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ObjectiveTo investigate the effect of triptolide on fibrosis in mice with cerulein-induced chronic pancreatitis (CP) based on the nuclear factor-kappa B (NF-κB)/interleukin-6 (IL-6) signaling pathway. MethodsA total of 15 male C57BL/6 mice were randomly divided into control group (treated with normal saline), cerulein group (model mice with cerulein-induced CP), and triptolide group (induced by cerulean and treated with triptolide), with 5 mice in each group. Samples were collected for detection after 6 weeks. The weight of the pancreas was measured; HE staining, picrosirius red staining, and Masson staining were used to observe pancreatic histomorphology and collagen deposition; ELISA was used to measure the expression of IL-6 in serum; immunohistochemistry was used to measure the expression of alpha-smooth muscle actin (α-SMA) and NF-κB/p65; Western blot was used to measure the expression of NF-κB/p65, IL-6, and α-SMA. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsThere were significant differences between the three groups in the weight of the pancreas, IL-6 concentration, pathological score, picrosirius red staining results, Masson staining results, and mean optical density of positive α-SMA signal and positive NF-κB/p65 signal(F=64.87, 15.85, 145.33, 141.80, 121.77, 250.22, and 69.22, all P＜0.001). Compared with the cerulein group, the triptolide group had a significant increase in the weight of the pancreas and significant reductions in the expression of IL-6, pancreatic fibrosis, collagen deposition, and the expression of α-SMA and NF-κB/p65 (all P＜0.05). Western blotting showed that there were significant differences in the expression of NF-κB/p65, IL-6, and α-SMA between the three groups (F=8.86, 6.74, and 16.23, all P＜0.05), and compared with the cerulein group, the triptolide group had significantly lower expression of NF-κB/p65, IL-6, and α-SMA in the pancreatic tissue (all P＜0.05). ConclusionTriptolide alleviates fibrosis in mice with cerulein-induced CP and inhibit the protein expression of NF-κB/p65 and IL-6.
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Objective To observe the time dependent changes of pancreas and lung injury in mice acute pancrea-titis induced by caerulein,and compare the effects of mice acute pancreatitis model induced by different injection protocols of caerulein and lipopolysaccharides. Methods Acute pancreatitis were induced by different injection frequency of caerulein or caerulein combined with lipopolysaccharides, and plasma amylase activity was detected by starch-iodine colorimetry,and pancreas and lung injury severity was observed on paraffin section after HE stai-ning. Results The injury of pancreas and lung in mice acute pancreatitis induced by caerulein was most severe at 4 h, which began to repair at 24 h, and basically returned to normal at 5 d. 4 h after last injection, the amylase activity in CAE7 group and CAE9 group was higher than that in the control group(6 461±1 078 U/dl vs 3 093± 331 U/dl;6 821 ± 495 U/dl vs 3 093 ± 331 U/dl, all P<0.001), and CAE7+LPS group was higher than in CAE9 group[(8 912±465)U/dl vs (6 821±495)U/dl,P<0.001],the histological score in CAE7 group and CAE9 group was higher than that in the control group(4.750 ± 0.524 vs 0 ± 0; 4.917 ± 0.664 vs 0 ± 0, all P<0.001), and that in CAE7+LPS group was higher than in CAE9 group(7.167 ± 0.258 vs 4.917 ± 0.664, P<0.001). Conclusions Mice acute pancreatitis can be successfully induced by 7 i.p. injections of caerulein (50 μg/kg body weight) at 1-hour intervals,caerulein combined with lipopolysaccharides.
