高氧诱导慢性肺疾病早产鼠肺组织中氧化及抗氧化系统的动态研究

目的　探讨肺组织中氧化及抗氧化系统在高氧致早产儿慢性肺疾病 (CLD)中的动态变化规律。　方法　采用高浓度氧致早产鼠CLD模型为研究对象 ,分为实验组和对照组 ,各 4 0只。应用分光光度计比色法分别测定肺组织中脂质过氧化产物丙二醛 (MDA)的含量及超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶 (GSH Px)和过氧化氢酶 (CAT)的活性。　结果　实验组和对照组各时间点肺组织中SOD、GSH Px及CAT的活性差异均无显著性 (P >0 .0 5 ) ;而实验组MDA水平早期即明显升高 [(5 5 .92± 5 .5 3)nmol/mg肺组织 ,P <0 .0 1],7d达高峰 [(94 .30± 12 .4 0 )nmol/mg肺组织 ,P <0 .0 0 1],持续 1周后逐渐下降 ,2 1d时仍高于正常水平 [(47.95± 7.4 8)nmol/mg肺组织 ,P <0 .0 1]。　结论　肺组织中抗氧化酶系统的活性不足及氧自由基损伤始终伴随高氧致CLD发生、发展的全过程。     

超氧化物歧化酶对实验性骨折愈合的影响

目的：探讨超氧化物歧化酶（ＳＯＤ）对实验性骨折愈合的作用。方法：应用３Ｈ－脯氨酸和４５Ｃａ－氯化钙无载体放射性核素体内示踪法，检测了ＳＯＤ对实验性骨折过程中基质形成与钙化的影响，并观察了骨痂组织细胞的形态学变化。结果：ＳＯＤ使３Ｈ－脯氨酸和４５Ｃａ－氯化钙参入早期骨痂的量增加（与对照组相比，Ｐ＜０．０１），４５Ｃａ／３Ｈ的比率提高迅速。早期骨痂内毛细血管含量增加；软骨细胞、成纤维细胞功能活跃，基质钙化迅速、完全，骨性骨痂发生早。结论：ＳＯＤ可作为促进骨折愈合的辅助疗法。     

Silymarin protects dopaminergic neurons against lipopolysaccharide-induced neurotoxicity by inhibiting microglia activation

An inflammatory response in the central nervous system mediated by activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. Silymarin is a polyphenolic flavanoid derived from milk thistle that has anti-inflammatory, cytoprotective and anticarcinogenic effects. In this study, we first investigated the neuroprotective effect of silymarin against lipopolysaccharide (LPS)-induced neurotoxicity in mesencephalic mixed neuron-glia cultures. The results showed that silymarin significantly inhibited the LPS-induced activation of microglia and the production of inflammatory mediators, such as tumour necrosis factor-alpha and nitric oxide (NO), and reduced the damage to dopaminergic neurons. Therefore, the inhibitory mechanisms of silymarin on microglia activation were studied further. The production of inducible nitric oxide synthase (iNOS) was studied in LPS-stimulated BV-2 cells as a model of microglia activation. Silymarin significantly reduced the LPS-induced nitrite, iNOS mRNA and protein levels in a dose-dependent manner. Moreover, LPS could induce the activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase but not extracellular signal-regulated kinase. The LPS-induced production of NO was inhibited by the selective p38 MAPK inhibitor SB203580. These results indicated that the p38 MAPK signalling pathway was involved in the LPS-induced NO production. However, the activation of p38 MAPK was not inhibited by silymarin. Nevertheless, silymarin could effectively reduce LPS-induced superoxide generation and nuclear factor kappaB (NF-kappaB) activation. It suggests that the inhibitory effect of silymarin on microglia activation is mediated through the inhibition of NF-kappaB activation.     

Linoleic acid and tumor necrosis factor-α increase manganese superoxide dismutase activity in intestinal cells

Manganese superoxide dismutase (MnSOD) protects mitochondria from oxidative damage. Alterations in the regulation of MnSOD plays an important role in the development of many types of cancer. Activity of this enzyme is induced by inflammatory cytokines and other conditions that increase oxygen radical production. High levels of dietary lipid have been shown to decrease MnSOD activity. This study was designed to define the effect of various type of fatty acids on MnSOD activity and MnSOD induction. IEC-6 cells were treated with 40 μmol/l of either linoleic acid (LA), eicosapentaenoic acid (EPA), or oleic acid (OA) in the presence or absence of 10 ng/ml tumor necrosis factor-α (TNF-α). Fatty acid supplementation increased MnSOD activity. MnSOD activity was greater in the LA group than in the EPA or OA groups. TNF-α induced MnSOD activity equally in all fatty acid-supplemented groups. High levels of MnSOD activity may be an indicator of chronic inflammation resulting from fatty acid, particularly LA, supplementation.     

