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Objective:To investigate the effects of NOD-like receptor protein 3(NLRP3) inflammasome activation on the proliferation, migration and extracellular matrix desposition of activated pancreatic stellate cells(PSCs).Methods:The rat PSCs were isolated, cultured and identified, and were divided into control group or LPS group based on the pretreatment with LPS (10 μg/ml for 24 hours) or without. The expression of NLRP3 inflammasome associated molecules in PSCs culture medium was detected by ELISA. The PSCs with NLRP3 inhibition were constructed by shRNA carrying lentivirus infection and were divided into LPS+ negative control group and LPS+ lentivirus group based on whether the cells were treated with LPS and infected by lentivirus or not. The alteration in cell proliferation and migration were detected by CCK-8 kit and transwell chamber method. The expression of extracellular matrix α-SMA and collagen in PSCs was detected by immunofluorescence staining and the expression of TGF-β mRNA was analyzed by RT-qPCR.Results:The cytoplasm of PSCs which were cultured for 24 hours was rich in bright annular lipid droplets, and the cells expressed desmin. After 7 days of culture, the cell became larger in size, the lipid droplets basically disappeared, and the cells were activated and expressed α-SMA. The expression of caspase-1, IL-1β and IL-18 in the supernatant of PSCs culture medium in LPS group were significantly higher than those in control group (1.55±0.04 vs 0.65±0.03), (2.02±0.04 vs 1.05±0.05) and (1.70±0.05 vs 0.97±0.03), respectively. After inhibiting by lentivirus infection, the expression of NLRP3 in the lentivirus group (0.25±0.04) was significantly lower than that in negative control group (0.68±0.05). In control group, LPS group, LPS+ negative control group and LPS+ lentivirus group, the A490 values was 0.61±0.02, 1.15±0.06, 0.96±0.05, and 0.56±0.01, respectively; the migrating PSCs number was (64.12±4.58), (121.67±8.02), (111.67±4.67) and (69.67±8.08)/HF, respectively; the relative expression of α-SMA was 0.78±0.05, 4.12±0.04, 3.81±0.06 and 0.88±0.05, respectively; the relative expression of collagen was 0.65±0.03, 3.43±0.02, 2.67±0.02 and 0.48±0.03, respectively; and the expression of TGF-β mRNA was 0.22±0.03, 0.89±0.01, 0.86±0.03 and 0.43±0.02, respectively. The A490 value, the migrating cells number, the expression of α-SMA, collagen and the expression of TGF-β mRNA in LPS group and LPS+ negative control group was significantly higher than those in control group and LPS+ lentivirus group, and all the differences were statistically significant (all P value <0.05). Conclusions:NLRP3 inflammasome activation may accelerate the extracellular matrix deposition and pancreatic fibrogenesis by promoting PSCs proliferation and migration ability via regulating the biological functions.
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Objective:To investigate the role and mechanism of pancreatic stellate cells (PSCs) and pancreatic cancer cells (PCCs) in the angiogenesis of pancreatic cancer.Methods:The experimental study was conducted. The human PSCs and PCCs and human umbilical vein endothelial cells (HUVECs) were cultured in vitro. HUVECs was treated with PSCs/PCCs supernatants and matrix metalloproteinase (MMP) inhibitor of different types and concentrations. As controls, HUVECs treated with complete endoprime medium (C/E) and DMEM/Ham's F12 medium (D/F) were set as the C/E group and the D/F group, respectively. Observation indicators: (1) proliferation of HUVECs under different conditions; (2) tube formation of HUVECs under different conditions; (3) migration of HUVECs under different conditions; (4) expression of MMP-2 in the supernatants of PSCs and PCCs; (5) effect of MMP inhibitor GM6001 on migration of HUVECs. Measurement data with normal distribution were represented as Mean± SD, comparison among groups was conducted using the one way ANOVA and comparison between groups was conducted using the