The influence of epidermal growth factor on the course of ischemia-reperfusion induced pancreatitis in rats.

Acute pancreatitis is accompanied by the enhanced expression of EGF in the pancreas and the administration of EGF was found to exhibit the beneficial effect on edematous cerulein-induced pancreatitis. Therefore, we decided to determine the influence of EGF on necro-hemorrhagic pancreatitis induced by ischemia and reperfusion (I/R). Acute pancreatitis was induced in rats by restricting the pancreatic blood flow (PBF) in the inferior splenic artery for 30 min using microvascular clips. EGF was administered three times daily (10 microg/kg per dose s.c.) starting immediately after the clips removal. Rats were sacrificed on day 1, 3, 5, 10 and 21 following ischemia. PBF was measured using a laser Doppler flowmeter. Morphological signs of pancreatitis, as well as the levels of plasma amylase, lipase, interleukin-1beta and interleukin-10 concentration and pancreatic cell proliferation were examined. Results: Ischemia with reperfusion caused acute necro-hemorrhagic pancreatitis with a histological and biochemical manifestation of pancreatic damage, followed by a spontaneous regeneration. The administration of EGF caused the reduction in the histological signs of pancreatic damage, such as necrosis, edema and leukocyte infiltration, and accelerated the pancreatic repair. Also, EGF treatment significantly attenuated the reduction in pancreatic blood flow and DNA synthesis. The activity of plasma amylase and lipase, as well as plasma interleukin-1beta and interleukin-10 concentrations were decreased in EGF treated animals. CONCLUSIONS: EGF exerts beneficial influence on the course of I/R induced pancreatitis and this effect seems to be related to the reduction in the activation of pro-inflammatory interleukin cascade, the improvement of PBF, and the increase in pancreatic cell growth.     

Immunohistochemical expression of FGF-2, PDGF-A, VEGF and TGF beta RII in the pancreas in the course of ischemia/reperfusion-induced acute pancreatitis.

Acute pancreatitis leads to pancreatic damage followed by subsequent regeneration. The aim of our study was to evaluate the presence of growth factors in the course of spontaneous pancreatic regeneration after ischemia/reperfusion (I/R)-induced pancreatitis. METHODS: In rats, I/R was evoked by clamping of splenic artery for 30 min followed by reperfusion. Rats were sacrificed 1, 5, 12 h or 1, 2, 3, 5, 7, 9 or 21 days after removal of vascular clips. Pancreatic blood flow (PBF), plasma lipase, interleukin-1beta (IL-1beta), interleukin-10, pancreatic cells proliferation and morphological signs of pancreatitis were determined. Pancreatic presence of fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), platelet-derived growth factor-A (PDGF-A) and transforming growth factor-beta type II receptor (TGFbeta RII) was detected by immunohistochemisty. RESULTS: Exposure to I/R led to the development of acute necrotizing pancreatitis followed by regeneration. Morphological features showed maximal pancreatic damage between the 1(st) and 2(nd) day of reperfusion. It was correlated with a maximal increase in plasma lipase, and pro-inflammatory IL-1beta concentration, as well as, a reduction in PBF and pancreatic DNA synthesis. I/R increased FGF-2 content in pancreatic acinar cells between the 12(th) and 24(th) h, and between 5(th) and 9(th) day of reperfusion. At the 2(nd) day the presence of FGF-2 in pancreatic acinar cells was reduced. After I/R PDGF-A appeared in pancreatic vessels from the 12(th) h to 5 (th) day of reperfusion. PDGF-A was not observed in pancreatic acinar cells in the control or in I/R group. In pancreatic ducts, the presence of PDGF-A was reduced between the 1(st) and 3(rd), and between 7(th) and 9(th) day of reperfusion. In acinar cells, VEGF content was increased after I/R at the time between the 1(st) and 24(th) h, and between 3(rd) and 7(th) day of reperfusion. At the 2(nd) day of reperfusion, VEGF was not detected in the pancreatic acinar cells. Moreover, VEGF was found in the inflammatory infiltration, in the tubular complexes between the 2(nd) and 5(th) day, and in granulation tissue at the 9(th) day of reperfusion. In pancreatic acinar cells, I/R caused an increase in TGFbeta RII presence between the 5(th) and 24(th) h, and between 7(th) and 9(th) day of reperfusion. Between the 2(nd) and 5(th) day of reperfusion the acinar presence of TGFbeta RII was reduced. In the pancreatic ducts, the presence of TGFbeta RII was increased after I/R from the 1(st) h to 9(th) day of observation. Four weeks after induction of acute pancreatitis, the pancreatic regeneration was completed and the presence of growth factors tested returned to control value. CONCLUSIONS: The presence of FGF, VEGF, PDGF-A and TGFbeta RII is modified in the course of I/R-induced acute pancreatitis. Maximal content of FGF, VEGF and TGFbeta RII has been observed in early stage of pancreatic regeneration suggesting the involvement these factors in pancreatic recovery.     

