Development Of Pancreatic Fibrosis Research Articles

The role of predictive and prognostic values of inflammatory markers in acute pancreatitis: a narrative review

Pancreatitis is an inflammatory condition affecting the pancreas and is classified into 2 types, acute and chronic, which can manifest in various forms. This review article summarizes the role of predictive and prognostic values of inflammatory markers in the pathogenesis of acute pancreatitis, mainly focused on preclinical and clinical studies. It includes serum amyloid A (SAA), monocyte chemotactic protein-1 (MCP-1), erythrocyte sedimentation rate (ESR), interleukin-6 (IL-6), C-reactive protein (CRP), IL-10, myeloperoxidase, pentraxin 3, and plasminogen activator inhibitor 1. SAA3 plays a crucial role in developing acute pancreatitis by triggering a receptor-interacting protein 3–dependent necroptosis pathway in acinar cells. Targeting SAA3 could be a potential strategy for treating acute pancreatitis. The recruitment of monocytes/macrophages and the activation of the systemic MCP-1 signaling pathway play a role in the progression of pancreatitis, and blocking MCP-1 may have a suppressive effect on the development of pancreatic fibrosis. The ESR can predict severe acute pancreatitis with slightly lower accuracy than CRP. When ESR and CRP levels are combined at 24 hours, they predict severe acute pancreatitis accurately. IL-6 plays a crucial role in activating the Janus kinase/signal transducers and activators of the transcription pathway, exacerbating pancreatitis and contributing to the initiation and progression of pancreatic cancer. Endogenous IL-10 plays a crucial role in controlling the regenerative phase and limiting the severity of fibrosis and glandular atrophy induced by repeated episodes of acute pancreatitis in mice. The predictive and diagnostic roles of these inflammatory factors in pancreatitis were introduced in detail in this review.

Pancreatic Stellate Cells and Metabolic Alteration: Physiology and Pathophysiology.

Pancreatic stellate cells play a pivotal role in the development of pancreatic fibrosis. A wide variety of external stimuli can cause PSC activation accompanied by metabolic changes, which alters the tissue microenvironment by producing extracellular matrix proteins, cytokines, growth factors, and other mediators. Several metabolites aggravate fibrosis and inflammation by acting as key activating factors for PSCs. In other words, PSCs sense systemic metabolic changes. The detrimental effects of PSC activation on normal pancreatic cells, especially islet cells, further complicate metabolic imbalance through the dysregulation of glucose metabolism. PSC activation promotes cancer by altering the metabolism in pancreatic cancer cells, which collaborate with PSCs to efficiently adapt to environmental changes, promoting their growth and survival. This collaboration also contributes to the acquisition of chemoresistance. PSCs sequester chemotherapeutic agents and produce competing molecules as additional resistance mechanisms. The application of these metabolic targets for novel therapeutic strategies is currently being explored. This mini-review summarizes the role of PSCs in metabolic regulation of normal and cancerous cells.

Lnc-PFAR facilitates autophagy and exacerbates pancreatic fibrosis by reducing pre-miR-141 maturation in chronic pancreatitis

Chronic pancreatitis (CP) is described as progressive inflammatory fibrosis of pancreas, accompanied with irreversible impaired endocrine and exocrine insufficiency. Pancreatic stellate cells (PSCs) are widely distributed in the stroma of the pancreas and PSCs activation has been shown as one of the leading causes for pancreatic fibrosis. Our previous study has revealed that autophagy is dramatically activated in CP tissues, which facilitates PSCs activation and pancreatic fibrosis. Long non-coding RNAs (LncRNAs) have been recognized as crucial regulators for fibrosis-related diseases. LncRNAs interact with RNA binding protein or construct competitive endogenous RNA (ceRNA) hypothesis which elicited the fibrotic processes. Until now, the effects of lncRNAs on PSCs activation and pancreatic fibrosis have not been clearly explored. In this study, a novel lncRNA named Lnc-PFAR was found highly expressed in mouse and human CP tissues. Our data revealed that Lnc-PFAR facilitates PSCs activation and pancreatic fibrosis via RB1CC1-induced autophagy. Lnc-PFAR reduces miR-141 expression by suppressing pre-miR-141 maturation, which eventually upregulates the RB1CC1 and fibrosis-related indicators expression. Meanwhile, Lnc-PFAR enhanced PSCs activation and pancreatic fibrosis through trigging autophagy. Our study interrogates a novel lncRNA-induced mechanism in promoting the development of pancreatic fibrosis, and Lnc-PFAR is suggested to be a prospective therapeutic target in clinical scenarios.

