孤啡肽和阿片类配体与阿片孤儿受体相互作用的机制研究
摘要： 研究孤啡肽和阿片类配体与阿片孤儿受体相互作用的分子机制。方法：用分子动力学方法计算孤啡肽的低能构象；通过分子对接程序将孤啡肽、阿片类配体对接到阿片孤儿受体的结合口袋中；通过结合能的计算研究配体对受体的亲和力与它们的结合能之间的关系。结果：孤啡肽(1-4)残基位于结合口袋的底部，孤啡肽(5-7)残基位于结合口袋的顶部，孤啡肽(8-17)残基与孤儿受体的第二膜外环区结合；阿片类配体和孤儿受体的结合方式与孤啡肽的情况类似，区别在于孤儿受体参与配体结合的残基种类和数量不同，因而亲和力不同；配体-受体的结合能与配体的亲和力之间有很好的相关性；预测了洛芬太尼四个异构体与阿片孤儿受体的亲和力。结论：该研究能够解释许多实验事实，有助于进一步理解阿片受体与配体相互作用的分子机制并设计新的分子生物学实验。
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大鼠腹腔肥大细胞脱颗粒过程中磷脂酶D活性的变化
AIM: To study the changes of phospholipase D (PLD) activity of actively sensitized rat peritoneal mast cells (RPMC) in degranulation. METHODS: Degranulation of RPMC was determined by measurement of β-hexosaminidase release. PLD activity assay was carried out by measurement of PLD product, choline, with chemiluminescent oxidation of luminol. RESULTS: Actively sensitized RPMC challenged with ovalbumin (0.5-8 mg/L for 120 s, 4 mg/L for 15-120 s) resulted in significant activation of PLD accompanied with the increment of β-hexosaminidase release. PLD activity of sensitized RPMC was increased by more than 2-fold compared with that of unsensitized RPMC which contained low levels of PLD activity [(35+ 13) pmol choline/min in 1 x 106cells], but β-hexosaminidase releases of the sensitized cells were as low as spontaneous releases. After challenge with ovalbumin 4 mg/L for 120 s, PLD activity of sensitized RPMC was increased to (155+43) pmol choline/min in lx 106cells and β-hexosaminidase release was also elevated significantly (4.5-fold of spontaneous release, n=6, P＜0.05). When unsensitized RPMC were stimulated with antigen, PLD activity and β-hexosaminidase release of the cells were hardly changed.Sensitized RPMC were treated with 1% 1-butanol or 2,3- disphosphoglycerate l0 mmol/L before challenge with ovalbumin, these drugs induced an inhibition of PLD activity and a reduction of β-hexosaminidase release to basal level. 1-Butanol 0.1% also worked. CONCLUSION: Phospholipase D plays an important role in the regulation of β-hexosaminidase release in actively sensitized rat peritoneal mast cells.
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活性炭血液灌流对重度敌敌畏中毒的治疗作用
AIM: To assess the efficacy of hemoperfusion (HP) in the treatment of the patients with acute severe dichlorvos (DDVP) poisoning. METHODS: One hundred and eight patients with acute severe DDVP poisoning in the two teaching hospitals were enrolled. Sixty-seven patients were treated with HP (HP group) and forty-one patients accepted traditional treatment only as the control. Serum concentration of DDVP was determined by gas chromatography. RESULTS: The duration of coma, impaired consciousness, ICU stay, and mechanical ventilation was significantly shorter in the HP group than that in the control. The cumulative dosages (mg) of atropine required either in the first 24 h on admission (442±436 vs 899±485 in the control, P＜0.01) or within the hospital (568±574 vs 1228±982 in the control, P＜0.01) were markedly reduced in the HP patients. The lower incidence of mechanical ventilation required (13.4 ％ vs 36.6 ％ P＜0.01), respiratory muscular paralysis (4.5 ％ vs 17.1％, P＜0.05) and the lower mortality of death (7.5 ％ vs 34.1％, P＜0.01) were observed in the HP group. HP could accelerate the recovery of suppressed cholinesterase activity. After the procedure, the DDVP level was decreased from (11±±4) to (7±±3) mg/L in parallel with a decline in APACHE Ⅱ Score or dopamine dose and a rise in Glasgow Coma Scale (P＜0.05). In addition, the mean values of peak clearance and reduction rate were (87±17) Ml/min and 44 ％±±11％,respectively. CONCLUSION: The rapid fall in blood DDVP level and the dramatic clinical response suggest that HP is effective in the treatment of acute severe DDVP poisoning.