The influence of sensory nerves and CGRP on the pancreatic regeneration after repeated episodes of acute pancreatitis in rats.

Stimulation of capsaicin sensitive nerves or administration of calcitonin gene-related peptide (CGRP) before induction of acute pancreatitis (AP) attenuates pancreatic damage, whereas CGRP administration after development of AP aggravates lesion of pancreatic tissue. The aim of this study was to determine the effect of prolonged activity of sensory nerves or CGRP administration on the pancreatic repair after repeated episodes of AP. Five episodes of acute caerulein-induced pancreatitis (10 microg/kg/h for 5 h s.c.) were performed at weekly intervals in rats receiving either vehicle or capsaicin at the sensory nerve stimulatory dose (0.5 mg/kg, 3 times daily), or CGRP (10 microg/kg, 3 times daily). Two weeks after the last induction of AP morphological signs of pancreatic damage, pancreatic blood flow (PBF), serum and pancreatic amylase activity, fecal chymotrypsin activity, pancreatic weight, pancreatic RNA and DNA content, as well as, serum interleukin-1beta (Il-1beta ) were assessed. Pancreata of animals receiving vehicle alone showed almost full recovery within two weeks after last episode of pancreatitis induction. In capsaicin-treated group of rats, we observed the increase in PBF by 44% and in serum Il-1beta concentration by 91%. The pancreatic amylase activity, fecal activity of chymotrypsin, pancreatic nucleic acids content and DNA synthesis were decreased. In rats treated with CGRP the alterations in PBF, serum Il-1beta concentration, as well as, in pancreatic and fecal activity of enzymes were similar to capsaicin treated group but less pronounced. We conclude that prolonged activity of capsaicin-sensitive sensory nerves and the presence of their main mediator-CGRP during pancreatic regeneration after AP leads to pancreatic functional insufficiency typical for chronic pancreatitis.     

Calcitonin gene-related peptide can attenuate or augment pancreatic damage in caerulein-induced pancreatitis in rats.

We have recently shown that treatment with calcitonin gene-related peptide (CGRP) before and during induction of acute pancreatitis exhibits a protective effect against pancreatic damage evoked by overdose of caerulein. Studies in the stomach have shown that administration of CGRP exhibits dual action on gastric mucosa, CGRP administration before induction of gastric lesions, protects gastric mucosa against damage, whereas treatment with this peptide after development of gastric ulcer exacerbates mucosal injury. These observations prompt us to determine the influence of CGRP administrated before and after induction of pancreatitis on development and evolution of pancreatic tissue damage. METHODS: Acute pancreatitis was induced by s.c. infusion of caerulein (10 microg/kg/h) for 5 h. CGRP was administrated (10 microg/kg s.c. per dose) 30 min prior to caerulein infusion and 3 h later during caerulein infusion or at the time 1 h, 4 h and 7 h after the end of caerulein infusion. Rats were sacrificed at the time 0 h, 3 h or 9 h after cessation of caerulein administration. The pancreatic blood flow (PBF), plasma activity of amylase, plasma interleukin-1beta concentration, cell proliferation, biochemical and morphological signs of pancreatitis were examined. RESULTS: Caerulein-induced pancreatitis (CIP) led to 42% decrease in DNA synthesis, 30% inhibition of PBF, as well as, a significant increase in pancreatic weight, plasma amylase activity, plasma interleukin-1beta concentration, and development of the histological signs of pancreatic damage (edema, leukocyte infiltration and vacuolization). Treatment with CGRP prior and during induction of CIP attenuated the pancreatic damage what was manifested by partial reversion of the drop in DNA synthesis (40.9+1.7 v. 34.2+2.0 dpm/microg DNA) and PBF (83+3% v. 70+3%). Increases in pancreatic weight and plasma interleukin-1beta were reduced. Morphology showed improvement of pancreatic integrity. Administration of CGRP after induction of CIP aggravated pancreatic damage what was manifested by additional decrease in PBF and DNA synthesis. Also pancreatic weight as well as histological signs of pancreatic damage were increased. CONCLUSIONS: (1) Administration of CGRP before and during induction of pancreatitis protects pancreas against pancreatic damage. (2) Treatment with CGRP after development of CIP aggravates pancreatic damage.     

Effect of ischemic preconditioning on pancreatic regeneration and pancreatic expression of vascular endothelial growth factor and platelet-derived growth factor-A in ischemia/reperfusion-induced pancreatitis.