Структурна трансформація протокової системи підшлункової залози у хворих на хронічний панкреатит

Актуальність. Клітини проток становлять до 30 % від маси підшлункової залози (ПЗ) людини, а збільшення кількості бранчів проток відноситься до одних із перших сонологічних ознак хронічного панкреатиту (ХП). Мета дослідження — визначення морфологічних й імуногістохімічних особливостей протокової системи ПЗ хворих на ХП, яким були виконані резекційні та дренуючі операції з приводу ускладнень хронічного панкреатиту. Матеріали та методи. Проведено імуногістохімічне дослідження операційного матеріалу 40 хворих з ускладненим хронічним панкреатитом з використанням реакцій на a-ізоформу гладком’язового актину (a-SMA), десмін, фібронектин й колаген I типу. Результати. Характерна морфологічна картина розвитку ХП є поєднанням фіброзування з деформацією протокової системи за рахунок перидуктального фіброзу з розширенням міждолькових фіброзних септ, потовщенням інтралобулярних сполучно­тканинних тяжів, а також значною звивістістю і розширенням проток з ретенційними кістами. Значна структурна перебудова протокової системи ПЗ у хворих на ХП спостерігалася в 87,5 % випадків, в 17,5 % випадків картина доповнювалася явищами ацинарно-протокової метаплазії з трансформацією ацинарних клітин у протокові і формуванням дрібних дуктулоподібних структур, так званих тубулярних комплексів. У 7,5 % випадків в головній панкреатичній протоці визначалися вогнища атрофії протокового епітелію, які чергувалися з вогнищами папілярної інтраепітеліальної гіперплазії епітелію (PanIN). Розвитку панкреатичного фіброзу при ХП сприяє накопичення a-SMA+-активованих панкреатичних зірчастих клітин, що синтезують надлишок компонентів екстрацелюлярного матриксу, зокрема колагену I типу. Середня площа колагену І типу та інтенсивність його експресії були вірогідно нижче при зниженій експресії a-SMA (p < 0,05) і вірогідно вище у випадках вираженої експресії фібронектину (p < 0,05). Висновки. Вивчення структурних змін протокової системи ПЗ з використанням методів імуногістохімії є одним із способів покращення ранньої діагностики ускладнень ХП.

Role of NLR family pyrin domain-containing 3 inflammasome in the activation of pancreatic stellate cells

NLRP3 inflammasome activation plays an important role in the development of pancreatic fibrosis. However, it is unclear whether the activation of the NLRP3 inflammasome is directly involved in the activation of Pancreatic stellate cells (PSCs). The aim of this study was to investigate the role and mechanism of the NLRP3 inflammasome in the activation of PSCs. In vivo, a rat model of chronic pancreatitis (CP) was induced by intravenous injection of dibutyltin dichloride (DBTC). In vitro, rat primary PSCs were isolated from pancreatic tissues and incubated with the NLRP3 inflammasome activator LPS, the NLRP3 inhibitor MCC950, or NLRP3 siRNA. The results showed that the expression of NLRP3, pro-Caspase-1, Caspase-1 and IL-18 was increased in the rat model of CP and during PSCs activation. LPS increased the protein levels of NLRP3, ASC, Caspase-1, IL-1β and IL-18 accompanied by the upregulation of α-SMA, Col I and FN expression. Moreover, MCC950 or NLPR3 siRNA decreased the expression of α-SMA, Col I, FN, TGF-β1 and p-Smad3. Furthermore, MCC950 reversed the LPS-induced upregulation of α-SMA, FN and Col Ⅰ expression in PSCs.This study revealed that the NLRP3 inflammasome is directly involved in the activation of PSCs in vivo and in vitro. Inhibiting NLRP3 suppresses the activation of PSCs through the TGF-β1/Smad3 pathway.

Baicalin Ameliorates Pancreatic Fibrosis by Inhibiting the Activation of Pancreatic Stellate Cells in Mice with Chronic Pancreatitis.

