白芍水煎剂对老龄小鼠抗衰老作用的实验研究

中药白芍水煎剂给予老龄小鼠灌胃(10g/kg),每日一次,连用4周。采用邻苯三酚自氧化法、DTNB和TBA比色法测定红细胞超氧化物歧化酶(SOD)活性,全血谷胱甘肽过氧化物酶(GSH-Px)活力和红细胞中及血浆中MDA含量。结果表明,白芍水煎剂能显著增强老龄小鼠红细胞SOD活性、全血GSH-Px的活力,而且可显著降低红细胞和血浆中MDA含量,故具有延缓衰老作用。     

白细胞介素—2脂质体的抗肿瘤作用试验

本文建立Ｃ５７ＢＬ／６小鼠荷瘤动物模型，通过给荷瘤小鼠腹腔注射空白脂质体、单纯ＩＬ－２及ＩＬ－２脂质体来比较其在肿瘤生长中的抑制作用。结果是空白脂质体组抑瘤率为４．２６％，单纯ＩＬ－２和脂质体ＩＬ－２的抑瘤率分别为３４．０４％和５４．６０％。空白脂质体组与单纯对照组相比无显著性差异（Ｐ＞０．０５），脂质体ＩＬ－２组与单纯ＩＬ－２组之间存在显著性差异（Ｐ＜０．０５），脂质体ＩＬ－２的抑瘤率比单纯ＩＬ－２抑瘤率提高了２０．６％。     

酵母多糖脂质体的免疫增强作用

本文比较了酵母多糖脂质体、酵母多糖及脂质体对小鼠免疫功能的影响。结果表明,前两者对小鼠免疫功能均有促进作用,但经脂质体包封后的酵母多糖(多糖脂质体)表现出更强的促进能力,其差异有显著性(P＜0.01)。单纯脂质体的免疫增强作用不明显。     

超氧化物歧化酶脂质体在大鼠体内的药代动力学和组织分布

目的考察3种超氧化物歧化酶(SOD)脂质体静脉给药后在大鼠体内的药代动力学和组织分布。方法 用反相蒸发法制备SOD脂质体，采用黄嘌呤氧化酶法检测SOD活力，静脉注射给药后，测定大鼠血中SOD含量变化和不同组织中SOD含量变化。结果在血浆中，SOD水溶液、SOD普通脂质体、用DSPE-PEG2000修饰的SOD脂质体、用Tween 80修饰的SOD脂质体的半衰期分别为0.25，0.34，0.66和0.41 h；AUC分别为12.48，24.66，41.16和33.02 μg·h·mL^-1。与普通脂质体比较，经过DSPE-PEG和Tween 80修饰后的脂质体，使肝、脾中SOD的含量有不同程度的降低，脑中含量有所提高。结论3种SOD脂质体均可不同程度地延长SOD的血浆半衰期，并以用DSPE-PEG2000修饰的SOD脂质体效果最好。与普通脂质体相比，用Tween 80修饰的SOD脂质体可以提高进入脑中的SOD量，用DSPE-PEG2000修饰的SOD脂质体可以减少肝脾对SOD的摄取。     

白细胞介素2脂质体的制备及其抗肿瘤作用的研究

采用逆相蒸发法制备稳定的白细胞介素2（IL－2）大单层脂质体，对其包封率、稳定性及活性进行了测定。建立C57BL／6小鼠荷瘤动物模型，通过给荷瘤小鼠腹腔注射空白脂质体、单纯IL-2及IL-2脂质体来比较其在肿瘤生长中的抑制作用，结果3组抑癌率分别为4．26％、34．04％和54.60％。空白脂质体组与单纯对照组相比尤显著性差异（P＞0．05），脂质体-IL-2组与单纯IL-2组之间存在显著性差异（P＜0．05），抑瘤率提高了20.6％。     

