Soluble complement receptor 1 preserves endothelial barrier function and microcirculation in postischemic pancreatitis in the rat

Components of the activated complement cascade are considered to play a pivotal role in ischemia-reperfusion-induced organ injury. With the use of intravital epifluorescence microscopy, we investigated the effect of complement inhibition by the recombinant soluble complement receptor 1 (sCR1; TP10) on the effect of macromolecular microvascular permeability, functional capillary perfusion, and leukocyte endothelium interaction in postischemic pancreatitis. Anaesthetized Sprague-Dawley rats were subjected to 60 min of normothermic pancreatic ischemia induced by microclipping of the blood-supplying arteries of the organ. Rats who received sCR1 (15 mg/kg body wt iv; n = 7) during reperfusion showed a significant reduction of permeability (1.77 +/- 1.34 x 10(-8) cm/s; n = 7) of tetramethylrhodamine isothiocyanate-labeled albumin injected 90 min after the onset of reperfusion compared with vehicle-treated animals (6.95 +/- 1.56 x 10(-8) cm/s; n = 7). At 120 min after the onset of reperfusion, the length of red blood cell-perfused capillaries (functional capillary density) was significantly improved (from 279 +/- 15.7 to 330 +/- 3.7 cm(-1); n = 7) and the number of leukocytes adherent to postcapillary venules was significantly reduced (from 314 +/- 87 to 163 +/- 71 mm(-2); n = 7) by sCR1 compared with vehicle treatment. Complement inhibition by sCR1 effectively ameliorates pancreatic ischemia-reperfusion-induced microcirculatory disturbances and might be considered for treatment of postischemic pancreatitis.     

Prophylactic and therapeutic treatments with AT 1 and AT 2 receptor antagonists and their effects on changes in the severity of pancreatitis

Previous studies showed that a local pancreatic renin-angiotensin system (RAS) was upregulated in experimental acute pancreatitis. RAS inhibition could attenuate pancreatic inflammation and fibrosis, which casts a new light on the role of the pancreatic RAS in pancreatitis. The present study explores the prophylactic and therapeutic potentials, and possible molecular mechanism for the antagonism of angiotensin II receptors on the changes in the severity of pancreatic injury induced by acute pancreatitis. Experimental pancreatitis was induced by an intraperitoneal injection of supra-maximal dose of cerulein. The differential effects of angiotensin II receptors inhibitors losartan and PD123319 on the pancreatic injury were assessed by virtue of using the pancreatic water content, biochemical and histological analyses. Blockade of the AT(1) receptor by losartan at a dose of 200microg/kg could markedly ameliorate the pancreatic injury induced by cerulein, as evidenced by biochemical and histopathological studies. However, blockade of the AT(2) receptor by PD123319 appeared not to provide any beneficial role in cerulein-induced pancreatic injury. Both prophylactic and therapeutic treatments with losartan were effective against cerulein-induced pancreatic injury. The protective action of losartan was linked to an inhibition of NAD(P)H oxidase activity, thus consequential oxidative modification of pancreatic proteins in the pancreas. Inhibition of the AT(1) receptor, but not AT(2) receptor, may play a beneficial role in ameliorating the severity of acute pancreatitis. The differential effects of AT(1) and AT(2) inhibitors on cerulein-induced pancreatic injury might be due to the distinctive mechanism of the AT(1) and AT(2) receptors on the activation of NAD(P)H oxidase. Thus the protective role of AT(1) receptor antagonist, losartan, could be mediated by the inhibition of NAD(P)H oxidase-dependent generation of reactive oxygen species (ROS).     

Diphenyleneiodonium suppresses apoptosis in cerulein-stimulated pancreatic acinar cells