Pancreatic inflammation and fibrosis are typical pathological features in chronic pancreatitis (CP). Activated pancreatic stellate cells (PSCs) have been regarded as the core event in the development of pancreatic fibrosis and are considered to be the key target for treatment of CP. Baicalin (C21H18O11), the main chemical composition of Baikal skullcap in the traditional Chinese medicines Dachaihu decoction (DCHD) and Xiaochaihu decoction (XCHD), has shown significant effects in the treatment of pancreatic fibrosis in CP mice; however, whether baicalin can inhibit the activation of PSCs and its underlying mechanism remain unclear. In this study, the influence of baicalin on activated PSCs in vitro and in vivo was investigated, and the results showed that Baicalin could significantly ameliorate the degree of pancreatic inflammation and fibrosis, while decreasing the levels of alpha-smooth muscle actin (α-SMA), F4/80 (surface markers of mouse macrophages), nuclear factor kappa-B (NF-κB), monocyte chemotactic protein 1 (MCP-1), and collagen type I alpha 1 (COL1A1)in the pancreas. Moreover, NF-κB and α-SMA were co-expressed in the pancreas of CP mice. Baicalin treatment markedly reduced the expression of co-location of α-SMA and NF-κB. In vitro, the protein expression levels of transforming growth factor-β receptor 1 (TGF-βR1), phosphorylated TGF-β activated kinase 1 p-TAK 1, and NF-κBp65 in PSCs were all remarkably reduced after treatment with baicalin. In addition, baicalin could inhibit MCP-1 mRNA expression in supernatant of activated PSCs, as well as the excessive migration of macrophages. Taken together, our findings indicated that baicalin could inhibit the TGF-β1/TGF-βR1/TAK1/NF-κB signaling pathway of activated PSCs, reduce the secretion of MCP-1, and further decrease the infiltration of macrophages and inflammation cells of the local microenvironment of the pancreas. Thus, this study provides a reliable experimental basis for baicalin in the prevention and treatment of CP.

Hic-5 is required for activation of pancreatic stellate cells and development of pancreatic fibrosis in chronic pancreatitis

Accumulated evidence suggests that activated pancreatic stellate cells (PSCs) serve as the main source of the extracellular matrix proteins accumulated under the pathological conditions leading to pancreatic fibrosis in chronic pancreatitis (CP). However, little is known about the mechanisms of PSC activation. PSCs have morphologic and functional similarities to hepatic stellate cells, which are activated by hydrogen peroxide-inducible clone-5 (Hic-5), a TGF-β1-induced protein. In this study, we investigated whether Hic-5 activates PSCs, which promote pancreatic fibrosis development in CP. Hic-5-knockout and wild type mice were subjected to caerulein injection to induce CP. Hic-5 expression was strongly upregulated in activated PSCs from human CP tissue and from mouse pancreatic fibrosis in caerulein-induced CP. Hic-5 deficiency significantly attenuated mouse pancreatic fibrosis and PSC activation in the experimental murine CP model. Mechanistically, Hic-5 knock down significantly inhibited the TGF-β/Smad2 signaling pathway, resulting in reduced collagen production and α-smooth muscle actin expression in the activated PSCs. Taken together, we propose Hic-5 as a potential marker of activated PSCs and a novel therapeutic target in CP treatment.

PANCREATIC STEATOSIS: A NEW DIAGNOSIS AND THERAPEUTIC CHALLENGE IN GASTROENTEROLOGY.

Fat infiltration in the pancreas is called pancreatic steatosis and it has several synonyms such as pancreatic lipomatosis, non-alcoholic fatty pancreatic disease, lipomatous pseudohypertrophy, fatty replacement, fatty pancreas and fatty infiltration. Pancreatic steatosis describes a disease ranging from infiltration of fat in the pancreas to pancreatic inflammation, and development of pancreatic fibrosis. There are multiple aetiologies of this condition, such as metabolic syndrome, alcohol intake, viral infections, toxins, congenital syndromes, etc. Pancreatic steatosis is usually diagnosed by trans-abdominal ultrasound, computed tomography scan and magnetic resonance imaging. Fatty infiltration in pancreas may lead to pancreatitis, diabetes mellitus and may be a predisposing cause of pancreatic cancer. Now a day, pancreatic steatosis is a common incidental finding during abdominal ultrasonography for other reasons and is a new challenge in Gastroenterology. But there is no guideline for pancreatic steatosis till now. In this review article, we are trying to give an overall idea (aetiologies, diagnosis, management, clinical significances) on pancreatic steatosis.

