活性氧在骨性关节炎发病中的作用机制（英文）

近年来越来越多的研究表明活性氧在骨性关节炎的发病中起到关键作用,主要涉及到软骨细胞的凋亡和细胞外基质的退化,后者包括基质合成减少、分解增加以及钙化形成。本文就活性氧在骨性关节炎发病机制研究中的进展作一综述。     

Inhibitory effect of local anaesthetics on reactive oxygen species production by human neutrophils

Background: Reactive oxygen species (ROS) generated from neutrophils accumulated in various major organs are thought to play a pivotal role in the pathogenesis of host auto-injury. Lidocaine has been shown to reduce the injury. We investigated the effect of local anaesthetics (lidocaine, mepivacaine and bupivacaine) on ROS production by neutrophils using an in vitro system.
Methods: We measured the production of superoxide (ferric-ytochrome c method), hydrogen peroxide (H₂O₂: scopoletin fluorescence technique), and hydroxyl radical (OH.: ethylene gas method) by neutrophils isolated from human adult volunteers in the absence and presence of lidocaine (2–200 μg/mL), mepivacaine (3–300 μg/mL), and bupivacaine (3–300 μg/mL). We also measured the ROS generation in a cell-free (xanthinexanthine oxidase) system.
Results: Lidocaine and mepivacaine at higher levels significantly decreased the production of ROS by neutrophils. However, these local anaesthetics at clinically relevant blood concentrations had no effect on the levels of ROS. Furthermore, lidocaine and mepivacaine failed to reduce ROS generated by the cell-free system. Bupivacaine did not decrease ROS generation by either generating system.
Conclusion: In conclusion, in the present in vitro system, only concentrations of lidocaine and mepivacaine 100-fold higher than clinically feasible ones reduced ROS production by human neutrophils. However, the local anaesthetics at clinically relevant blood concentrations had no suppressive effect. Further studies using in vivo systems are required to elucidate the inhibitory effects of local anaesthetics on ROS generation in clinical settings.     

The role of reactive oxygen species in homeostasis and degradation of cartilage

OBJECTIVES: The metabolism of cells in articular joint tissues in normal and pathological conditions is subject to a complex environmental control. In addition to soluble mediators such as cytokines and growth factors, as well as mechanical stimuli, reactive oxygen species (ROS) emerge as major factors in this regulation. ROS production has been found to increase in joint diseases, such as osteoarthritis and rheumatoid arthritis, but their role in joint diseases initiation and progression remains questionable. METHOD: This review is focused on the role of ROS, mainly nitric oxide, peroxynitrite and superoxide anion radicals, in the signaling mechanisms implied in the main cellular functions, including synthesis and degradation of matrix components. The direct effects of ROS on cartilage matrix components as well as their inflammatory and immunomodulatory effects are also considered. RESULTS: Some intracellular signaling pathways are redox sensitive and ROS are involved in the regulation of the production of some biochemical factors involved in cartilage degradation and joint inflammation. Further, ROS may cause damage to all matrix components, either by a direct attack or indirectly by reducing matrix components synthesis, by inducing apoptosis or by activating latent metalloproteinases. Finally, we have highlighted the uncoupling effect of ROS on tissue remodeling and synovium inflammation, suggesting that antioxidant therapy could be helpful to treat structural changes but not to relieve symptoms. CONCLUSIONS: This review of the literature supports the concept that ROS are not only deleterious agents involved in cartilage degradation, but that they also act as integral factors of intracellular signaling mechanisms. Further investigation is required to support the concept of antioxidant therapy in the management of joint diseases.     

In vivo scavenging effect of ethylcysteine on reactive oxygen species in human semen]

