO-1 and Occludin expressions were significantly reduced in the SAP model group and siRNA-SAP group, but the expressions in the siRNA-SAP group was higher than that in the SAP model group. Western blotting showed that the expression level of GSDMD protein in the intestinal tissues of the SAP model group was significantly higher than that of the NS group and the siRNA-NS group [GSDMD protein (GSDMD-N/β-actin): 1.99±0.46 vs. 1, 1.00±0.78, both P < 0.05]. Compared with the SAP model group, the expression of GSDMD protein in the siRNA-SAP group was significantly decreased [GSDMD protein (GSDMD-N/β-actin): 1.42±0.42 vs. 1.99±0.46, P < 0.05]. The systemic inflammatory response and intestinal mucosal barrier damage of SAP mice may be related to the increase of GSDMD expression in intestinal tissues. GSDMD mediates cell pyrolysis to promote the release of inflammatory factors, cause intestinal injury, and down-regulate the expression of intestinal epithelial cell tight junction proteins such as ZO-1 and Occludin, resulting in intestinal mucosal damage.

N-(3',4'-dimethoxycinnamonyl) anthranilic acid alleviates severe acute pancreatitis by inhibiting intestinal barrier dysfunction and NF-κB activation.

Severe acute pancreatitis (SAP) can affect intestinal barrier with a high mortality. To date, effective therapies for SAP are still in urgently need. The purpose of this study was to investigate the role of anthranilic acid active synthetic derivative, N-(3',4'-dimethoxycinnamonyl) anthranilic acid (3,4-DAA), in intestinal barrier dysfunction of SAP. In this study, SAP mice model was induced by caerulein combined with lipopolysaccharide (LPS). SAP mice were pretreated with 3,4-DAA orally. Histological structures of pancreatic and intestinal tissues were observed via hematoxylin-eosin (H&E) staining. Pancreas myeloperoxidase (MPO), serum lipase, and amylase were detected using corresponding kits. Western blot analysis and reverse transcription and quantitative PCR (RT-qPCR) were employed to determine the levels of inflammatory factors in both pancreatic and intestinal tissues. Moreover, the levels of intestinal barrier-related proteins, NLRP3 inflammasome and NF-κB pathway were examined by western blot analysis. Result revealed that 3,4-DAA significantly attenuated pancreas and intestine damage, inhibited the release of inflammatory factors and intestinal barrier dysfunction. Moreover, the expression of NLRP3 and phospho-NF-κB p65 in pancreatic and intestinal tissues was notably suppressed by 3,4-DAA. To sum up, these results demonstrated that 3,4-DAA could ameliorate SAP, partly attributing to the inhibition of intestinal barrier dysfunction and the release of inflammatory factors. These findings may provide a new mechanism support for 3,4-DAA application in the clinical treatment of SAP.

ATF6 aggravates acinar cell apoptosis and injury by regulating p53/AIFM2 transcription in Severe Acute Pancreatitis

Background: There is no curative therapy for severe acute pancreatitis (SAP) due to poor understanding of its molecular mechanisms. Endoplasmic reticulum (ER) stress is involved in SAP and increased expression of ATF6 has been detected in SAP patients. Here, we aimed to investigate the role of ATF6 in a preclinical SAP mouse model and characterize its regulatory mechanism.Methods: Pancreatic tissues of healthy and SAP patients were collected during surgery. Humanized PRSS1 transgenic mice were treated with caerulein to mimic the SAP development, which was crossed to an ATF6 knockout mouse line, and pancreatic tissues from the resulting pups were screened by proteomics. Adenovirus-mediated delivery to the pancreas of SAP mice was used for shRNA-based knockdown or overexpression. The potential functions and mechanisms of ATF6 were clarified by immunofluorescence, immunoelectron microscopy, Western blotting, qRT-PCR, ChIP-qPCR and luciferase reporter assay.Results: Increased expression of ATF6 was associated with elevated apoptosis, ER and mitochondrial disorder in pancreatic tissues from SAP patients and PRSS1 mice. Knockout of ATF6 in SAP mice attenuated acinar injury, apoptosis and ER disorder. AIFM2, known as a p53 target gene, was identified as a downstream regulatory partner of ATF6, whose expression was increased in SAP. Functionally, AIFM2 could reestablish the pathological disorder in SAP tissues in the absence of ATF6. p53 expression was also increased in SAP mice, which was downregulated by ATF6 knockout. p53 knockout significantly suppressed acinar apoptosis and injury in SAP model. Mechanistically, ATF6 promoted AIFM2 transcription by binding to p53 and AIFM2 promoters.Conclusion: These results reveal that ATF6/p53/AIFM2 pathway plays a critical role in acinar apoptosis during SAP progression, highlighting novel therapeutic target molecules for SAP.