Saikosaponin A inhibits the activation of pancreatic stellate cells by suppressing autophagy and the NLRP3 inflammasome via the AMPK/mTOR pathway

Pancreatic stellate cells (PSCs) are the main effector cells in the development of pancreatic fibrosis. Finding substances that inhibit PSC activation is an important approach to inhibiting pancreatic fibrosis. Saikosaponin A (SSa) has numerous pharmacological activities, but its effect on PSCs remains unknown. This study was conducted to explore the effects of SSa on PSC activation in cultured rat PSCs. Cell viability, proliferation, migration and apoptosis were evaluated by MTT assays, the iCELLigence System, Transwell assays and flow cytometry. Markers of PSC activation, autophagy and the NLRP3 inflammasome were measured by real-time PCR, immunofluorescence and western blotting. Rapamycin and phenformin hydrochloride were used to determine the effect of SSa via the AMPK/mTOR pathway. The results showed that SSa suppressed PSC viability, proliferation, and migration and promoted apoptosis. SSa inhibited PSC activation, restrained PSC autophagy and suppressed the NLRP3 inflammasome. In addition, there was interaction between autophagy and the NLRP3 inflammasome during SSa inhibition of PSCs. Moreover, promotion of p-AMPK increased autophagy and the NLRP3 inflammasome. Inhibition of p-mTOR increased autophagy and decreased the NLRP3 inflammasome. Our results indicated that SSa inhibited PSC activation by inhibiting PSC autophagy and the NLRP3 inflammasome via the AMPK/mTOR pathway. These findings provide a theoretical basis for the use of SSa to treat pancreatic fibrosis and further suggest that targeting autophagy and the NLRP3 inflammasome may provide new strategies for the treatment of pancreatic fibrosis.

Role of stellate cells in the morphogenesis of chronic pancreatitis

This review presents an analysis of the literature on the role of pancreatic stellate cells (PSCs) in the pathogenesis of fibrosis, a predominant histological feature of chronic alcoholic pancreatitis. It is shown that ethanol and toxic products of its metabolism can affect PSCs directly and indirectly, facilitating their transformation from a quiescent to an activated state. During the pathological process, PSCs interact with parenchymal and immune cells of the pancreas through cytokines and growth factors. In activated PSCs, the proliferative and migratory activity, as well as the synthesis of extracellular matrix (ECM) proteins increases. A continuous activation of PSCs during the disease promotes the maintenance of inflammation, the deposition of excessive amounts of ECM proteins and the development of pancreatic fibrosis.

FTY720 inhibits the activation of pancreatic stellate cells by promoting apoptosis and suppressing autophagy via the AMPK/mTOR pathway

AimsPancreatic stellate cells (PSCs) play a critical role in the development of pancreatic fibrosis. Any agents that can affect PSC activation could become potential candidates for treating pancreatic fibrosis. FTY720 can attenuate chronic pancreatic fibrosis by suppressing T-cell infiltration, but its effect on PSCs remains unknown. This study was conducted to investigate the effects of FTY720 on PSC activation in cultured rat PSCs. Main methodsThe viability of PSCs after FTY720 treatment was detected by MTT. Cell proliferation and migration analysis was performed using the iCELLigence System and a Transwell assay. Cell apoptosis was assessed by flow cytometry, western blot and an activity assay. The mitochondrial membrane potential (MMP) was assessed by JC-1 staining. The expression of α-SMA, collagen I, fibronectin, Beclin-1, Atg5, P62 and LC3B were analysed by immunofluorescence, quantitative real-time PCR and western blot. Rapamycin and phenformin hydrochloride were used to determine whether FTY720 inhibits PSC autophagy by the AMPK/mTOR pathway. Key findingsFTY720 supressed PSC viability, proliferation and migration. FTY720 inhibited PSC activation, induced PSC apoptosis and supressed PSC autophagy. We also confirmed that FTY720 inhibited PSC autophagy via the AMPK/mTOR pathway. SignificanceOur results indicated that FTY720 inhibited PSC activation by promoting cell apoptosis and inhibiting PSC autophagy by suppressing AMPK and activating the mTOR pathway. These findings may explain the therapeutic mechanisms of FTY720 in treating pancreatic fibrosis and further suggest that targeting autophagy and the related signalling pathways may provide new strategies for the treatment of pancreatic fibrosis.