PURPOSE: The production of reactive oxygen species (ROS) is a normal physiological event in various organs including the testis. Overproduction of ROS, however, can be detrimental to sperm, being associated with male infertility. In vivo experiments using vitamin E (Vit. E), one of the major membrane protectants against ROS and lipid peroxidation, have shown its significant potential in treating ROS-associated male infertility. There has been no study that the scavenging drugs reduce the level of ROS in human semen. Previously we reported the in vitro scavenging effectiveness of ethylcysteine (EC) against ROS in human semen. The present study was performed in order to determine the effectiveness of the in vivo administration of EC as treatment for ROS-associated male infertility. PATIENTS AND METHODS: Ten cases of male infertility, with the exceptions of azoospermia and pyospermia, were chosen. Patients were divided randomly into two groups. Each group received either 600 mg/day of EC (Group A) or Vit. E 600 mg/day (Group B) for 3 months. Then, after a 1-month wash-out period, the patients were switched to another treatment. Conventional semen analysis, computerized motility assessment, measurement of ROS generation and sperm function assessment by triple stain were performed before and after administration of EC and Vit. E. The levels of EC and Vit. E were also assessed in patients' blood serum and seminal plasma before and after administration of EC and Vit. E. RESULTS: Sperm density and sperm motility did not improve but sperm function had a significant tendency toward improvement after administration of EC and Vit. E. ROS levels significantly decreased only after administration of EC After administration of EC and Vit. E, their levels significantly increased in patients' blood serum but were unchanged in patients' seminal plasma. CONCLUSION: Since our study showed that oral administration of EC produced results similar to those of Vit. E, we conclude that EC is an effective treatment agent for ROS-associated male infertility.     

Myocardial ischaemia and reperfusion injury: reactive oxygen species and the role of neutrophil

A growing body of evidence suggests that oxygen radicals can mediate myocardial tissue injury during ischaemia and, in particular, during reperfusion. This review focuses on the role of neutrophil as a mediator of myocardial damage. Upon reperfusion, neutrophils accumulate and produce an inflammatory response in the myocardium that is responsible, in part, for the extension of tissue injury associated with reperfusion. It has shown that the inhibition of neutrophil accumulation and adhesion is associated with decreased infarct size. This strongly suggests that myocardial cells at risk region undergo irreversible changes upon reperfusion and accumulation of neutrophils. Several pharmacological agents (ibuprofen, allopurinol, prostacyclin, and prostaglandin E analogues) protect the myocardium from reperfusion injury. In addition, the mechanisms by which these agents act and directions of research that may lead to therapeutically useful approaches are also discussed in this review.     

The role of reactive oxygen species in testicular dysfunction associated with varicocele

OBJECTIVE: To determine the level of malondialdehyde (MDA), an indirect indicator of lipid peroxidation-induced injury by reactive oxygen species, in testicular biopsy specimens from infertile patients with and without varicocele. PATIENTS AND METHODS: Levels of MDA were measured in the testicular biopsy specimens from 25 infertile men (15 with varicocele, mean age 30.0 years, SD 5.7, range 23-45, and 10 without, mean age 28.7 years, SD 4.2, range 21-34). All patients were evaluated by a detailed history, physical examination, semen analysis (at least twice), serum follicle-stimulating hormone and free testosterone levels, testicular biopsy and contact imprint. Scrotal colour Doppler ultrasonography was used to confirm suspected varicocele. The level of MDA in testicular biopsy specimens was measured using the thiobarbituric acid test and the results expressed per unit tissue weight. RESULTS: As a causal factor for infertility, varicocele was identified in 15 men (60%), testicular failure in four (16%), idiopathic infertility in four (16%) and obstruction in two (8%). Of the 15 patients with varicocele, eight had bilateral varicocele and it was subclinical in three; the varicocele was grade I in four, grade II in six and grade III in two. The mean (SD) MDA level in the men with a subclinical varicocele was 15.7 (3.1) pmol/mg tissue, while in those with grade I-III varicocele it was 32.9 (12.25), 37.1 (12.25) and 86.9 (2.89) pmol/mg tissue, respectively. The levels in patients with grade III varicocele were significantly greater than in the other groups (P < 0.05). The mean MDA level in patients with or without varicocele was 38.3 (22.92) and 33.5 (18.93) pmol/mg tissue, respectively (P > 0.05). CONCLUSION: These results suggest that increasing levels of MDA are associated with higher grades of varicocele and support a possible rationale for controlled trials in infertile men with varicocele.     