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鞘内注射NMDA和AMPA受体激动剂或拮抗剂对异丙酚抗伤害作用的影响
AIM: To study the effects of intrathecal (it) agonists and antagonists of N-methyl-D-aspartate (NMDA) and alphaamino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors and NMDAR1 antisenseoligodeoxynucleotides (AS ODN) on the antinociception of propofol. METHODS: Hot-plate test (HPPT) and acetic acid-induced writhing test were used to measure the nociceptive thresholds in mice. The effects of intrathecal NMDA, AMPA, MK-801, NBQX, or NMDAR1 AS ODN on the antinociception of propofol were observed.RESULTS: Propofol (25, 50 mg/kg, ip) displayed an appreciable antinociceptive effect in hot-plate test and acetic acid-induced writhing test. NMDA (12.5, 25 ng, it) or AMPA (1.25, 2.5 ng, it) exhibited no effects on the behavior but decreased HPPT significantly compared with basal HPPT and aCSF group (P＜0.05, P＜0.01). No effects on behavior and HPPT were obtained in NMDA (6.25 ng, it) or AMPA (0.625 ng, it) groups. NMDA (6.25, 12.5, and 25 ng, it) dose-dependently decreased the HPPT in propofol-treated group. AMPA (1.25, 2.5 ng, it) also decreased HPPT significantly. MK-801 (0.25, 0.5 μg, it) or NBQX (0.25, 0.5 μg, it) groups had no behavioral changes, two antagonists 0.5 μg but not 0.25 μg increased HPPT in conscious or propofol-treated mice. AS ODN (5, 10, and 20 μg, it) groups exhibited dose-dependent increased in HPPT in propofol-treated groups compared with aCSF group (P＜0.05, P＜0.01). CONCLUSION: Both agonists NMDA and AMPA reversed the antinociception of propofol.MK-801, NBQX, and NMDAR1 AS ODN potentiated the antinociceptive effects of propofol. Propofol produced antinociception through an interaction with spinal NMDA and AMPA receptors in mice.
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15-deoxy-△12,14-prostaglandin J2对ECV304细胞增殖与凋亡的影响
AIM: To investigate the effects of 15-deoxy-△12,14-prostaglandin J2 (15d-PGJ2) on cell proliferation and apoptosis in ECV304 endothelial cells and related molecular mechanism. METHODS: MTT, Hoechst33258, TUNEL, Flow cytometry, DNA ladder, RT-PCR, Western blot, and electrophoretic mobility shift assay (EMSA) analysis were employed. RESULTS: The 15d-PGJ2 induced apoptosis in ECV304 endothelial cells in a dose-dependent manner (the percentage of apoptosis was enhanced from 10.0 %+1.3 % to 32.8 %+1.6%), which was accompanied by inhibition of NF-κB and AP-1 DNA binding activity, down-regulation of c-myc, upregulation of Gadd45 and p53,and activation of p38 kinase. However, the expression of p21 was found no significant change. CONCLUSION:peroxisome proliferator-activated receptor gamma ligand, 15d-PGJ2, can inhibit proliferation and induce apoptosis in ECV304 endothelial cells through different mechanisms.
