二丁基二氯化物尾静脉注射建立大鼠慢性胰腺炎模型

目的 探讨经尾静脉注射二丁基二氯化物建立大鼠慢性胰腺炎模型的方法,为慢性胰腺炎纤维化机制研究提供一种合适的动物模型.方法 将SD大鼠随机分为实验组和对照组,每组再分为1,7,14,21,28,60 d 6个观察点,每个时间点各5只大鼠.实验组大鼠尾静脉注射二丁基二氯化物8 mg/kg体重,对照组注射相同剂量的乙醇和甘油溶剂.上述时间点分别处死大鼠,收集血液和胰腺标本.检测血清淀粉酶、脂肪酶、透明质酸浓度.观察胰腺形态,病理改变,胶原染色评价纤维化程度.结果 造模后1 d胰腺组织水肿,表现为急性中度间质性水肿性胰腺炎;7 d炎症加重,表现为腺泡肿胀,散在的腺泡细胞坏死;14 d广泛的炎症细胞浸润,伴轻度纤维化;21 ～ 28 d胶原沉积,纤维化加重,可见大量的纤维结缔组织;60 d,胰腺小叶结构破坏,腺泡消失,广泛间质纤维化.结论 尾静脉注射二丁基二氯化物是一种简便、有效的大鼠慢性胰腺炎模型制作方法,能为慢性胰腺炎纤维化的研究提供合适的动物模型.     

二丁基二氯化物尾静脉注射建立大鼠慢性胰腺炎模型

目的探讨经尾静脉注射二丁基二氯化物建立大鼠慢性胰腺炎模型的方法,为慢性胰腺炎纤维化机制研究提供一种合适的动物模型。方法将SD大鼠随机分为实验组和对照组,每组再分为1,7,14,21,28,60d6个观察点,每个时间点各5只大鼠。实验组大鼠尾静脉注射二丁基二氯化物8mg/kg体重,对照组注射相同剂量的乙醇和甘油溶剂。上述时间点分别处死大鼠,收集血液和胰腺标本。检测血清淀粉酶、脂肪酶、透明质酸浓度。观察胰腺形态,病理改变,胶原染色评价纤维化程度。结果造模后1d胰腺组织水肿,表现为急性中度间质性水肿性胰腺炎;7d炎症加重,表现为腺泡肿胀,散在的腺泡细胞坏死;14d广泛的炎症细胞浸润,伴轻度纤维化;21~28d胶原沉积,纤维化加重,可见大量的纤维结缔组织;60d,胰腺小叶结构破坏,腺泡消失,广泛间质纤维化。结论尾静脉注射二丁基二氯化物是一种简便、有效的大鼠慢性胰腺炎模型制作方法,能为慢性胰腺炎纤维化的研究提供合适的动物模型。     

Anti-monocyte chemoattractant protein 1 gene therapy attenuates experimental chronic pancreatitis induced by dibutyltin dichloride in rats

BACKGROUND: Monocyte chemoattractant protein 1 (MCP-1) is a member of the C-C chemokine family and exerts strong chemoattractant activity in monocytes, macrophages, and lymphocytes. Rat pancreatic fibrosis induced by dibutyltin dichloride (DBTC) is considered to be an appropriate chronic pancreatitis model histologically and enzymatically, as has demonstrated in a previous study. AIM: We examined the effect of human dominant negative inhibitor of MCP-1 (mutant MCP-1) on progression of chronic pancreatitis induced by DBTC in a rat model. METHODS: We used the experimental model of chronic pancreatitis induced by DBTC in rats. Mutant MCP-1 or empty plasmid at a dose of 50 microg/body weight was administrated into rat thigh muscles on days 4, 11, and 18 after administration of DBTC. On days 14 and 28, we evaluated the effect of mutant MCP-1 morphologically and biochemically. RESULTS: The mutant MCP-1 treated group inhibited early pancreatic inflammation and later pancreatic fibrosis histologically, and showed a decrease in serum MCP-1 concentration, intrapancreatic hydroxyproline, alpha-smooth muscle actin, and an increase in intrapancreatic amylase and protein content compared with the empty plasmid treated group. The mutant MCP-1 group also inhibited intrapancreatic mRNA expression of cytokines and chemokines. CONCLUSIONS: : Our findings suggest that monocyte/macrophage recruitment and the systemic MCP-1 signal pathway contribute to progression of chronic pancreatitis, and that blockade of MCP-1 may suppress the development of pancreatic fibrosis.     