Respective roles of the mitogen-activated protein kinase (MAPK) family members in pancreatic stellate cell activation induced by transforming growth factor-β1 (TGF-β1)

Activated pancreatic stellate cells (PSCs) play a crucial role in the progression of pancreatic fibrosis. Transforming growth factor-β (TGF-β) 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-β1 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-β1 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-β1 and the development of pancreatic fibrosis. p38 MAPK participate in the progression of CP, but it does not respond to TGF-β1 directly and may not be regarded as the target of TGF-β1 induced PSC activation.

Caralluma fimbriata and metformin protection of rat pancreas from high fat diet induced oxidative stress

A high fat diet promotes oxidative stress, which contributes to the development of pancreatic fibrosis. We compared the protective effects of a hydroalcoholic extract of Caralluma fimbriata (CFE) to metformin (Met) in the pancreas of Wistar rats fed a high fat diet. The experimental animals were divided into five groups: control (C), treated with CFE (C + CFE), treated with high fat diet (HFD), high fat diet treated with CFE (HFD + CFE), and high fat diet treated with metformin (Met) (HFD + Met). CFE was administered orally to groups C + CFE and HFD + CFE rats for 90 days. Met was given to the HFD + Met group. After 90 days, oxidative stress markers in the pancreas including reduced glutathione (GSH), lipid oxidation (LO), protein oxidation (PO), and activities of antioxidant and polyol pathway enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH) were assayed and tissue histology was examined. Establishment of oxidative stress in high fat diet fed rats was verified by elevated LO and PO, decreased GSH, decreased activities of antioxidants and increased activities of polyol pathway enzymes. Oxidative stress was prevented in HFD + CFE and HFD + Met groups. Group C + CFE exhibited improved antioxidant status compared to group C. CFE treatment prevented high fat diet induced acinar cell degeneration, necrosis, edema and hemorrhage. CFE could be used as adjuvant therapy for preventing or managing high fat diet induced pancreatic damage.

Pancreatic Stellate Cells Have Distinct Characteristics From Hepatic Stellate Cells and Are Not the Unique Origin of Collagen-Producing Cells in the Pancreas.

The origin of collagen-producing myofibroblasts in pancreatic fibrosis is still controversial. Pancreatic stellate cells (PSCs), which have been recognized as the pancreatic counterparts of hepatic stellate cells (HSCs), are thought to play an important role in the development of pancreatic fibrosis. However, sources of myofibroblasts other than PSCs may exist because extensive studies of liver fibrosis have uncovered myofibroblasts that did not originate from HSCs. This study aimed to characterize myofibroblasts in an experimental pancreatic fibrosis model in mice. We used transgenic mice expressing green fluorescent protein via the collagen type I α1 promoter and induced pancreatic fibrosis with repetitive injections of cerulein. Collagen-producing cells that are negative for glial fibrillary acidic protein (ie, not derived from PSCs) exist in the pancreas. Pancreatic stellate cells had different characteristics from those of HSCs in a very small possession of vitamin A using mass spectrometry and a low expression of lecithin retinol acyltransferase. The microstructure of PSCs was entirely different from that of HSCs using flow cytometry and electron microscopy. Our study showed that characteristics of PSCs are different from those of HSCs, and myofibroblasts in the pancreas might be derived not only from PSCs but also from other fibrogenic cells.

Pancreatic stellate cells: A dynamic player of the intercellular communication in pancreatic cancer

