单发佐剂性关节炎大鼠脊髓3种神经肽的变化规律

目的:观察慢性佐剂性关节炎大鼠脊髓3种神经肽含量及释放量的动态变化,以阐明其与炎症痛的关系.方法:采用完全弗氏佐剂在大鼠右后肢造成单发性关节炎模型,用放射免疫分析法检测致炎前及致炎后1～9周大鼠脊髓背半侧和脊髓灌流液中与疼痛有关的神经肽:八肽胆囊收缩素(cholecystokinin octapeptid,CCK-8)、孤啡肽(orphanin FQ,OFQ)、内吗啡肽-2(endornorphin-2,EM-2)的含量及释放量的动态变化.结果:(1)炎症期间,脊髓中CCK-8-ir释放量显著降低,脊髓背半侧含量于炎症第2周呈一过性增高;(2)OFQ-ir释放量于炎症后的第3、7周明显升高,而含量则在3周后降至正常水平以下,并以患侧变化更为明显;(3)EM-2-ir释放量于第1～2周明显降低,此后恢复正常,患侧脊髓含量1～9周均显著降低.结论:单发佐剂性关节炎大鼠脊髓背角和脊髓灌流液中有关的神经肽CCK-8、OFQ、EM-2均发生了可塑性变化.     

孤啡肽受体拮抗剂对机械性损伤神经元存活率的影响

目的既往研究表明,孤啡肽在脑损伤后的表达明显升高,本研究通过特异性阻断孤啡肽受体(opioid-receptor-like receptor,ORL-1),观察对受损神经元是否具有保护作用。方法建立神经元机械性损伤模型,用孤啡肽受体特异性阻断剂([Nphe1]-NC(1-13)-NH2,Nphe)阻断孤啡肽受体,通过四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法、乳酸脱氢酶(lactate dehydrogenase,LDH)活性,钙离子水平测定,研究Nphe对机械性损伤神经元存活率的影响。结果 MTT法测定神经元机械性损伤后12 h细胞存活率显示:单纯损伤组细胞存活率为46%±4%,与对照组相比显著下降(P<0.05),不同剂量Nphe(30、300、1 200 nM)干预组的细胞存活率分别为56%±5%、67%±7%、72%±8%,与单纯损伤组存活率46%±4%相比差异显著(P<0.05)。LDH活性检测提示损伤后12 h和48 h,Nphe干预组LDH活性与损伤组相比有显著差异(P<0.05)。神经元机械性损伤后12 h,Nphe能够降低损伤后细胞内钙离子水平(P<0.05)。结论 ORL-1的特异性拮抗剂Nphe能够减少机械性损伤后继发性神经元损害,对神经元具有一定的保护作用。     

Effects of receptor density on Nociceptin/OrphaninFQ peptide receptor desensitisation: studies using the ecdysone inducible expression system

Pretreatment of the G-protein coupled nociceptin receptor (NOP) with nociceptin/orphaninFQ (N/OFQ) produces desensitisation. The influences of receptor expression and genomic effects are largely unknown. We have used an ecdysone-inducible NOP expression system in a CHO line (CHO_INDhNOP) to examine the effects of N/OFQ pretreatment upon receptor density, GTPγ[³⁵S] binding, cAMP formation and NOP-mRNA. CHO_INDhNOP induced with 5 and 10 μM PonasteroneA (PonA) for 20 h produced NOP densities (B _max) of 194 and 473 fmol. mg^-1 protein, respectively. This was accompanied by decreased NOP mRNA. The lower B _max is typical of the central nervous system. Pretreatment with 1 μM N/OFQ significantly (p < 0.05) reduced B _max at 5 and 10 μM PonA to 100 and 196 fmol. mg^-1 protein, respectively. There was no change in binding affinity. Along with the reduction in B _max, potency and efficacy for N/OFQ-stimulated GTPγ[³⁵S] binding were also reduced (5 μM PonA: pEC₅₀-control = 8.55 ± 0.06, pretreated = 7.88 ± 0.07; E _max-control = 3.52 ± 0.43, pretreated = 2.48 ± 0.10; 10 μM PonA: pEC₅₀-control = 8.41 ± 0.18, pretreated = 7.76 ± 0.03; E _max-control = 5.07 ± 0.17, pretreated = 3.38 ± 0.19). For inhibition of cAMP formation, there was a reduction in potency (5 μM PonA: pEC₅₀-control = 9.78 ± 0.08, pretreated = 8.92 ± 0.13; 10 μM PonA: pEC₅₀-control = 9.99 ± 0.07, pretreated = 9.04 ± 0.14), but there was no reduction in efficacy. In addition, there were 39 and 31% reductions in NOP mRNA at 5 and 10 μM PonA, respectively, but these measurements were made following concurrent N/OFQ challenge and PonA induction. In CHO_INDhNOP, we have shown a reduction in cell surface receptor numbers and a reduction in functional coupling after N/OFQ pretreatment. This was observed at pseudo-physiological and supraphysiological receptor densities. Moreover, we also report a reduction in NOP mRNA, but further studies are needed which include ‘pulsing’ PonA and desensitizing following wash-out.     

