布地奈德干预对卵白蛋白致敏小鼠抗原激发后气道炎症及气道重塑的影响

目的　观察早期及延迟应用布地奈德对支气管哮喘 (简称哮喘 )小鼠气道炎症和气道重塑的防治作用。方法　40只小鼠分为 5组,每组 8只。鸡卵白蛋白(OVA)致敏 /激发组 (A组 ),生理盐水对照组(B组),布地奈德 (BUD)早期治疗组 (C组 ),BUD延迟治疗组 (D组 ),OVA延迟对照组(E组)。小鼠于第 0、14天以OVA致敏,从第 1次致敏后第 24天开始雾化吸入 2.5%的OVA激发并持续 18d,建立气道重塑模型;分别在早期(抗原激发前 1d始)和延迟(第 1次抗原激发后第 18天始)雾化吸入BUD(0.5mg/ml),观察抗原激发及BUD应用后支气管肺泡灌洗液(BALF)中嗜酸粒细胞(EOS)数、上清液白细胞介素 5(IL-5)和γ干扰素 (IFN-γ)水平的变化,同时对肺组织切片行苏木精 伊红(HE)、过碘酸雪夫(PAS)、Masson三色染色,测定支气管壁周围EOS计数、定量杯状细胞百分比及黏液分泌评分并测定气道平滑肌增生高度及胶原面积。结果　经过反复抗原激发,A组BALF中EOS数为(57.460±11 060)×104 /ml,B组为[ (0.050±0.020)×104 /ml],两组比较差异有统计学意义(P<0.01);A组BALF中IL- 5水平及IFN -γ水平分别为(52.9±2.8)pg/ml、(39.5±3.2)pg/ml,B组分别为(16.8±1.5)pg/ml、(63.8±3.3)pg/ml,两组比较差异有统计学意义 (P<0.01 );A、B组支气管壁周围EOS数分别     

降钙素基因相关肽在哮喘患者气道中的表达

目的　探讨降钙素基因相关肽(CGRP)在哮喘患者气道中的表达。方法　收集轻度持续哮喘患者,通过纤支镜于右中叶嵴取活检,用S P免疫组化法检测CGRP的表达。结果　10例轻度持续哮喘患者的气道表现为黏膜上皮脱落,嗜酸细胞浸润,在黏膜下、大血管周围可见丝状、条状、串珠状或簇状CGRP表达,阳性占区域比( 0. 1286±0. 0271 )和阳性综合分析( 0. 1299±0. 0188),与对照组比较(0. 0108±0. 0013, 0. 0124±0. 0041),差异具有显著性(P<0. 01)。结论　轻度持续哮喘患者气道CGRP高表达。     

Serotonin,calcitonin and calcitonin gene-related peptide in acute pancreatitis

Objective: The aim of this study was to investigate plasma levels of serotonin, calcitonin and calcitonin gene-related peptide (CGRP) in the course of acute pancreatitis (AP) taking organ failure, etiology and severity into consideration.

Material and methods: Sixty consecutive patients with alcohol- or gallstone-induced AP were included over a 15-month period. Patients were treated according to a standardized algorithm and monitored for organ specific morbidity and mortality. Organ functions and blood samples were assessed on days 0, 1, 2 and 14 after hospital admission. Twenty healthy volunteers, matched for age and gender, comprised the reference group.

Results: Lower levels of serotonin were observed in patients at admission compared to healthy volunteers (p?=?.021). Serotonin levels increased from day 2 to 14 (p?<?.001), but with no relation to severity, etiology or organ failure. No difference in calcitonin levels was found in patients at admission compared to healthy volunteers. However, calcitonin levels decreased over time (p?<?.001) and higher levels were found in patients with respiratory failure (p?=?.039). No difference was observed in relation to severity or etiology. CGRP levels in patients at admission did not differ from healthy volunteers, nor did CGRP change over time or show any relationship to severity, etiology or organ failure.

Conclusion: Our data suggest serotonin and calcitonin levels to be associated to time-course of AP, and calcitonin levels to organ dysfunction. We hypothesize that serotonin plays a pathogenic role in the compromised pancreatic microcirculation, and calcitonin a role as a biomarker of severity in AP.     