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p44/p42 MAPK反义寡脱氧核苷酸对血管紧张素Ⅱ诱导的培养乳鼠心肌细胞肥大反应的抑制作用
AIM: To explore the inhibitory effect of antisense oligonucleotide (ODN) to mitogen activated protein kinase (MAPK) on cardiomyocyte hypertrophy induced by angiotensin Ⅱ (Ang Ⅱ). METHODS: A 17-mer phosphorothioateprotected antisense ODN directed against the initiation of translation sites of the p42 and p44 MAPK isoforms by liposomal transfection was applied to inhibit the translation of p44/p42 MAPK mRNA. The sense and random ODNs to p44/p42MAPK were used as sequence controls. Neonatal cardiac myocytes were exposed to Ang Ⅱ (10 nmol/L) for 5 min and then harvested in lysis buffer for the measurement of the activity and the phosphorylated protein content of p44/p42MAPK that were tested by P-81 phosphocellulose filter paper method and Western blotting, respectively. The rate of protein synthesis by [3H]leucine incorporation and the diameter of cell were measured after exposure to Ang Ⅱ for 24 h and 72 h, respectively. RESULTS: In cardiac myocyte Ang Ⅱ increased p44/p42MAPK activity and phosphorylated protein content by 140 % and 699 %, and also increased [3H]leucine incorporation and cell diameter by 40 % and 27 %. c-fos and c-myc mRNAs were induced significantly after exposure to Ang Ⅱ. Antisense ODN to p44/p42MAPK (0.2 μmol/L) reduced Ang Ⅱ-induced MAPK activity by 30 %,and phophorylated MAPK protein expression by 59 % in cardiac myocyte, and inhibited c-fos and c-myc mRNA expression induced by Ang Ⅱ by 44 % and 43 %, respectively. The diameter and the rate of protein synthesis of cardiac myocyte induced by Ang Ⅱ were decreased by 16 % and 22 % after pretreatment with antisense ODN to p44/p42MAPK. CONCLUSION: Antisense ODN to p44/p42 MAPK inhibited the increase of rate of protein synthesis,and the augmentation of cell diameter and expression of c-fos and c-nyc mRNA induced by Ang Ⅱ in cultured cardiac myocytes, p44/p42 MAPK played a critical role in the hypertrophic response induced by Ang Ⅱ in cultured neonatal rat cardiac myocytes.
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托吡酯对Lewis肺癌转移的抑制作用与水通道蛋白1的关系
AIM: To study the effect of topiramate on tumor metastasis and its relation with aquaporin 1 (AQP1) water channel. METHODS: Lewis lung carcinoma metastatic model was used to determine the effect of topiramate on tumor growth and metastasis. Colorimetric estimation was used to investigate the action of topiramate on carbonic anhydrase (CA) activity. Western blotting and immunohistochemical analysis were used to study the influence of topiramate on AQP1 water channel expression in lungs or tumor tissues of mice bearing Lewis lung carcinoma.cantly (P＜0.05). Its inhibitory rate of metastasis was 81.25 %. Topiramate inhibited CA activity in lungs of mice in a dose-dependent manner. Topiramate apparently decreased AQP1 protein expression and immunostaining in lungs or in tumor microvessel endothelial cells of mice. CONCLUSION: Suppression of AQP1 water channel expression may be an important pathway for the inhibitory effect of topiramate on tumor metastasis.
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前胡甲素对缺血再灌注心肌NF-κB活性和TNF-α表达的影响
AIM: To study the effects of dl-praeruptorin A (Pd-Ia) on nucleus factor-κB (NF-κB) activativity and tumor necrosis factor-α (TNF-α) expression in ischemia-reperfusion (I/R) myocardium. METHODS: Langendorff's isolated rat heart was subjected to a 10-min ischemia followed by a 30-min reperfusion. NF-κB activity in nucleus was analyzed by Sandwich Enzyme-Linked Immunosorbent Assay (ELISA). TNF-α level in cytoplasm was measured by radioimmunoassay. Infiltration of neutrophils was observed using Hematoxylin-Eosin staining under optical microscope. RESULTS: Pd-Ia 1.0 μmol/L with 30-min preventive perfusion decreased NF-κB activity from 0.98±0.13 to 0.65±0.17 (P＜0.05 vs solvent) and down-regulated TNF-α expression from 13.7±6.1 μg/L to 9.4±2.7 μg/L (P＜0.01 vs solvent) under conditions with increase of coronary flow, negative inotropic action,inhibition of creatine kinase and without chronotropic action, whereas, infiltration of neutrophils was mild.CONCLUSION: Pd-Ia inhibited NF-κB activativity in I/R myocardium and led to down-regulation of TNF-α expression, which might be one of molecular mechanisms of Pd-Ia in cardioprotection.