聚(2-乙基丙烯酸)脂肪酰胺衍生物构建的酸敏脂质体

本实验合成了系列聚(2-乙基丙烯酸)长链脂肪酰胺衍生物，并采用高分子插入法制备了聚(2-乙基丙烯酸)酸敏高分子脂质体。应用荧光指示剂、粒径仪、荧光显微镜及细胞实验，系统研究了高分子修饰和脂肪胺的链长对高分子衍生物嵌入脂质体的效率和质量的影响。结果表明，高分子插入法可以制备聚(2-乙基丙烯酸)酸敏高分子脂质体。(1) 高分子嵌入量与高分子脂肪胺的链长无关，但与高分子修饰度相关。(2) 高分子嵌入量与起始的高分子-脂质体比例成正比。(3) 在酸性条件下聚(2-乙基丙烯酸)脂质体可产生显著的脂质体融合及释药行为。(4) 聚(2-乙基丙烯酸)脂质体在细胞内呈现出良好的酸敏诱导释药特性。实验证明这种方法制备的脂质体具有良好的酸敏释药性能，并且制备方法简便，可控性好，实用性强。     

不同脂质载体对超氧化物歧化酶(SOD)渗入小白鼠红细胞能力的观察

<正> 对SOD减少的疾病,放疗的肿瘤病人或超氧阴离子自由基(O_2增多的疾病,如心肌缺血再灌注损伤等,都可以用SOD来治疗。由于SOD分子量较大,不易进入细胞内。因此在临床应用中必须首先克服这个缺点。Michelson发现带正电荷的硬脂酰胺或烷酰基卵磷脂包裹的SOD进入细胞的能力要比单纯SOD大。为将SOD应用于临床,我们对不同的脂质载体携带SOD渗入小白鼠红细胞的能力进行了观察,现报道如下。材料和方法     

多胺胆固醇缀合物纳米粒作为反义核苷酸传递系统的研究

目的研究多胺胆固醇缀合物传递反义核苷酸的能力。方法通过对自制脂质体的载药量、红细胞毒性、M3骨髓瘤细胞的转染实验。结果自制脂质体具有比常规脂质体良好的载药量,并且对红细胞毒性相对偏小,对M3骨髓瘤细胞具有一定的转染能力。结论自制的脂质体具有一定的应用前景。     

油酸多相脂质体(139)注射液包封率测定方法的研究

本文提出了油酸多相脂质体139注射液中脂质体的药物包封率和药物含量的测定方法,以凝胶过滤法Sephadex G-50柱测定139注射液中多相脂质体的重量包封率Qw平均为94.2%;同时又以显微镜照像及统计方法测量了脂质体的体积包封率Qv平均为97.1%。并讨论了影响脂质体中药物包封率的各种因素。     

SOD和Cu（Ⅱ）络合物脂质体的SOD样活性

用Cyt.c-HX-XO法分别测定了SOD和4种Cu（Ⅱ）络合物及脂质体的SOD样活性，结果表明：所有体系均有某种程度的SOD样活性，其中组氨酸酮脂质体的SOD样活性最高，Cu(Ⅱ）络合物与脂质体具有一些协同作用。     

Role of superoxide dismutase enzymes and ascorbate in protection of nitrergic relaxation against superoxide anions in mouse duodenum

Aim: The aim of this study was to investigate whether superoxide dismutase (SOD) enzymes and ascorbate play a role in the protection of the nitrergic relaxation against superoxide anion inhibition in the mouse duodenum. Methods: The effects of exogenous SOD, N , N '-bis(salicylidene) ethylenediamine chloride (EUK-8; a synthetic cell-permeable mimetic of the manganese SOD [Mn SOD] and ascorbate on relaxant responses induced by nitrergic nerve stimulation), exogenous nitric oxide (NO), and nitroglycerin were investigated in isolated mouse duodenum tissues. Results: Diethyldithiocarbamate (DETCA) inhibited the relaxation to exogenous NO and nitroglycerin, but not relaxation to electrical field stimulation (EFS). SOD and ascorbate partially prevented the inhibitory effect of DETCA on relaxation to NO, abut not to nitroglycerin. The DETCA-induced inhibition on nitroglycerin was prevented by EUK-8. Hemoglobin, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazolinel-oxyl-3-oxide, and hydroxo-cobalamin inhibited the relaxation to NO, but not to EFS and nitroglycerin in the presence of DETCA. Pyrogallol and hydroquinone inhibited the relaxation to NO, but not to EFS and nitroglycerin. This inhibition was prevented by exogenous SOD and ascorbate, but was not prevented by EUK-8. Pyrogallol and hy-droquinone did not inhibit the EFS-induced relaxation in the presence of DETCA. Duroquinone and 6-anilino-5.8-quinolinedione inhibited the relaxation to EFS, NO, and nitroglycerin, and this inhibition was prevented by EUK-8. Conclusion: These results suggest that the nitrergic neurotransmission in the mouse duodenum is protected by endogenous tissue antioxidants against superoxide anions, and Mn SOD, in addition to copper/zinc SOD, can protect NO from attack from superoxide anion generators intracellularly. Also, the possibility that the endogenous neurotransmitter may not be the free NO but a NO-containing or NO-generating molecule in the mouse duodenum remains open.     