Reduced (+/-)-3,4-methylenedioxymethamphetamine ("Ecstasy") metabolism with cytochrome P450 2D6 inhibitors and pharmacogenetic variants in vitro

"Ecstasy" [(+/-)-3,4-methylenedioxymethamphetamine or MDMA] is a CNS stimulant, whose use is increasing despite evidence of long-term neurotoxicity. In vitro, the majority of MDMA is demethylenated to (+/-)-3,4-dihydroxymethamphetamine (DHMA) by the polymorphic cytochrome P450 2D6 (CYP2D6). We investigated the demethylenation of MDMA and dextromethorphan (DEX), as a comparison drug, in reconstituted microsomes expressing the variant CYP2D6 alleles (*)2, (*)10, and (*)17, all of which have been linked to decreased enzyme activity. With MDMA, intrinsic clearances (V(max)/K(m)) in CYP2D6.2, CYP2D6.17, and CYP2D6.10 were reduced 15-, 13-, and 135-fold, respectively, compared with wild-type CYP2D6.1. With DEX, intrinsic clearances were reduced by 37-, 51-, and 164-fold, respectively. It was evident that CYP2D6.17 displayed substrate-specific changes in drug affinity (K(m)). Compounds potentially used with MDMA [fluoxetine, paroxetine, (-)-cocaine] demonstrated significant inhibition of MDMA metabolism in both human liver and CYP2D6.1-expressing microsomes. These data demonstrate that individuals possessing the CYP2D6(*)2, (*)17, and, particularly, (*)10 alleles may show significantly reduced MDMA metabolism. These individuals, and those taking CYP2D6 inhibitors, may demonstrate altered acute and/or long-term MDMA-related toxicity.     

Autoschizis: a novel cell death

Vitamin C (VC) and vitamin K(3) (VK(3)) administered in a VC:VK(3) ratio of 100:1 exhibit synergistic antitumor activity and preferentially kill tumor cells by autoschizis, a novel type of necrosis characterized by exaggerated membrane damage and progressive loss of organelle-free cytoplasm through a series of self-excisions. During this process, the nucleus becomes smaller, cell size decreases one-half to one-third of its original size, and most organelles surround an intact nucleus in a narrow rim of cytoplasm. While the mitochondria are condensed, tumor cell death does not result from ATP depletion. However, vitamin treatment induces a G(1)/S block, diminishes DNA synthesis, increases H(2)O(2) production, and decreases cellular thiol levels. These effects can be prevented by the addition of catalase to scavenge the H(2)O(2). There is a concurrent 8- to 10-fold increase in intracellular Ca(2+) levels. Electrophoretic analysis of DNA reveals degradation due to the caspase-3-independent reactivation of deoxyribonuclease I and II (DNase I, DNase II). Redox cycling of the vitamins is believed to increase oxidative stress until it surpasses the reducing ability of cellular thiols and induces Ca(2+) release, which triggers activation of Ca(2+)-dependent DNase and leads to degradation of DNA. Recent experiments indicate that oral VC:VK(3) increases the life-span of tumor-bearing nude mice and significantly reduces the growth rate of solid tumors without any significant toxicity by reactivating DNase I and II and inducing autoschizis. This report discusses the mechanisms of action employed by these vitamins to induce tumor-specific death by autoschizis.     

Oxidative stress and potassium channel function

1. Modulation of K+ channel activities by cellular oxidative stress has emerged as a significant determinant of vasomotor function in multiple disease states. 2. Evidence from in vitro and in vivo studies suggest that superoxide (O2-) and hydrogen peroxide (H2O2) enhance BKCa channel activity in rat and cat cerebral arterioles; however, activity is decreased by peroxynitrite (ONOO-) in rat cerebral arteries. The mechanisms of changes in BKCa channel properties are not fully understood and may involve oxidation of cysteine residues that are located in the cell membranes. 3. Studies further suggest that O2- increases KATP channel activity in guinea-pig cardiac myocytes, but decreases opening in cerebral vasculature. Both H2O2 and ONOO- enhance KATP channel activity in the myocardium and in coronary, renal, mesenteric and cerebral vascular beds. Alteration of KATP channels by free radicals may be due to oxidation of SH groups or changes in the cytosolic concentration of ATP. 4. It does appear that O2- produced by either reaction of xanthine and xanthine oxidase or elevated levels of glucose reduces Kv channel activity and the impairments can be partially restored by free radical scavengers, superoxide dismutase and catalase. 5. Thus, redox modulation of potassium channel activity is an important mechanism regulating cell vascular smooth muscle membrane potential.     

Kennedy’s disease: a triplet repeat disorder or a motor neuron disease?