Orphanin FQ, a novel neuropeptide with anti-stress-like activity

Potential anxiolytic-like properties of intracerebroventricular (i.c.v.) infusion of orphanin FQ (OFQ), a recently discovered neuropeptide, were investigated in the mouse defense test battery, a well-validated anxiolytic screening test. In this model, Swiss mice are directly confronted with a natural threat (a rat) as well as situations associated with this threat. Primary measures taken during and after rat confrontation were flight, risk assessment, defensive attack and escape attempts. Unlike the anxiolytic drug diazepam (3–10 μg/5 μl, i.c.v.), which affected all defensive responses, OFQ (0.3–3 nM/5 μl) only clearly reduced defensive upright postures and biting reactions. Subjects displayed these latter defensive behaviors upon forced contact with the threat stimulus, a situation which is considered to be highly stressful. These results suggest that the OFQ system may not be primarily involved in anxiety-related responses including cognitive aspects (i.e., risk assessment), while it may play a role in the adaptative responses to unavoidable or extreme stress stimuli.     

实验性脑损伤大鼠血浆及肾脏组织孤啡肽水平的变化

目的研究脑损伤大鼠血浆及肾组织匀浆中孤啡肽含量变化及急性肾损害中的作用。方法提高建立大鼠脑损伤模型,用RIA方法测定血浆及肾组织匀浆中孤啡肽含量。结果大鼠脑外伤组血浆及肾组织匀浆中孤啡肽含量明显高于对照组。结论孤啡肽含量变化在脑外伤后急性肾损害过程中起重要作用。     

Pharmacological attenuation of chronic alcoholic pancreatitis induced hypersensitivity in rats

AIM:To characterize an alcohol and high fat diet induced chronic pancreatitis rat model that mimics poor human dietary choices.METHODS:Experimental rats were fed a modified Lieber-De Carli alcohol(6%) and high-fat(65%) diet(AHF) for 10 wk while control animals received a regular rodent chow diet.Weekly behavioral tests determinedmechanical and heat sensitivity.In week 10 a fasting glucose tolerance test was performed,measuring blood glucose levels before and after a 2 g/kg bodyweight intraperitoneal(i.p.) injection of glucose.Post mortem histological analysis was performed by staining pancreas and liver tissue sections with hematoxylin and eosin.Pancreas sections were also stained with Sirius red and fast green to quantify collagen content.Insulinexpressing cells were identified immunohistochemically in separate sections.Tissue staining density was quantified using Image J software.After mechanical and heat sensitivity became stable(weeks 6-10) in the AHF-fed animals,three different drugs were tested for their efficacy in attenuating pancreatitis associated hypersensitivity:a Group Ⅱ metabotropic glutamate receptor specific agonist(2R,4R)-4-Aminopyrrolidine-2,4-dicarboxylate(APDC,3 mg/kg,ip; Tocris,Bristol,United Kingdom),nociceptin(20,60,200 nmol/kg,ip; Tocris),and morphine sulfate(3 mg/kg,μ-opioid receptor agonist; Baxter Healthcare,Deerfield,IL,United States).RESULTS:Histological analysis of pancreas and liver determined that unlike control rats,AHF fed animals had pancreatic fibrosis,acinar and beta cell atrophy,with steatosis in both organs.Fat vacuolization was significantly increased in AHF fed rats(6.4% ± 1.1% in controls vs 23.8% ± 4.2%,P 0.05).Rats fed the AHF diet had reduced fasting glucose tolerance in week 10 when peak blood glucose levels reached significantly higher concentrations than controls(127.4 ± 9.2 mg/d L in controls vs 161.0 ± 8.6 mg/d L,P 0.05).This concurred with a 3.5 fold higher incidence of single and small 2-10 cell insulin-positive cell clusters(P 0.05).Insulin expressing islet of Langerhans cells appeared hypertrophied while islet number and area measurements were not different from controls.Weekly behavioral tests determined that mechanical and heat sensitivities were significantly increased by 4 wk on AHF diet compared to controls.Hypersensitivitywas attenuated with efficacy similar to morphine with single dose treatment of either metabotropic glutamate receptor 2/3 agonist APDC,or nociceptin,the endogenous ligand for opioid-receptor-like 1 receptor.CONCLUSION:The AHF diet induces a chronic alcoholic pancreatitis in rats with measurable features resembling clinical patients with chronic pancreatitis and type 3c diabetes mellitus.     

