锰超氧化物歧化酶基因转染小鼠小肠上皮细胞的放射损伤保护作用

目的探讨治疗和预防直肠癌患者围手术期放射性小肠损伤的有效方法及其机制。方法应用复制缺陷型单纯疱疹病毒(HSV)作为锰超氧化物歧化酶(MnSOD)和(或)绿色荧光蛋白(GFP)基因载体转染小鼠小肠上皮;应用逆转录聚合酶链反应(RT-PCR)测定MnSOD在小鼠小肠上皮细胞内的表达,酶活性检测法测定MnSOD活性;全腹腔照射(0,7.5,10.0,12.5,15 Gy)24 h和72 h后取标本,应用SZ-PT光学系统测量小肠绒毛的长度。结果MnSOD在小鼠小肠上皮细胞过度表达,酶活性检测表明,MnSOD活性显著增高,转染组MnSOD对超氧阴离子的抑制率达39.33%±0.67%。全腹腔照射24 h和72h后,对照组小鼠小肠绒毛长度分别降低了40.1%~59.3%和44.2%~65.1%;而MnSOD组小鼠小肠绒毛长度分别降低了3.1%~12.4%和6.3%~29.1%,两者差异具有统计学意义(P<0.01)。结论复制缺陷型HSV作为载体可有效转染MnSOD。小肠上皮内MnSOD的过度表达具有显著的放射性保护作用。     

Protective effect of polyethylene glycol-superoxide dismutase on leukocyte dynamics in rat retinal microcirculation under lipid hydroperoxide-induced oxidative stress

The levels of lipid hydroperoxide (LHPs) in vitreous are elevated in a variety of retinal disorders. Recently, we have shown that increased levels of LHPs in the vitreous enhanced leukocyte-endothelium interaction in the retina, which should contribute to the initial disturbance of the retinal microcirculation. Based upon the previous work, the purpose of the present study was to investigate the effect of polyethylene glycol-superoxide dismutase (PEG-SOD), one of the important enzyme antioxidants, on leukocyte-endothelial interaction in the retinal microcirculation under LHP-induced oxidative stress. Male Brown-Norway rats weighing approximately 250 g were used. LHP(18:2) was made from linoleic acid (LA) with lipoxygenase and 10 microg of LHP dissolved in 5 microl of sodium borate buffer (SBB, 0.02 m) was slowly injected into the vitreous using a 33-gauge needle. PEG-SOD (5000 units/kg) was given intravenously 5 min before LHP injection. At 2, 4, 6, 12, 24 and 48 hr after the vitreous injections, we evaluated the number of rolling leukocytes along the major retinal veins and the number of leukocytes that accumulated in the retinal microvasculature with acridine orange digital fluorography. In LHP-treated rats, leukocyte rolling along the major retinal veins was maximal at 6 hr after LHP injection. The number of rolling leukocytes in the PEG-SOD-treated rats was decreased to 5.5% of those in the LHP-treated rats at 6 hr after LHP injection (P<0.01). No rolling leukocytes were observed in either control or vehicle-treated eyes. The number of accumulated leukocytes in LHP-treated eyes started to increase at 12 hr, and peaked at 24 hr which was significantly higher than in both control and vehicle-treated eyes (P<0.01). The number of accumulated leukocytes in the PEG-SOD-treated rats was reduced by 88.0% at 24 hr (P<0.01). Intravenous injection of PEG-SOD significantly inhibited the leukocyte rolling and its accumulation under LHP-induced oxidative stress. These results suggest that PEG-SOD might attenuate various retinal microcirculatory disorders associated with LHP.     

Biological activity of for-Met-Leu-Phe-OMe analogs: relevant substitutions specifically trigger killing mechanisms in human neutrophils

Two analogs of the prototypical peptide for-Met-Leu-Phe-OMe (fMLP-OMe), for-Gln-Tyr-Phe-OMe (1) and for-Gln-Tyr-Tyr-OMe (2), carrying unusual hydrophilic residues, were synthesized in order to investigate whether they provoked specific biological responses, as well as intracellular calcium mobilization, in human neutrophils. Whereas neither compound stimulates chemotaxis, both are able to elicit lysosomal enzyme production. However compound 1 is able to trigger copious superoxide anion production while compound 2 only elicits minor superoxide anion production. In binding experiments on formylpeptide receptors, the newly synthesized compounds for-Gln-Tyr-Phe-OMe (1) and for-Gln-Tyr-Tyr-OMe (2) showed affinity values in the micromolar range. These derivatives demonstrate inability to find a positive contribute from single substitutions. A very important result of this research is the evidence of the ability of the formyl group alone to trigger the primary target of the human neutrophil activity, i.e. killing mechanisms, by activating the specific receptor conformation.     

