ABSTRACT
Aim: Chronic pancreatitis (CP) is a complex and intractable disease mainly manifested as chronic inflammation and fibrosis. Aspirin(acetylsalicylic acid, ASA) has been reported to be used in the treatment of acute pancreatitis (AP), but its effectiveness on CP is unclear. This study aimed to investigate the therapeutic effects of ASA in CP mice. Methods: A murine model of CP was induced by intraperitoneal injection with 20% L-arginine. After one week of L-arginine administration, mice in the ASA treatment group were administered aspirin (100mg/kg/d) by intragastric gavage. At two, four, and six weeks after the first injection of L-arginine, mice were euthanized and the pancreas was collected for histological and molecular analysis. A second model of CP (caeruelin-induced) was used as a validation experiment to test the effect of ASA. Results: L-arginine-induced CP resulted in over-expression of the inflammatory enzyme cyclooxygenase (COX)-2. COX-2 expression decreased after ASA treatment. Pancreatic-injury inflammatory response (measured by changes in amylase, CK-19, F4/80, CD3, MCP-1, IL-6) and fibrosis degree (measured by expression of COL1A1, MMP-1 and TIMP-1) was reduce in ASA -treated mice model. The therapeutic effect of ASA was also observed in caeruelin-induced CP. Conclusion: ASA has an ameliorating effect in murine models of CP through inhibition of pancreatic inflammation and fibrosis, which may be a promising option for clinical treatment.
ABSTRACT
The activation of pancreatic stellate cells (PSCs) plays a critical role in the progression of pancreatic fibrosis. Nuclear factor-kappa B (NF-κB) is associated with chronic pancreatitis (CP). Previous evidence indicated that NF-κB in acinar cells played a double-edged role upon pancreatic injury, whereas NF-κB in inflammatory cells promoted the progression of CP. However, the effects of NF-κB in PSCs have not been studied. In the present study, using two CP models and RNAi strategy of p65 in cultured PSCs, we found that the macrophage infiltration and MCP-1 expression were increased, and the NF-κBp65 protein level was elevated. NF-κBp65 was co-expressed with PSCs. In vitro, TGF-ß1 induced overexpression of the TGF-ß receptor 1, phosphorylated TGF-ß1-activated kinase 1 (p-TAK1) and NF-κB in the PSCs. Moreover, the concentration of MCP-1 in the supernatant of activated PSCs was elevated. The migration of BMDMs was promoted by the supernatant of activated PSCs. Further knockdown of NF-κBp65 in PSCs resulted in a decline of BMDM migration, accompanied by a lower production of MCP-1. These findings indicate that TGF-ß1 can induce the activation of NF-κB pathway in PSCs by regulating p-TAK1, and the NF-κB pathway in PSCs may be a target of chronic inflammation and fibrosis.
Subject(s)
NF-kappa B/metabolism , Pancreatic Stellate Cells/metabolism , Pancreatitis, Chronic/etiology , Pancreatitis, Chronic/metabolism , Animals , Biomarkers , Chemokine CCL2/biosynthesis , Disease Models, Animal , Disease Susceptibility , Fibrosis , Gene Expression , Gene Knockdown Techniques , Immunohistochemistry , Macrophages/immunology , Macrophages/metabolism , Mice , Models, Biological , NF-kappa B/genetics , Pancreatitis, Chronic/pathology , RNA Interference , RNA, Small Interfering/genetics , Transforming Growth Factor beta1/metabolismABSTRACT
Activated pancreatic stellate cells (PSCs) play a crucial role in the progression of pancreatic fibrosis. Transforming growth factor-ß (TGF-ß) is one of the strongest stimulator inducing fibrosis. The mitogen-activated protein kinase (MAPK) proteins (including ERK, JNK and p38 MAPK) are known to contribute to PSC activation and pancreatic fibrosis. Previous studies have identified PSC activation induced by TGF-ß1 is related to MAPK pathway, but the respective role of MAPK family members in PSC activation still unclear, and which family member may be the key mediator in mice PSC activation still controversial. In this study, we investigated the influence of different MAPK family member (JNK, ERK, and p38 MAPK) on mice PSC activation using an in vivo and in vitro model. The results showed p-JNK, p-ERK and p-p38 MAPK were all over-expressed in CP group, and p-JNK, p-ERK, and p-p38 MAPK were co-expressed with activated PSC. In vitro, TGF-ß1 induced JNK and ERK over-expression in PSCs. In contrast, p38 MAPK expression in PSC showed only a very weak increase. JNK- and ERK-specific inhibitors inhibited FN and α-SMA mRNA expression in PSCs, and a p38 MAPK inhibitor had no effect on PSC activation. These findings indicate that JNK and ERK were directly involved in the PSCs activation induced by TGF-ß1 and the development of pancreatic fibrosis. p38 MAPK participate in the progression of CP, but it does not respond to TGF-ß1 directly and may not be regarded as the target of TGF-ß1 induced PSC activation.
Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Pancreatic Stellate Cells/metabolism , Pancreatitis, Chronic/pathology , Transforming Growth Factor beta1/pharmacology , Animals , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Gene Expression Regulation, Enzymologic/drug effects , MAP Kinase Signaling System/drug effects , Male , Mice , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/genetics , Pancreatic Stellate Cells/drug effects , Pancreatitis, Chronic/metabolism , Protein Kinase Inhibitors/pharmacology , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1/metabolismABSTRACT
AIM: To explore the role of macrophages in chronic pancreatitis (CP) and the effect of Dachaihu decoction (DCHD) on pancreatic fibrosis in mice. METHODS: KunMing mice were randomly divided into a control group, CP group, and DCHD group. In the CP and DCHD groups, mice were intraperitoneally injected with 20% L-arginine (3 g/kg twice 1 d/wk for 6 wk). Mice in the DCHD group were administered DCHD intragastrically at a dose of 14 g/kg/d 1 wk after CP induction. At 2 wk, 4 wk and 6 wk post-modeling, the morphology of the pancreas was observed using hematoxylin and eosin, and Masson staining. Interleukin-6 (IL-6) serum levels were assayed using an enzyme-linked immunosorbent assay. Double immunofluorescence staining was performed to observe the co-expression of F4/80 and IL-6 in the pancreas. Inflammatory factors including monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α) and IL-6 were determined using real time-polymerase chain reaction. Western blot analysis was used to detect fibronectin levels in the pancreas. RESULTS: Compared with the control group, mice with 20% L-arginine-induced CP had obvious macrophage infiltration and a higher level of fibrosis. IL-6 serum concentrations were significantly increased. Double immunofluorescence staining showed that IL-6 and F4/80 were co-expressed in the pancreas. With the administration of DCHD, the infiltration of macrophages and degree of fibrosis in the pancreas were significantly attenuated; IL-6, MCP-1 and MIP-1α mRNA, and fibronectin levels were reduced. CONCLUSION: The dominant role of macrophages in the development of CP was mainly related to IL-6 production. DCHD was effective in ameliorating pancreatic fibrosis by inhibiting macrophage infiltration and inflammatory factor secretion in the pancreas.