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Objective To observe the time dependent changes of pancreas and lung injury in mice acute pancrea-titis induced by caerulein,and compare the effects of mice acute pancreatitis model induced by different injection protocols of caerulein and lipopolysaccharides. Methods Acute pancreatitis were induced by different injection frequency of caerulein or caerulein combined with lipopolysaccharides, and plasma amylase activity was detected by starch-iodine colorimetry,and pancreas and lung injury severity was observed on paraffin section after HE stai-ning. Results The injury of pancreas and lung in mice acute pancreatitis induced by caerulein was most severe at 4 h, which began to repair at 24 h, and basically returned to normal at 5 d. 4 h after last injection, the amylase activity in CAE7 group and CAE9 group was higher than that in the control group(6 461±1 078 U/dl vs 3 093± 331 U/dl;6 821 ± 495 U/dl vs 3 093 ± 331 U/dl, all P<0.001), and CAE7+LPS group was higher than in CAE9 group[(8 912±465)U/dl vs (6 821±495)U/dl,P<0.001],the histological score in CAE7 group and CAE9 group was higher than that in the control group(4.750 ± 0.524 vs 0 ± 0; 4.917 ± 0.664 vs 0 ± 0, all P<0.001), and that in CAE7+LPS group was higher than in CAE9 group(7.167 ± 0.258 vs 4.917 ± 0.664, P<0.001). Conclusions Mice acute pancreatitis can be successfully induced by 7 i.p. injections of caerulein (50 μg/kg body weight) at 1-hour intervals,caerulein combined with lipopolysaccharides.
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Objective To investigate the effects of pioglitazone pre-treating on pancreatic acinar cell (AR42J cells) apoptosis induced by caerulein.Methods AR42J cells were divided into blank control group (with normal culture),pioglitazone group (40 μmol/L),caerulein control group (1 × 10-8 mol/L),pioglitazone+ caerulein group (40 μmol/L pioglitazone + 1 × 10-8 mol/L caerulein) and pioglitazone + GW9662+caerulein group (40 μmol/L pioglitazone+ 5 μmol/L GW9662 + 1 × 10-8 mol/L caerulein).Pioglitazone and GW9662 were added 30 minutes earlier than caerulein.Cell proliferation rate of each group was determined by MTT assay at three,six,12 and 24 hour.The cell apoptosis rate was detected by flow cytometry with Annexin Ⅴ/PI staining and terminal dexynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining.The activity of Caspase 3,8 and 9 of each group was measured.Mitochondrial membrane potential (MMP) was detected by flow cytometry with JC-1 staining.Single factor analysis of variance and LSD test were performed for data analysis.Results At six,12 and 24 hour,the cell proliferation rate of pioglitazone group and pioglitazone + caerulein group was 0.19±0.02,0.22±0.02,0.36±0.02 and 0.20±0.04,0.23±0.02,0.38±0.02,respectively,which were significantly lower than those of blank control group (0.25 ±0.04,0.28 ± 0.03 and 0.46±0.02) and caerulein group (0.23±0.02,0.29±0.01 and 0.46±0.05,t lgroup=-3.16,-4.61 and-6.25,tcaerulein group =-1.58,-4.61 and-6.15,all P＜0.05).And the cell proliferation rates of pioglitazone+GW9662+caerulein group at six,12 and 24 hour (0.23±0.02,0.27±0.02 and 0.45±0.01) were significantly higher than those of pioglitazone+caerulein group (t=2.25、3.87、4.56,all P＜0.05).There was no significant difference in cell apoptosis rate detected by flow cytometry with Annexin Ⅴ/PI staining between pioglitazone group ((11.80 ± 0.47) ％,(9.62 ± 2.63) ％ and (14.92 ± 2.52) ％) and pioglitazone+caerulein group ((8.78±0.47)％,(11.89±2.80)％ and (14.25±2.67)％,all P＞0.05),but cell apoptosis of these two groups were higher than those of control group ((5.52± 0.64)％,(5.30±0.97)％ and (5.47±0.88)％) and caerulein group ((5.98±1.21)％,(7.47± 0.58) ％ and (8.11 ± 1.32) ％) respectively,and the differences were statistically significant (t l group =9.81,4.45 and 10.74,tcaerulein group =4.38,7.62 and 6.98,all P ＜0.05).There was no significant difference in apoptosis rate between pioglitazone+GW9662+caerulein group ((5.82±0.26) ％,(6.05± 0.83) ％ and (9.23±0.90)％) and caerulein group; while significantly higher when compared with those of pioglitazone+ caerulein group (t=-4.63,-10.07 and-5.70,all P＜0.05).At 12 hour,the apoptosis rate detected by TUNEL staining of pioglitazone group ((3.93 ± 0.40)％) was significantly higher than that of control group ((2.73 ±0.68) ％),the apoptosis rate of pioglitazone+ caerulein group ((8.43 ± 1.65)％) was significantly higher than that of caerulein group ((2.80 ± 0.56)％),the apoptosis rate of pioglitazone+GW9662+caerulein group ((3.87±0.35)％) was lower than that of