LSD- t test. Results:(1) Proliferation of HUVECs under different conditions. Results of HUVECs proliferation assay using 5-ethynyl-2′-deoxyuridine (EdU) labeling showed that the binding rate of EdU in the HUVECs of D/F group and HUVECs treated with supernatants of different concentration (25%, 50%, 75%, 95%) of PSCs was 12.4%±1.0%, 24.5%±2.9%, 25.3%±3.0%, 22.8%±2.0%, 22.9%±2.8%, respectively, showing a significant difference among them ( F=8.60, P<0.05). There were significant differences in the binding rate of EdU between HUVECs in the D/F group and HUVECs treated with supernatants of different concentration (25%, 50%, 75%, 95%) of PSCs, respectively ( P<0.05). The binding rate of EdU between HUVECs in the D/F group and HUVECs treated with supernatants of different concentration (25%, 50%, 75%, 95%) of PCCs was 12.4%±1.0%, 30.0%±3.2%, 32.1%±1.0%, 32.3%±3.5%, 26.2%±5.6%, respectively, showing a significant difference among them ( F=11.93, P<0.05). There were significant differences in the binding rate of EdU between HUVECs in the D/F group and HUVECs treated with supernatants of different concentration (25%, 50%, 75%, 95%) of PSCs, respectively ( P<0.05). (2) Tube formation of HUVECs under different conditions. Number of tube formation, length of tube in the HUVECs of D/F group and HUVECs treated with PSCs supernatants was 15.2±2.3, (12.1±1.5)mm and 49.7±3.2, (39.8±2.3)mm, respectively, showing significant differences between the two groups of HUVECs ( P<0.05). (3) Migration of HUVECs under different conditions. Results of single cell tracing experiment showed that the migration rate of HUVECs treated with supernatants of different ratio of PSCs and PCCs was faster than that of HUVECs in the D/F group, and the enhancement effect of supernatants of PSCs and PCCs was dose-dependent. The migration rate of HUVECs treated with mix supernatants of different concentration of PSCs and PCCs and supernatants of co-cultured PSCs and PCCs was faster than that of HUVECs in the D/F group. The migration rate of HUVECs treated supernatants of co-cultured PSCs and PCCs was faster than that of HUVECs treated with mix supernatants of different concentration of PSCs and PCCs, showing a synergistic effect in the HUVECs treated supernatants of co-cultured PSCs and PCCs. (4) Expression of MMP-2 in the supernatants of PSCs and PCCs. Results of gelatine zymography showed that the MMP-2 expression levels decreased sequentially in super-natants of co-cultured PSCs and PCCs, supernatants of PSCs, mix supernatants of PSCs and PCCs and supernatants PCCs. (5) Effect of MMP inhibitor GM6001 on migration of HUVECs. Results of single cell tracing experiment showed that the migration rate of HUVECs treated with PSCs supernatants combined with different concentration of GM6001 (0, 1, 10, 25 μmol/L) was (25.70±2.06)μm/h, (18.37±1.61)μm/h, (16.20±0.26)μm/h, (15.99±0.58)μm/h, respectively, showing a significant difference among them ( F=11.39, P<0.05). There were significant differences in the migration rate between HUVECs treated with PSCs supernatants combined with 1, 10, 25 μmol/L GM6001 and HUVECs treated with PSCs supernatants ( P<0.05). The migration rate of HUVECs treated with mix super-natants of PSCs and PCCs combined with different concentration of GM6001 (0, 1, 10, 25 μmol/L) was (30.06±3.70)μm/h, (22.76±1.56)μm/h, (23.87±2.84)μm/h, (22.10±2.35)μm/h, respectively, showing a significant difference among them ( F=4.06, P<0.05). There were significant differences in the migration rate between HUVECs treated with mix supernatants of PSCs and PCCs combined with 1, 10, 25 μmol/L GM6001 and HUVECs treated with mix supernatants of PSCs and PCCs ( P<0.05). Conclusions:Both PSCs and PCCs can promote the proliferation, migration and angiogenesis of HUVECs in vitro experiment. Releasing of MMP-2 by interaction between PSCs and PCCs is an important factor to stimulate endothelial cell migration, which increases the stimulating activity of angiogenesis, especially the migration ability of HUVECs.