Pathophysiology of chronic pancreatitis induced by dibutyltin dichloride joint ethanol in mice

AIM: To search for a new chronic pancreatitis model in mice suitable for investigating the pathophysiological processes leading to pancreatic fibrosis.METHODS: The mice were randomly divided into 2 groups(n = 50), control group and model group. The mice in model group were given ethanol(10%) in drinking water after injection of dibutyltin dichloride(DBTC)(8 mg/kg BW) in tail vein. The mice in control group were injected with only solvent into tail vein( 60 % ethanol, 20% glycerine and 20% normal saline) and drank common water. At days 1, 7, 14, 28, and 56 after application of DBTC or solvent, 10 mice in one group were killed at each time point respectively. Blood was obtained by inferior vena cava puncture. The activity of amylase, concentration of bilirubin and hyaluronic acid in serum were assayed. The pancreas was taken to observe the pancreatic morphology by HE staining, and to characterize the pancreatic fibrosis by Masson staining. The expression of F4/80, CD3 and fibronectin(FN) were assayed by immuno-histochemistry or Immunofluorescence technique. Collagen typeⅠ(COL1A1) in pancreas were detected by Western blot. The expression of matrix metalloproteinase-1(MMP-1) and tissue inhibitor of metalloproteinases-1(TIMP-1) m RNA in the pancreas was assessed by real time PCR.RESULTS: DBTC induced an acute edematous pancreatitis within 1 d. The dilated acini, scattered acinar cell necrosis, and inflammatory cells were found at day 7. Extensive infiltration with inflammatory cells following deposition of connective tissue was observed at day 14. At day 28, level of pancreatic fibrosis was aggravated. The pancreatic tissue was replaced by an extended interstitial fibrosis at the end of 2 mo. There was significant difference in the level of amylase, bilirubin and hyaluronic acid in serum between control group and model group(P 0.05). The level of COL1A1 and FN in pancreas increased. The expression of MMP-1 m RNA in pancreas decreased, but TIMP-1 m RNA increased at model group.CONCLUSION: DBTC joint Ethanol drinking can induce chronic pancreatitis in accordance with the pathophysiological modification of human. DBTC joint Ethanol-induced pancreatitis in mice is an effective and handy experimental method. The model is suitable to study the mechanism of pancreatic fibrosis in chronic pancreatitis.     

Cytokine mRNA levels and lymphocyte infiltration in pancreatic tissue during experimental chronic pancreatitis induced by dibutyltin dichloride

There is little information available regarding the role of inflammatory cells in the pathogenesis of chronic pancreatitis. Therefore, we analyzed the local cytokine profile and infiltrating lymphocytes in a rat model of chronic pancreatitis. Experimental pancreatitis was induced by a single intravenous application of dibultyltin dichloride (DBTC). During a time course of two months we observed the mRNA expression of cytokines using competitive RT-PCR. Lymphocytes were characterized by immunohistochemistry, FACS analysis, and the lymphocyte proliferation test. IL-1, IL-6, IL-5, and IL-10 were immediately up-regulated in the acute phase of disease, while lymphocyte-restricted expression of IL-2, IL-2R, and IFN- was only found in the chronic course. Among the infiltrating lymphocytes, CD4⁺ cells dominated, but during the chronic process there was an increase of CD8⁺ cells, resulting in a reduced CD4/CD8 ratio. Mitogen-induced activation of isolated mesenteric lymph node cells increased during the chronic inflammation. Our results suggest that in experimental pancreatitis acute inflammatory reactions are followed by a T-lymphocyte-mediated process.     