Two definite genetic causes of adult motor neuron degeneration have been identified to date: CAG repeat expansion in the androgen receptor gene in Kennedy’s disease and point mutations in the SOD1 gene, encoding the enzyme, Cu/Zn superoxide dismutase, in some familial forms of amyotrophic lateral sclerosis. Although both have unrelated genetic causes, Kennedy’s disease and SOD1-linked amyotrophic lateral sclerosis share several pathogenic features. First, expanded androgen receptor and mutant Cu/Zn superoxide dismutase have a propensity to aggregate into insoluble complexes and form inclusion bodies in affected neurons. Deposits of mutant proteins could be detrimental to neuronal viability by interfering with the normal housekeeping functions of chaperones and of the ubiquitin/proteasome system. Secondly, cytoskeletal function may be impaired in both diseases as decreased transactivational activity of expanded androgen receptor may cause an abnormal pattern of tubulin expression in motor neurons in Kennedy’s disease and disruption of neurofilament organisation is a hallmark of amyotrophic lateral sclerosis. The concept of activation of overlapping cell death cascades by two distinct genetic defects could help elucidating downstream pathogenic processes and may provide novel targets for pharmacological intervention or gene therapy for the treatment of motor neuron disorders.     

Regional Vulnerability after Traumatic Brain Injury: Gender Differences in Mice That Overexpress Human Copper, Zinc Superoxide Dismutase

Neuronal loss was quantified in both cortical and subcortical brain regions after traumatic brain injury in male and female nontransgenic (nTg) and transgenic (Tg) mice that overexpress human copper, zinc superoxide dismutase. Mice were euthanized at 7 days after a controlled cortical impact injury. Sections of brain were processed for immunolocalization of NeuN, a neuronal nuclear antigen, and the complement type 3 receptor, a marker of microglia/macrophages, and stained for iron. Cortical lesion volume and neuronal loss in the medial and/or lateral ventroposterior thalamic nuclei were significantly less in the nTg female compared to the nTg male (P = 0.0373 and P = 0.0023, respectively). In contrast, in CA3 of the hippocampus and laterodorsal thalamic nucleus (LD), there were no gender differences in neuronal loss between these nTg groups. Cortical lesion volume was significantly reduced in Tg males compared to nTg males (P = 0.0137) and was unchanged in the Tg females compared to the nTg females. Neuronal loss was attenuated in the CA3 and LD in the Tg females compared to the nTg females (P = 0.0252 and P = 0.0244, respectively). A similar protection was not observed in the Tg males. Microglial activation paralleled the pattern of neuronal loss and was most consistently aligned with iron deposition in the cortex and hippocampus. No overt differences were found in the pattern of microglial activation or iron staining between nTg and Tg mice nor between genders. Our findings demonstrate that neuroprotection, afforded by overexpression of copper, zinc superoxide dismutase, exhibits both regional and gender specificity.     

Formation of advanced glycation end‐product‐modified superoxide dismutase‐1 (SOD1) is one of the mechanisms responsible for inclusions common to familial amyotrophic lateral sclerosis patients with SOD1 gene mutation,and transgenic mice expressing human SOD1 gene mutation

Neuronal Lewy body‐like hyaline inclusions (LBHI) and astrocytic hyaline inclusions (Ast‐HI) are morphological hallmarks of certain familial amyotrophic lateral sclerosis (FALS) patients with superoxide dismutase‐1 (SOD1) gene mutations, and transgenic mice expressing the human SOD1 gene mutation. The ultrastructure of inclusions in both diseases is identical: the essential common constituents are granule‐coated fibrils approximately 15– 25 nm in diameter and granular materials. Detailed immunohistochemical analyses have shown that the essential common protein of the inclusions in both diseases is an SOD1 protein. This finding, together with the immunoelectron microscopy finding that the abnormal granule‐coated fibrils comprising the inclusions are positive for SOD1, indicates that these granule‐coated fibrils containing SOD1 are important evidence for mutant SOD1‐linked disease in human and mouse. For im‐munoelectron microscopy, the granule‐coated fibrils are modified by advanced glycation endproducts (AGE) such as N^?‐carboxymethyl lysine, pyrraline and pentosidine (Maillard reaction). Based on the fact that AGE themselves are insoluble molecules with direct cytotoxic effects, the granule‐coated fibrils and granular materials are not digested by the lysosomal and ubiquitin systems. The neurons and astrocytes of the normal individuals and non‐transgenic mice show no significant immunoreactivity for AGE. Considered with the mutant‐SOD1 aggregation toxicity, a portion of the SOD1 comprising both types of the inclusion is modified by the AGE, and the formation of the AGE‐modified SOD1 (probably AGE‐modified mutant SOD1) is one of the mechanisms responsible for the aggregation (i.e. granule‐coated fibril formation).