Loss of endothelium-derived nitric oxide in rabbit aorta by oxidant stress: restoration by superoxide dismutase mimetics

1. Structurally distinct superoxide dismutase (SOD) mimetics were examined for their ability to protect nitric oxide (NO) from destruction by oxidant stress in rabbit aorta.
2. These were the spin traps, PTIYO (4-phenyl-2,2,5,5-tetramethyl imidazolin-1-yloxy-5-oxide), tempol (4-hydroxy 2,2,6,6,-tetramethylpiperidine-1-oxyl) and tiron (4,5-dihydroxy-1,3-benzene-disulphonic acid), the metal salts, CuSO₄ and MnCl₂, and the metal-based agents CuDIPS (Cu (II)-[diisopropylsalicylate]₂) and MnTMPyP (Mn (III) tetrakis [1-methyl-4-pyridyl]porphyrin).
3. Oxidant stress was generated in isolated aortic rings by inactivating endogenous Cu/Zn SOD with diethyldithiocarbamate (DETCA; 60 min) either alone at 3 mM or at 0.3 mM in combination with superoxide generation using xanthine oxidase (XO; 4.8 mu ml⁻¹) and hypoxanthine (HX; 0.1 mM).
4. Acetylcholine (ACh)-induced relaxation was inhibited by DETCA (3 mM, 60 min) and was not restored by exogenous SOD (250 u ml⁻¹), suggesting the oxidant stress was intracellular. MnTMPyP (600 μM and 1 mM) and MnCl₂ (100 μM) were the only agents to reverse the blockade of ACh-induced relaxation.
5. Addition of XO/HX to DETCA (0.3 mM)-treated tissues powerfully impaired ACh-induced relaxation and exogneous SOD (250 u ml⁻¹) fully reversed the blockade, suggesting the oxidant stress was extracellular. CuDIPS (0.1–3 μM), CuSO₄ (0.3–3 μM), MnCl₂ (1–100 μM) and MnTMPyP (100–600 μM) also reversed blockade powerfully, tempol (30 μM–1 mM) and tiron (0.3–10 mM) reversed blockade weakly and PTIYO (10–300 μM) enhanced the blockade.
6. Thus, MnTMPyP was the only SOD mimetic to restore NO-dependent relaxation in conditions of both extracellular and intracellular oxidant stress. This agent may, therefore, provide a lead in the development of SOD mimetics for the treatment of pathologies associated with oxidant stress.

     

Decline in the expression of copper/zinc superoxide dismutase in the kidney of rats with endotoxic shock: Effects of the superoxide anion radical scavenger,tempol, on organ injury