A sustained prostacyclin analog,ONO-1301, attenuates pancreatic fibrosis in experimental chronic pancreatitis induced by dibutyltin dichloride in rats

BackgroundONO-1301, a novel sustained-release prostacyclin agonist, has an anti-fibrotic effect on the lungs, heart, and kidneys that is partly associated with the induction of hepatocyte growth factor (HGF). This study examined the anti-fibrotic effect of ONO-1301 on chronic pancreatitis (CP) progression.MethodsCP was induced in rats in vivo by dibutyltin dichloride (DBTC). Seven days after DBTC injection (day 7), a slow-release form of ONO-1301 (10 mg/kg; ONO-1301–treated group) or vehicle (DBTC-treated group) was injected. On days 14 and 28, we evaluated the histopathological CP score and mRNA expressions of HGF, cytokines, and collagen in the pancreas by real-time RT-PCR. In vitro, monocytes and pancreatic stellate cells (PSCs) were isolated from normal rat spleen and pancreas, respectively. The cytokine and collagen expressions of monocytes and PSCs were detected by real-time RT-PCR, and PSCs proliferation was examined by BrdU assay.ResultsHistopathological CP scores in vivo improved in the ONO-1301–treated group compared to the DBTC-treated group, particularly inflammatory cell infiltration on day 14 and interstitial fibrosis on day 28. HGF mRNA increased significantly after ONO-1301 administration, whereas IL-1β, TNF-α, TGF-β, MCP-1, and collagen mRNA decreased significantly. Cytokine expression in monocytes was suppressed in vitro not only by HGF, but also ONO-1301 alone. However, neither ONO-1301 nor HGF affected the proliferation, or cytokine or collagen expression of PSCs.ConclusionsONO-1301 suppresses pancreatic fibrosis in the DBTC-induced CP model by inhibiting monocyte activity not only with induction of HGF but also by ONO-1301 itself.     

依达拉奉对大鼠重症急性胰腺炎的保护作用

目的:探讨依达拉奉对L-精氨酸诱导的重症急性胰腺炎(SAP)大鼠的治疗作用及机制.方法:将60只大鼠随机分为三组( n = 20). 采用大鼠腹腔内分次注射大剂量L-精氨酸(2.5g/k g×2, 间隔1 h)的方法制备SAP大鼠模型, 治疗组大鼠腹腔内注射依达拉奉注射液3 mg/kg, bid×3 d. 72 h后比较三组大鼠胰腺组织病理变化、腹水性状及量、血清淀粉酶(AMY)、TNF-α、IL-6水平和胰腺组织丙二醛(MDA)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)含量及预后.结果:72 h后, 与对照组相比, SAP组出现典型的SAP病理形态改变, 腹水量较多(5.16±1.52vs 0.50±0.10, P<0.01), 且大鼠血清AMY、TNF-α、I L-6及胰腺组织M DA均显著升高(8967.5±298.4 vs 720.1±119.7; 103.98±10.56 vs 41.59±3.79; 548.57±10.45 vs 198.34±2.10; 35.6±3.8 vs 7.9±2.2, 均P<0.01), 胰腺组织抗氧化物质GSH、SOD明显降低(7.2±0.6 vs 17.1±2.1; 7300±1800 vs 28400±2700,均P<0.01); 与SAP组相比, 治疗组胰腺组织病理损害减轻, 腹水量减少(4.05±1.22 vs 5.16±1.52, P<0.05), 且组织病理评分较低( P<0.05),血清AMY、TNF-α、IL-6及胰腺组织MDA明显降低(7809.5±158.3 vs 8967.5±298.4; 79.80±14.23 vs 103.98±10.56; 467±6.64 vs 548.57±10.45; 29.1±2.6 vs 35.6±3.8, 均P<0.05), 胰腺组织GSH、SOD升高(8.7±1.3 vs 7.2±0.6;114 000±27 000 vs 7300±1800, 均P<0.05).结论:依达拉奉能清除氧自由基、提高胰腺组织抗氧化物质SOD和GSH含量, 降低促炎细胞因子水平, 可减轻胰腺组织的病理损害,并且有可能降低死亡率.     