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Objective:To study the effects of naringenin on pancreatic fibrosis in the mouse model of chronic pancreatitis (CP) and its effects on the activation, proliferation and apoptosis of pancreatic stellate cells (PSCs).Methods:Eighteen C57BL/6 mice were randomly divided into control group, CP group and naringenin group, with 6 mice in each group. The CP mouse model was established by intraperitoneal injections of caerulein. Naringenin group was given naringenin (200 mg/kg/day) by gavage once a day from the first day of the fourth week of modeling process to the day before the killing; the control group and CP group were treated by gavage with an equivalent amount of drug solvent containing 0.5% sodium carboxymethyl cellulose (CMC-Na). Mice were killed 5 days after the last caerulein injection, and their pancreatic tissues were collected for hematoxylin-eosin staining and Sirius Red staining, pathological scoring and collagen sedimentation detection. Naringenin with different concentrations (0, 5, 10, 20, 50, 100, 150, 200 μmol/L) were used to intervene HPSC for 24 hours, and CCK-8 method was used to detect the cell activity. TGF-β1 recombinant protein (2 ng/ml) was used to induce PSCs for 1 hour (TGF-β1 stimulation group), and naringenin with low (50 μmol/L), middle (100 μmol/L) and high (150 μmol/L) concentration was used to intervene for 36 hours after TGF-β1 stimulation, respectively. Western Blotting was used to detect the expression of PSC activation related proteins FN and COL1A1, cell proliferation marker p21, anti-apoptotic protein Bcl-xL, pro-apoptotic protein Bax and Bid.Results:The pathological scores of pancreatic tissue [(7.33±1.15), (4.67±1.15)] and the percentage of collagen positive areas [(46±4), (28±2)%] in CP group and naringenin group were higher than those in the control group [0, (4±2)%]. However, these indexes in the naringenin group were lower than those in CP group, and the differences were all statistically significant (all P value <0.05). The relative expression of FN in control group, TGF-β1 stimulation group and low, medium and high naringenin group was 0.02, 0.76, 0.67, 0.34 and 0.07, respectively; the expression of COL1A1 in these groups was 0.51, 1.71, 1.34, 0.84 and 0.11. The expression of FN and COL1A1 in TGF-β1 stimulation group was significantly higher than that in control group, and the expression of FN and COL1A1 in low, medium and high naringenin group was significantly lower than that in TGF-β1 stimulation group, and the differences were all statistically significant (all P value <0.05). The expression of p21 in the above five groups was 0.87, 1.18, 1.27, 1.22 and 1.00. The expression of p21 in TGF-β1 stimulation group was higher than that in control group, and the expression of p21 in high naringenin group was obviously lower than that in TGF-β1 stimulation group, and the differences were all statistically significant (all P value <0.05). In addition, the expression of Bcl-xL in these groups was 2.09, 2.21, 2.38, 2.50 and 2.12; the expression of Bax was 0.98, 0.88, 0.98, 1.00 and 0.88; the expression of Bid was 1.15, 1.09, 1.14, 1.18 and 1.18. There was no statistically significant difference among these groups (all P value >0.05). Conclusions:Naringenin could significantly alleviate the inflammation, atrophy and fibrosis in the CP mouse model, and inhibit the activation and proliferation of PSCs. However, naringenin had no significant effect on the apoptosis of PSCs, indicating that naringenin may be potentially used to treat pancreatic fibrosis in CP.
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Activated pancreatic stellate cells (PSCs) have been widely accepted as a key precursor of excessive pancreatic fibrosis, which is a crucial hallmark of chronic pancreatitis (CP) and its formidable associated disease, pancreatic cancer (PC). Hence, anti-fibrotic therapy has been identified as a novel therapeutic strategy for treating CP and PC by targeting PSCs. Most of the anti-fibrotic agents have been limited to phase I/II clinical trials involving vitamin analogs, which are abundant in medicinal plants and have proved to be promising for clinical application. The use of phytomedicines, as new anti-fibrotic agents, has been applied to a variety of complementary and alternative approaches. The aim of this review was to present a focused update on the selective new potential anti-fibrotic agents, including curcumin, resveratrol, rhein, emodin, green tea catechin derivatives, metformin, eruberin A, and ellagic acid, in combating PSC in CP and PC models. It aimed to describe the mechanism(s) of the phytochemicals used, either alone or in combination, and the associated molecular targets. Most of them were tested in PC models with similar mechanism of actions, and curcumin was tested intensively. Future research may explore the issues of bioavailability, drug design, and nano-formulation, in order to achieve successful clinical outcomes with promising activity and tolerability.