活性氧在肝硬化门静脉高压症高动力循环中的作用

肝硬化门静脉高压症机体氧化应激反应亢进,并且与肝硬化门静脉高压症高动力循环存在密切联系.本文阐述了不同种类活性氧(超氧阴离子和过氧化氢)及其抗氧化剂,如NAD(P)H氧化酶抑制剂、超氧阴离子和过氧化氢清除剂以及维生素C或E在肝硬化门静脉高压症高动力循环发生和发展中的作用.研究显示肝硬化门静脉高压症机体活性氧产物增多,抑制或清除活性氧能缓解门静脉高动力血液循环状态,降低门静脉高压症并发症,具有潜在的临床应用价值.     

Effects of H2O2 exposure on human sperm motility parameters,reactive oxygen species levels and nitric oxide levels

Research has revealed that reactive oxygen species (ROS) negatively affect sperm function, both in vivo and in vitro. Sperm preparation techniques for assisted reproductive technologies (ART) are potential causes for additional ROS production. This study aimed to correlate the concentration of exogenous H₂O₂ with sperm motility parameters and intracellular ROS and nitric oxide (NO) levels to reiterate the importance of minimising ROS levels in ART. Human spermatozoa from 10 donors were incubated and exposed to different exogenous H₂O₂ concentrations (0, 2.5, 7.5 and 15 μm ). Subsequently, motility was determined using computer‐aided semen analysis, while ROS (2,7‐dichlorofluorescin diacetate) and NO (diaminofluorescein‐2/diacetate) were analysed using fluorescence‐activated cell sorting. Results showed that H₂O₂ did affect the sperm parameters. Exogenous H₂O₂ was detrimental to motility and resulted in a significant increase in overall ROS and NO levels. A significant increase in static cells was seen as well. It is important to elucidate the mechanisms between intracellular ROS levels with sperm motility parameters. While this experiment demonstrated a need to reduce exogenous ROS levels during ART, it did not illustrate the cause and effect relationship of intracellular ROS and NO levels with sperm motility. Further research needs to be conducted to define a pathological level of ROS.     

The cardiovascular action of oxygen breathing: effect on adrenergic stimulation

20 anesthetized mechanically ventilated dogs received an infusion of epinephrine, 2 micrograms/kg/min, for 4 h. 10 dogs were ventilated with air for the entire 4-hour period and 10 were ventilated with oxygen for the first hour and then air for 3 h. During the first hour of the epinephrine infusion in the group ventilated with oxygen, as compared to the group ventilated with air, the heart rate decreased (it increased in the dogs ventilated with air); the cardiac output increased less; the systemic vascular resistance decreased less; the pulmonary vascular resistance decreased more; and the pulmonary artery wedge pressure increased (it decreased in the dogs ventilated with air). During the next 3 h when the animals were ventilated with air, the effects of oxygen on the hemodynamic changes induced by epinephrine persisted for variable periods. The epinephrine-induced pulmonary arteriovenous shunt was significantly less at all times in the group ventilated with oxygen, not only during the oxygen breathing but following it for 3 h. Oxygen breathing has been known to exert powerful effects on the heart, and the pulmonary and systemic circulations in normal man and animals and in patients with pulmonary hypertension and acute respiratory failure. This study demonstrates that oxygen breathing significantly alters the cardiovascular effects of epinephrine during oxygen breathing and reduces the epinephrine-induced pulmonary shunt during and for several hours after oxygen is discontinued. These finding add further evidence that hyperoxia affects, by unknown mechanisms, the autonomic regulation of the cardiovascular system.     

The role of reactive oxygen species in animal models of glomerular disease.

Reactive oxygen metabolites have been demonstrated to play a pathobiologic role in a number of experimental models of both immune and nonimmune glomerular injury. Using scavenging substances, enzyme inhibitors, and transition metal chelators, all of the major reactive oxygen metabolites have been implicated in glomerular injury. In addition, in neutrophil-dependent models, interaction between neutrophil-derived myeloperoxidase and halide anions has been shown to be involved in glomerular damage, as well as halogenation of the glomerular basement membrane (GBM). Finally, recent attention has focused on the role of cytokines (perhaps elaborated by infiltrating monocytes/macrophages) in stimulating mesangial cells (MC) to produce reactive oxygen species. Theoretically, this pathobiologic sequence could further enhance an inflammatory state within the glomerular tuft and enable the propagation of initial glomerular injury, which may be associated with an increase in monocyte infiltration into the mesangium, to glomerulosclerosis in experimental models that manifest this transition.     