依达拉奉对大鼠急性坏死性胰腺炎的保护作用

目的:探讨自由基清除剂依达拉奉对大鼠急性坏死性胰腺炎的保护作用及其机制.方法:90只♂SD大鼠随机分为假手术组(SHAM组)、坏死性胰腺炎组(ANP组)、依达拉奉治疗组(EDA组),每组30只.SHAM组为开腹后只翻动十二指肠及胰腺后关腹;ANP组胰胆管内逆行输注1.5%脱氧胆酸钠制备急性坏死性胰腺炎模型;EDA组为ANP造模后立即尾静脉注射依达拉奉(6mg/kg).分别于术后6、12、24h处死大鼠(每个时点10只),观察胰腺病理形态改变并评分;检测血清淀粉酶、TNF-α、ET-1、sICAM-1含量;检测胰腺组织中丙二醛(MDA)含量及总超氧化物歧化酶(T-SOD)活力.结果:与ANP组比较,EDA治疗组在胰腺病理改变、血清TNF-α水平(6h:109.6ng/L±49.0ng/Lvs190.2ng/L±46.6ng/L,12h:405.4ng/L±116.3ng/Lvs559.7ng/L±203.9ng/L,24h:415.4ng/L±164.6ng/Lvs648.7ng/L±222.1ng/L,均P<0.05)、血清ET-1水平(6h:45.6ng/L±13.5ng/Lvs66.0ng/L±16.0ng/L,12h:83.5ng/L±15.4ng/Lvs96.8ng/L±23.0ng/L,24h:85.1ng/L±25.8ng/Lvs103.9ng/L±28.9ng/L),血清sICAM-1水平(6h:0.58ng/L±0.13ng/Lvs0.78ng/L±0.14ng/L,12h:0.78ng/L±0.10ng/Lvs0.94ng/L±0.12ng/L,24h:0.96ng/L±0.16ng/Lvs1.24ng/L±0.30ng/L,均P<0.05)、胰腺组织MDA含量(6h:4.22nmol/mgprot±0.40nmol/mgprotvs8.79nmol/mgprot±0.80nmol/mgprot,12h:5.90nmol/mgprot±0.51nmol/mgprotvs12.30nmol/mgprot±1.02nmol/mgprot,24h:9.10nmol/mgprot±0.84nmol/mgprotvs17.88nmol/mgprot±1.43nmol/mgprot)均有不同程度减轻(均P<0.05),T-SOD活力增强(6h:88.6U/mgprot±7.1U/mgprotvs68.8U/mgprot±10.5U/mgprot,12h:77.6U/mgprot±6.8U/mgprotvs46.0U/mgprot±8.9U/mgprot,24h:45.5U/mgprot±5.3U/mgprotvs27.8U/mgprot±4.3U/mgprot,均P<0.05);血清淀粉酶变化无显著差异.与SHAM组比较,ANP组胰腺组织病理评分、血清淀粉酶、TNF-α、ET-1、sICAM-1明显升高,胰腺组织MDA含量升高,T-SOD活力下降,差异均有统计学意义.结论:依达拉奉可以清除坏死性胰腺炎体内过量生成的氧自由基并减少炎性因子的表达,减轻胰腺组织损伤.     

Increased heat shock protein 70 expression attenuates pancreatic fibrosis induced by dibutyltin dichloride

Abstract

Objectives: Heat shock protein (HSP) 70 performs a chaperoning function and protects cells against injury. Although the effect of HSPs against acute inflammatory change has been proven, the relationship between HSP70 and chronic pancreatitis remains unclear. This study aimed to investigate the protective effect of increased HSP70 expression induced by thermal stress against pancreatic fibrosis in experimental chronic pancreatitis.

Materials and Methods: Two experiments to evaluate pancreatic HSP70 expression induced by thermal stress and determine the effect of increased HSP70 expression against pancreatic fibrosis were performed. To investigate HSP70 expression, rats were immersed in a warm bath and sequentially killed, and pancreatic HSP70 expression was measured. To study the effect of increased HSP70 expression, pancreatic fibrosis was induced by intravenous injection of dibutyltin dichloride (DBTC) and analyzed under repeated thermal stress. The severity of pancreatic fibrosis was measured.