There is accumulating evidence that activated pancreatic stellate cells (PSCs) play a pivotal role in the development of pancreatic fibrosis within the pancreatic cancer tissue. Not only do they produce extracellular matrix components, PSCs dynamically interact with other cell types to constitute the cancer-conditioned microenvironment. There exist bidirectional interactions between PSCs and pancreatic cancer cells. Pancreatic cancer cells promote the proliferation, migration, extracellular matrix production and degradation, and angiogenetic responses in PSCs. In turn, PSCs promote the proliferation and migration, and inhibit the apoptosis of pancreatic cancer cells. PSCs also induce epithelial-mesenchymal transition and stem cell like phenotypes in pancreatic cancer cells, resulting in resistance to conventional therapies, distant metastasis, and recurrence. PSCs interact with endothelial cells, neural cells and β-cells, leading to angiogenesis, neurogenesis and β-cell dysfunction and apoptosis. PSCs cause impaired immune responses and help pancreatic cancer cells escape from host immune-surveillance. PSCs induce the differentiation of myeloid-derived suppressor cells, induce the apoptosis of T cells, inhibit the infiltration of T cells, and induce the activation of mast cells. Overall, these interactions appear to promote the progression of pancreatic cancer, and anti-stroma therapies targeting PSCs are under intense investigation. Further elucidation of these interactions could lead to the identification of novel therapeutic targets in pancreatic cancer.

Inhibition of pancreatic stellate cell activity by adipose-derived stem cells

Pancreatic stellate cells (PSCs) play a critical role in the development of pancreatic fibrosis. In this study we used a novel method to isolate and culture rat PSCs and then investigated the inhibitory effects of adipose-derived stem cells (ADSCs) on activation and proliferation of PSCs. Pancreatic tissue was obtained from Sprague-Dawley rats for PSCs isolation. Transwell cell cultures were adopted for co-culture of ADSCs and PSCs. PSCs proliferation and apoptosis were determined using CCK-8 and flow cytometry, respectively. alpha-SMA expressions were analyzed using Western blotting. The levels of cytokines [nerve growth factor (NGF), interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1)] in conditioned medium were detected by ELISA. Gene expression (MMP-2, MMP-9 and TIMP-1) was analyzed using qRT-PCR. This method produced 17.6+/-6.5X10(3) cells per gram of the body weight with a purity of 90%-95% and a viability of 92%-97%. Co-culture of PSCs with ADSCs significantly inhibited PSCs proliferation and induced PSCs apoptosis. Moreover, alpha-SMA expression was significantly reduced in PSCs+ADSCs compared with that in PSC-only cultures, while expression of fibrinolytic proteins (e.g., MMP-2 and MMP-9) was up-regulated and anti-fibrinolytic protein (TIMP-1) was down-regulated. In addition, NGF expression was up-regulated, but IL-10 and TGF-beta1 expressions were down-regulated in the co-culture conditioned medium compared with those in the PSC-only culture medium. This study provided an easy and reliable technique to isolate PSCs. The data demonstrated the inhibitory effects of ADSCs on the activation and proliferation of PSCs in vitro.

Effect of Saikokeishito, a Kampo medicine, on hydrogen peroxide-induced premature senescence of normal human dermal fibroblasts

Saikokeishito (TJ-10) is a Kampo (traditional Japanese herbal) medicine, clinically used for hundreds of years in East Asia. Among its various mechanisms elucidated so far, TJ-10 inhibits the production of transforming growth factor-β1 (TGF-β1) and development of pancreatic fibrosis in vivo. Oxidative damage of normal human dermal fibroblasts (NHDFs) in the corium is a cause of human dermal senescence. Our aim was to determine whether TJ-10 protects NHDFs from premature senescence by hydrogen peroxide (H₂O₂). Premature senescence was induced in NHDFs by 200 μmol/L H₂O₂ for 4 h. Cell viability and the expressions of p53, AMP-activated protein kinase α1 (AMPKα1), AMPKα2, and 14-3-3 protein sigma (14-3-3 σ) were measured in NHDFs treated with TJ-10 for 48 h before exposure to H₂O₂for 4 h. Cell viability after treatment with 200 μmol/L H₂O₂ for 4 h was similar (about 80%) to after pre-treatment with TJ-10. Ascorbic acid as a control did not protect NHDFs from damage by 200 μmol/L H₂O₂. Treatment with 200 μmol/L H₂O₂tended to up-regulate p53 and to down-regulate SIRT1 and AMPKα1, but had no effect on AMPKα2 and 14-3-3 σ expression. Pretreatment with TJ-10 inhibited H₂O₂-induced up-regulation of p53 and enhanced AMPKα1 expression. It is suggested that Saikokeishito has a protective effect on oxidative stress-induced senescence of NHDFs.