1. Endotoxaemia causes an enhanced formation of reactive oxygen species (ROS) which contribute to the multiple organ dysfunction syndrome (MODS) in septic shock. Here we investigate (i) the effects of endotoxin on the expression of two isoforms of superoxide dismutase (SOD), namely Cu/Zn-SOD (cytosol) and Mn-SOD (mitochondria) in the rat kidney, and (ii) the effects of the radical scavenger tempol on the MODS caused by lipopolysaccharide (LPS, E. coli, 6 mg kg⁻¹ i.v.) in the rat.
2. Endotoxaemia resulted in a rapid, but transient, decline in the expression of both mRNA and protein of Cu/Zn-SOD as well as an increase in the expression of the mRNA of Mn-SOD in the kidney. Endotoxaemia for 6 h also caused hypotension, acute renal dysfunction, hepatocellular injury, pancreatic injury and an increase in the plasma levels of nitrite/nitrate.
3. Pretreatment of rats with tempol (100 mg kg⁻¹ i.v. bolus injection, 15 min prior to LPS followed by an infusion of 30 mg kg⁻¹ i.v., n=9) did not affect the circulatory failure, but attenuated the renal dysfunction and the hepatocellular injury/dysfunction caused by LPS. Tempol did not affect the rise in nitrite/nitrate caused by endotoxin.
4. These results imply that an enhanced formation of ROS (including superoxide anions) in conjunction with inadequate defences against such ROS contributes to the injury and dysfunction of the kidney and the liver in endotoxic shock.

     

Inhibition of nitrergic neurotransmission in the bovine retractor penis muscle by an oxidant stress: effects of superoxide dismutase mimetics

1. A number of superoxide dismutase (SOD) mimetics were examined both biochemically for their ability to inhibit the superoxide-catalyzed reduction of cytochrome c and nitro blue tetrazolium, and functionally for their ability to mimic authentic Cu/Zn SOD in restoring nitrergic neurotransmission in bovine retractor penis (BRP) muscle following its inhibition by oxidant stress.
2. The SOD mimetics investigated were CuSO₄, MnCl₂, CuDIPS (copper [II] [diisopropylsalicylate]₂), MnTBAP (manganese [III] tetrakis 4-benzoic acid porphyrin), MnTMPyP (manganese [III] tetrakis 1-methyl-4-pyridyl porphyrin pentachloride), tiron (4,5-dihydroxy-1,3-benzene disulphonic acid), PTIYO (4-phenyl,2,2,5,5,-tetramethyl-3-imidazolin-1-yloxy-3-oxide) and tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl).
3. The rank order of potency in inhibiting the reduction of cytochrome c was: CuSO₄⩾MnCl₂⩾CuDIPS⩾MnTMPyP>MnTBAP>tempol⩾tiron>PTIYO.
4. The requirement for EDTA (0.1 mM) prevented assessment of the activity of CuSO₄, MnCl₂ and CuDIPS in the assay involving inhibition of reduction of nitro blue tetrazolium. However, the rank order of potency for those agents which could be examined (MnTMPyP>MnTBAP>tiron⩾tempol>PTIYO) was essentially similar to that seen in the cytochrome c assay.
5. Inhibition of endogenous Cu/Zn SOD with diethyldithiocarbamate (DETCA, 3 mM, 120 min) in BRP muscle strips, followed by addition of the superoxide anion generator, LY 83583 (1 μM), resulted in almost complete abolition of nitrergic relaxation (4 Hz, 10 s).
6. Authentic Cu/Zn SOD (1–300 u ml⁻¹), CuSO₄ (0.1–300 μM), MnCl₂ (0.1–100 μM) and MnTMPyP (10–300 μM) each restored nitrergic transmission by around 50%. However, CuDIPS (0.1–30 μM), MnTBAP (0.1–100 μM), tempol (10 μM–3 mM), PTIYO (1–300 μM) and tiron (10 μM–10 mM) all failed to restore nitrergic transmission.
7. The ability of MnTMPyP to restore nitrergic neurotransmission may therefore provide a lead in the development of SOD mimetics as therapeutic agents in the treatment of neuropathies associated with oxidant stress.