NADPH oxidase has been considered a major source of reactive oxygen species in phagocytic and non-phagocytic cells. Apoptosis linked to oxidative stress has been implicated in pancreatitis. Recently, we demonstrated that NADPH oxidase subunits Nox1, p27phox, p47phox, and p67phox are constitutively expressed in pancreatic acinar cells, which are activated by cerulein, a cholecystokinin analogue. Cerulein induces an acute and edematous form of pancreatitis. We investigated whether inhibition of NADPH oxidase by diphenyleneiodonium suppresses the production of reactive oxygen species and apoptosis by determining viable cell numbers, DNA fragmentation, TUNEL staining, caspase-3 activity, and the expression of apoptosis-inducing factor in pancreatic acinar AR42J cells stimulated with cerulein. Inhibition on NADPH oxidase by diphenyleneiodonium was assessed by the alterations in NADPH oxidase activity and translocation of the cytosolic subunits p67phox and p47phox to the membrane. Intracellular Ca2+ level was monitored to investigate the relationship between NADPH oxidase and Ca2+ in cells stimulated with cerulein. As a result, cerulein induced the activation of NADPH, increased production of reactive oxygen species, and apoptotic indices determined by the expression of apoptosis-inducing factor, caspase-3 activation, TUNEL staining, DNA fragmentation, and cell viability. Treatment with DPI inhibited cerulein-induced activation of NADPH oxidase, the production of reactive oxygen species, and apoptosis, but not the increase of intracellular Ca2+ levels in pancreatic acinar cells. These results demonstrate that the cerulein-induced increase in intracellular Ca2+ level may be an upstream event of NADPH oxidase activation. Diphenyleneiodonium, an NADPH oxidase inhibitor, inhibits the expression of apoptosis-inducing factor and caspase-3 activation, and thus apoptosis in pancreatic acinar cells.     

Fibroblast growth factor 21 alleviates acute pancreatitis via activation of the Sirt1‐autophagy signalling pathway

Fibroblast growth factor 21 (FGF21), a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity, alleviates the process of acute pancreatitis (AP). However, its mechanism remains elusive. The pathological and physiological characteristics of FGF21 are observed in both patients with AP and cerulein‐induced AP models, and the mechanisms of FGF21 in response to AP are investigated by evaluating the impact of autophagy in FGF21‐treated mice and cultured pancreatic cells. Circulating levels of FGF21 significantly increase in both AP patients and cerulein‐induced AP mice, which is accompanied by the change of pathology in pancreatic injury. Replenishment of FGF21 distinctly reverses cerulein‐induced pancreatic injury and improves cerulein‐induced autophagy damage in vivo and in vitro. Mechanically, FGF21 acts on pancreatic acinar cells to up‐regulate Sirtuin‐1 (Sirt1) expression, which in turn repairs impaired autophagy and removes damaged organs. In addition, blockage of Sirt1 accelerates cerulein‐induced pancreatic injury and weakens the regulative effect in FGF21‐activated autophagy in mice. These results showed that FGF21 protects against cerulein‐induced AP by activation of Sirtuin‐1‐autophagy axis.     

Protective effects of soluble CR1 in complement- and neutrophil-mediated tissue injury.

Complement activation is an important step for triggering of acute inflammatory reactions. Soluble human recombinant complement receptor type 1 (sCR1) blocks complement activation by both classical and alternative pathways. In addition to glycogen-induced peritonitis, three models of complement-dependent acute inflammatory injury have been used to assess the protective effects of sCR1: lung and dermal injury after intraalveolar or intradermal deposition of IgG immune complexes; acute lung injury resulting from intravascular activation of complement after the i.v. injection of cobra venom factor; and acute skin and lung injury (at 4 h) after thermal trauma involving 25 to 30% total body surface area. Vascular injury was quantified by increases in vascular permeability, hemorrhage, neutrophil infiltration, and, as indicated, tissue water content. Intravenous infusion of sCR1 reduced lung and dermal vascular injury in all models studied. In glycogen-induced peritoneal exudates sCR1-reduced neutrophil accumulation by 79%. In animals undergoing IgG immune complex-induced alveolitis, sCR1 treatment reduced vascular permeability and hemorrhage by 72 and 71%, respectively, and tissue accumulation of neutrophils was reduced by 68%. After cobra venom factor injection, sCR1 reduced increases in lung vascular permeability by 67%, hemorrhage by 73%, and lung myeloperoxidase content by 55%. Four hours after thermal injury of skin, sCR1-treated animals demonstrated significant protection against lung injury; increases in vascular permeability and hemorrhage were reduced by 45 and 46%, respectively, and myeloperoxidase content was lowered by 39%. In thermal injury of the skin, sCR1 injection reduced dermal vascular permeability by 25% at 1 h (p = NS) and 44% at 4 h. Water content in skin biopsies was also decreased. There was a dose-response relationship between the amount of sCR1 infused and the extent of protection in each of the injury models. These data demonstrate that sCR1 offers significant protection against complement-dependent tissue injury in the animal models studied and that the protective effects are related to reduced neutrophil content.     