Results: Thermal stress significantly increased HSP70 expression in the pancreas. HSP70 expression peaked at 6–12?h after warm bathing, and the increased HSP70 expression was associated with the attenuation of pancreatic fibrosis. Although pancreatic fibrosis was induced by DBTC injection, HSP70 expression induced by repeated thermal stress diminished the severity of atrophy and fibrosis. On western blot analysis, collagen type 1 expression was diminished in the increased HSP70 expression group, but not α-smooth muscle actin expression.

Conclusions: Thermal stress could increase pancreatic HSP70 expression, and induced HSP70 expression showed a protective effect against pancreatic fibrosis. Modulation of HSP70 expression could be a potential therapeutic target in the treatment of chronic pancreatitis.     

Protective effects of rhubarb on experimental severe acute pancreatitis

AIM: To investigate the effects of rhubarb on severe acute pancreatitis (SAP) in rats. METHODS: Severe acute pancreatitis was induced by two intraperitoneal injections of cerulein (40 μg/kg body weight) plus 5-h restraint water-immersion stress. Rhubarb (75-150 mg/kg) was orally fed before the first cerulein injection. The degree of pancreatic edema, serum amylase level, local pancreatic blood flow (PBF), and histological alterations were investigated. The effects of rhubarb on pancreatic exocrine secretion in this model were evaluated by comparing with those of somatostatin. RESULTS: In the Cerulein+Stress group, severe edema and diffuse hemorrhage in the pancreas were observed, the pancreatic wet weight (11.60&#177;0.61 g/Kg) and serum amylase (458 490&#177;43 100 U/L) were markedly increased (P&lt;0.01 vs control). In the rhubarb (150 mg/kg) treated rats, necrosis and polymorphonuclear neutrophil (PMN) infiltration in the pancreas were significantly reduced (P&lt;0.01), and a marked decrease (50%) in serum amylase levels was also observed (P&lt;0.01). PBF dropped to 38% (93&#177;5 mL/min per 100 g) of the control in the Cerulein+Stress group and partly recovered in the Cerulein+Stress+Rhubarb 150 mg group (135&#177;12 mL/min per 100 g) (P&lt;0.01). The pancreatic exocrine function was impaired in the SAP rats. The amylase levels of pancreatic juice were reduced in the rats treated with rhubarb or somatostatin, comparing with that of untreated SAP group. The bicarbonate concentration of pancreatic juice was markedly elevated only in the rhubarbtreated group (P&lt;0.01). CONCLUSION: Rhubarb can exert protective effects on SAP, probably by inhibiting the inflammation of pancreas, improving pancreatic microcirculation, and altering exocrine secretion.     

Influence of selenium treatment on the acute toxicity of dibutyltin dichloride in rats.

BACKGROUND/AIMS: Dibutyltin dichloride (DBTC) is an organotin compound used as a model for acute pancreatitis.The aim was to determine the effect of various doses of Na-selenite on the pathogenesis and course of DBTC-induced toxic changes in organs and serum of rats. METHODS: Experimental pancreatitis was induced by intravenous administration of 6 mg kg(-1) BW DBTC. Na-selenite was applied as a single intravenous dose of 5 mg kg(-1) BW and as daily oral dose of 1 mg kg(-1) BW. Malondialdehyde (MDA) was detected to observe the level of oxidative stress. The tin concentration in bile and urine shows the elimination of DBTC. Organ changes were indicated by serum parameters as well as histology. RESULTS: DBTC causes an acute pancreatitis, cholestasis and liver lesions determined by specific elevated serum parameters and several histological lesions. Na-selenite significantly diminished MDA concentration, lipase, bilirubin and transaminases as well as organ injuries compared to only DBTC-treated rats. CONCLUSIONS: The treatment with Na-selenite in the scope of DBTC-induced pancreatitis points to a reduced oxidative stress characterized by diminished MDA serum levels and a milder course of pancreatitis. The generation of DBTC-Na-selenite complexes could also be a mechanism to decrease the toxicity of organotin compounds like DBTC.     