Chrm3 protects against acinar cell necrosis by stabilizing caspase-8 expression in severe acute pancreatitis mice model.

Acinar cells in acute pancreatitis (AP) die through apoptosis and necrosis, the impacts of which are quite different. Early clinical interference strategies on preventing the progress of AP to severe acute pancreatitis (SAP) are the elimination of inflammation response and inhibition of necrosis. Muscarinic acetylcholine receptor M3 was encoded by Chrm3 gene. It is one of the best-characterized receptors of pancreatic β cells and regulates insulin secretion, but its function in AP remains unclear. In this study, we explored the function of Chrm3 gene in the regulation of cell death in l-arginine-induced SAP animal models. We found that Chrm3 was upregulated in pancreatitis, and we further confirmed the localization of Chrm3 resided in both pancreatic islets and acinar cell membranes. The reduction of Chrm3 decreased the pathological lesion of SAP and reduced amylase activities in serum. Consistently, Chrm3 can suppress acinar cells necrosis markedly, but has no effect on regulating apoptosis after l-arginine treatment. It was shown that Chrm3 attenuated acinar cells necrosis at least in part by stabilizing caspase-8. Thus, this study indicates that Chrm3 is critical participants in SAP, and regulation of Chrm3 expression might be a useful therapeutic strategy for preventing pathologic necrosis.

Iguratimod (T-614) attenuates severe acute pancreatitis by inhibiting the NLRP3 inflammasome and NF-κB pathway

Severe acute pancreatitis (SAP) is an acute abdominal disease that can develop locally to the multiple organs. It is characterized by pancreatic tissue self-digestion, and the rapid release of inflammatory cytokines, which play a dominant role in local or even systemic inflammation. In this study, we investigate the protective effect of T-614 against SAP induced by cerulein plus LPS in mice. Biochemical markers associated with pancreatitis in serum such as inflammatory cytokines, amylase and lipase activities were measured. Related proteins of NLRP3 inflammasome and NF-κB signaling pathway were evaluated by western blotting. Hematoxylin-eosin staining (HE) and immunohistochemistry (IHC) were used to evaluate changes of inflammation in pancreatic tissue. T-614 significantly alleviated the elevation markers of pancreatitis and suppresses the pancreatic tissue damage, including histopathological and molecular manifestations. In conclusion, T-614 plays a protective role in experimental SAP mice model via anti-inflammatory effects.

Dexamethasone protects the glycocalyx on the kidney microvascular endothelium during severe acute pancreatitis.

This study demonstrated that dexamethasone (DEX) protects the endothelial glycocalyx from damage induced by the inflammatory stimulus tumor necrosis factor-α (TNF-α) during severe acute pancreatitis (SAP), and improves the renal microcirculation. Ninety mice were evenly divided into 3 groups (Sham, SAP, and SAP+DEX). The SAP mice model was established by ligature of pancreatic duct and intraperitoneal injection of cerulein. Renal perfusion and function, and morphological changes of the glycocalyx were evaluated by laser Doppler velocimetry, electron microscopy, and histopathology (hematoxylin and eosin (H&E) staining), respectively. Serum levels of syndecan-1 and TNF-α were assessed by enzyme-linked immunosorbent assay (ELISA). The protective effects of dexamethasone on the glycocalyx and renal microcirculation were evaluated. Significantly high levels of serum TNF-α were detected 3 h after the onset of SAP. These levels might induce degradation of the glycocalyx and kidney hypoperfusion, resulting in kidney microcirculation dysfunction. The application of dexamethasone reduced the degradation of the glycocalyx and improved perfusion of kidney. Dexamethasone protects the endothelial glycocalyx from inflammatory degradation possibly initiated by TNF-α during SAP. This is might be a significant discovery that helps to prevent tissue edema and hypoperfusion in the future.