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Objective To explore the effect of osteopontin (OPN) on pancreatic stellate cell (PSC) and its mechanisms. Methods We transfected PSC with OPN lentiviral overexpression vector (OPN-O/E) and constructed empty vector control cells (control group). After PSCs were treated with OPN-O/E or OPN-O/E in combination with Akt inhibitor LY294002 (10 µmol/L and 50 µmol/L), the proliferation ability and chemotactic activity were detected by CCK-8 and Transwell assays, respectively. The expression levels of a-smooth muscle actin (α-SMA) and related proteins of PI3K/Akt signal pathway were determined by Western blotting. Results Compared with the control group, proliferation ability and chemotactic activity of PCS were significantly increased in the OPN-O/E group, and the expression levels of phosphorylated- PI3K (p-PI3K), phosphorylated-Akt (p-Akt) and α-SMA were also significantly increased (P0.05). Compared with the OPN-O/E group, the expression levels of a-SMA and p-Akt were significantly inhibited in the PCS treated with 10 µmol/L or 50 µmol/L LY294002 (all P<0.01); however, there was no significant difference in the Akt expression. Conclusion OPN can activate PSC through the PI3K/Akt signaling pathway, and the proliferation ability and chemotactic activity of activated PSC are also increased.
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Objective To explore a new method for the separation of human pancreatic stellate cells.Methods Single-cell suspension of normal pancreatic tissue and pancreatic cancer tissue was prepared by gentle MACSTM tissue processor-constant temperature shaking digestion.Human pancreatic stellate cells of quiescent and activated state were isolated by density gradient centrifugation.Results A new type of isolation method could obtain about (2.6 ± 0.7) × 106 quiescent pancreatic stellate cells in 1 g of human normal pancreatic tissue,with a viability of about 90.0％.The morphology of the cells were conformed to the representative for the quiescent state characteristics and transient blue-green autofluorescence was observed at the 328 nm excitation wavelength;1 g of human pancreatic cancer was able to obtain approximately (4.1 ± 1.1) × 106 activated PSCs with a viability of 92.0％,and all of the activated cells expressed α-SMA vimentin,FSP-1 and other characteristic markers.Conclusions The new separation method of this experiment is suitable for both human resting and activated human pancreatic stellate cells.At the same time,the purity is high and the separation time is greatly shortened,which is worth promoting.
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Pancreatic cancer is one of the most common digestive tract malignant tumors with poor prognosis and low rate of the surgical resection,which has been a major difficulty in clinical surgeons.In recent years,the study founds that the interaction relations activated pancreatic stellate cell in the pancreas tumor microenvironment and cancer cells plays a very important role in the development of tumor.The cancer cells promote the activation of pancreatic stellate cells,secretion,and metastasis,at the same time,the pancreatic stellate cells can promote the proliferation,invasion,metastasis of cancer cells,and has a dual function of promotion and suppression the formation of new blood vessels around the tumor and immune suppression.Therefore,deeply study of the PSC will help us figure out the regulation of tumor development and initiate a new way for treatment of pancreas tumor at the cell and molecular level.
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Objective To investigate the role of fibromodulin (FMOD) in the xidative stress induced activation of pancreatic stellate cells (PSCs).Methods Lentivirus containing ShRNA targeting FMOD (sh-FMOD) was transfected into PSCs,and then the prooxidant menadione (MND) was used to treat PSCs for 24 h (MND + sh-FMOD group).Lentivirus transfected PSCs cells treated by a equal volume of DMSO served as shFMOD group,parent cells as control group and PSCs treated by MND as MND group.RT-PCR were used to detect the mRNA expression of the markers of activated PSCs including α-SMA,Col3α1,Col1α1,TIMP1 and α1-integrin.Chronic pancreatitis (CP) rat model was induced by DBTC infusion into the tail vein.Immunohistochemical (IHC) staining was used to detect the protein expression of FMOD,FN,α-SMA,SOD and MDA in normal pancreatic tissue and CP tissue.Results FMOD mRNA expression of the PSCs in FMOD group was obviously lowerer than that in control group (0.16 ±0.03 vs 1),and the difference was statistically significant (P ＜ 0.01),indicating that FMOD was successfully silenced.The mRNA expression of FMOD,α-SMA,Col3α1,Col1α1,TIMP1 and α1-Integrin of PSCs in MND group was obviously higher than those in control group,which in MND + sh-FMOD group was lower than those in MND group,and the difference was statistically significant (P ＜0.05 or ＜0.01).Compared with those in normal pancreatic tissue,the protein expression of FMOD,α-SMA,SOD and MDA in CP tissue was up-regulated,and the difference was statistically significant (all P ＜ 0.05).Conclusions Oxidative stress can facilitate the activation of PSCs through the induction of fibromodulin expression.
