Heterozygous Spink1 Deficiency Promotes Trypsin-dependent Chronic Pancreatitis in Mice.

BACKGROUND & AIMS: Heterozygous SPINK1 mutations are strong risk factors for chronic pancreatitis in humans, yet heterozygous disruption of mouse Spink1 yielded no pancreatic phenotype. To resolve this contradiction, we used CRISPR/Cas9-mediated genome editing to generate heterozygous Spink1-deleted mice (Spink1-KOhet) in the C57BL/6N strain and studied the effect of this allele in trypsin-independent and trypsin-dependent pancreatitis models. METHODS: We investigated severity of acute pancreatitis and progression to chronic pancreatitis in Spink1-KOhet mice after transient (10 injections) and prolonged (2 × 8 injections) cerulein hyperstimulation. We crossed Spink1-KOhet mice with T7D23A and T7D22N,K24R mice that carry strongly autoactivating trypsinogen mutants and exhibit spontaneous chronic pancreatitis. RESULTS: Prolonged but not transient cerulein stimulation resulted in increased intrapancreatic trypsin activity and more severe acute pancreatitis in Spink1-KOhet mice relative to the C57BL/6N control strain. After the acute episode, Spink1-KOhet mice developed progressive disease with chronic pancreatitis-like features, whereas C57BL/6N mice recovered rapidly. Trypsinogen mutant mice carrying the Spink1-KOhet allele exhibited strikingly more severe chronic pancreatitis than the respective parent strains. CONCLUSIONS: Heterozygous Spink1 deficiency caused more severe acute pancreatitis after prolonged cerulein stimulation and promoted chronic pancreatitis after the cerulein-induced acute episode, and in two strains of trypsinogen mutant mice with spontaneous disease. In contrast, acute pancreatitis induced with limited cerulein hyperstimulation was unaffected by heterozygous Spink1 deletion, in agreement with recent observations that trypsin activity does not mediate pathologic responses in this model. Taken together, the findings strongly support the notion that loss-of-function SPINK1 mutations in humans increase chronic pancreatitis risk in a trypsin-dependent manner.

Interleukin-22 Alleviates Caerulein-Induced Acute Pancreatitis by Activating AKT/mTOR Pathway.

BACKGROUND: Acute pancreatitis (AP) is one of the most common acute abdominal disorders; due to the lack of specific treatment, the treatment of acute pancreatitis, especially serious acute pancreatitis (SAP), is difficult and challenging. We will observe the changes of Interleukin -22 levels in acute pancreatitis animal models, and explore the mechanism of Interleukin -22 in acute pancreatitis. OBJECTIVE: This study aims to assess the potential protective effect of Interleukin -22 on caerulein-induced acute pancreatitis and to explore its mechanism. METHODS: Blood levels of amylase and lipase and Interleukin -22 were assessed in mice with acute pancreatitis. In animal model and cell model of caerulein-induced acute pancreatitis, the mRNA levels of P62 and Beclin-1 were determined using PCR, and the protein expression of P62, LC3-II, mTOR, AKT, p-mTOR, and p-AKT were evaluated through Western blot analysis. RESULTS: Interleukin -22 administration reduced blood amylase and lipase levels and mitigated tissue damage in acute pancreatitis mice model. Interleukin -22 inhibited the relative mRNA levels of P62 and Beclin-1, and the Interleukin -22 group showed a decreased protein expression of LC3-II and P62 and the phosphorylation of the AKT/mTOR pathway. Furthermore, we obtained similar results in the cell model of acute pancreatitis. CONCLUSION: This study suggests that Interleukin -22 administration could alleviate pancreatic damage in caerulein-induced acute pancreatitis. This effect may result from the activation of the AKT/mTOR pathway, leading to the inhibition of autophagy. Consequently, Interleukin -22 shows potential as a treatment.

Prescription opioids induced microbial dysbiosis worsens severity of chronic pancreatitis and drives pain hypersensitivity.