Colocalization of calcitonin gene-related peptide and somatostatin in pancreatic islet cells and inhibition of insulin secretion by calcitonin gene-related peptide in the rat

Calcitonin gene-related peptide (CGRP)- and somatostatin (SRIF)-containing cells were identified by immunocytochemical techniques in pancreatic islet cells of the rat. CGRP-containing cells were found primarily in the peripheral portion of the pancreatic islets. In addition, CGRP-containing cells also contained somatostatin, which identifies the islet CGRP-containing cells as D cells. In the present study, we also tested the effect of CGRP on gastrin-releasing peptide (GRP; 10(-9) M)- or cholecystokinin (CCK-8, 10(-9) M)-stimulated release of insulin from isolated rat islets in vitro. At concentrations of 10(-8)-10(-11) M, CGRP inhibited GRP- and CCK-8-stimulated release of insulin significantly when compared with GRP or CCK-8 alone. At the lowest concentration of CGRP (10(-11) M), the inhibitory effect of CGRP on CCK-8-stimulated release of insulin was statistically significant (p less than 0.05) and exceptionally potent (65-90% inhibition). We have also found that CGRP does not stimulate the release of SRIF from isolated islet cells. These findings suggest that CGRP may play a regulatory role in the release of insulin.     

Effect of calcitonin and calcitonin gene-related peptide on pancreatic functions in man.

Calcitonin gene-related peptide (CGRP) has recently been identified in central and peripheral nerve fibres, including those of blood vessels supplying the exocrine pancreas, and in pancreatic islet cells. Moreover, receptors have been characterised in the same tissue. The present study examined the effects of human CGRP and of calcitonin on exocrine pancreatic secretion and on islet cell function in nine healthy volunteers. CGRP (300 ng/kg/h) caused, respectively, a 25% and 31% inhibition of caerulein stimulated trypsin and amylase output which was similar to that seen with calcitonin (300 ng/kg/h). Arginine stimulated insulin and glucagon release was unaffected by either CGRP, or calcitonin. Calcitonin gene-related peptide caused cutaneous flushing, but did not affect the pulse rate or arterial blood pressure in the doses tested. Calcitonin gene-related peptide inhibits exocrine pancreatic secretion in vivo in man, but does not affect islet cell hormone release.     

Regional differences of adenylate cyclase stimulation by calcitonin and calcitonin gene-related peptide in the human kidney

Calcitonin (CT) gene-related peptide (CGRP)-like immunoreactivity was detected in both the cortex and medullo-papillary portion of human kidneys. The two forms of human CGRP as well as rat CGRP were capable of stimulating renal cortical adenylate cyclase activity in a concentration-related manner, with a half-maximally effective concentration (EC50) similar to that of human CT and approximately 100-1000 times higher than that of salmon CT. However, in the medullo-papillary portion, in which both salmon CT and human CT were inactive, the two forms of human and rat CGRP increased adenylate cyclase activity by 100%, with EC50 values ranging from 36 nmol/L to 1 mumol/L. In cortical membrane preparations the effect of CGRP was additive to that of salmon CT. We concluded that regional differences exist in the effect of CT and CGRP in human renal tissue and that in the medullo-papillary portion and possibly in the cortex, CGRP stimulates adenylate cyclase activity through a CT-independent mechanism.     

Activation of the renin-angiotensin system in alpha-calcitonin gene-related peptide/calcitonin gene knockout mice

OBJECTIVE: To test the hypotheses that circulating or tissue renin-angiotensin system (RAS) activity is increased in alpha-calcitonin gene-related peptide (alpha CGRP) knockout mice, and that this contributes to the increased blood pressure in these mice. DESIGN AND METHODS: Three- to six-month-old male alpha CGRP/calcitonin knockout mice and wild-type controls were studied. Mean arterial pressure (MAP) and its response to an angiotensin II type 1 (AT1) receptor blocker, losartan (3 mg/kg intravenously), were determined in conscious, unrestrained knockout mice and wild-type mice. Radioimmunoassay and western blot were used, respectively, to determine plasma renin activity (PRA) and AT1 receptor protein content in tissues. RESULTS: Basal MAP and PRA were significantly greater in the knockout mice than in the wild-type mice. In contrast, AT1 receptor content in the renal medulla was significantly decreased in the knockout mice compared with that in wild-type mice. AT1 receptor content in the renal cortex and mesenteric resistance arteries was not different in the knockout and wild-type mice. Losartan produced a significant decrease in MAP in the knockout mice compared with that in wild-type mice. CONCLUSION: Activity of the circulating RAS, but not tissue AT1 receptor expression, is increased in alpha CGRP/calcitonin knockout mice, which may contribute to the increase in blood pressure in this mouse model. The mechanism(s) responsible for the increased activity of the circulating RAS in the absence of alpha CGRP throughout the developmental stages of these animals remains to be determined.     