Role of nitric oxide during coronary endothelial dysfunction after myocardial infarction

This study aimed to investigate whether permanent ischaemia influences subacute vasodilatation responses of non-infarcted rat coronary vasculature, and to characterise these coronary changes. Ischaemia led to a significant impairment of the endothelium-dependent vasodilator response, while coronary vasodilatory capacity remained unaltered. In normal coronary circulation, nitric oxide (NO) and prostanoids contributed to vasodilatation, while basal involvement of endothelium-derived hyperpolarising factor was limited. Vasodilatory impairment following myocardial infarction did not originate from alterations in the prostanoid pathway, and only a slightly increased influence of K+ channels was observed. However, NO-mediated vasodilatation was significantly increased after ischaemia, as also confirmed by higher mRNA and protein levels of iNOS and eNOS. Additionally, the amount of superoxide was enhanced following infarction. We conclude that subacute postinfarction remodeling is accompanied by endothelial dysfunction in non-infarcted coronary arteries. Although the NO-mediated response is increased after ischaemia, its final action is restricted due to the presence of superoxide.     

Spin adduct formation from lipophilic EMPO-derived spin traps with various oxygen- and carbon-centered radicals

Free radicals are involved in the onset of many diseases, therefore the availability of adequate spin traps is crucial to the identification and localization of free radical formation in biological systems. In recent studies several hydrophilic compounds of 2-ethoxycarbonyl-2-methyl-pyrroline-N-oxide (EMPO) have been found to form rather stable superoxide spin adducts with half-lives up to twenty minutes at physiological pH. This is a major improvement over DMPO (t_1/2 = ca. 45 s), and even over DEPMPO (t_1/2 = ca. 14 min), the best commercially available spin trap for the unambiguous detection of superoxide radicals. In order to allow the detection of superoxide and also other radicals in lipid environment a series of more lipophilic derivatives of EMPO was synthesized and their structure unambiguously characterized by ¹H and ¹³C NMR spectroscopy. In this way, six different compounds with a n-butyl group in position 5 and either an ethoxy- (EBPO), propoxy- (PBPO), iso-propoxy- (iPBPO), butoxy- (BBPO), sec-butoxy- (sBBPO) or tert-butoxycarbonyl group (tBBPO) in position 5 of the pyrroline ring were obtained and fully analytically characterized (NMR, IR). The stability of the superoxide adducts of all investigated spin traps were comparable with EMPO (t_1/2 = ca. 8 min), except for the two compounds bearing an additional methyl group in position 3 or 4 of the pyrroline ring, 5-butyl-5-ethoxycarbonyl-3-methyl-pyrroline-N-oxide (BEMPO-3) and 5-butyl-5-ethoxycarbonyl-4-methyl-pyrroline-N-oxide (BEMPO-4), of which the superoxide adducts were stable for more than 30 min. Spin adducts of other carbon- and oxygen-centered radicals were also investigated.     

Poly(ADP-ribose) polymerase regulates myocardial calcium handling in doxorubicin-induced heart failure

Reactive oxygen and nitrogen species are overproduced in the cardiovascular system in response to the exposure to doxorubicin, a cardiotoxic anticancer compound. Oxidant-induced cell injury involves the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) and pharmacological inhibition of PARP has recently been shown to improve myocardial contractility in doxorubicin-induced heart failure models. The current investigation, by utilizing an isolated perfused heart system capable of beat-to-beat intracellular calcium recording, addressed the following questions: (1) is intracellular calcium handling altered in hearts of rats after 6-week doxorubicin treatment, under baseline conditions, and in response to oxidative stress induced by hydrogen peroxide exposure in vitro; and (2) does pharmacological inhibition of PARP with the phenanthridinone-based PARP inhibitor PJ34 affect the changes in myocardial mechanical performance and calcium handling in doxorubicin-treated hearts under normal conditions and in response to oxidative stress. The results showed a marked elevation in intracellular calcium in the doxorubicin-treated hearts which was normalized by pharmacological inhibition of PARP. PARP inhibition also prevented the myocardial contractile disturbances and calcium overload that developed in response to hydrogen peroxide in the doxorubicin-treated hearts. We conclude that PARP activation contributes to the development of the disturbances in cellular calcium handling that develop in the myocardium in response to prolonged doxorubicin exposure.     