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Objective To investigate the effect of antioxidants including PDTC on pancreatic fibrosis of rats with chronic pancreatitis.Methods The rats were randomly divided into 5 groups including control group, CP group, PDTC treatment group, vitamin E treatment group and vitamin C treatment group.The CP model was in ducad by using intraperitoneal injection of DETC (750 mg/kg), twice a week.The control group received no treatment.After DETC injection, the treatment groups received an intraperitoneal injection of PDTC (100 mg/kg), vitamin E (15 mg/kg), vitamin C (15 mg/kg), respectively.Rats were sacrificed at 90 min, 24 h, 48 h, 72 h, 2 w, 3 w, 4 w, 6 w after first injection of DETC.Pancreatic tissue was taken for routine pathological examination.The activity of SOD, GSH-PX and MDA content were detected by spectrophotometric ratio method.α-SMA, desmin collagen Ⅰ, Ⅲ, TGF-β1, FN were detected by immunohistochemical assay.The expression of TGF-β1, FN mRNA was measured by RT-PCR.Results At 6w, the fibrosis and the parameters for damage of the pancreas in the three treatment groups were significantly better than that in CP group (P ＜0.01), the vacuolar degeneration index in vitamin E group and vitamin C group was also better than that in CP group (P ＜0.01).From the 2nd week, the activity of SOD, GSH PX in PDTC group, Vit C group and Vit E group was higher than that in CP group, while the MDA activity was lower than that in CP group, and the difference was statistically significant (P ＜ 0.01 or P ＜ 0.05).No significant difference was found among the three treatment groups.The mRNA levels of TGF-β1 and FN of the treatment groups were lower than those of CP group (P ＜0.05 or P ＜0.01), but higher than those of the control group (P ＜ 0.05).There was no significant difference among the three treatment groups (P ＞ 0.05).Conclusions PDTC and the other antioxidants can reduce oxygen free radicals by increasing the activity of SOD,suppressing the activation of PSCs, reducing the secretion of TGF-β1, Collagen Ⅰ , Ⅲ, FN and eventually inhibit the progress of pancreatic fibrosis.
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Pancreatic cancer is still a dismal disease.Angiogenesis is very important for the development of pancreatic cancer.Pancreatic stellate cells (PSCs) are the main source of extra-cellular matrix of pancreatic cancer and they provide advantageous microenvironment for cancer cells.PSC could promote the angiogenesis of pancreatic cancer both in vitro and in vivo.Further studies of the angiogenesis of pancreatic cancer are helpful in learning the characteristics of development and metastasis of pancreatic cancer,and provide new treatment method in the cellular and molecular levels.