pioglitazone+ caerulein group (t=7.93,8.92,-5.35,all P＜0.05).At 24 hour,the activity of Caspase 3,8 and 9 of pioglitazone+ caerulein group (1.28 ± 0.05,1.38 ± 0.04 and 1.53 ± 0.09) significantly increased compared with those of caerulein group (1.12±0.88,1.22±0.02 and 0.53±0.07,t=3.20,8.62 and 1.29,all P＜0.05).After treated with GW9662,part of activity of Caspase enzymes recovered.The number of cells with potential change of mitochondrial membrane in pioglitazone group and pioglitazone + caerulein group was more than that of caerulein group (28.50±0.91)％ and (28.20±2.56)％ vs (15.00±3.67)％) and part of membrane potential recovered after GW9662 added ((20.67 ± 2.20) ％).Conclusions Pioglitazone might promote AR42J cell apoptosis through the activation of caspases enzymes and changing membrane potential.And the antagonist GW9662 would partially inhibit the apoptosis induced by pioglitazone.
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AIM:To investigate the effects of grape polyphenol ( GP) on caerulein-induced acute pancreatitis (AP) in mice.METHODS:Two-month-old female mice of ICR strains (n=21) were randomly divided into 3 groups:normal control ( NC) group, AP group, and GP-treated AP group.Before AP induction, the mice in GP-treated AP group were continuously administrated with 1.5 g/kg GP aqueous solution by gavage for 7 d, while those in NC group and AP group were treated with saline as a vehicle control.On the 7th day, the mice in AP group and GP-treated AP group were in-traperitoneally injected with caerulein (50μg/kg) in 1 h interval for 7 serial injections in total.The mice in NC group were treated with saline according to the same procedure in experimental group.All the mice were sacrificed 24 h after AP induc-tion, and the pancreatic tissues and lung tissues were harvested for further investigation of the pathological changes, macro-phages infiltration, myeloperoxidase ( MPO ) activity and expression of inflammatory and oxidative stress factors.RE-SULTS:Compared with AP group, the mice in GP-treated AP group showed milder morphological changes and lower path-ological scores, including the scores of edema, inflammation and vacuolization (P<0.05), but the necrosis scores and to-tal scores showed no statistical difference between these 2 groups.Besides, the mice in GP-treated AP group had fewer macrophage infiltration, lower lung MPO activity (P<0.01), and lower expression of inflammatory factors, tumor necrosis factor α(TNF-α)and monocyte chemotactic protein 1 (MCP-1) (P<0.05), and oxidative stress factors, superoxide dis-mutase (SOD)-1, SOD-2 and NADPH oxidase 2 (NOX-2) (P<0.01).CONCLUSION:Grape polyphenol has remark-able protective effect on pancreatic tissues of mice with caerulein-induced acute pancreatitis, and the mechanisms may be related to down-regulation of inflammatory and oxidative stress factors.
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Objective To establish a mouse model of acute necrotizing pancreatitis.Methods Thirty six male ICR mice were randomly divided into control group ( n =6) and experimental group ( n =30). Each of the animals in the experimental group received 7 intraperitoneal injections of caerulein (50 ?g/kg body weight) in 0.9% NaCl at hourly intervals over 6 hours. The animals in the experimental group were killed at 9,18,24,48 and 72 hours respectively after the first caerulein injection. The control animals received the same volume of 0.9% NaCl without caerulein. The animals in the control group were killed at the 18th hour after the first intraperitoneal injection. The severity of acute necrotizing pancreatitis was evaluated in terms of amylase level, pancreatic weight/body weight and the histological changes. Variance analysis was employed in the processing of these data. Results Both amylase level and pancreatic weight elevated 9 hours after the first caerulein injection, and correlated with the course of pancreatitis. The maximums of both alterations were observed at the same time point (18 hours after the first injection of caerulein). Prominent interstitial inflammation and acinar cell necrosis occurred at the 18th hour, and the histological score for pancreatitis reached a maximum ( P
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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% and 68.24 ± 10.47% in control group and caerulein group, respectively. Caerulein group had a mean decrease of 31.75 ± 16.79%. The serum amylase was 1323.70 ± 239.10U.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% 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.