Immunolocalization of orphanin FQ in rat cochlea

Orphanin FQ/nociceptin (OFQ/N) and its receptor (ORL-1) have been proposed to play a role in the regulation of hearing. In this study, we investigate the localization of OFQ/N-like immunoreactivity in the mammalian cochlea. Sprague-Dawley rat temporal bones were harvested and decalcified. The organ of Corti was microdissected, and indirect immunohistochemistry was performed using a rabbit polyclonal antibody raised against OFQ/N. Immunoreactivity was seen in the tunnel crossing fibers and the large boutons terminating onto outer hair cells, and in the fibers terminating onto the afferents to the inner hair cells. The findings are consistent with OFQ/N expression in lateral and medial olivocochlear efferents.     

Orphanin FQ and glutamate connection in the regulation of gonadotropin-releasing hormone secretion in the preoptic area of conscious male rats

Utilizing the method of push-pull perfusion and radioimmunoassay (RIA), the secretory profile of gonadotropin-releasing hormone (GnRH) in the preoptic area (POA) and serum-luteinizing hormone (LH) levels were examined in conscious male rats after administration of [Nphe(1)]NC(1-13)NH(2), a competitive antagonists of the opioid receptor-like 1 receptor (ORL1 receptor) which is endogenous receptor for Orphanin FQ (OFQ). Glutamate release in the POA was also measured by high-performance liquid chromatography (HPLC) after perfusion of [Nphe(1)]NC(1-13)NH(2), i.e. NC13. The results showed that GnRH secretion from the POA and serum LH levels was increased significantly 40 min and 60 min, respectively after perfusion of 2 and 20 mmol/L NC13 in freely moving male rats (p<0.05). Pretreatment with a glutamate, N-methyl-D-aspartate (NMDA) receptor antagonist (MK-801, s.c., 0.2 mg/kg) abolished the increase of GnRH release in the POA induced by 2 mmol/L NC13. Additionally, 20 mmol/L NC13 significantly enhanced glutamate release in the POA at 40 min post-perfusion in a dose-dependent manner. These findings suggest that hypothalamic OFQ/ORL1 receptor system plays a role in the physiological inhibitory control of GnRH secretion in the POA of male rats, and provide evidence for involvement of an OFQ and glutamate pathway in the control of GnRH secretion.     

Immunological challenge modulates brain orphanin FQ/nociceptin and nociceptive behavior

Neuromedin B (NMB) is a mammalian bombesin-like peptide distributed widely in the central nervous system. This peptide exerts its function via the NMB receptor (NMB-R). Female NMB-R-deficient mice were used to study the role that NMB/NMB-R may play in 5-HT neuron function since this relationship was suggested in previous in vitro studies. As 5-HT neurons are thought to modulate marble burying behavior, a role for NMB-R in this behavior was assessed. Relative to wild-type mice, NMB-R-deficient mice showed decreased marble burying behavior. However, depletion of 5-HT by treatment with p-chlorophenylalanine (p-CPA) increased burying behavior in NMB-R-deficient mice suggesting that increased levels of 5-HT in the brain cause a decrease in burying behavior in NMB-R-deficient mice. While HPLC analysis showed that 5-HT content in the whole brain does not differ between NMB-R-deficient and wild-type mice, an immunohistochemical analysis of brain sections showed that 5-HT expression in the dorsal raphe (DR) nucleus is elevated in NMB-R-deficient mice. Furthermore, a quantitative RT-PCR analysis revealed that 5-HT(1A)-receptor gene expression is downregulated in NMB-R-deficient mice at the whole brain level. These behavioral and biological results suggest that NMB/NMB-R may modulate 5-HT neuronal activity by affecting DR function.