Ischemic preconditioning has been shown to protect several organs from ischemia/reperfusion-induced injury. In the pancreas, protective effect of ischemic preconditioning has been shown against pancreatitis evoked by ischemia/reperfusion, as well as by caerulein. However, the effect of ischemic preconditioning on the course of acute pancreatic is unclear. The aim of our study was to evaluate the influence of ischemic preconditioning on pancreatic regeneration and pancreatic presence of platelet-derived growth factor-A (PDGF-A) and vascular endothelial growth factor (VEGF) in the course of ischemia/reperfusion-induced pancreatitis. METHODS: In male Wistar rats, ischemic preconditioning of the pancreas was performed by short-term clamping of celiac artery (twice for 5 min with 5 min interval). Acute pancreatitis was induced by clamping of inferior splenic artery for 30 min followed by reperfusion. Rats were sacrificed 1, 5, 12 h or 1, 2, 3, 5, 7, 9 and 21 days after the start of reperfusion. Severity of acute pancreatitis and pancreatic regeneration were determined by biochemical and morphological examination, expression of growth factors was determined by immunohistochemical analysis. RESULTS: In ischemia/reperfusion-induced pancreatitis, the pancreatic damage reached the maximal range between the first and second day of reperfusion, and was followed by subsequent pancreatic regeneration. Ischemic preconditioning alone caused mild passing pancreatic damage and an increase in plasma concentration of pro-inflammatory interleukin-1 and anti-inflammatory interleukin-10. Ischemic preconditioning applied before ischemia/reperfusion-induced pancreatitis reduced morphological and biochemical signs of the pancreatitis-evoked pancreatic damage and accelerated pancreatic regeneration. This effect was associated with improvement of pancreatic blood flow. Ischemic preconditioning, ischemia/reperfusion-induced pancreatitis and their combination increased the presence of VEGF in acinar and islet cells, and immunostaining for PDGF-A in blood vessels. This effect was maximally pronounced after combination of ischemic preconditioning plus pancreatitis and occurred earlier than after pancreatitis alone. CONCLUSIONS: Ischemic preconditioning reduces pancreatic damage and accelerates pancreatic healing in the course of ischemia/reperfusion-induced pancreatitis. This effect is associated with the increase in plasma concentration of anti-inflammatory interleukin-10, improvement of pancreatic blood flow and alteration of pancreatic immunohistochemical expression of PDGF-A and VEGF.     

IGF-1 stimulates production of interleukin-10 and inhibits development of caerulein-induced pancreatitis.

BACKGROUND/AIM: Insulin-like growth factor-1 (IGF-1) and other growth factors overexpression was reported in acute pancreatitis. Previous studies have shown the protective effect of epidermal growth factor (EGF), Hepatocyte Growth Factor (HGF) and Fibroblast Growth Factor (FGF) in the course of experimental acute pancreatitis. The aim of our studies was to determine the effect of IGF-1 administration on the development of caerulein-induced pancreatitis. METHODS: Acute pancreatitis was induced by infusion of caerulein (10 micro/kg/h) for 5 h. IGF-1 was administrated twice at the doses: 2, 10, 50, or 100 micro/kg s.c. RESULTS: Administration of IGF-1 without induction of pancreatitis increased plasma interleukin-10 (IL-10). Infusion of caerulein led to development of acute edematous pancreatitis. Histological examination showed pancreatic edema, leukocyte infiltration and vacuolization of acinar cells. Also, acute pancreatitis led to an increase in plasma lipase and interleukin 1beta (IL-1beta) level, whereas pancreatic DNA synthesis and pancreatic blood flow were decreased. Treatment with IGF-1, during induction of pancreatitis, increased plasma IL-10 and attenuated the pancreatic damage, what was manifested by histological improvement of pancreatic integrity, the partial reversion of the drop in pancreatic DNA synthesis and pancreatic blood flow, and the reduction in pancreatitis-evoked increase in plasma amylase, lipase and IL-1beta level. Protective effect of IGF-1 administration was dose-dependent. Similar strong protective effect was observed after IGF-1 at the dose 2 x 50 and 2 x 100 microg/kg. CONCLUSIONS: (1) Administration of IGF-1 attenuates pancreatic damage in caerulein-induced pancreatitis; (2) This effect is related, at least in part, to the increase in IL-10 production, the reduction in liberation of IL-1beta and the improvement of pancreatic blood flow.     

Ghrelin attenuates the development of acute pancreatitis in rat.