Role of calcitonin gene-related peptide in hypertension-induced renal damage

Calcitonin gene-related peptide, a potent vasodilator neuropeptide, is localized in perivascular sensory nerves. We have reported that alpha-calcitonin gene-related peptide knockout mice have elevated baseline blood pressure and enhanced hypertension-induced renal damage compared with wild-type controls. Thus, the aim of this study was to determine the mechanism and functional significance of this increased hypertension-induced renal damage. We previously demonstrated by telemetric recording that the deoxycorticosterone-salt protocol produces a 35% increase in mean arterial pressure in both alpha-calcitonin gene-related peptide knockout and wild-type mice. Both strains of mice were studied at 0, 14, and 21 days after deoxycorticosterone-salt hypertension. Renal sections from hypertensive wild-type mice showed no pathological changes at any time point studied. However, on days 14 and 21, hypertensive knockout mice displayed progressive increases in glomerular proliferation, crescent formation, and tubular protein casts, as well as the inflammatory markers intercellular adhesion molecule-1, vascular adhesion molecule-1, and monocyte chemoattractant protein-1. There was a significant increase in 24-hour urinary isoprostane, a marker of oxidative stress-induced lipid peroxidation, levels at days 14 and 21 in the hypertensive knockout compared with hypertensive wild-type mice. Urinary microalbumin was significantly higher (2-fold) at day 21 and creatinine clearance was significantly decreased 4-fold in the hypertensive knockout compared with hypertensive wild-type mice. Therefore, in the absence of alpha-calcitonin gene-related peptide, deoxycorticosterone-salt hypertension induces enhanced oxidative stress, inflammation, and renal histopathologic damage, resulting in reduced renal function. Thus, sensory nerves, via alpha-calcitonin gene-related peptide, appear to be renoprotective against hypertension-induced damage.     

Hypotension- and endotoxin-induced alterations in calcitonin gene-related peptide: modulation by dexamethasone

Plasma levels of calcitonin gene-related peptide (CGRP) were studied in anesthetized rats during and after recovery from hemorrhagic hypotension as well as following administration of bacterial endotoxin. Hypotension of 35-40 mmHg maintained for 2 hr resulted in significant (P less than .05) elevation of plasma CGRP levels. Plasma levels at 120 min of hypotension were 49.5 +/- 5.9 pg/ml, over 4-fold above average control levels (11.1 +/- 1.1 pg/ml). Plasma CGRP levels at 90 min of hypotension (20.2 +/- 2.4 pg/ml) or before were not more than 2.5-fold above control levels. Ninety minutes after the return of shed blood in the 30 min group, blood pressure (83 +/- 3 mmHg) and CGRP (17.2 +/- 2.2 pg/ml) were not different from saline controls (88 +/- 3 mmHg and 15.1 +/- 1.7 pg/ml CGRP). However, if hypotension was maintained for 120 min before the return of shed blood, blood pressure following 90 min recovery was significantly lower (59 +/- 5 mmHg) and CGRP levels significantly higher (39.2 +/- 5.6 pg/ml) than saline control values (82 +/- 5 mmHg and 19.5 +/- 2.7 pg/ml). Dexamethasone treatment of the 120 min hypotension group when shed blood was returned resulted in CGRP values not different from saline treated, but hypotension persisted. Administration of bacterial endotoxin (16.7 mg/kg) to anesthetized rats caused significant elevations (P less than .05 vs. saline treatment at 3 hr) in plasma CGRP levels from aorta (82.2 +/- 5.0 pg/ml), vena cava (79.4 +/- 12.9), and portal vein (117.7 +/- 29 pg/ml) compared to levels in saline-treated control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics and organ-specific metabolism of calcitonin gene-related peptide in sheep