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采用RNA干扰技术抑制A549细胞表皮生长因子受体表达
AIM: To investigate the biological features of A549 cells in which epidermal growth factor (EGF) receptors expression were suppressed by RNA interference (RNAi). METHODS: A549 cells were transfected using short small interfering RNAs (siRNAs) formulated with Lipofectamine 2000. The EGF receptor numbers were determined by Western blotting and flowcytometry. The antiproliferative effects of sequence specific double stranded RNA(dsRNA) were assessed using cell count, colony assay and scratch assay. The chemosensitivity of transfected cells to cisplatin was measured by MTT. RESULTS: Sequence specific dsRNA-EGFR down-regulated EGF receptor expression dramatically. Compared with the control group, dsRNA-EGFR reduced the cell number by 85.0 %,decreased the colonies by 63.3 %, inhibited the migration by 87.2 %, and increased the sensitivity of A549 to cisplatin by four-fold. CONCLUSION: Sequence specific dsRNA-EGFR were capable of suppressing EGF receptor expression, hence significantly inhibiting cellular proliferation and motility, and enhancing chemosensitivity of A549 cells to cisplatin. The successful application of dsRNA-EGFR for inhibition of proliferation in EGF receptor overexpressing cells can help extend the list of available therapeutic modalities in the treatment of non-small-cell lung carcinoma (NSCLC).
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野生型p53增强药物耐药的人肝癌细胞Bel7402/5-FU的化疗敏感性
AIM: To study the effect of wild type (wt) p53 gene transfection on drug resistant human hepatocellular carcinoma (HCC) cells induced by 5-Fluorouracil (5-FU). METHODS: The cytotoxicity of anticancer drugs on Be17402 and Be17402/5-FU cells was assessed using SRB assay. p53 expression was detected at its mRNA level by RT-PCR assay and at its protein level Western blot or immunocytochemistry assay in Be17402/5-FU cells transfected with either control vector or wt p53. AnnexinV-FITC/PI double labeled assay was performed to detect apoptosis. The chemosensitivity of Be17402/5-FU cells transfected with wt p53 was assessed using SRB assay. RESULTS: Be17402/5-FU cells exhibited cross-resistance to vincristine, doxorubicin, paclitaxel, and so on. wt p53 gene transfection upregulated the expression of p53 in Be17402/5-FU cells. wt p53 was able to greatly inhibit cell proliferation and significantly induce apoptosis in Bel7402/5-FU cells. Moreover, wt p53 gene transfection increased the chemosensitivity of Be17402/5-FU cells to some anticancer drugs. CONCLUSION: These results indicated that the wt p53 gene transfection not only induced suppression of cell growth, but also increased the sensitivity of Be17402/5-FU cells to 5-FU, vincristine, and doxorubicin.
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维拉帕米在大鼠肝微粒体中代谢产物的体外鉴定
AIM: To investigate the metabolism of verapamil at low concentrations in rat liver microsomes. METHODS: Liver microsomes of Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of verapamil was studied with the rat liver microsomal incubation at concentration of 1.0 μmol/L and 5.0 μmol/L. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MSn), and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. RESULTS: Eight metabolites, including two novel metabolites (M4 and M8), were found in rat liver microsomal incubates. They were identified as O-demethyl-verapamil isomers (M1 - M4), N-dealkylated derivatives of verapamil (M5-M7), and N, O-didemethyl-verapamil (M8). CONCLUSION: O-Demethylation and N-dealkylation were the main metabolic pathways of verapamil at low concentrations in rat liver microsomes, and the relative proportion of them in verapamil metabolism changed with different substrate concentrations.
