实验性急性胰腺炎大鼠胰腺腺泡细胞超微结构的观察

目的：了解实验性急性胰腺炎大鼠胰腺腺泡细胞超微结构的改变。并探讨雨蛙素致急性胰腺炎的机理。方法：采用皮下注射超大剂量雨蛙素建立大鼠急生水肿性胰腺炎模型,用透射电镜观察了雨蛙素刺激１ｈ,３ｈ,６ｈ,１２ｈ及２４ｈ后胰腺腺泡细胞的形态学改变。结果：①腺泡细胞质空泡形成。在雨蛙素刺激后１小时即可见到,随着雨蛙素刺激时间的延长,空泡数量显著增加,６ｈ达高峰;体积变显著增大,空泡之间或空泡与酶原颗粒间互相融     

A Highly Specific and Sensitive Radioimmunoassay of Caerulein,An Analogue of Cholecystokinin-8

Abstract

A highly specific and sensitive competitive radioimmunoassay was developed for caerulein (CLN), an analogue of cholecystokinin-8 (CCK-8), in plasma and brain. Antiserum was produced in rabbit by immunization with Nδ-[CLN-(1-6)]-ornithine amide conjugated with bovine serum albumin by the glutaraldehyde method. Nα-[CLN-(1-6)]-lysine amide was labelled with ¹²⁵I-Bolton & Hunter reagent and used as a labelled antigen after purification by high-performance liquid chromerography. This assay was highly specific for CLN, and cross reactivities for other related peptides, CCK-4, CCK-8, gastrin-I, and gastrin-(14–17), were not observed (<0.01%). The limits of determination in biological specimens after CLN administration were 11 pg/ml in human plasma and rat plasma and 80 pg/g in rat brain. This study showed that the slight structure difference between hapten and ¹²⁵I-labelled antigen is important to the assay performance.     

p38 MAPK抑制剂SB203580对雨蛙肽诱导的小鼠胰腺组织损伤的影响

 目的：探讨p38 MAPK抑制剂SB203580（SB）对雨蛙肽（caerulein,CAE）诱导的小鼠离体胰腺组织损伤的影响,并探讨其可能的机制。方法：分离小鼠离体胰腺组织后培养4 h,用CAE(10-5mol/L)刺激,加用或不加用SB (10-5mol/L)进行干预,以生理盐水（NS）作为对照。在刺激1 h和4 h后测定胰腺组织活力（MTT）、培养上清液中淀粉酶和脂肪酶活性（生化法）以及白细胞介素6（IL-6）和细胞因子诱导的中性粒细胞趋化因子1（CINC-1）的水平（ELISA法）;同时测定胰腺组织中热休克蛋白60（HSP60)和HSP70蛋白水平（ELISA法）,并用Western blotting测定胰腺组织p38及磷酸化p38 （p-p38）MAPK蛋白的表达。结果：CAE刺激后胰腺组织活力与NS组比较有所下降,尤其在刺激后4 h明显降低（P<0.05）;CAE刺激后1 h,离体胰腺组织培养上清液中淀粉酶、脂肪酶、IL-6和CINC-1水平较NS组均明显升高（P<0.05）;胰腺组织中HSP60、HSP70、p38及p-p38 MAPK蛋白的表达较NS组有所升高（P<0.05）,而SB可不同程度干预CAE引起的这些指标的改变。结论：CAE对离体胰腺组织具有损伤作用,SB可减轻CAE造成的胰腺组织的损伤,其机制可能与其对p38 MAPK抑制进而使炎症反应受到抑制有关;HSP60和HSP70的变化是因为炎症反应减轻而使细胞应激程度降低所致,还是失去了p38 MAPK的调控作用所致,还有待进一步研究。     

Wirkung von Caerulein auf die exokrine Pankreasfunktion des Menschen

Zusammenfassung Untersucht wurde die Sekretionsantwort des exokrinen Pankreas des Menschen auf verschiedene Dosen des synthetischen Dekapeptid Caerulein (Takus). 5, 10 und 20 ng/kg Caerulein während einer Infusion von 0,5 CU/kg/h Sekretin (GIH) i.v. injiziert bewirkten eine lineare Steigerung der Enzymabgabe (Amylase, Lipase, Trypsin und Chymotrypsin) sowie der Sekretin-induzierten Wasser-und Bikarbonatsekretion des Pankreas. Die Injektion von 40 ng/kg Caerulein führte zu keiner weiteren Steigerung der ekbolen Funktion. Intravenös injiziert sind 1 Ivy Hunde-Einheit (IDU/kg) sowie 20 und 40 ng/kg Caerulein in ihrer Wirkung auf das exokrine Pankreas equivalent, es fanden sich keine statistischen Unterschiede.     