Calcitonin gene-related peptide (CGRP) is a product of the calcitonin gene with a widespread distribution in neural tissue of the brain, gut and perivascular nerves. Infusion of CGRP produces multiple biological effects, but the physiological significance of these findings will be influenced by the sites and rates of CGRP metabolism. The metabolic clearance rate and half-life of disappearance of human CGRP were estimated in conscious sheep after infusing CGRP at 1 or 5 pmol/kg per min to steady-state conditions. The particular organs involved in the clearance of CGRP were assessed by measuring the inflow and outflow concentrations across the liver, gut, kidney, lung and brain. The metabolic clearance rate at steady state was 22.6 +/- 2.1 (S.E.M.) and 15.0 +/- 17 ml/kg per min for the 1 and 5 pmol/kg per min doses respectively. The half-life of disappearance was bi-exponential: 3.6 +/- 0.3 min for the first phase and 13.6 +/- 1.0 min for the second phase. High-pressure liquid chromatography of plasma at equilibrium revealed only a single peak coeluting with CGRP(1-37): no immunoreactive metabolites were detected. These pharmacokinetic values are intermediate between that of a neurotransmitter and a hormone and are therefore consistent for a peptide with both circulatory and neurotransmitter modes of action. The kidney, with an arterial-renal vein gradient of 14%, and the liver, with a portal-hepatic vein gradient of 25%, were the major organs involved in the clearance of CGRP.(ABSTRACT TRUNCATED AT 250 WORDS)     

肠易激综合征血清中降钙素基因相关肽的变化

目的探讨降钙素基因相关肽(CGRP)在肠易激综合征(IBS)中的变化,为进一步探讨其在IBS中的作用提供证据。方法 SPF级大鼠30只,随机分为3组:模型组大鼠以0.85 mL含三硝基苯磺酸(TNBS)的50%乙醇灌肠1次诱发远端结肠炎,对照组仅以等体积的生理盐水灌肠,正常组不作处理。正常饲养30 d后处死。造模前后均以腹部回撤反射(AWR)测定内脏感觉阈值用以评价模型。造模成功后行心脏取血,以ELISA试剂盒测定血清中CGRP浓度。结果 TNBS灌肠法复制模型成功,腹部回撤反射内脏感觉阈值测定模型组造模后内脏感觉阈值较造模前及正常组下降(P<0.05)。模型组血中CGRP浓度较对照组及正常组明显升高(P<0.05),对照组较正常组中CGRP浓度也有升高但差异无统计学意义(P>0.05)。结论模型组血清中CGRP浓度升高,可能在IBS的发生中起着一定作用。     

Specific receptor and cardiovascular effects of calcitonin gene-related peptide

Specific binding sites for calcitonin gene-related peptide (CGRP) were demonstrated in the rat heart and spleen. Autoradiography revealed rat [125I]iodo CGRP binding associated with the intima and media of the aorta, the coronary arteries and the heart valves, and the red pulp of the spleen. Half-maximal inhibition of rat [125I]iodo-CGRP binding to membranes of the rat atria and the spleen was obtained with, respectively, 5 and 0.35 nM unlabeled rat CGRP; these values correspond to EC50 values of 3 and 0.14 nM for activation of adenylate cyclase by CGRP. In the isolated, spontaneously beating right atrium, the EC50 values of stimulation of the force and rate of contraction by rat CGRP were 120 and 70 nM, respectively. Rat CGRP caused relaxation of splenic strips, precontracted with noradrenaline; the EC50 was 50 nM. The beta-adrenergic blocking agent metoprolol, while obliterating the increase in the force and rate of contraction evoked by noradrenaline in the right atrium, did not significantly change the action of CGRP. Similarly, preserved action of CGRP in the presence of indomethacin as well as mepyramine and cimetidine argues against a role of prostaglandins or histamine in the functional responses of CGRP. Much like CGRP, capsaicin, which releases mediators from sensory neurons, caused stimulation of the force and rate of contraction of the isolated right rat atrium. After tachyphylaxis to CGRP, the response to noradrenaline was intact, while the positive chronotropic and inotropic effects of capsaicin were suppressed. The results indicate that the cardiac effects of capsaicin may be due to the release of endogenous CGRP through a local mode of action.     