BACKGROUND: Ghrelin, a circulating growth hormone-releasing peptide isolated from human and rat stomach, stimulates growth hormone secretion, food intake and exhibits gastroprotective properties. Ghrelin is predominantly produced by a population of endocrine cells in the gastric mucosa, but its presence in bowel, pancreas, pituitary and hypothalamus has been reported. In human fetal pancreas, ghrelin is expressed in a prominent endocrine cell population. In adult pancreatic islets the population of these cell is reduced. The aim of present study was to investigate the influence of ghrelin administration on the development of acute pancreatitis. METHODS: Acute pancreatitis was induced in rat by caerulein injection. Ghrelin was administrated twice (30 min prior to the first caerulein or saline injection and 3 h later) at the doses: 2, 10 or 20 nmol/kg. Immediately after cessation of caerulein or saline injections the following parameters were measured: pancreatic blood flow, plasma lipase activity, plasma interleukin-1beta (IL-1beta) and interleukin 10 (IL-10) concentration, pancreatic DNA synthesis, and morphological signs of pancreatitis. RESULTS: Administration of ghrelin without induction of pancreatitis did not affect significantly any parameter tested. Caerulein led to the development of acute edematous pancreatitis. Treatment with ghrelin at the dose 2 nmol/kg, during induction of pancreatitis, was without effect on pancreatic histology or biochemical and functional parameters. Treatment with ghrelin at the dose 10 and 20 nmol/kg attenuated the development of pancreatitis and the effects of both doses were similar. Administration of ghrelin (10 or 20 nmol/kg) reduced inflammatory infiltration of pancreatic tissue and vacuolization of acinar cells. Also, plasma lipase activity and plasma IL-1beta concentration were reduced, and caerulein-induced fall in pancreatic DNA synthesis was reversed. Administration of ghrelin at the dose 10 and 20 nmol/kg was without effect on caerulein-induced pancreatic edema and pancreatitis-related fall in pancreatic blood flow. CONCLUSIONS: (1) Administration of ghrelin attenuates pancreatic damage in caerulein-induced pancreatitis; (2) Protective effect of ghrelin administration seems Background: Ghrelin, a circulating growth hormone-releasing peptide isolated from human and rat stomach, stimulates growth hormone secretion, food intake and exhibits gastroprotective properties. Ghrelin is predominantly produced by a population of endocrine cells in the gastric mucosa, but its presence in bowel, pancreas, pituitary and hypothalamus has been reported. In human fetal pancreas, ghrelin is expressed in a prominent endocrine cell population. In adult pancreatic islets the population of these cell is reduced. The aim of present study was to investigate the influence of ghrelin administration on the development of acute pancreatitis. Methods: Acute pancreatitis was induced in rat by caerulein injection. Ghrelin was administrated twice (30 min prior to the first caerulein or saline injection and 3 h later) at the doses: 2, 10 or 20 nmol/kg. Immediately after cessation of caerulein or saline injections the following parameters were measured: pancreatic blood flow, plasma lipase activity, plasma interleukin-1beta (IL-1beta) and interleukin 10 (IL-10) concentration, pancreatic DNA synthesis, and morphological signs of pancreatitis. Results: Administration of ghrelin without induction of pancreatitis did not affect significantly any parameter tested. Caerulein led to the development of acute edematous pancreatitis. Treatment with ghrelin at the dose 2 nmol/kg, during induction of pancreatitis, was without effect on pancreatic histology or biochemical and functional parameters. Treatment with ghrelin at the dose 10 and 20 nmol/kg attenuated the development of pancreatitis and the effects of both doses were similar. Administration of ghrelin (10 or 20 nmol/kg) reduced inflammatory infiltration of pancreatic tissue and vacuolization of acinar cells. Also, plasma lipase activity and plasma IL-1beta conc; concentration were reduced, and caerulein-induced fall in pancreatic DNA synthesis was reversed. Administration of ghrelin at the dose 10 and 20 nmol/kg was without effect on caerulein-induced pancreatic edema and pancreatitis-related fall in pancreatic blood flow. Conclusions: (1) Administration of ghrelin attenuates pancreatic damage in caerulein-induced pancreatitis; (2) Protective effect of ghrelin administration seems to be related the inhibition in inflammatory process and the reduction in liberation of pro-inflammatory IL-1beta.     

Influence of ischemic preconditioning on blood coagulation, fibrinolytic activity and pancreatic repair in the course of caerulein-induced acute pancreatitis in rats.