OBJETIVO: O fluxo capilar pancreático (FCP) foi estudado para determinar suas alterações durante a pancreatite aguda induzida por ceruleína, em ratos. MÉTODOS: Vinte ratos foram divididos em grupo controle e grupo ceruleína. Um laser-Doppler fluxímetro foi empregado para determinar, continuamente, o FCP durante 120 minutos. A pressão arterial média (PAM) e a freqüência cardíaca (FC) foram determinadas, durante o experimento. Análise bioquímica sérica e estudo histopatológico, por microscopia ótica, do tecido pancreático foram realizados, ao final do experimento. RESULTADOS: O FCP foi em média 109,08 ± 2,17% e 68,24 ± 16,79% nos grupos controle e ceruleína , respectivamente. No grupo ceruleína, houve uma diminuição média de 31,75 ± 16,79%. Os níveis de amilase sérica foram de 1323,70 ± 239,10U.I-1 e 2184,60 ± 700,46U.I-1 nos grupos controle e ceruleína, respectivamente. Houve diferença significante (p 0,05) no FCP e na amilasemia, quando comparado o grupo controle com o grupo ceruleína. Embora micro e macrovacuolização estivessem presentes no grupo ceruleína, não houve diferença histológica entre os grupos. CONCLUSÃO: A diminuição do FCP parece um evento precoce, antecedendo o aparecimento de alterações histopatológicas, por microscopia ótica, que caracterizam este modelo de pancreatite edematosa aguda.
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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. Serum biochemistry and histopathological analyses were performed. RESULTS: PCBF measured a mean of 109.08 ± 14.54%, 68.24 ± 10.47%, 102.18 ± 10.23% and 87.73 ± 18.72% in groups 1, 2, 3 and 4, respectively. PCBF in groups 2 and 4 decreased 31.75 ± 16.79% and 12.26 ± 15.24%, respectively. Serum amylase was 1323.70 ± 239.10 U/l, 2184.60 ± 700.46 U/l, 1379.80 ± 265.72 U/l and 1622.10 ± 314.60 U/l in groups 1, 2, 3 and 4, respectively. There was a significant difference in the PCBF and serum 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 serum amylase increase during caerulein-induced pancreatitis. ROS effect may be one of the leading causative events in this model of acute pancreatitis.
OBJETIVO: A inativação de radicais livres (RL) foi estudada para determinar as alterações do fluxo capilar pancreático (FCP) na pancreatite aguda induzida por ceruleína em ratos. MÉTODOS: Um laser-Doppler fluxímetro determinou o FCP e o composto N-t-Butyl-Phenylnitrone (PBN), para inativar os RL, foi utilizado. Quarenta ratos foram divididos em 4 grupos: 1) controle; 2)ceruleína; 3) PBN; 4)ceruleína+PBN. Dosagens bioquímicas e análise histopatológica foram realizadas. RESULTADOS: O FCP foi em média 109.08 ± 14.54%, 68.24 ± 10.47%, 102.18 ± 10.23% e 87.73 ± 18.72% nos grupos 1, 2, 3 and 4, respectivamente. O FCP nos grupos 2 e 4 diminuíram em média 31.75 ± 16.79% e 12.26 ± 15.24%, respectivamente. A média da amilase sérica foi de 1323,70 ± 239.10 U/l, 2184,60 ± 700,46 U/l, 1379,80 ± 265,72 U/l e 1622,10 ± 314,60 U/l nos grupos 1, 2, 3 e 4, respectivamente. Observou-se diferença significante no FCP e na amilase sérica quando comparados os grupos 2 e 4. Vacuolização citoplasmática estava presente nos grupos 3 e 4. Não foram observadas outras alterações qualitativas. CONCLUSÃO: A inativação de RL melhorou o FCP e minimizou a elevação da amilase sérica na pancreatite aguda induzida por ceruleína. A presença de RL parece ser um evento precoce neste modelo de pancreatite aguda experimental.