Potential role of reactive oxygen species on testicular pathology associated with infertility

Aim: To investigate the level of malondialdehyde (MDA), a direct indicator of lipid peroxidation-induced injury by reactive oxygen species (ROS), in testicular biopsy specimens from infertile patients. Methods: Levels of MDA were measured in testicular biopsy specimens from 29 consequent-randomized infertile men, aged 29.58±4.76 (21-45) years. All patients were evaluated by a complete medical and reproductive history, physical examination, semen analysis (at least two), serum follicle-stimulating hormone and free testosterone levels, testicular biopsy and contact imprint. Scrotal colour Doppler ultrasonography was used to confirm suspected varicocele. The testicular MDA level was measured using the thiobarbituric acid test and the results were expressed per unit tissue weight. Results: As a causal factor in infertility, varicocele was identified in 17 (58.6 %) patients, and idiopathic infertility, testicular failure and obstruction in 4 (13.8 %) patients each. The testicular MDA level was 13.56 (6.01),     

低浓度活性氧逆转肾癌先天性多药耐药的实验研究

目的探讨低浓度活性氧对肾癌786-O细胞先天性多药耐药的逆转作用及相关机制。方法采用WST-1细胞增殖及细胞毒性检测试剂盒确定活性氧H2O2的非细胞毒性剂量,以及对786-O细胞药物敏感性的影响。罗丹明123实验检测细胞P糖蛋白(P-gp)功能,Western blot方法检测经典多药耐药基因(mdr1)产物P-gp的表达。结果在0.00001～0.1mmol/L浓度范围内,H2O2具有明显的促细胞生长作用,且显著增加了阿霉素及长春新碱的细胞毒性,0.02mmol/LH2O2孵育786-O细胞72h后其对阿霉素及长春新碱的药物敏感性分别增加至对照组的5.43及4.47倍,荧光染料罗丹明123的蓄积量显著增加,Western blot检测结果显示0.02mmol/LH2O2可抑制肾癌786-O细胞P-gp的表达。结论低浓度活性氧H2O2可部分逆转人肾癌786-O细胞先天性多药耐药对阿霉素的耐药性,其逆转机制与增加细胞内化疗药物浓度、抑制P-gp的表达有关。     

The role of mitochondrial reactive oxygen species in pH regulation in articular chondrocytes

OBJECTIVE: To examine the effect of O(2) and the role, and source, of reactive oxygen species (ROS) on pH regulation in articular chondrocytes. METHODS: Cartilage from equine metacarpo/tarsophalangeal joints was digested (collagenase) to isolate chondrocytes and loaded with 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein, a pH-sensitive fluorophore. O(2) tension was maintained using Eschweiler tonometers and a Wosthoff gas mixer. Cells were exposed to agents which alter ROS levels, mitochondrial inhibitors and/or inhibitors of protein phosphorylation. ROS levels were determined by dichlorofluorescein and mitochondrial membrane potential measured using JC-1. RESULTS: pH homeostasis was dependent on ROS. Na(+)/H(+) exchanger (NHE) activity was inhibited at low O(2) tension (acid efflux reducing from 2.30+/-0.05 to 1.27+/-0.11mMmin(-1) at 1%). NHE activity correlated with ROS levels (r(2)=0.65). ROS levels were increased by antimycin A (with levels at 1% O(2) tension increasing from 59+/-9% of the value at 20% to 87+/-7%), but reduced by rotenone, myxothiazol and diphenyleneiodonium. Hypoxia induced depolarisation of the mitochondrial membrane potential (with JC-1 red-green fluorescence ratio at 1% O(2) tension decreasing to 40+/-10% of the value at 20%). The response to changes in O(2) and to antimycin A was inhibited by staurosporine, wortmanin and calyculin A. CONCLUSION: The fall in ROS levels in hypoxia reduces the ability of articular chondrocytes to regulate pH, inhibiting NHE activity via changes in protein phosphorylation. The site of ROS generation is likely to be mitochondrial electron transport chain complex III. These effects are important to understanding normal chondrocyte function and response to altered O(2) tension.     