Identification of calcitonin and calcitonin gene-related peptide messenger ribonucleic acid in medullary thyroid carcinomas by hybridization histochemistry

Synthesis and secretion of calcitonin and calcitonin gene-related peptide (CGRP) were studied in medullary thyroid carcinomas (MTC) by hybridization histochemistry on tissue sections and by Northern gel analysis of mRNA. Five patients with MTC and elevated serum levels of calcitonin and CGRP were studied. Surgically obtained tumor samples (four primary and three lymph node metastases) were extracted after freezing, and the RNA was fractionated on Northern gels. Hybridization was carried out with 32P-labeled synthetic oligodeoxyribonucleotides coding specifically for calcitonin and CGRP. Calcitonin- and CGRP-specific mRNAs approximately 1000 nucleotides in length were demonstrated in all 7 tumor samples. However, neither calcitonin nor CGRP mRNA was detected in a pheochromocytoma from 1 of the patients who had multiple endocrine neoplasia type II. A series of unselected lung carcinomas yielded the same result. Hybridization histochemistry was carried out on sections from the same tumors using the same probes. The mRNAs for calcitonin and CGRP were located in all cells of neoplastic MTC appearance, with CGRP mRNA at significantly lower levels. This demonstrated that both calcitonin and CGRP mRNA were present within the same tumor cells. The lung tumors and pheochromocytoma were negative with both probes. Hybridization histochemistry is likely to be of use in diagnosis of medullary thyroid cancer and in studying the calcitonin-CGRP mRNA processing mechanism in whole cells.     

降钙素基因相关肽衍生物的优化表达及纯化

目的优化重组降钙素基因相关肽衍生物(CGRP-VY)蛋白的表达体系,并利用亲和层析获得高纯度的重组蛋白。方法在前期研究的基础上,将构建的pET-32a(+)-rhCGRP-VY在大肠埃希菌中表达,分别对时间、温度和IPTG的浓度进行优化。采用Ni-NTA亲和层析柱分离纯化可溶性蛋白,YLN凝胶影像分析系统和Bradford法检测纯化蛋白的纯度与含量。结果重组蛋白的最佳表达条件为诱导时间4h、诱导温度37℃、IPTG浓度0.75mmol/L,重组蛋白的最高表达量占菌体总蛋白的70%～80%,高效表达的CGRP-VY融合蛋白大部分可溶,纯化后蛋白纯度可达90%以上,蛋白浓度约为0.09mg/ml。结论成功优化了蛋白表达条件,并用亲和层析法纯化出大量可溶性TrxA-rhCGRP-VY。     

The failure of interleukin-10-deficient mice to develop airway hyperresponsiveness is overcome by respiratory syncytial virus infection in allergen-sensitized/challenged mice

Interleukin-10-deficient mice develop a robust pulmonary inflammatory response but no airway hyperresponsiveness (AHR) to inhaled methacholine (MCh) following allergen sensitization and challenge. In the present study, we investigated the effect of respiratory syncytial virus (RSV) infection on AHR and pulmonary inflammation in allergic IL-10-/- mice. Unlike littermate control mice, RSV-infected or ovalbumin (OVA)-sensitized/challenged IL-10-/- mice failed to develop significant AHR. In contrast, sensitized/challenged IL-10-/- mice infected with RSV did develop AHR accompanied by increased eosinophil numbers, both in bronchoalveolar lavage (BAL) and pulmonary tissue, and mucin production in airway epithelium. The cytokine profile in OVA-sensitized/challenged IL-10-/- mice was skewed toward a Th1 response but after RSV infection, this response was more of a Th2 type, with increased IL-5 levels in the BAL. Studies with an RSV mutant that lacks the G and SH genes showed equal enhancement of the AHR response as the parental wild-type strain, indicating that G protein is not essential to this response. These data suggest that RSV infection can overcome the failure of development of AHR in allergic IL-10-/- mice.     