Organ microcirculatory disturbances in experimental acute pancreatitis. A role of nitric oxide

Microcirculatory disturbances are important early pathophysiological events in various organs during acute pancreatitis (AP). The aim of the study was to investigate an influence of L-arginine (nitric oxide substrate) and N(G)-nitro-L-arginine (L-NNA, nitric oxide synthase inhibitor) on organ microcirculation in experimental acute pancreatitis induced by four consecutive intraperitoneal cerulein injections (15 microg/kg/h). The microcirculation of pancreas, liver, kidney, stomach, colon and skeletal muscle was measured by laser Doppler flowmeter. Serum interleukin 6 and hematocrit levels were analyzed. AP resulted in a significant drop of microperfusion in all examined organ. L-arginine administration (2 x 100 mg/kg) improved the microcirculation in the pancreas, liver, kidney, colon and skeletal muscle, and lowered hematocrit levels. L-NNA treatment (2 x 25 mg/kg) caused aggravation of edematous AP to the necrotizing situation, and increased IL-6 and hematocrit levels. A further reduction of blood perfusion was noted in the stomach only. It is concluded that L-arginine administration has a positive influence on organ microcirculatory disturbances accompanying experimental cerulein-induced AP. NO inhibition aggravates the course of pancreatitis.     

Endothelial targeting and enhanced antiinflammatory effects of complement inhibitors possessing sialyl Lewisx moieties

The complement inhibitor soluble complement receptor type 1 (sCR1) and a truncated form of sCR1, sCR1[desLHR-A], have been generated with expression of the selectin-reactive oligosaccharide moiety, sialyl Lewisx (sLex), as N-linked oligosaccharide adducts. These modified proteins, sCR1sLex and sCR1[desLHR-A]sLex, were assessed in the L-selectin- and P-selectin-dependent rat model of lung injury following systemic activation of complement by cobra venom factor and in the L-selectin-, P-selectin-, and E-selectin-dependent model of lung injury following intrapulmonary deposition of IgG immune complexes. In the cobra venom factor model, sCR1sLex and sCR1[desLHR-A]sLex caused substantially greater reductions in neutrophil accumulation and in albumin extravasation in lung when compared with the non-sLex-decorated forms. In this model, increased lung vascular binding of sCR1sLex and sCR1[desLHR-A]sLex occurred in a P-selectin-dependent manner, in contrast to the absence of any increased binding of sCR1 or sCR1[desLHR-A]. In the IgG immune complex model, sCR1[desLHR-A]sLex possessed greater protective effects relative to sCR1[desLHR-A], based on albumin extravasation and neutrophil accumulation. Enhanced protective effects correlated with greater lung vascular binding of sCR1[desLHR-A]sLex as compared with the non-sLex-decorated form. In TNF-alpha-activated HUVEC, substantial in vitro binding occurred with sCR1[desLHR-A]sLex (but not with sCR1[desLHR-A]). This endothelial cell binding was blocked by anti-E-selectin but not by anti-P-selectin. These data suggest that sLex-decorated complement inhibitors have enhanced antiinflammatory effects and appear to have enhanced ability to localize to the activated vascular endothelium.     

Morphometric measurements to quantify the cerulein induced hyperstimulatory pancreatitis of rats under the protective effect of lectins.

In preceding papers we demonstrated an inhibitory effect of wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA) on the cholecystokinin (CCK) binding to the CCK receptor of rat pancreatic cells and also on the CCK induced Ca2+ release and alpha-amylase secretion in vitro as well as on pancreatic secretion of intact rats in vivo. In the present study we show the same inhibitory effect of both lectins on the cerulein pancreatitis of rats. This acute pancreatitis was induced by supramaximal injections (5 microg/kg/h i.v. or 10 microg/kg/h i.p.) of the CCK analogue cerulein in rats every hour. To monitor the degree of pancreatitis, we measured the number and diameter of injury vacuoles in the pancreatic acinar cells as one of the most important signs of this type of pancreatitis by light microscopic morphometry with two different systems on paraffin sections. Furthermore, the serum alpha-amylase activity was measured biochemically. We found a correlation between the diameter of vacuoles inside the acinar cells and the serum enzyme activity up to 24 h. The simultaneous i.p. administration of cerulein and WGA or UEA in a dosage of 125 microg/kg/h for 8 h led to a reduction of vacuolar diameter from 13.1+/-2.0 microm (cerulein) to 7.5+/-1.1 microm (cerulein + WGA) or 7.2+/-1.3 microm (cerulein + UEA). The serum amylase activity was reduced from 63.7+/-15.8 mmol/l x min (cerulein) to 37.7+/-11.8 (cerulein + WGA) or 39.4; +52.9; -31.1 (cerulein + UEA-I). Both parameters allow the grading this special type of pancreatitis to demonstrate the protective effect of the lectins.     