Opioids, such as morphine and oxycodone, are widely used for pain management associated with chronic pancreatitis (CP); however, their impact on the progression and pain sensitivity of CP has never been evaluated. This report investigates the impact of opioid use on the severity of CP, pain sensitivity, and the gut microbiome. C57BL/6 mice were divided into control, CP, CP with morphine/oxycodone, and either morphine or oxycodone alone groups. CP was induced by administration of caerulein (50ug/kg/h, i.p. hourly x7, twice a week for 10 weeks). The mouse-to-pancreas weight ratio, histology, and Sirius red staining were performed to measure CP severity. Tail flick and paw pressure assays were used to measure thermal and mechanical pain. DNA was extracted from the fecal samples and subjected to whole-genome shotgun sequencing. Germ-free mice were used to validate the role of gut microbiome in sensitizing acute pancreatic inflammation. Opioid treatment exacerbates CP by increasing pancreatic necrosis, fibrosis, and immune-cell infiltration. Opioid-treated CP mice exhibited enhanced pain hypersensitivity and showed distinct clustering of the gut microbiome compared to untreated CP mice, with severely compromised gut barrier integrity. Fecal microbiota transplantation (FMT) from opioid-treated CP mice into germ-free mice resulted in pancreatic inflammation in response to a suboptimal caerulein dose. Together, these analyses revealed that opioids worsen the severity of CP and induce significant alterations in pain sensitivity and the gut microbiome in a caerulein CP mouse model. Microbial dysbiosis plays an important role in sensitizing the host to pancreatic inflammation.

Qing Xia Jie Yi Formula granules alleviated acute pancreatitis through inhibition of M1 macrophage polarization by suppressing glycolysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal formulas from Traditional Chinese Medicine are common and well-established practice for treating acute pancreatitis (AP) patients. However, little is known about their bioactive ingredients and mechanisms, such as their targets and pathways to inhibit inflammation. AIM OF THE STUDY: This study aimed to evaluate the effect of Qing Xia Jie Yi Formula (QXJYF) granules on AP and discuss the molecular mechanisms involved. MATERIALS AND METHODS: Major compounds in QXJYF granules were identified using UPLC-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap MS). The effect of QXJYF granules on experimental AP models both in vitro and in vivo, and detailed mechanisms were clarified. Two AP models were induced in mice by intraperitoneally injections of caerulein or L-arginine, and QXJYF granules were used to treat AP mice in vivo. Histological evaluation of pancreas and lung, serum amylase and lipase levels, serum inflammatory cytokines, inflammatory cell infiltration and macrophage phenotype were assessed. Bone marrow derived macrophages (BMDMs) were cultured and treated with QXJYF granules in vitro. BMDM phenotype and glycolysis levels were measured. Lastly, clinical effect of QXJYF granules on AP patients was verified. Predicted severe AP (pSAP) patients eligible for inclusion were assessed for enrollment. RESULTS: Nine major compounds were identified in QXJYF granules. Data showed that QXJYF granules significantly alleviated AP severity both in caerulein and L-arginine-induced AP models in vivo, pancreatic injury and inflammatory cell infiltration, systematic inflammation, lung injury and inflammatory cell infiltration were all improved after QXJYF treatment. QXJYF granules significantly reduced M1 macrophages during AP both in vivo and in vitro; besides, the mRNA expression levels of M1 genes such as inos, Tnfα, Il1ß and Il6 were significantly lower after QXJYF treatment in M1 macrophages. Mechanistically, we found that HK2, PFKFB3, PKM, LDHα levels were increased in M1 macrophages, but significantly decreased after QXJYF treatment. Clinical data indicated that QXJYF granules could significantly reduce CRP levels and shorten the duration of organ failure, thereby reducing the incidence of SAP and preventing pSAP patients from progressing to SAP. CONCLUSION: QXJYF granules alleviated AP through the inhibition of M1 macrophage polarization by suppressing glycolysis.

Tectoridin alleviates caerulein-induced severe acute pancreatitis by targeting ERK2 to promote macrophage M2 polarization.

Severe acute pancreatitis (SAP) is an inflammatory disease of the pancreas with a high mortality rate. Macrophages play a crucial role in the pathogenesis of pancreatitis. Tectoridin (Tec) is a highly active isoflavone with anti-inflammatory pharmacological activity. However, the role of Tec in the SAP process is not known. The purpose of this study was to investigate the therapeutic effect and potential mechanism of Tec on SAP. To establish SAP mice by intraperitoneal injection of caerulein and Lipopolysaccharide (LPS), the role of Tec in the course of SAP was investigated based on histopathology, biochemical indicators of amylase and lipase and inflammatory factors. The relationship between Tec and macrophage polarization was verified by immunofluorescence, real-time quantitative PCR and Western blot analysis. We then further predicted the possible targets and signal pathways of action of Tec by network pharmacology and molecular docking, and validated them by in vivo and in vitro. In this study, we demonstrated that Tec significantly reduced pancreatic injury in SAP mice, and decreased serum levels of amylase and lipase. The immunofluorescence and Western blot analysis showed that Tec promoted macrophage M2 polarization. Network pharmacology and molecular docking predicted that Tec may target ERK2 for the treatment of SAP, and in vivo and in vitro experiments proved that Tec inhibited the ERK MAPK signal pathway. In summary, Tec can target ERK2, promote macrophage M2 polarization and attenuate pancreatic injury, Tec may be a potential drug for the treatment of SAP.