Previous studies have shown that ischemic preconditioning protects several organs, including the pancreas, from ischemia/reperfusion-induced injury. The aim of the investigation was to determine whether ischemic preconditioning affects the course edematous pancreatitis. METHODS: In rats, ischemic preconditioning was performed by short-term clamping the celiac artery. Acute pancreatitis was induced by caerulein. The severity of acute pancreatitis was evaluated between the first and tenth day of inflammation. RESULTS: Ischemic preconditioning applied alone caused a mild pancreatic damage. Combination of ischemic preconditioning with caerulein attenuated the severity of pancreatitis in histological examination and reduced the pancreatitis-evoked increase in plasma lipase and pro-inflammatory interleukin-1beta. This effect was associated with an increase in plasma level of anti-inflammatory interleukin-10 and partial reversion of the pancreatitis-evoked drop in pancreatic DNA synthesis and pancreatic blood flow. In secretory studies, ischemic preconditioning in combination with induction of acute pancreatitis attenuated the pancreatitis-evoked decrease in secretory reactivity of isolated pancreatic acini to stimulation by caerulein. In the initial period of acute pancreatitis, ischemic preconditioning alone and in combination with caerulein-induced acute pancreatitis prolonged the activated partial thromboplastin time (APTT), increased plasma level of D-dimer and shortened the euglobulin clot lysis time. The protective effect of ischemic preconditioning was observed during entire time of experiment and led to acceleration of pancreatic regeneration. CONCLUSIONS: Ischemic preconditioning reduces the severity of caerulein-induced pancreatitis and accelerates pancreatic repair; and this effect is related to the activation of fibrinolysis and reduction of inflammatory process.     

Involvement of thrombopoietin in acinar cell necrosis in l-arginine-induced acute pancreatitis in mice

Thrombopoietin (TPO) plays an important role in injuries of different tissues. However, the role of TPO in acute pancreatitis (AP) is not yet known. The aim of the study was to determine the involvement of TPO in AP. Serum TPO was assayed in necrotizing pancreatitis induced by l-arginine in mice. Recombinant TPO and anti-TPO antibody were given to mice with necrotizing pancreatitis. Amylase, lipase, lactate dehydrogenase, myeloperoxidase activity and pancreatic water content were assayed in serum and tissue samples. Pancreas and lung tissue samples were also collected for histological evaluation. Immunohistochemistry of amylase α and PCNA were applied for the study of acinar regeneration and TUNEL assay for the detection of apoptosis in the pancreas. Increased levels of serum TPO were found in necrotizing pancreatitis. After TPO administration, more severe acinar necrosis was found and blockade of TPO reduced the acinar necrosis in this AP model. Acinar regeneration and apoptosis in the pancreas were affected by TPO and antibody treatment in necrotizing pancreatitis. The severity of pancreatitis-associated lung injury was worsened after TPO treatment, but attenuated after Anti-TPO antibody treatment. In conclusion, serum TPO is up-regulated in the necrotizing pancreatitis induced by l-arginine in mice and may be a risk factor for the pancreatic acinar necrosis in AP. As a pro-necrotic factor, blockade of TPO can attenuate the acinar necrosis in AP and may be a possible therapeutic intervention for AP.     

Effects of gabexate mesilate on serum inflammatory cytokines in rats with acute necrotizing pancreatitis

Gabexate mesilate is a synthetic protease inhibitor. The effectiveness of gabexate mesilate in patients with acute pancreatitis is controversial. Proinflammatory cytokines are associated with systemic inflammatory response syndrome (SIRS) in acute pancreatitis. A compensatory anti-inflammatory response occurs in parallel with SIRS. We investigated the effects of gabexate mesilate on acute necrotizing pancreatitis in rats, emphasizing the changes in serum levels of proinflammatory and anti-inflammatory cytokines. Acute necrotizing pancreatitis was induced by retrograde infusion of sodium taurodeoxycholate into the pancreatobiliary duct in rats. The rats were divided into three groups. Group I was given gabexate mesilate 2 mg/kg/h i.v. continuously 1 h before the induction of acute pancreatitis. Group II was given gabexate mesilate the same dose immediately after the induction of acute pancreatitis. Group III was given normal saline as the controls. Serum levels of amylase, lipase, tumor necrosis factor alpha, interleukin-6, and interleukin-10, pancreatic histopathology and hemodynamics were examined at 5h after the induction of acute pancreatitis. Gabexate mesilate significantly reduced serum levels of amylase, lipase, tumor necrosis factor alpha and interleukin-6 at 5 h. Serum levels of interleukin-10 significantly increased in Group I, as compared with Groups II and III. The severity of pancreatic histopathology, the reduction of mean arterial pressure, the volume of ascites and pancreatic wet weight/body weight ratios were also significantly improved by the administration of gabexate mesilate. The beneficial effects of gabexate mesilate on acute pancreatitis may be, in part, due to the modulation of inflammatory cytokine responses.     