Effect of endothelin-1 receptor antagonists on histological and ultrastructural changes in the pancreas and trypsinogen activation in the early course of caerulein-induced acute pancreatitis in rats

AIM: To assess the effect of non-selective ETA/B (LU 302872) and selective ETA (LU 302146) antagonist on pancreatic histology and ultrastructure of acinar cells in connection with trypsinogen activation in early caerulein-induced AP. METHODS: Male Wistar rats with caerulein-induced AP, lasting 4 h, were treated i.p. with 10 and 20 mg/kg b.w. of each antagonist. Edema, inflammatory infiltration, necrosis and vacuolization of acinar cells in the pancreas were scored at 0-3 scale. Free active trypsin (FAT), total potential trypsin (TPT) after activation with enterokinase, and index of trypsinogen activation (%FAT/TPT) were assayed in pancreatic homogenates. RESULTS: In untreated AP, the edema, inflammatory infiltration, necrosis and vacuolization increased as compared to control healthy rats (P<0.01). None of the treatment exerted any meaningful effect on the edema and inflammatory infiltration. The selective antagonist increased slightly the necrosis score to 0.82±0.06 at higher dose (P<0.05) vs 0.58±0.06 in untreated AP. The non selective antagonist increased slightly the vacuolization score to 2.41±0.07 at higher dose (P<0.01) vs 1.88±0.08 in untreated AP. The decrease in the number of zymogen granules, disorganization of endoplasmic reticulum, autophagosomes and cytoplasmic vacuoles were more prominent in treated AP than in untreated AP groups. %FAT/TPT in untreated AP increased about four times (18.4±3.8 vs 4.8±1.3 in control group without AP, P<0.001). Treatment of AP with both antagonists did not affect significantly augmented trypsinogen activation. CONCLUSION: The treatment with endothelin-1 receptors (non-selective ETA/Band selective ETA) antagonists has essential effect neither on the edema and inflammatory infiltration nor on trypsinogen activation observed in the early course of caerulein-induced AP. Nevertheless a slight increase of the necrosis and vacuolization score and some of the ultrastructural data could suggest the possibility of their undesired effects in caerulein-induced AP at investigated doses.     

A caerulein-sensitive potentiation of the behavioral effects of apomorphine by dibutyryl-cAMP

The dopaminergic behavioral effects of apomorphine in rats were evaluated using a rating scale. Caerulein, a decapeptide physiologically similar to cholecystokinin, enhanced at lower doses and inhibited at higher doses the behaviors induced by apomorphine. Dibutyryl-cAMP, but not dibutyryl-cGMP, potentiated apomorphine behaviors. The potentiation was inhibited by a high dose of caerulein. These data provide evidence for an opposing effect of cAMP and caerulein or cholecystokinin in modulating dopaminergic systems.     

Peptides with CCK-like activity administered intracranially elicit satiety in sheep

Cholecystokinin (CCK) peptides and receptors have been shown to be present in the brain as well as in gastrointestinal organs. While functions for peripheral CCK are well recognized, those for central CCK peptides are only now being investigated. We have shown previously that CCK-octapeptide (CCK-OP) is a very potent and specific suppressor of feeding when administered in the cerebral ventricles of sheep. In the present study the objective was to determine the relative potency of several CCK analogues in inhibiting feeding when administered as 75 min continuous injections into the lateral cerebral ventricles of 2-hr fasted sheep. In comparing feeding response during CCK-OP injections to that during caerulein injections, it was found that feed intakes were similar only at an equal molar dose (0.638 pmole/min); whereas three times as much CCK-33 (1.91 pmoles/min) as CCK-OP (0.638 pmoles/min) was required to produce similar feed intakes. Both caerulein (0.638 pmoles/min) and CCK-33 (1.91 pmoles/min) caused significant decreases in feeding compared to control (sCSF). Desulfated CCK-OP had no effect on feeding at a dose (0.638 pmoles/min) that causes 80–100% decreases in feeding when the C-7 tyrosine is sulfated. Feed intake was significantly less with 2.55 pmoles/min CCK-OP than with an equal dose of desulfated CCK-OP. These results concur with those of previous studies on specific CCK receptors in the pancreas and in the brain, and therefore support the concept of specific CCK receptors in brain having a role in satiety.     