Protective effect of dextromethorphan against endotoxic shock in mice

Dextromethorphan (DM) is a dextrorotatory morphinan and an over-the-counter non-opioid cough suppressant. We have previously shown that DM protects against LPS-induced dopaminergic neurodegeneration through inhibition of microglia activation. Here, we investigated protective effects of DM against endotoxin shock induced by lipopolysaccharide/d-galactosamine (LPS/GalN) in mice and the mechanism underlying its protective effect. Mice were given multiple injections of DM (12.5 mg/kg, s.c.) 30 min before and 2, 4 h after an injection of LPS/GalN (20 microg/700 mg/kg). DM administration decreased LPS/GalN-induced mortality and hepatotoxicity, as evidenced by increased survival rate, decreased serum alanine aminotransferase activity and improved pathology. Furthermore, DM was also effective when it was given 30 min after LPS/GalN injection. The protection was likely associated with reduced serum and liver tumor necrosis factor alpha (TNF-alpha) levels. DM also attenuated production of superoxide and intracellular reactive oxygen species in Kupffer cells and neutrophils. Real-time RT-PCR analysis revealed that DM administration suppressed the expression of a variety of inflammation-related genes such as macrophage inflammatory protein-2, CXC chemokine, thrombospondin-1, intercellular adhesion molecular-1 and interleukin-6. DM also decreased the expression of genes related to cell-death pathways, such as the DNA damage protein genes GADD45 and GADD153. In summary, DM is effective in protecting mice against LPS/GalN-induced hepatotoxicity, and the mechanism is likely through a faster TNF-alpha clearance, and decrease of superoxide production and inflammation and cell-death related components. This study not only extends neuroprotective effect of DM, but also suggests that DM may be a novel compound for the therapeutic intervention for sepsis.     

Very stable superoxide radical adducts of 5-ethoxycarbonyl- 3,5-dimethyl-pyrroline N-oxide (3,5-EDPO) and its derivatives

Oxygen radicals are involved in the onset of many diseases. Adequate spin traps are required for identification and localisation of free radical formation in biological systems. Superoxide spin adducts with half-lives up to 20 min at physiological pH have recently been reported to be formed from derivatives of the spin trap 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO). This is a major improvement over DMPO (t(1/2) ca. 45 s), and even DEPMPO (t(1/2) ca. 14 min). In this study, an additional methyl group was introduced into position 3 or 4 of the pyrroline ring which greatly increases the stability of the respective superoxide spin adducts. In addition, the ethoxy group of EMPO was exchanged by either a propoxy- or an iso-propoxy group in order to test the influence of increasing lipophilic properties of the investigated spin traps. The structure of all compounds was confirmed by (1)H and (13)C-NMR with full signal assignment. In comparison with EMPO (t(1/2) ca. 8 min) or DEPMPO (t(1/2) ca. 14 min), the superoxide adducts of all novel spin traps were considerably higher (t(1/2) ca. 12-55 min). In addition, various other spin adducts obtained from oxygen-centered as well as carbon-centered radicals (e.g. derived from methanol or linoleic acid hydroperoxide) were also detected.     

Is it a safe practice to administer oxygen during uncomplicated delivery: A randomized controlled trial?

Background

Newborns exposed to oxygen suffer from an oxidative stress with significant alterations in the concentrations of superoxide dismutase (SOD) and glutathione (GSSG).

Objective

To investigate the biological and clinical effects of oxygen administration to delivering mothers.

Methods

We conducted a randomized, double-blinded, controlled trial on a cohort of delivering women (n = 56) with an uncomplicated term pregnancy. Women were randomly assigned to one of two groups: Oxygen group or Room Air group. The Oxygen group received 100% oxygen (2 l/min) via nasal cannula for at least 30 min before delivery. Subjects in the Room Air group were connected to a nasal cannula while on room air. Concentrations of SOD (μg/g of Hb) and GSSG (μM/ml) were measured in maternal and umbilical cord blood. Bivariate and multivariate analyses were used to compare the two groups using the SAS system.

Results

Maternal SOD and GSSG did not differ between the two groups at baseline or after delivery. Concentrations of SOD and GSSG in umbilical cord blood did not differ between groups. More infants in Oxygen Group required delivery room resuscitation (20% vs. 0%, P = 0.03). This difference could not be explained by mode of delivery, infant sex, or other confounders.

Conclusions

Maternal exposure to oxygen during delivery is not associated with changes in umbilical cord SOD or GSSG. Further studies are needed to explore mechanisms responsible for the need of resuscitation in the oxygen group.     

慢性肾炎患者血浆leptin和血清IL-6、EL-18及SOD检测的临床意义

目的：探讨慢性肾炎患者治疗前后血浆leptin和血清IL-6、IL．18及SOD水平的变化及临床意义。方法：应用放射免疫分析和酶联免疫法对33例慢性肾炎患者进行了血浆leptin和血清IL-6、IL．18及SOD检测,并与35名正常健康人作比较。结果：慢性肾炎患者在治疗前血浆leptin和血清IL-6、IL．18均显着地高于正常人组（P〈0．01）,而血清SOD水平则显着地低于正常人组（P〈0．01）。经中西医结合治疗3个月后与正常人组比较仍有显著性差异（P〈0．05）,血浆leptin水平与血清IL-6、IL．18水平呈正相关（r=0．5718、0．4916,P〈0．01）,而与SOD水平呈负相关（r=-0．6018,P〈0．01）。结论：检测慢性肾炎患者治疗前后血浆leptin和血清IL-6、IL-18及SOD水平的变化对临床观察和预后有重要的临床价值。