电针联合ICE抑制剂在急性胰腺炎的治疗中的作用及机制

观察和探讨电针联合白细胞介素(IL-1β)转化酶(ICE)抑制剂对炎症介质抑制和诱导急性胰腺炎腺泡细胞凋亡的作用。选取90只SD大鼠随机分为对照组、模型组和干预组,每组30只。各组造模后在第6小时、第12小时和第24小时各时间点分别检测10只大鼠。抑制剂组于造模前48 h采用电针并开始腹腔注射ICE抑制剂2.5 mg/100 mg体质量,间隔12 h注射1次。胰腺炎组仅在相同时间注射同等量的生理盐水。应用细胞凋亡原位标记(TUNEL)染色、ELISA方法等,检测血清中淀粉酶、肿瘤坏死因子-α(TNF-α)、IL-1β和胰腺细胞凋亡。HE染色见胰腺组织中典型的细胞核固缩及凋亡小体形成。干预组各个时间点病理学评分和血淀粉酶、TNF-α、IL-1β浓度均低于胰腺炎组,胰腺细胞凋亡指数高于胰腺炎组(p<0.05)。电针联合ICE抑制剂能减轻急性胰腺炎严重程度,其机制可能与其对炎症介质的抑制和诱导胰腺细胞的凋亡有关。     

清胰汤对大鼠急性坏死性胰腺炎IL-6,IL-10及肠道通透性影响

目的：探讨清胰汤改善大鼠急性坏死性胰腺炎（acute necrotizing pancreatitis）ANP炎症反应及肠道通透性功能的治疗效果及机制。方法：将72只雄性SD大鼠随机分为3组,其中2组大鼠采用从胰腺被膜下多点缓慢均匀注入3.8%牛黄胆酸钠（0.5ml/100g）建立大鼠急性坏死性胰腺炎模型,再分为急性坏死性胰腺炎常规治疗组（A组）、清胰汤干预治疗组（B组）,其他24只大鼠为假手术组（S组）,每组再随机分为24h、48h、72h组。各组于12h后给于肠内营养,B组肠内营养后给于2次清胰汤2.5ml/100g,A组、S组给于同等剂量生理盐水。各组于建模后24h、48h、72h处死,腹腔动脉取血检测血清淀粉酶浓度、IL-6、IL-10、D-乳酸水平。结果：48h时点B组IL-10水平较A组高（P〈0.05）;72时点B组血清淀粉酶水平较A组低（P〈0.01）,IL-6水平较A组低（P〈0.01）,IL-10水平较A组高（P〈0.01）,D-乳酸水平较A组低（P〈0.01）。结论：清胰汤可以上调IL-10改善大鼠急性胰腺炎炎症反应从而降低肠道通透性。     

Prevention of ischemia-reperfusion injury in rat ovarian tissue with the on-off method

Ischemia is defined as cell death caused by insufficient perfusion of the tissue due to reduction in arterial or venous blood flow, depletion of cellular energy storages, and accumulation of toxic metabolites. The positive effects of controlled reperfusion are known and are used clinically. But the positive effects of controlled reperfusion on ovarian tissue have not been seen in the literature yet. The biochemical and histopathological comparative investigation of rat ovaries that were experimentally exposed to ischemia (IG), ischemia-reperfusion (I/R), and ischemia-controlled reperfusion (ICR) was aimed. Forty rats were divided into four groups (10 rats per group). First group: 3 h ischemia by vascular clips on ovarian tissue. Second group: 3 h ischemia + 1 h reperfusion. Third group: 3 h ischemia + 1 h controlled reperfusion (on-off method: controlled reperfusion by opening and closing the clips (on/off) in 10-second intervals, for 5 times for a total of 100 seconds). Fourth group: healthy rats. Biochemical (tGSH, MDA, and DNA damage level and SOD activity) and histopathological analysis were performed. The highest glutathione and superoxide dismutase measurements were found in ischemia/controlled reperfusion group among the ischemia or ischemia/reperfusion groups. Similarly the damage indicators (malondialdehyde, DNA damage level and histopathological damage grade) were the lowest in ischemia/controlled reperfusion group. These results indicate that controlled reperfusion can be helpful in minimizing ischemia-reperfusion injury in ovarian tissue exposed to ischemia for various reasons (ovarian torsion, tumor, etc.).     

Interleukin-1 beta is a potent growth inhibitor of adult rat hepatocytes in primary culture

Interleukin-1 beta (IL-1 beta) strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin and epidermal growth factor (EGF). Its effect was dose-dependent and was maximal at 2 ng/ml. IL-1 beta had no cytotoxic effect but changed the cells from a flat to a spindle shape as shown by phase-contrast microscopy. The inhibition of DNA synthesis by IL-1 beta was closely correlated with a decrease in the labeling index. This inhibitory effect was observed only when IL-1 beta was added for 10 h to cultured hepatocytes in the G1 phase within 12 h after addition of insulin and EGF: it was not observed in the S phase, which starts about 24 h after addition of the mitogens. Exposure of the hepatocytes to IL-1 beta for two 1-h periods, one at an early stage (0-6 h) and one at a late stage (6-12 h) of the G1 phase, resulted in the same marked inhibition of DNA synthesis as exposure to IL-1 beta for 10 h in the G1 phase. This requirement of IL-1 beta at two stages in the G1 phase for inhibition of DNA synthesis of hepatocytes is different from that with transforming growth factor-beta, which is required for only 1 h in the early G1 phase for a similar inhibition. These findings suggest that IL-1 beta acts at two distinct stages in the G1 phase and that its cooperative actions are necessary to inhibit growth of adult rat hepatocytes in primary culture. Other cytokines, such as IL-6/B-cell stimulating factor-2, were less potent, but caused significant inhibition of DNA synthesis of adult rat hepatocytes at 2 ng/ml, whereas IL-2 and tumor necrosis factor did not affect hepatocyte growth. From these results it is suggested that Kupffer cells in liver lobules and macrophages in the blood may play important roles, mainly via IL-1, in repair of liver damage and regeneration.     