TSG-6 secreted by human adipose tissue-derived mesenchymal stem cells ameliorates severe acute pancreatitis via ER stress downregulation in mice

BackgroundThrough recent studies, the onset of acute pancreatitis in pancreatic acinar cells (PACs) and the regulatory role of PACs in severe acute pancreatitis (SAP) have been revealed. During the early stages of pancreatitis, the endoplasmic reticulum (ER) in PACs undergoes significant changes, including swelling and vacuolization. In response to an increase in the extracellular stress in ER, PACs lose their functions, leading to cell apoptosis and inflammation response. The beneficial effects of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) on SAP have been well documented in previous studies. However, the underlying mechanism of their action remains controversial.MethodsIn this study, the therapeutic effects of intraperitoneally administered hAT-MSCs in a caerulein (50 μg/kg)- and lipopolysaccharide (LPS) (10 mg/kg)-co-induced SAP mouse model were evaluated. Inflammatory response and ER stress were measured in pancreatic tissue samples, and the beneficial effects were evaluated through quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot, and immunofluorescence analysis.ResultsInflammatory response and ER stress were ameliorated following hAT-MSC injection, and the beneficial effects were observed in the absence of significant engraftment of hAT-MSCs. hAT-MSCs transfected with siRNA-targeting tumour necrosis factor-α-induced gene/protein 6 (TSG-6) were unable to inhibit ER stress and inflammation. In addition, TSG-6 from hAT-MSCs significantly suppressed ER stress-induced apoptosis and nuclear factor kappa B (NF-κB) activity in SAP model mice.ConclusionsTSG-6 secreted by hAT-MSCs protects PACs in SAP model mice via the inhibition of ER stress, as well as inflammatory responses. This study has revealed a new area for ER stress-targeted therapy in SAP patients.Graphical abstract

Histidine-Rich Glycoprotein Suppresses Hyperinflammatory Responses of Lung in a Severe Acute Pancreatitis Mouse Model.

ObjectivesSevere acute pancreatitis is a highly lethal disease caused by systemic inflammatory response syndrome, leading to multiple organ failure. We recently showed that histidine-rich glycoprotein (HRG) supplemental therapy ameliorated septic acute respiratory distress syndrome due to unnecessary neutrophil activation and immunothrombosis formation. Here, we evaluated the effect of HRG on lung inflammation followed by pancreatitis in a severe acute pancreatitis mouse model.MethodsMice received intraperitoneal injections of cerulein 7 times (100 μg/kg each) at 1-hour intervals to induce acute pancreatitis. Immediately after the first cerulein injection, phosphate-buffered saline, human serum albumin (20 mg/kg), or HRG (20 mg/kg) was intravenously injected. One hour after the last cerulein injection, phosphate-buffered saline or lipopolysaccharide (5 mg/kg) was intravenously injected into the tail vein. We evaluated lung inflammatory level after pancreatitis.ResultsWe observed significantly decreased plasma HRG levels in an acute pancreatitis mouse model. Histidine-rich glycoprotein treatment inhibited lung edema and the accumulation of neutrophil in severe acute pancreatitis, but HRG did not directly affect pancreatitis. Moreover, HRG suppressed tumor necrosis factor α, inducible nitric oxide synthase, interleukin 6, and neutrophil elastase mRNA expression and myeloperoxidase activity in the lung.ConclusionsThese data suggested that HRG ameliorated lung inflammation secondary to pancreatitis.