Chitin induces steroid-resistant airway inflammation and airway hyperresponsiveness in mice

BackgroundPrevious reports have shown that pathogen-associated patterns (PAMPs) induce the production of interleukin (IL)-1β in macrophages. Moreover, studies using mouse models also suggest that chitin, which acts as a PAMP, induces adjuvant effects and eosinophilic infiltration in the lung. Thus, we investigated the effects of inhaled chitin in mouse models.MethodsWe developed mouse models of inhaled chitin particle-induced airway inflammation and steroid-resistant ovalbumin (OVA)-induced airway inflammation. Some experimental groups of mice were treated additionally with dexamethasone (DEX). Murine alveolar macrophages (AMs), which were purified from bronchoalveolar lavage (BAL) fluids, were incubated with chitin, and treated with or without DEX.ResultsThe numbers of total cells, AMs, lymphocytes, eosinophils, and neutrophils among BAL-derived cells, as well as the IL-1β levels in BAL fluids and the numbers of IL-1β-positive cells in lung, were significantly increased by chitin stimulation. Airway hyperresponsiveness (AHR) was aggravated in mice of the chitin inflammation model compared to control animals. The production of IL-1β was significantly increased in murine AMs by chitin treatment, but DEX administration did not inhibit this chitin-induced IL-1β production. Furthermore, in mouse models, DEX treatment inhibited the OVA-induced airway inflammation and AHR but not the airway inflammation and AHR induced by chitin or the combination of OVA and chitin.ConclusionsThese results suggest that inhaled chitin induces airway inflammation, AHR, and the production of IL-1β. Furthermore, our findings demonstrate for the first time that inhaled chitin induces steroid-resistant airway inflammation and AHR. Inhaled chitin may contribute to features of steroid-resistant asthma.     

CGRP在大鼠胃痛觉过敏形成机制中的作用

目的:探索降钙素基因相关肽(CGRP)相关的干预措施对胃痛觉过敏的影响,了解CGRP在胃痛觉过敏形成过程中发挥的作用．方法:成年SD古大鼠,均植入胃内气囊．观察伤害性扩张或CGRP iv对大鼠疼痛阈值的影响:观察由上述措施诱发内脏过敏的大鼠在给予CGRP受体特异性拮抗剂hCGRP8-37后疼痛阈值的变化:观察不同剂量CGRP和hCGRP8-37对疼痛阈值的影响．结果:CGRP iv后胃疼痛阈值为11．7±2．6 mmHg,对照组疼痛阈值为19．2±2．0 mmHg,生理盐水对照组则为18．3±2．5 mmHg,实验组与其他两组比较尸均<0．05．CGRP使大鼠的疼痛阈值降低．hCGRP8-37能逆转伤害性扩张和CGRP引起的内脏敏感性增高,该作用呈剂量依赖性(r=0．821,P<0．01)．结论:胃扩张刺激能引起胃敏感性增高,在此过程中CGRP具有重要的作用．     

Inhibitory effect of calcitonin gene-related peptide and calcitonin on opossum esophageal smooth muscle

Calcitonin gene-related peptide (CGRP) is widely distributed in the gastrointestinal nerves, including those of the esophagus. The present investigation was undertaken to examine the effect and the mechanism of action of CGRP on the lower esophageal sphincter and esophageal contractions. This peptide caused dose-dependent relaxation of the lower esophageal sphincter. The D50 for inhibitory effect of intraarterial CGRP on the sphincter was 5.0 X 10(-13) mol/kg. Calcitonin gene-related peptide is 3000 times more potent than calcitonin. The effect of CGRP on the lower esophageal sphincter was partially antagonized by tetrodotoxin or black widow spider venom. The inhibitory effect of CGRP on the sphincter appears to be exerted at two levels: (a) at the sphincteric smooth muscle, and (b) at the noncholinergic, nonadrenergic inhibitory neurons. Calcitonin gene-related peptide also exerts a potent inhibitory effect on the peristaltic contraction of the esophageal body in response to swallowing and vagal efferent stimulation. Using immunohistochemical studies we also showed the presence of CGRP-immunoreactive neurons within the myenteric ganglia of the esophagus. These studies suggest that CGRP may play an important role as an inhibitory neurotransmitter in the esophagus.