肠缺血/再灌注损伤后白介素1-β水平与磷脂酶A2激活的关系

为了探讨肠缺血/再灌注损伤后IL-1β基因表达和蛋白含量变化与磷脂酶A2抑制之间的关系,采用大鼠肠缺血/再灌注损伤模型,在对照组,损伤组和磷脂酶A2抑制剂处理组动物中收集血清,肺灌洗液,腹腔灌洗液及全身重要脏器组织样品,采用放射免疫法测定IL-1β含量,并且RT-PCR法测定肺组织中IL-1β和Ⅱ型PLA2基因表达,结果表明,损伤后6h血清中IL-1β含量明显高于对照组;损伤后1和3h,腹腔注保IL-1β也明显高于对照组;损伤后肝组织中IL-1β水平有明显增加,而肺,肾、肠组织中IL-1β没有明显变化。损伤后肺灌洗液中IL-1β也明显高于对照组水平,肺组织中IL-1βmRNA表达增加,而Ⅱ型PLA2mRNA在损伤后表达反而有所下降,采用磷脂酶A2抑制剂氯喹,环氧化物酶抑制剂消炎痛,血小板活化因子受体阻断剂SR27417后,IL-1β蛋白和基因表达有不同的改变,提示肠缺血/再灌注损伤后一定时间内,肝内IL-1βmRNA表达和血中IL-1β水平明显增高,但是否与磷脂酶A2激活或其代谢产物的释放有关尚需进一步证明。     

Extract of grapefruit-seed reduces acute pancreatitis induced by ischemia/reperfusion in rats: possible implication of tissue antioxidants.

Grapefruit seed extract (GSE) has been shown to exert antibacterial, antifungal and antioxidant activity possibly due to the presence of naringenin, the flavonoid with cytoprotective action on the gastric mucosa. No study so far has been undertaken to determine whether this GSE is also capable of preventing acute pancreatic damage induced by ischemia/reperfusion (I/R), which is known to result from reduction of anti-oxidative capability of pancreatic tissue, and whether its possible preventive effect involves an antioxidative action of this biocomponent. In this study carried out on rats with acute hemorrhagic pancreatitis induced by 30 min partial pancreatic ischemia followed by 6 h of reperfusion, the GSE or vehicle (vegetable glycerin) was applied intragastrically in gradually increasing amounts (50-500 microl) 30 min before I/R. Pretreatment with GSE decreased the extent of pancreatitis with maximal protective effect of GSE at the dose 250 microl. GSE reduced the pancreatitis-evoked increase in serum lipase and poly-C specific ribonuclease activity, and attenuated the marked fall in pancreatic blood flow and pancreatic DNA synthesis. GSE administered alone increased significantly pancreatic tissue content of lipid peroxidation products, malondialdehyde and 4-hydroxyalkens, and when administered before I/R, GSE reduced the pancreatitis-induced lipid peroxidation. We conclude that GSE exerts protective activity against I/R-induced pancreatitis probably due to the activation of antioxidative mechanisms in the pancreas and the improvement of pancreatic blood flow.     

Endotoxemia in newborn rats attenuates acute pancreatitis at adult age.

