Role of PERK pathway in morphine-induced myocardial protection of H9c2 cells

AIM: To explore whether morphine protects oxidative stress-damaged myocardial cells by inhibiting the PERK pathway to reduce endoplasmic reticulum stress and prevent mitochondrial permeability transition pore (mPTP) opening. METHODS: Rat myocardial H9c2 cells were cultured to establish an oxidative stress model, and then randomly divided into control group, H₂O₂ group, H₂O₂+morphine group, H₂O₂+morphine+PERK pathway inhibitor GSK2656157 group, morphine group and GSK2656157 group. Immunohistochemical method was used to detect the effects of morphine on expression of glucose-regulated protein (GRP) 78 and GRP94 induced by oxidative stress. The protein levels of PERK signaling pathway-related molecules were determined by Western blot. Confocal microscopy was used to observe the effects of morphine on mPTP opening and endoplasmic reticulum induced by oxidative stress. Cellular toxicity was detected by lactate dehydrogenase (LDH) kit and cell viability was measured by MTT assay. RESULTS: Compared with control group, GRP78 and GRP94 proteins in H₂O₂ group were strongly expressed, and the brown-yellow particles were significantly increased, but morphine significantly inhibited this process. Compared with control group, the phosphorylation of PERK was significantly reduced with GSK2656157 treatment at different concentrations, among which 2 μmol/L had the most significant effect (P < 0.05). Oxidative stress significantly increased the protein levels of GRP78, GRP94, p-PERK and CHOP, but significantly decreased p-GSK-3β level. These changes were inhibited by morphine, and the effects of morphine were further enhanced by GSK2656157 (P < 0.05). Compared with control group, oxidative stress significantly reduced the fluorescence intensity of mitochondrial TMRE and ER-Tracker Red. Morphine significantly inhibited this effect even when mitochondrial membrane potential was reduced, mPTP was open, and endoplasmic reticulum was damaged, while GSK2656157 further enhanced the effect of morphine (P < 0.05). Compared with control group, H₂O₂ significantly increased cellular toxicity and decreased the cell viability. Morphine inhibited this effect and GSK2656157 significantly enhanced the effect of morphine (P < 0.05). CONCLUSION: Morphine protects cardiac H9c2 cells under oxidative condition by inhibiting endoplasmic reticulum stress through PERK pathway and preventing the mPTP opening via GSK-3β inactivation.     

不同灌水模式下罂粟吗啡含量的变化

本文初步探讨了水分调控对罂粟不同生育时期地上主要器官吗啡含量的影响,实验结果表明,冬灌区现蕾期到盛花前期,随着灌水频率加大,罂粟壳中吗啡含量明显提高,盛花后期的连续灌水使罂粟壳中吗啡含量急剧下降,免冬灌区恰好相反,发育后期适当加大灌水频率和提高灌水量能明显的提高罂粟壳中的吗啡含量     

Effects of CCK-8 and its receptor antagonists on expression of CREB and pCREB in prefrontal cortex and hippocampus of morphine withdrawal rats

AIM：To observe the effects of cholecystokinin octapeptide (CCK-8) and its receptor antagonists on cAMP response element binding protein (CREB) and phosphorylated CREB (pCREB） expression in frontal cortex and hippocampus of morphine withdrawal rats, which aim to explore the post-receptor mechanism through which CCK-8 regulates morphine withdrawal. METHODS：After the morphine dependence and naloxone-precipitated withdrawal animal models were established, the effects of CCK-8, L-364718 (CCK1 receptor antagonist) and LY-288513 (CCK2 receptor antagonist) pretreatment on CREB and pCREB expression in frontal cortex and hippocampus were observed by Western blotting and immunohistochemistry. RESULTS：In rat frontal cortex neuron, CREB was expressed in both cytoplasm and nucleus, but pCREB was only highly expressed in the nucleus. In the pyramidal cell layer of hippocampal CA1 region, CREB showed high expression in the cytoplasm and low expression in the nucleus, while pCREB was only expressed in the nu-cleus. No obvious change of CREB was observed after either chronic morphine treatment or naloxone withdrawal. The pCREB expression was increased after chronic morphine treatment and further increased after naloxone withdrawal. Compared with the withdrawal group, chronic pretreatment with CCK-8, L-364718 and LY-288513 had no effect on CREB expression in the frontal cortex, but obviously decreased the pCREB expression. In the hippocampus, pretreatment with L-364718 and LY-288513 decreased CREB and pCREB expression, but only the pCREB expression was decreased after CCK-8 treatment. CONCLUSION：CCK-8 and CCK receptor antagonists may alleviate morphine withdrawal symptoms by regulating CREB, with specificity in different brain regions.     