     

Recovery by ascorbate of impaired nitric oxide-dependent relaxation resulting from oxidant stress in rat aorta

1. In this study we investigated the ability of ascorbate to protect nitric oxide from destruction by superoxide anion.
2. Ascorbate produced concentration-dependent relaxation of rings of rat aorta, comprising two components: the first, seen at 1–300 μM, reached a maximum of 45.3±2.8%, and was abolished by endothelial removal or treatment with L-NAME (100 μM), demonstrating involvement of nitric oxide. The second occurred at concentrations of 1 mM and above and was associated with falls in the pH of the bathing fluid.
3. Pretreatment with ascorbate at concentrations up to 3 mM had no effect on the relaxation to acetylcholine (10 nM–10 μM) on endothelium-containing rings or adenosine (0.1 μM–3 mM) on endothelium-denuded rings.
4. An oxidant stress was applied to aortic rings, comprising inhibition of endogenous Cu/Zn superoxide dismutase by diethyldithiocarbamate (0.1 mM) followed by generation of superoxide anion by hypoxanthine (0.1 mM/xanthine oxidase (16 u ml⁻¹). This reduced maximal acetylcholine-induced relaxation from 96.7±1.3% to 42.4±3.5% (P<0.001). Treatment with ascorbate (30 μM–3 mM) reversed this blockade in a concentration-dependent manner.
5. Our findings show that ascorbate has the ability to protect nitric oxide from destruction by superoxide anion. This action is seen with ascorbate at levels normally present in plasma, suggesting that this antioxidant may exert a tonic protective effect on nitric oxide within the vasculature.

     

Interaction between superoxide anion and nitric oxide in the regulation of vascular endothelial function

1. Nitric oxide (NO)-mediated, endothelium-dependent vasodilator function in rat aortic smooth muscle was investigated in an in vitro model of endogenous vascular superoxide anion stress, generated by pretreatment with the Cu/Zn superoxide dismutase (SOD, EC 1.15.1.1) inhibitor, diethyldithiocarbamate (DETCA).
2. Contraction to noradrenaline (NA, 1 nM–1 μM) in endothelium-intact vessels was augmented after a 30 min pretreatment with DETCA (10 mM) followed by 30 min washout. This effect was abolished by N^G-nitro-L-arginine methyl ester (L-NAME, 0.3 mM) and removal of the endothelium and partially reversed by exogenous Cu/Zn SOD (200 u ml⁻¹).
3. Endothelium- and basal NO-dependent vasorelaxation to the phosphodiesterase (PDE) type V inhibitor ONO-1505 (4-[2-(2-hydroxyethoxy)ethylamino]-2-(1H-imidazol-1-yl)-6-methoxyquinazoline methanesulphonate) (0.1–10 μM) was inhibited after DETCA (10 mM) pretreatment. In addition, the ability of L-NAME (0.3 mM) to enhance established contractile tone was effectively absent.
4. In contrast, DETCA pretreatment did not significantly affect vasorelaxation to acetylcholine (ACh, 1 nM–3 μM) or S-nitroso-N-acetyl penicillamine (SNAP, 0.03–30 μM). However, L-NAME (0.3 mM) unmasked an inhibitory effect of DETCA pretreatment on vasorelaxation to SNAP in endothelium-intact vessels while markedly potentiating vasorelaxation to SNAP in control tissue.
5. L-NAME (0.3 mM)- and exogenous catalase (200 u ml⁻¹)-sensitive vasorelaxation to exogenous Cu/Zn SOD (200 u ml⁻¹) was greater after DETCA (10 mM) pretreatment in endothelium-intact aortic rings. This difference was abolished by catalase (200 u ml⁻¹).
6. In conclusion, tissue Cu/Zn SOD inhibition elicited a selective lesion in basal endothelial function in rat isolated aortic smooth muscle, consistent with the inactivation of basal NO by superoxide anion. The resulting leftward shift in nitrovasodilator reactivity, due to the loss of the tonic depression by basal NO, is likely to mask the inhibitory effect of superoxide anion on agonist-stimulated endothelial function and nitrovasodilator-derived NO, thereby accounting for the differential pattern of endothelial dysfunction after DETCA pretreatment.

     

Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists, 1994: FREE RADICAL TOXICOLOGY AND ANTIOXIDANT DEFENCE

1. A large number of compounds that have toxic effects can be metabolized to free radicals and secondary reactive oxygen species. These may be directly damaging or may affect cell function by altering regulatory mechanisms through changing redox status. 2. Protection is provided by an integrated system of anti-oxidant defences. This includes reduced glutathione, one of the functions of which is to scavenge free radicals. It acts by channelling radicals to superoxide so that the one enzyme, superoxide dismutase, has a major control over radical reactions in the cell.     