PIAS1基因沉默对雨蛙肽诱导胰腺腺泡细胞凋亡的影响

活化STAT蛋白抑制物(PIAS)1基因沉默可增强雨蛙肽诱导的胰腺腺泡细胞炎症反应。目的:探讨PIAS1基因沉默对雨蛙肽刺激胰腺腺泡细胞凋亡的影响。方法:AR42J胰腺腺泡细胞在Lipofectamine~(TM)2000介导下转染PIAS1-siRNA和阴性siRNA。将细胞分为PIAS1-siRNA+雨蛙肽组、阴性siRNA+雨蛙肽组、脂质体+雨蛙肽组、PBS+雨蛙肽组和对照组。以DNA Ladder、Hoechst 33258染色检测细胞凋亡情况,流式细胞术测定细胞周期和细胞凋亡率,RT-PCR和蛋白质印迹法检测1353、Bax、Bcl-2、caspase-3 mRNA和蛋白表达。结果:与其余各组相比,PIAS1-siRNA+雨蛙肽组DNA梯度裂解条带明显增加,荧光着色阳性细胞数增多;G1期细胞数增多,S期细胞数减少,细胞凋亡率增加;p53、Bax、caspase-3 mRNA和蛋白表达明显上调,Bcl-2 mRNA和蛋白表达下调(P均0.05)。结论:PIAS1基因沉默可增强雨蛙肽活化的caspase-3凋亡途径诱导的胰腺腺泡细胞凋亡,为通过调控PIAS1表达治疗急性胰腺炎提供新的理论依据。     

Differential effects of endothelins on histological and ultrastructural changes and trypsinogen activation in the secretagogue-induced acute pancreatitis in rats

The role of endothelins in acute pancreatitis remains obscure. To assess the effects of endothelins (ETs) in early (4 h) caerulein-induced acute pancreatitis (AP) in rats, ET-1, ET-2 and ET-3 (0.5 or 1.0 nmol/kg) were applied twice with i.p. caerulein (2×40 μg/kg) at 1 h interval. Histological and ultrastructural examinations of pancreases and the assay of trypsinogen activation in whole homogenate were performed. All ETs, especially ET-1 at the higher dose, decreased inflammatory cell infiltration despite an increase in the edema score. The vacuolization and necrosis of acinar cells were slightly increased after the lower dose of ET-1 and ET-2. Ultrastructural changes were generally improved after the higher dose of ETs. Trypsinogen activation increased from 4.8±1.3% in control to 18.4±3.8% in AP (p<0.01). It was attenuated to 6.4±1.3% (p<0.01) by the higher dose of ET-1 and to 8.8±1.5% (p<0.05) by the lower dose of ET-3. In summary, ETs, especially ET-1 at the higher dose, were found to have some beneficial effects on morphological changes and trypsinogen activation in the pancreas in early caerulein-induced AP.     

Activities of seasonably variable caerulein and rothein skin peptides from the tree frogs Litoria splendida and Litoria rothii

Two species of tree frog of the genus Litoria, namely L. splendida and L. rothii have been reported to change the compositions of their host-defence skin peptide profiles in summer and winter. L. splendida produces the potent smooth muscle active caerulein [pEQDY(SO₃H)TGWMDF-NH₂] in summer, but in winter much of the caerulein is hydrolysed to the less active desulfated form; in addition, caerulein 1.2 [pEQDY(SO₃H)TGWFDF-NH₂] (which has only some 50% of the smooth muscle activity of caerulein) is released and acts via CCK2R. In contrast, Litoria rothii shows a most unexpected seasonal change of peptides. In summer it exudes caerulein together with a range of potent caerin antimicrobials and nNOS active peptides. In winter, none of the antibiotic or nNOS active caerin peptides are expressed. The major peptides produced by the skin glands in winter are caerulein 1.2 and rothein 1 (SVSNIPESIGF-OH). Like L. splendida, L. rothii has reduced the smooth muscle potency of caerulein by replacing it with caerulein 1.2. Rothein 1 is a lymphocyte proliferator acting via CCK2R. Activity testing and 2D NMR spectra of rothein 1 and some synthetic modifications indicate that both hydrophobic and hydrophilic interactions between rothein 1 and CCK2R are important.