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An enhanced formation of nitric oxide(NO), due to the induction of inducible nitric oxide synthase(iNOS), has been implicated in the pathogenesis of shock and inflammation, but its role in acute pancreatitis still remains controversial. To clarify the role of NO in acute pancreatitis, the present experiment investigated the expression of iNOS and the effect of NOS inhibition on cerulein-induced pancreatitis in rats. Group I received intraperitoneal (ip) injection of normal saline. Group II received two ip injections of cerulein (20 microgram/kg). Group III received injections of N(G)-nitro-L-arginine methyl este(L-NAME) (30 mg/kg) with cerulein. Group IV received L-arginine(250 mg/kg) with cerulein and L-NAME. The expression of iNOS in the pancreas was examined by western blot analysis. The plasma concentration of NO metabolites was measured. The severity of pancreatitis was assessed by measuring serum amylase, pancreas water content and histopathological examination. Compared with controls, the cerulein group displayed significantly increased expression of iNOS and raised plasma NO metabolites. Treatment with L-NAME significantly decreased hyperamylasemia, plasma NO level, and the extent of pancreatic injury. Treatment with L-arginine reversed the effects of L-NAME. These findings suggest that an enhanced formation of NO by iNOS plays an important role in the development of acute pancreatitis, and inhibition of NO production has the beneficial effects in reducing pancreas injury.
Subject(s)
Animals , Male , Rats , Amylases/blood , Arginine/pharmacology , Blotting, Western , Ceruletide/pharmacology , Enzyme Inhibitors/pharmacology , Inflammation , NG-Nitroarginine Methyl Ester/pharmacology , Necrosis , Nitric Oxide/metabolism , Nitric Oxide Synthase/metabolism , Pancreatitis/chemically induced , Rats, Sprague-DawleyABSTRACT
ObjectiveTo investigate the effect of triptolide on fibrosis in mice with cerulein-induced chronic pancreatitis (CP) based on the nuclear factor-kappa B (NF-κB)/interleukin-6 (IL-6) signaling pathway. MethodsA total of 15 male C57BL/6 mice were randomly divided into control group (treated with normal saline), cerulein group (model mice with cerulein-induced CP), and triptolide group (induced by cerulean and treated with triptolide), with 5 mice in each group. Samples were collected for detection after 6 weeks. The weight of the pancreas was measured; HE staining, picrosirius red staining, and Masson staining were used to observe pancreatic histomorphology and collagen deposition; ELISA was used to measure the expression of IL-6 in serum; immunohistochemistry was used to measure the expression of alpha-smooth muscle actin (α-SMA) and NF-κB/p65; Western blot was used to measure the expression of NF-κB/p65, IL-6, and α-SMA. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsThere were significant differences between the three groups in the weight of the pancreas, IL-6 concentration, pathological score, picrosirius red staining results, Masson staining results, and mean optical density of positive α-SMA signal and positive NF-κB/p65 signal(F=64.87, 15.85, 145.33, 141.80, 121.77, 250.22, and 69.22, all P＜0.001). Compared with the cerulein group, the triptolide group had a significant increase in the weight of the pancreas and significant reductions in the expression of IL-6, pancreatic fibrosis, collagen deposition, and the expression of α-SMA and NF-κB/p65 (all P＜0.05). Western blotting showed that there were significant differences in the expression of NF-κB/p65, IL-6, and α-SMA between the three groups (F=8.86, 6.74, and 16.23, all P＜0.05), and compared with the cerulein group, the triptolide group had significantly lower expression of NF-κB/p65, IL-6, and α-SMA in the pancreatic tissue (all P＜0.05). ConclusionTriptolide alleviates fibrosis in mice with cerulein-induced CP and inhibit the protein expression of NF-κB/p65 and IL-6.