Bacterial endotoxin (lipopolysaccharide, LPS), at high concentration is responsible for sepsis, and neonatal mortality, however low concentration of LPS protected the pancreas against acute damage. The aim of this study was to investigate the effect of exposition of suckling rats to LPS on the course of acute pancreatitis at adult age. Suckling rat (30-40g) received intraperitoneal (i.p.) injection of saline (control) or LPS from Escherichia coli or Salmonella typhi (5, 10 or 15 mg/kg-day) during 5 consecutive days. Two months later these rats have been subjected to i.p. cearulein infusion (25 microg/kg) to produce caerulein-induced pancreatitis (CIP). The following parameters were tested: pancreatic weight and morphology, plasma amylase and lipase activities, interleukin 1beta (IL-1 beta), interleukin 6 (IL-6), and interleukin 10 (IL-10) plasma concentrations. Pancreatic concentration of superoxide dismutase (SOD) and lipid peroxidation products; malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) have been also measured. Caerulein infusion produced CIP in all animals tested, that was confirmed by histological examination. In the rats, which have been subjected in the neonatal period of life to LPS at doses 10 or 15 mg/kg-day x 5 days, all manifestations of CIP have been reduced. In these animals acute inflammatory infiltration of pancreatic tissue and pancreatic cell vacuolization have been significantly diminished. Also pancreatic weight, plasma lipase and alpha-amylase activities, as well as plasma concentrations of IL-1beta and IL-6 have been markedly decreased, whereas plasma anti-inflammatory IL-10 concentration was significantly increased in these animals as compared to the control rats, subjected in the infancy to saline injection instead of LPS. Caerulein-induced fall in pancreatic SOD concentration was reversed and accompanied by significant reduction of MDA + 4 HNE in the pancreatic tissue. The effects of LPS derived from E. coli or S. typhi were similar. Pretreatment of suckling rats with LPS at dose of 10 mg/kg-day x 5 days resulted in the most prominent attenuation of acute pancreatitis at adult age, whereas LPS at dose of 5 mg/kg-day x 5 days given to the neonatal rats failed to affect significantly acute pancreatitis induced in these animals 2 months later. We conclude that: 1/ Prolonged exposition of suckling rats to bacterial endotoxin attenuated acute pancreatitis induced in these animals at adult age. 2/ This effect could be related to the increased concentration of antioxidative enzyme SO in the pancreatic tissue and to the modulation of cytokines production in these animals.     

The recovery of acute pancreatitis depends on the enzyme amount stored in zymogen granules at early stages

Little is known about the changes in pancreatic enzyme storage in acute pancreatitis. We have performed flow cytometric studies of zymogen granules from rats with acute pancreatitis induced by hyperstimulation with caerulein. A comparison was made with rats treated with hydrocortisone (10 mg/kg/day) over 7 days before inducing pancreatitis in order to find out whether the amount of enzymes stored in the pancreas plays a key role in the development of pancreatitis. The potentially therapeutic effect of L-364,718 (0.1 mg/kg/day, for 7 days), a CCK receptor antagonist, was assayed in the rats with caerulein-induced pancreatitis which had previously received the hydrocortisone treatment. A significant increase in the intragranular enzyme content was observed 5 h after hyperstimulation with caerulein. The highest values were reached in the rats previously treated with hydrocortisone. The greatest pancreatic enzyme load was parallel to the highest values in plasma amylase, edema and haematocrit observed. Acute pancreatitis was reversed seven days later. At this stage smaller granules appeared in the pancreas whose enzyme content was similar to that of controls when no treatment was applied after pancreatitis. In contrast, L-364,718 administration prevented the favourable evolution of pancreatitis since the antagonism exerted on CCK receptors induced a blockade of secretion of the large amounts of enzymes stored in the pancreas. Moreover, the enzyme content in zymogen granules was below normal values since the stimulatory CCK action on enzyme synthesis can be inhibited by L-364,718. Our results suggest that the efficiency of CCK antagonists, as potential therapy, would also depend on the load of enzymes in the pancreas when acute pancreatitis is produced.     

Pathophysiological role of platelets in acute experimental pancreatitis: influence of endothelin A receptor blockade

The potential pathophysiological role of platelet-endothelium interactions was investigated during ischemia/reperfusion (I/R), and the effect of a selective endothelin(A) receptor antagonist (ET(A)-RA) was evaluated in an acute pancreatitis model. Acute pancreatitis was induced by warm ischemia (60 min) in Wistar rats, and its effects with and without antagonist treatment were investigated. Equivalent sham-operated animals were also studied. Microcirculatory changes were assessed by in vivo microscopy, and serum levels for lipase/amylase and histological specimens were investigated. Capillary constriction to 83.7 +/- 6.7% of sham-operated diameters was observed after 60 min of ischemia. A capillary density of 56.8 +/- 9.3% of the sham-operated group (396.3 +/- 15.8 mm(-1)) was measured after reperfusion. Stagnant leukocytes (329.5 +/- 30.4%) and platelets (337.5 +/- 32.3%) increased in postcapillary venules (P < 0.05). Administration of the ET(A)-RA significantly reduced I/R injury. Capillary diameters were maintained (101.4 +/- 4.5%), and capillary density was improved to 73.3 +/- 7.6% of sham-operated animals (P < 0.05). Stagnant leukocytes (152.3 +/- 10.6%) and platelets (207.1 +/- 19.8%) in sinusoids and postcapillary venules were reduced (P < 0.05). The extent of acute pancreatitis was reduced in the therapy group as indicated by serum lipase/amylase values and histological tissue damage (P < 0.05). Thus, ET(A)-RA therapy was effective in reducing I/R-induced pancreatitis in this experimental model.