L- 精氨酸和雨蛙素诱导急性胰腺炎模型的对比研究

目的:研究L-精氨酸和雨蛙素分别诱导SD大鼠急性胰腺炎(AP)模型的差异,为进一步研究急性胰腺炎提供可靠模型。方法:L-精氨酸采用3次腹腔注射,间隔1 h,雨蛙素采用7次腹腔注射,间隔1 h诱导急性胰腺炎模型。碘-淀粉比色法检测血清淀粉酶水平,血清脂肪酶测定试剂盒检测脂肪酶活性,胰腺组织切片观察组织的破坏情况,TUNEL法检测腺泡细胞凋亡。结果:①L-精氨酸诱导的大鼠模型血清淀粉酶和脂肪酶水平在诱导成功后6 h即显著升高,蛙皮素诱导的大鼠模型在12 h显著升高,与正常对照组比较均有统计学差异(P<0.05),提示急性胰腺炎建模成功。②L-精氨酸诱导的模型中胰腺组织结构破坏,有大片出血坏死灶、大量炎细胞浸润;而蛙皮素诱导的模型组织腺泡、间质水肿,炎性细胞浸润,少量散在出血坏死灶,血管变化常不明显,渗液清亮。结论:L-精氨酸和雨蛙素均能诱导SD大鼠急性胰腺炎模型,L-精氨酸诱导重症急性胰腺炎,雨蛙素诱导轻型急性胰腺炎,是研究急性胰腺炎的良好模型。     

Probiotics enhance pancreatic glutathione biosynthesis and reduce oxidative stress in experimental acute pancreatitis

Factors determining severity of acute pancreatitis (AP) are poorly understood. Oxidative stress causes acinar cell injury and contributes to the severity, whereas prophylactic probiotics ameliorate experimental pancreatitis. Our objective was to study how probiotics affect oxidative stress, inflammation, and acinar cell injury during the early phase of AP. Fifty-three male Sprague-Dawley rats were randomly allocated into groups: 1) control, 2) sham procedure, 3) AP with no treatment, 4) AP with probiotics, and 5) AP with placebo. AP was induced under general anesthesia by intraductal glycodeoxycholate infusion (15 mM) and intravenous cerulein (5 microg.kg(-1).h(-1), for 6 h). Daily probiotics or placebo were administered intragastrically, starting 5 days prior to AP. After cerulein infusion, pancreas samples were collected for analysis including lipid peroxidation, glutathione, glutamate-cysteine-ligase activity, histological grading of pancreatic injury, and NF-kappaB activation. The severity of pancreatic injury correlated to oxidative damage (r = 0.9) and was ameliorated by probiotics (1.5 vs. placebo 5.5; P = 0.014). AP-induced NF-kappaB activation was reduced by probiotics (0.20 vs. placebo 0.53 OD(450nm)/mg nuclear protein; P < 0.001). Probiotics attenuated AP-induced lipid peroxidation (0.25 vs. placebo 0.51 pmol malondialdehyde/mg protein; P < 0.001). Not only was AP-induced glutathione depletion prevented (8.81 vs. placebo 4.1 micromol/mg protein, P < 0.001), probiotic pretreatment even increased glutathione compared with sham rats (8.81 vs. sham 6.18 miccromol/mg protein, P < 0.001). Biosynthesis of glutathione (glutamate-cysteine-ligase activity) was enhanced in probiotic-pretreated animals. Probiotics enhanced the biosynthesis of glutathione, which may have reduced activation of inflammation and acinar cell injury and ameliorated experimental AP, via a reduction in oxidative stress.     

Dysregulation of the calpain-calpastatin system plays a role in the development of cerulein-induced acute pancreatitis in the rat

Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed micro-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg i.p.), pancreatitis was induced by cerulein injections (10 microg/kg i.p.; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both micro-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity (P < 0.01), pancreatic edema formation (P < 0.05), histological score for eight parameters (P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.     

Tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone initiates and enhances pancreatitis responses

Clinical studies indicate that cigarette smoking increases the risk for developing acute pancreatitis. The nicotine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a major cigarette smoke toxin. We hypothesized that NNK could sensitize to pancreatitis and examined its effects in isolated rat pancreatic acini and in vivo. In acini, 100 nM NNK caused three- and fivefold activation of trypsinogen and chymotrypsinogen, respectively, above control. Furthermore, NNK pretreatment in acini enhanced zymogen activation in a cerulein pancreatitis model. The long-term effects of NNK were examined in vivo after intraperitoneal injection of NNK (100 mg/kg body wt) three times weekly for 2 wk. NNK alone caused zymogen activation (6-fold for trypsinogen and 2-fold for chymotrypsinogen vs. control), vacuolization, pyknotic nuclei, and edema. This NNK pretreatment followed by treatment with cerulein (40 μg/kg) for 1 h to induce early pancreatitis responses enhanced trypsinogen and chymotrypsinogen activation, as well as other parameters of pancreatitis, compared with cerulein alone. Potential targets of NNK include nicotinic acetylcholine receptors and β-adrenergic receptors; mRNA for both receptor types was detected in acinar cell preparations. Studies with pharmacological inhibitors of these receptors indicate that NNK can mediate acinar cell responses through an nonneuronal α(7)-nicotinic acetylcholine receptor (α(7)-nAChR). These studies suggest that prolonged exposure to this tobacco toxin can cause pancreatitis and sensitize to disease. Therapies targeting NNK-mediated pathways may prove useful in treatment of smoking-related pancreatitis.     

Moderation of skeletal muscle reperfusion injury by a sLe(x)-glycosylated complement inhibitory protein

Therole of the sialyl Lewis^x (sLe^x)-decoratedversion of soluble complement receptor type 1 (sCR1) in moderatingskeletal muscle reperfusion injury, by antagonizing neutrophilendothelial selectin interaction and complement activation, isexamined. Mice underwent 2 h of hindlimb ischemia and3 h of reperfusion. Permeability index (PI) was assessed byextravasation of ¹²⁵I-labeled albumin. Neutrophil depletionand complement inhibition with sCR1 reduced permeability by 72% (PI0.81 ± 0.10) compared with a 42% decrease (PI 1.53 ± 0.08)observed in neutropenic mice, indicating that part of thecomplement-mediated injury is neutrophil independent.sCR1sLe^x treatment reduced PI by 70% (PI 0.86 ± 0.06), an additional 20% decrease compared with sCR1 treatment (PI1.32 ± 0.08). Treatment with sCR1sLe^x 0.5 and 1 h after reperfusion reduced permeability by 63% (PI 0.09 ± 0.07)and 52% (PI 1.24 ± 0.09), respectively, compared with therespective decreases of 41% (PI 1.41 ± 0.10) and 32% (PI1.61 ± 0.07) after sCR1 treatment. Muscle immunohistochemistry stained for sCR1 only on the vascular endothelium ofsCR1sLe^x-treated mice. In conclusion, sCR1sLe^xis more effective than sCR1 in moderating skeletal muscle reperfusion injury.

     

Angiotensin II type 1 receptor interaction is an important regulator for the development of pancreatic fibrosis in mice

The renin-angiotensin system (RAS) plays important roles in various pathophysiological processes. However, the role of the RAS in pancreatic fibrosis has not been established. We investigated the role of angiotensin II (ANG II)-ANG II type 1 (AT(1)) receptor pathway in the development of pancreatic fibrosis with AT(1a) receptor-deficient [AT(1a)(-/-)] mice. To induce pancreatic fibrosis, AT(1a)(-/-) and wild-type (WT) mice were submitted to three episodes of acute pancreatitis induced by six intraperitoneal injections of 50 microg/kg body wt cerulein at hourly intervals, per week, for four consecutive weeks. Pancreatic fibrosis was assessed by histology and hydroxyproline content. Pancreatic stellate cell (PSC) activation and the localization of AT(1) receptors were assessed by Western blot analysis for alpha-smooth muscle actin and immunostaining. Transforming growth factor-beta(1) (TGF-beta(1)) mRNA expression in the pancreas was assessed by RT-PCR. Six intraperitoneal injections of cerulein induced acute pancreatitis in both AT(1a)(-/-) and WT mice. There were no significant differences between two groups with regard to serum amylase and histological changes. Pancreatic fibrosis induced by repeated episodes of acute pancreatitis was significantly attenuated in AT(1a)(-/-) mice compared with that in WT mice. This finding was accompanied by a reduction of activated PSCs. Dual-immunofluorescence staining in WT mice revealed that activated PSCs express AT(1) receptors. The level of TGF-beta(1) mRNA was lower in AT(1a)(-/-) mice than in WT mice. Our results demonstrate that the ANG II-AT(1) receptor pathway is not essential for the local pancreatic injury in acute pancreatitis but plays an important role in the development of pancreatic fibrosis through PSC activation and proliferation.