Comparison of Analgesic Effects of Caudal Epidural 0.25% Bupivacaine with Bupivacaine Plus Morphine or Bupivacaine Plus Ketamine for Analgesia in Conscious Horses

The aim of this study was to compare the effects of caudal epidural bupivacaine alone (BP), bupivacaine plus morphine (BPMP), and bupivacaine plus ketamine (BPKE) for perineal analgesia in horses. Each of the six saddle horses received a caudal epidural catheter and underwent 3 treatments: BP, 0.25% (0.04 mg/kg) bupivacaine hydrochloride without epinephrine; BPMP, 0.02 mg/kg of bupivacaine combined with 0.1 mg/kg of morphine-preservative free; and BPKE, 0.02 mg/kg of bupivacaine combined with 0.5 mg/kg of ketamine. The order of treatments was randomized. The cardiovascular system, respiratory rate, quality of analgesia, sedation, and motor blockade were assessed before drug administration (baseline), at 5, 10, 15, and 30 minutes, and every 30 minutes thereafter until loss of analgesia. The median time to onset of analgesia was 5 minutes after BP treatment, faster than after BPKE or BPMP treatments, which were 10 minutes and 15 minutes, respectively (P < .05). The BPMP treatment produced analgesia (315 minutes) for a longer duration than BP treatment (210 minutes) or BPKE treatment (240 minutes), in the regions of the tail, perineum, and upper hind limb in horses. All treatments presented mild sedation or motor blockade. There were minimal effects on the cardiovascular system and respiratory rate. BPMP may be preferable to a high dose of BP or BPKE. Caudal epidural BPMP can be an appropriate choice for regional perineal analgesia in horses.     

A dopamine receptor antagonist modulates or enhances the analgesia of morphine and analysis of their synergetic analgesic mechanism

AIM:To observe the synergetic analgesic effects of low dose of haloperidol, a dopamine antagonist and under-threshold dose of morphine on mice induced by thermal and acetic acid, and to analyze the major mechanism of their synergetic actions. METHODS:To examine the analgesic synergetic effect of haloperidol (0.315 mg/kg, 0.625 mg/kg, 1.25 mg/kg, ip respectively), morphine (3.125 mg/kg, 6.25 mg/kg, 12.5 mg/kg ip, respectively) or combining effect of haloperidol (0.3125 mg/kg) with morphine (3.125 mg/kg) on mice, we compared the change of pain threshold stimulated by thermal, latent period of twisting, the number of times of twisting by acetic acid, and we also estimated the antagonistic effect of d-amphetamine (10 mg/kg) and naloxone (5 mg/kg) on haloperidol and morphine group. RESULTS:Combination of haloperidol with morphine significantly enhanced pain threshold of mice induced by thermal,prolonged latent period of twisting and decreased the number of times of twisting.Naloxone markedly antagonized the combination of analgesic action of haloperidol and morphine and not d-amphetamine.CONCLUSION:Combination of haloperidol with morphine have synergetic analgesic effect and morphine is the dominant factor.     

Roles of Akt pathway and cell apoptosis in leptin-mediated chronic morphine antinociceptive tolerance

AIM: To explore the roles of Akt (also called protein kinase B) and active caspase-3 in the leptin-mediated chronic morphine antinociceptive tolerance in rats. METHODS: A model of chronic morphine antinociceptive tolerance was established in the SD rats. The protein levels of spinal Akt and cleaved caspase-3 were tested by Western blotting. The technique of immunohistochemical staining was used to detect the immunoreactivity positive cells of phosphorylated (p)-Akt and cleaved caspase-3 in the spinal cord. Double staining of immunohistochemistry was used to examine the cellular location of the p-Akt and cleaved caspase-3 positive cells. RESULTS: The chronic intrathecal injection of morphine (15 μg) for 7 d markedly upregulated the spinal protein levels of p-Akt and cleaved caspase-3 in the rats. Thirty min before injection of morphine, intrathecal injection of leptin antagonist (3 μg) for 7 d significantly attenuated the upregulation of the protein levels of p-Akt and cleaved caspase-3 induced by chronic morphine treatment. The p-Akt was exclusively observed in the spinal neurons. The cleaved caspase-3 was only localized with the spinal astrocytes. Intrathecal injecting the inhibitors of leptin, Akt and caspase-3 ameliorated the chronic antinociceptive tolerance. CONCLUSION: The spinal Akt pathway and active caspase-3 are involved in the leptin-mediated chronic morphine antinociceptive tolerance in rats.     