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BACKGROUND/AIMS: Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) and peroxisome proliferator-activated receptor gamma (PPARgamma) ligands can modulate cellular differentiation, proliferation, and apoptosis through various pathways. It has been shown that HMG-CoA reductase inhibitors and PPARgamma agonists separately inhibit pancreatic stellate cell (PaSC) activation. We studied the effects of a combination of both types of drugs on activated PaSCs via platelet-derived growth factor (PDGF), which has not previously been reported. The present study was performed to elucidate the underlying mechanisms of these effects by focusing on the impact of the signaling associated with cell-cycle progression. METHODS: Primary cultures of rat PaSCs were exposed to simvastatin and troglitazone. Proliferation was quantified using the BrdU method, and cell-cycle analysis was performed using a fluorescent activated cell sorter. The protein expression levels of smooth muscle actin (SMA), extracellular signal-regulated kinase (ERK), and a cell cycle machinery protein (p27Kip1) were investigated using Western blot analysis. RESULTS: Simvastatin reversed the effects of PDGF on cell proliferation in a dose-dependent manner. The combination of a low concentration of simvastatin (1 mM) and troglitazone (10 mM) synergistically reversed the effects of PDGF on cell proliferation but had no effect on cell viability. The expression of a-SMA was markedly attenuated by combining the two drugs, which blocked the cell cycle beyond the G0/G1 phase by reducing the levels of phosphorylated ERK and reversed the expression of p27Kip1 interrupted by PDGF. CONCLUSIONS: Simvastatin and troglitazone synergistically inhibited cell proliferation in activated PaSCs by blocking the cell cycle beyond the G0/G1 phase. This inhibition was due to the synergistic modulation of the ERK pathway and the cell cycle machinery protein p27Kip1.
Subject(s)
Animals , Rats , Actins , Acyl Coenzyme A , Apoptosis , Blotting, Western , Bromodeoxyuridine , Cell Cycle , Cell Proliferation , Cell Survival , Chromans , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Ligands , MAP Kinase Signaling System , Muscle, Smooth , Oxidoreductases , Pancreatic Stellate Cells , Phosphotransferases , Platelet-Derived Growth Factor , PPAR gamma , Simvastatin , ThiazolidinedionesABSTRACT
Aim To study the role of 5A/6A polymorphism of matrix metalloproteinase (MMP-3) and their levels in the pathogenesis of chronic pancreatitis (CP). Methods One hundred and twenty CP patients and an equal number of age and sex-matched healthy controls were included in the study. Genotypes were determined for 5A/6A allele of MMP-3 gene by allele specific PCR (AS-PCR). The serum MMP-3 levels were estimated using sandwich ELISA method. Results The distribution of the genotypes of the 5A/6A polymorphism in both control and study patients was similar (p=0.523). Within the disease group, patients with older age, early onset of the disease, and addictions such as smoking and alcohol consumption had higher levels as compared to those who did not have these features. Conclusion We conclude that functional polymorphism of MMP-3 (5A/6A) is not associated with CP. However, the higher levels within the disease group indicate its possible role in the disease process.
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Pancreatic cancer is a frequent malignant tumor in digestive tract with extremely poor prognosis,characterized by difficult early diagnosis,high malignancy,poor resective,limited response to chemotherapy and radiotherapy and an intense fibrotic reaction known as tumor desmoplasia.Pancreatic stellate cells play an important role in this reaction and can stimulate pancreatic cancer cells proliferation,invasion and metastasis through the interaction with pancreatic cancer cells.This review describes the role of pancreatic stellate cells in the process of pancreatic cancer progression.
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Objective To introduce the role of pancreatic stellate cells in pancreatic fibrosis and the progress in treatment of pancreatic fibrosis. Methods Relevant literatures were collected and reviewed. Results Pancreatic stellate cells activation was closely related to pancreatic fibrosis. Inhibition of pancreatic stellate cells activation could provide a new approach in clinical treatment of chronic pancreatitis. Conclusion Pancreatic stellate cells are the key to pancreatic fibrosis,which are becoming the target for anti-fibrosis of the pancreas and treatment of chronic pancreatitis.