丙酮酸乙酯对急性坏死性胰腺炎大鼠肺损伤的保护作用

目的:观察丙酮酸乙酯(EP)对急性坏死性胰腺炎(ANP)肺损伤大鼠血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和肺组织高迁移率族蛋白B1(HMGB1)mRNA表达的影响,探讨丙酮酸乙酯治疗急性坏死性胰腺炎肺损伤的机制.方法:逆行性胰胆管注射50 g/L牛磺胆酸钠制作ANP模型.随机分成3组,对照组、ANP组和EP治疗组(40 mg/kg,每隔6h静脉注射一次).ELISA法检测血清TNF-α和IL-1β水平;半定量逆转录聚合酶链反应(RT-PCR)法检测肺组织HMGB1 mRNA表达,并观察血氧变化及肺组织的病理变化.结果:ANP组血清TNF-α和IL-1β水平在建模后6h达高峰,12h下降,在此两时点治疗组血清TNF-α和IL-1β水平明显低于ANP组(TNF-α:131.6±29.6 ng/L vs 196.3±16.3 ng/L,65.0±16.6 ng/L vs 90.2±20.1 ng/L,P<0.05;IL-1β:194.9±26.8 ng/L vs 223.0±34.8 ng/L,105.2±24.0 ng/L vs 130.4±23.0 ng/L,P<0.05).ANP组大鼠肺组织HMGB1 mRNA表达水平在ANP后12h明显升高,至24h仍维持在高水平.治疗组肺组织HMGB1 mRNA表达水平在各时间点均明显低于ANP组(0.68±0.11 vs 0.88±0.11,0.81±0.11 vs 1.04±0.10,1.08±0.08 vs 1.33±0.15,P<0.05),且同期肺损伤比ANP组轻.治疗组PaO_2均明显高于ANP组.结论:丙酮酸乙酯能显著抑制TNF-α、IL-1β和HMGB1等早晚期炎症因子,改善低氧血症,对ANP肺损伤有明显保护作用.     

Protective effects of endothelin-1 on acute pancreatitis in rats

Endothelin-1, a 21-residue peptide isolated from vascular endothelial cells, has a broad spectrum of actions. To clarify the involvement of endothelin-1 in acute pancreatitis, we examined the effects of endothelin-1 and its receptor antagonist BQ-123 on cerulein-induced pancreatitis in rats. Rats were infused intravenously with heparin-saline (control), endothelin-1 (100 pmol/kg/hr), cerulein (5 µg/kg/hr), or cerulein plus endothelin-1 for 3.5 hr. In another experiment, cerulein or cerulein plus BQ-123 (3 mg/kg/hr) was infused. Infusion of cerulein caused hyperamylasemia and pancreatic edema. Endothelin-1, when infused with cerulein, decreased the extent of pancreatic edema with a significant increase in the pancreatic dry- to wet-weight ratio. Histological changes induced by cerulein were markedly attenuated when endothelin-1 was given with cerulein. In contrast, endothelin-receptor blockade with BQ-123 further augmented pancreatic edema caused by cerulein. The extent of inflammatory cell infiltration was greater when BQ-123 was given with cerulein. Endothelin-1 or BQ-123 had no influence on hyperamylasemia. This study suggests that endothelin-1 has protective effects on experimental acute pancreatitis.Supported by a grant from the Ministry of Education, Japan (No. B-04454330).     

Experimental chronic interstitial pancreatitis induced by scorpion toxin in rats

Scorpion venom effects in the gastrointestinal system have been investigated both in men and experimental animals. Pancreatic flux and enzyme content are increased by TsTX, the purified venom from the scorpion Tityus serrulatus. In this study male rats received a single intravenous injection of TsTX. They were sacrificed 20 days later and their pancreas removed. Histopathological studies showed interstitial fibrosis, mononuclear infiltrate, acinar atrophy and ductal dilatation. There also appeared, although less frequently, eosinophil infiltrates, ductular hyperplasia and dense eosinophilic secretion in enlarged ducts. All lesions were multifocal. Islet hyperplasia and nesidioblastosis were also observed.