SOD酶生物传感器筛选清除超氧阴离子自由基的活性物

目的制备超氧化物歧化酶传感器，建立清除氧自由基药物的体外筛选方法。方法将固定化铜锌超氧化物歧化酶与光纤氧传感器通过特定装置组装为酶传感器。以邻苯三酚的自氧化作为超氧阴离子的发生源，预置的固定化酶响应为内标，以已知有氧自由基清除作用的Vit C为阳性对照，验证测定方法；通过对比加入样品前后邻苯三酚自氧化速度的变化情况考察样品清除超氧阴离子自由基能力。结果酶传感器检测限为7.0 U，使用寿命大于2周。以本传感器对15种样品进行了体外清除氧自由基活性实验，分别验证和发现了部分样品的清除活性。结论所研制的传感器信号稳定性较好，测定方法简便、快速，能直观地获得氧自由基清除的动力学信息，可用于大量药物的体外初筛实验。     

Suppression of respiratory burst of polymorphonuclear leukocytes by Azelastine hydrochloride (Azeptin)

The inhibitory action of azelastine hydrochloride (Azeptin) on the respiratory burst in peripheral polymorphonuclear leukocytes (PMN) and pulmonary alveolar macrophages (PAM) has been studied. Azeptin in vitro suppressed chemiluminescence and superoxide (O ₂ ^- ) generation by human PMN in a dose-and time-dependent manner. Phorbol myristyl acetate (PMA) and formyl-methionyl-leucyl-phenylalanine (FMLP)-induced O ₂ ^- generation were strongly suppressed by 10^–6M and 10^–5M Azeptin, respectively. PMN and PAM from rabbits injected with Azeptin 0.2 mg·kg^–1 for 5 days showed lower chemiluminescence and O ₂ ^- generation than cells from untreated rabbits. Nitroblue tetrazolium reduction activity in human PMN was suppressed by treatment of PMN with 10^–6M Azeptin for 6 h. Inositol trisphosphate, intracellular free calcium, and protein kinase C activity were decreased by 10^–6M to 10^–5M Azeptin. The tyrosine phosphorylation of many proteins, especially a 115 kDa protein, was suppressed by 10^–5M Azeptin. However, superoxide dismutase activity in PMN, PAM, and lung tissue samples was only slightly decreased, even when the rabbits were treated with 1.0 mg·kg^–1 Azeptin for 5 days. The results suggest that Azeptin suppresses multiple signal transduction steps in the respiratory burst of PMN. This suppressive action should be very useful in the prevention and treatment of reactive oxygen-associated disorders.Abbreviations Azeptin azelastine hydrochloride - PMN polymorphonuclear leukocytes - PAM pulmonary alveolar macrophages - PMA phorbol myristyl acetate - FMLP formyl-methionyl-leucyl-phenylalanine - PKC protein kinase C - ROS reactive oxygen species - O ₂ ^- superoxide - IP₃ inositol 1,4,5-trisphosphate - DG diacylglycerol - NBT nitroblue tetrazolium - SOD superoxide dismutase - TNF- tumor necrosis factor-alpha     

STUDIES ON THE CYTOTOXICITY OF PENICILLAMINE IN A RAT OSTEOGENIC SARCOMA

The effect of penicillamine on the growth rate of an osteogenic sarcoma of rats was investigated and compared with cyclophosphamide. Rats were inoculated with a readily transplantable osteogenic sarcoma subcutaneously into the left thigh and treated with penicillamine and cyclophosphamide alone or in combination. Cyclophosphamide inhibited tumour growth. Penicillamine did not delay the appearance or the growth rate of the tumour. Tumour sizes tended to be larger in the penicillamine-treated rats, but there was no evidence that penicillamine interfered with the antitumour effect of cyclophosphamide given in large doses (100 mg/kg).