食品中罂粟碱、那可丁、吗啡、可待因和蒂巴因的测定——高效液相色谱法

S本法建立了高效液相色谱法测定食品中罂粟碱、那可丁、吗啡、可待因和蒂巴因5种非法添加物的检测方法,色谱柱ZORBAX SB-C18(4.6×150 mm 5-Micron),流动相为0.1%甲酸的10 mmol/L乙酸铵溶液-乙腈(10:90),流速0.3 mL/min,检测波长280 nm,进样量10 μL,柱温30 ℃,进行定量分析。在浓度为0.01-10.00μg/mL范围内,标准曲线线性关系良好(相关系数R＞0.9990), 罂粟碱、那可丁、吗啡、可待因和蒂巴因的检出限分别是：0.05 μg/mL、0.03 μg/mL、0.1μg/mL、0.1μg/mL、0.05 μg/mL。五种生物碱加标水平在1.2、1.5 mg/kg时,回收率为88-93%,相对偏差为1.4-3.2%。该方法方便快捷,成本较低,操作简便,分析快速,可用于食品中5种非法添加物检测。     

紫金龙总提取物对吗啡依赖小鼠戒断症状的影响

[目的]观察紫金龙总提取物对吗啡依赖小鼠戒断症状的影响。[方法]以连续递增剂量方式皮下注射盐酸吗啡建立吗啡依赖小鼠模型,灌胃不同剂量（150、300、600 mg/kg）的紫金龙总提取物40 min后,用纳络酮（4 mg/kg）腹腔注射催瘾。[结果]紫金龙总提物低、中、高剂量组对吗啡依赖小鼠戒断跳跃症状有抑制作用（P〈0.05）;对扭体症状具有显著抑制作用（P〈0.01）;对小鼠体重减少症状具有抑制作用（P〈0.05）,并且中、高剂量组效果显著（P〈0.01）;然而仅低、中剂量组对小鼠抓脸的戒断症状有抑制作用（P〈0.05）,其中中剂量组效果显著（P〈0.01）,而高剂量组与自然戒断组比较无显著性差异。[结论]紫金龙总提取物在一定程度上可缓解吗啡依赖小鼠戒断症状。     

Evaluation of alkaloid profiles in hybrid generations of different poppy (Papaver somniferum L.) genotypes

Five varieties (‘Minoan’, ‘Medea’, ‘Korona’, ‘Przemko’, ‘Kozmosz’) of poppy representing different chemotypes were combined and the alkaloid profiles of F1-F3 progenies were studied during 2006-2009.In the crosses of high alkaloid containing varieties the content of total alkaloids and that of morphine and thebaine showed increased levels in the hybrid generations which persisted till F3. Narcotine (noscapine), however accumulated at lower level than the midparent values and showed a decreasing tendency over generations. A higher number of homogenous strains starts to appear in F3 progenies. The majority of codeine containing individuals concentrates to certain strains. A growing number of high thebaine containing individuals was observed in several combinations. In the crosses with low alkaloid containing parent (‘Przemko’) the F1 exhibits considerable heterosis for total alkaloid content. Low alkaloid containing recessive individuals segregate in F2 and stabilise in F3.Results of our crossing experiments reflected well the effects of genetic regulation at three levels of enzymatic processes during the biosynthesis of poppy alkaloids. Data support the recessive determination of transformations at TyDC (tyrosine decarboxylase) and BBE (berberine bridge enzyme) levels while more complex (polygenic) effects are supposed in controlling the quantity of narcotine (noscapine) and morphinanes. Selection for fixing very low alkaloid content and narcotine may be effective in early F2 generations, however a selection for morphinanes is not worthy before F3.