Monocytes Infiltrate The Pancreas Via MCP-1/CCR2 Signaling and Differentiate Into Pancreatic Stellate Cells During Pancreatic Injury

IntroductionWe previously reported that monocytes could differentiate into hepatic stellate cells during carbon tetrachloride (CCl4) injury. Pancreatic stellate cells (PaSCs), which play a central role in the development of pancreatic fibrosis, share many morphological and functional characteristics with those of hepatic stellate cells. Although PaSCs have been thought to be of mesenchymal origin, some authors have recently reported that they are derived from bone marrow (BM) cells during inflammation. Meanwhile, both monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) are thought to be involved in the development of pancreatic fibrosis. We hypothesized that PaSCs are also derived from monocytes and investigated the mechanism of how monocytes enter the injured pancreas, focusing on MCP-1 and Ang II. MethodsFirst, we generated chimeric mice by transplantation of clonal populations of cells derived from single lineage negative Sca-1+c-kit+CD34- cells or total-nucleated cells, which were sorted from the BM of enhanced green fluorescent protein (EGFP)-transgenic mice, into lethally irradiated C57BL/6 mice. Since 2 months after BM transplantation, the chimeric mice revealing high-level multilineage engraftment were intraperitoneally injected with CCl4 twice a week. Next, we intravenously transferred Ly6Chighc-kit– monocytes, which were isolated from the BM of EGFP-transgenic mice, into non-irradiated CCl4-treated mice. We analyzed the presence of EGFP+ PaSCs in the injured pancreas using immunofluorescence and laser confocal scanning microscopy. Finally, we investigated the effect of Ang II type 1 receptor (AT1R) antagonist, irbesartan, which is also an antagonist of MCP-1 receptor, CC chemokine receptor 2 (CCR2) on the occurrence of monocyte-derived PaSCs in the pancreases of CCl4-treated mice. ResultsAfter 12 weeks of injury by CCl4, EGFP+CD45- cells were detected in the pancreases of chimeric mice. They expressed PaSC-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin. Next, we found EGFP+ PaSCs in CCl4-treated mice with adoptively transferred Ly6Chigh monocytes but not other lineages. MCP-1 and Ang II were highly produced in the pancreas of CCl4-treted mice and their receptors CCR2 and AT1R were respectively expressed on Ly6Chigh monocytes isolated from the BM of EGFP-transgenic mice. Monocytes migrated toward MCP-1, but not Ang II, and irbesartan inhibited their chemotaxis in vitro. Irbesartan treatment significantly reduced the numbers of EGFP+CCR2+F4/80+ macrophages and EGFP+ PaSCs in the pancreas of CCl4-treated mice. We observed that a specific CCR2 antagonist RS504393 inhibited the monocyte migration toward MCP-1 in vitro and the occurrence of EGFP+ PaSCs in the injured mice like irbesartan. ConclusionWe propose that CCR2+ monocytes migrate into the injured pancreas via the MCP-1/CCR2 pathway and differentiate into PaSCs during inflammation. Disclosures:Masuya:Dainippon-Sumitomo Pharmaceutical: Research Funding.

Proteome Variations in Pancreatic Stellate Cells upon Stimulation with Proinflammatory Factors

Pancreatic stellate cells are key mediators in chronic pancreatitis and play a central role in the development of pancreatic fibrosis, stromal formation, and progression of pancreatic cancer. This study was aimed at investigating molecular changes at the level of the proteome that are associated with the activation of pancreatic stellate cells by proinflammatory factors, namely TNF-α, FGF2, IL6, and chemokine (C-C motif) ligand 4 (CCL4). They were added individually to cells growing in serum-free medium next to controls in medium supplemented with serum, thus containing a mixture of them all, or in serum-free medium alone. Variations were detected by means of a microarray of 810 antibodies targeting relevant proteins. All tested factors triggered increased proliferation and migration. Further analysis showed that TNF-α is the prime factor responsible for the activation of pancreatic stellate cells. CCL4 is associated with cellular neovascularization, whereas FGF2 and IL6 induction led to better cellular survival and decreased apoptotic activity of the stellate cells. The identified direct effects of individual cytokines on human pancreatic stellate cells provide new insights about their contribution to pancreatic cancer promotion.

The development of fibrosis in a novel model of chronic pancreatitis is mediated by complement factor C5

The development of fibrosis in a novel model of chronic pancreatitis is mediated by complement factor C5