Effect of rotation on the generation of reactive oxygen species in human semen

Summary. Rotation of semen after liquefaction is a standard procedure in an Andrology laboratory. During rotation under aerobic conditions the semen is exposed to oxygen. Oxygen toxicity induces lipid peroxidation which is responsible for the loss of sperm function. The generation of reactive oxygen species was significantly increased after rotation. This may have a detrimental effect on sperm function during assisted reproduction.     

The effects of pentoxifylline on the generation of reactive oxygen species and lipid peroxidation in human spermatozoa

Summary. The aims of this study were to compare the in vitro effects of 3.6 mM and 7.2 mM pentoxifylline on the ability of spermatozoa to generate reactive oxygen species (ROS) and on lipid peroxidation (LPO). Semen samples were obtained from 10 asthenozoospermic men who had been previously identified as producing ROS after addition of Phorbol 12-myristate 13-acetate (PMA) during the screening of patients attending with male factor infertility. Spermatozoa were prepared by a swim-up technique from unprocessed semen and divided into 3 aliquots. To the control aliquot [A] an equal volume of BWW medium was added. To aliquots B and C an equal volume of BWW medium containing pentoxifylline was added to obtain final concentrations of 3.6 and 7.2 mM, respectively. ROS production was measured from peak luminescence (mV 10⁻⁷ sperm) using a lucigenin chemiluminescent probe. LPO was also measured in the medium surrounding the spermatozoa after 30 min exposure to pentoxifylline using the thiobarbituric acid (TBA) assay for malondialdehyde (MDA). The reduction in ROS production was significantly greater in the samples exposed to 7.2 mM pentoxifylline as compared with the control and 3.6 mM pentoxifylline samples. There was no significant difference in peak luminescence between control and 3.6 mM pentoxifylline specimens. Both concentrations of pentoxifylline caused comparable reductions in MDA concentration in the medium ( P <0.05) surrounding the spermatozoa compared with control after 30 min exposure. Extracellular ROS generation may damage surrounding healthy spermatozoa. These findings suggest that higher concentrations of pentoxifylline are protective against ROS release in susceptible spermatozoa and may also reduce collateral LPO.     

The action of sevoflurane on vascular smooth muscle of isolated mesenteric resistance arteries (part 1): role of endothelium

BACKGROUND: The direct action of sevoflurane on systemic resistance arteries is not fully understood. METHODS: Isometric force was recorded in isolated rat small mesenteric arteries. RESULTS: Sevoflurane (2-5%) enhanced contractile response to norepinephrine only in the presence of endothelium, but inhibited it in its absence. Sevoflurane still enhanced the norepinephrine response after inhibitions of the nitric oxide, endothelium-derived hyperpolarizing factor, cyclooxygenase and lipoxygenase pathways, or after blockade of either endothelin-1 ET-1), angiotensin-II, or sevotonin receptors. Sevoflurane (3-5%) inhibited contractile response to potassium chloride only in the absence of endothelium but did not influence it in its presence. In the endothelium-intact strips, inhibition of the norepinephrine response, which was enhanced during application of sevoflurane, was observed after washout of sevoflurane and persisted for approximately 15 min. In the endothelium-denuded strips, the inhibition of norepinephrine response was similarly prolonged after washout of sevoflurane. However, no significant inhibitions of potassium chloride response were observed after washout of sevoflurane in both the endothelium-intact and the endothelium-denuded strips. CONCLUSIONS: The action of sevoflurane on norepinephrine contractile response consists of endothelium-dependent vasoconstricting and endothelium-independent vasodilating components. In the presence of endothelium, the former predominates over the latter, enhancing the norepinephrine response. The endothelium-independent component persisted after washout of sevoflurane, leading to prolonged inhibition of the norepinephrine response. The mechanisms behind the sevoflurane-induced inhibition of norepinephrine response are at least in part different from those behind its inhibition of potassium chloride response. Nitric oxide, endothelium-derived hyperpolarizing factor, cyclooxygenase products, lipoxygenase products, endothelin-1, angiotensin-II, and serotonin are not involved in the vasoconstricting action. (Key words: Halogenated volatile anesthetics; sympathetic nervous system; systemic hypotension; vascular endothelium.)