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To investigate the effects of Losartan,an angiotensinⅡ(AngⅡ)receptor(AT_1) antagonist,on pancreatic stellate cells(PSCs)and its possible mechanisms.Methods (1)PSCs were isolated from pancreatic cancerous samples to test the expressions of AT_1 and collagenⅠafter incubated with AngⅡor/and Losartan.(2)Ninety S-D rats were divided into normal group,control group and treatment group,with 30 rats in each.The rats in control and treatment groups were induced pancreatic fibrosis by injection of 2% trinitrobenenze sulfonic acid(TNBS)into biliopancreatic duct.Rats in treat- ment group were then treated with Losartan by garage daily and rats in control group were only given distilled water.The rats were sacrificed on day 3,7,14,21 and 28,respectively,and pancreas were removed.The histological abnormalities were observed by electron microscope.The mRNAs of trans- forming growth factor?_1(TGF?_1)and procollagenⅠwere detected by reverse transcription-polymerase chain reaction(RT-PCR).The expression of TGF?_1 and?-smooth muscle actin(?-SMA)proteins was assessed by immunohistochemistry and the level of?-SMA protein was quantified by Western blot. Results In vitro,there existed AT_1 expression in PSCs,and Losartan reduced expression of collagenⅠ.Losartan treatment reversed the histological abnormalities observed by electron microscope,com- pared to treatment with distill water.The expression of?-SMA,TGF?_1 and procollagenⅠwere signifi- cantly higher in the control group than those in normal group and were reduced by Losartan to different extent in treatment group.Conclusion AT_1 antagonist can inhibit the activation and the profibrogenic action of PSCs by blocking AT_1 receptor-mediated pathways.
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Objective To investigate the dynamic changes in expression of cell markers desmin, glial fibrillary acidic protein (GFAP), and (-smooth muscle actin (?-SMA) in primary cultures of human and rat pancreatic stellate cells (PSCs). Methods PSCs were isolated from human as well as rat pancreas using Nycodenz discontinuous density gradient centrifugation following digestion with combination of collagenase IV, Pronase E and DNase I, and purified by centrifugal elution techniques. Freshly isolated cells were examined by laser scanning confocal microscopy for vitamin A autofluorescence, by immunostaining for desmin, GFAP, and ?-SMA. Expression of ?-SMA was as well measured by Western analysis. Procollagen ?1(Ⅰ) mRNA expression was analyzed by Northern analysis. Results The purity of rat PSCs obtained by centrifugal elution were above 95%. More than 85% of either freshly-isolated human or rat PSCs displayed positive vitamin A autofluorescence. Rat PSCs stained positively for desmin and GFAP and negatively for ?-SMA, whereas human PSCs were negative for either desmin, GFAP or ?-SMA. During the process of primary culture, rat PSCs were positive for ?-SMA at 3d and completely transformed from quiescent state to myofibroblast-like phenotypes at 7d, which negatively or scarcely expressed desmin and GFAP, but fully expressed the ?-SMA protein and procollagen ?1(Ⅰ) mRNA, similarly to the settings of human PSCs. Conclusions Human and rat PSCs could be successively isolated in above 95% purity by combining gradient centrifugation with following centrifugal elution techniques. The results show some species differences in desmin and GFAP expression between freshly-isolated human and rat PSCs. Both of which, however, acquire a myofibroblast-like phenotype largely expressing ?-SMA protein and procollagen ?1(Ⅰ) gene in culture.
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Objective To investigate the impact of angiotension II (Ang II ) on the activation of nuclear factor-kappaB (NF-?B) signaling transduction pathway in cultured rat pancreatic stellate cells (PSCs). Methods Growth-arrested rat PSCs were incuhated for indicted time intervals with increasing concentrations of Ang II . The DNA binding activity of NF-?B was determined using electrophoretic mobility shift assay (EMSA). Western blot analysis was used to examine the degradation of inhibitory proteins of NF-?B (I?Bs). Expression of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein were assessed by Northern blot and enzyme-linked immunosorbent assay (ELISA), respectively. Results Treatment of cells with Ang II led to a biphasic activation of NF-?B, which peaked at 15 min and 6 h later, and it was correlated with differential degradation of I?B? and I?B?. Ang II -induced NF-?B activation was greatly inhibited by antioxidants pyrrolidine dithiocarbamate ( PDTC), diphenylene iodonium. and N-acetylcysteine, suggesting the involvement of oxidative stress in this process. In PSCs, Ang II induced a dose-dependent increase in the expression of MCP-1 and this effect was markedly inhibited by blocking NF-?B activation with MG132 and PDTC, indicating that Ang II -induced MCP-1 gene expression was NF-?B-dependent. Conclusion The present results suggest that Ang II , through an oxidative stress-dependent mechanism, may activate a functional NF-?B signaling pathway in PSCs, through which it may participate in pancreatic inflammation and fibrosis.