Pancreatic capillary blood flow during caerulein-induced pancreatitis evaluated by a laser-doppler flowmeter in rats

Purpose: The pancreatic capillary blood flow (PCBF) was studied to determine its alterations during caerulein-induced pancreatitis in rats. Methods: Twenty rats were divided in groups: control and caerulein. A laser-Doppler flowmeter to measure PCBF continuously was used. Blood pressure (BP) and heart rate (HR) were monitored. Serum biochemistry analyses were determined. Histopathological study was performed. Results: The PCBF measured a mean of 109.08 ± 14.54 percent and 68.24 ± 10.47 percent in control group and caerulein group, respectively. Caerulein group had a mean decrease of 31.75 ± 16.79 percent. The serum amylase was 1323.70 ± 239.l0U.I-1 and 2184.60 ± 700.46U.I-1 in control and caerulein groups, respectively. There was a significant difference in the PCBF (p<0.05) and serum amylase (p<0.05) when compared to control and caerulein groups. Although micro and microvacuolization were seen in 30 percent in caerulein group, no significant difference was seen between the groups. Conclusion: A decrease in the PCBF may be one of the leading events and it is present before histopathological tissue injury had been established in this model of acute pancreatitis.

Reactive oxygen species inactivation improves pancreatic capillary blood flow in caerulein-induced pancreatitis in rats

Purpose: Reactive oxygen species (ROS) inactivation was studied to determine alterations in the pancreatic capillary blood flow (PCBF) during caerulein-induced pancreatitis in rats. Methods: A laser-Doppler flowmeter to measure PCBF and N-t-Butyl-Phenylnitrone (PBN) compound to inactivate ROS were used. Forty rats were divided in groups: 1) control; 2) caerulein; 3) PBN; 4) caerulein+PBN. Serem biochemistry and histopathological analyses were performed. Results: PCBF measured a mean of 109.08 ± 14.54 percent, 68.24 t 10.47 percent, 102.18 ± 10.23 percent and 87.73 ± 18.72 percent in groups 1, 2, 3 and 4, respectively. PCBF in groups 2 and 4 decreased 31.75 ± 16.79 percent and 12.26 ± 15.24 percent, respectively. Serum amylase was 1323.70 ± 239.10 U/l, 2184.60 ± 700.46 U/1, 1379.80 t 265.72 U/1 and 1622.10 ± 314.60 U/1 in groups 1, 2, 3 and 4, respectively. There was a significant difference in the PCBF and serem amylase when compared groups 2 and 4. Cytoplasmatic vacuolation was present in groups 2 and 4. Otherwise, no qualitative changes were seen. Conclusion: ROS inactivation improves PCBF and minimizes the serem amylase increase during caerulein-induced pancreatitis. ROS effect may be one of the leading causative events in this model of acute pancreatitis.

Modelo simplificado de inducao de pancreatite aguda experimental com uso de dose supramaxima de ceruleina / Simplified model of produced experimental acute pancreatitis with use of supra-maximal dosis of cerulein

A administracao de ceruleina tanto por via endovenosa como intraperitonial, tem sido extensivamente utilizada para inducao de pancreatite aguda experimental. Com objetivo de se testar uma nova metodologia de inducao de pancreatite aguda, que fosse de facil e rapida execucao, assim como de boa reprodutibilidade , 40 ratos Wistar foram distribuidos em quatro grupos: GRUPO I - animais que receberam infusao continua de ceruleina (15µ/Kg) GRUPO II - animais que receberam infusao continua e salina GRUPO III - animais submetidos a duas injecoes (SC+CV) de ceruleina (40µ/Kg) GRUPO IV - animais submetidos a duas injecoes (SC+CV) de salina. Apos a realizacao de dosagens bioquimicas, nao houve diferenca estatisticamente significativa entre os grupos I e II nas concentracoes teciduaias de tripsionogenio, quimiotripsinogenio, proelastase , catepsina e tambem amilase serica; ocorrendo diferenca somente entre os valores de porcentagem de agua no tecido pancreatico...