Protection of melatonin against damage of sperm mito-chondrial function induced by reactive oxygen species

Aim: To study the mitochondrial function damage of sperm in-duced by reactive oxygen species (ROS) and the protection of melatonin (MLT) against the damage. Methods: Normal function spermatozoa were selected from semen samples by Percoll gradi-ent centrifugation technique. The ROS generated by the hypoxan-thine xanthine oxidase system was incubated with the normal sper-matozoa in the presence or absence of MLT (6 retool/L) for 30 and 60 minutes.     

肠缺血/再灌注对肝脏自由基的影响及亚甲蓝的抗损伤作用

目的 研究肠缺血/再灌注(I/R)后氧自由基(ROS)对远隔器官肝脏的损伤作用,探讨亚甲蓝(MB)抗肝脏损伤的作用机制。方法 新西兰白兔32只,体重2.3～2.9 kg,随机分为4组,(1)正常组:本组仅做假手术处理。(2)I/R组:本组通过夹闭兔肠系膜前动脉1h复制肠I/R模型。(3)治疗A组:本组于松夹后静脉内给予MB 3 mg·kg-1。(4)治疗B组:本组于松夹后静脉内给予MB 15 mg·kg-1。颈总动脉置管,连续监测血压。于夹闭前、松夹即刻、松夹后30 min、1h和2h取血测定血中MDA浓度。实验结束后取一小块肝组织用于测定超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、黄嘌呤氧化酶(XO)及MDA。结果与I/R前基础值比较,I/R组血中MDA水平I/R后显著增加,同时血压显著下降;而治疗A组及B组血中MDA水平于I/R后均未显著增加。与正常组比较,I/R组肝组织MDA水平显著增高,而正常组与两个治疗组间肝组织中MDA无显著差异。结论 MB能减少肠I/R后ROS的生成并能对抗ROS对肝脏的损伤作用。     

脱氢抗坏血酸对高糖诱导系膜细胞产生氧自由基的影响

目的 探讨脱氢抗坏血酸(DHA)对高糖诱导系膜细胞产生氧自由基(ROS)的影响。 方法 (1)原代培养大鼠系膜细胞；(2)以Fe3+还原法检测细胞内抗坏血酸(AA)和DHA浓度，观察系膜细胞摄取AA和DHA的情况及葡萄糖、葡萄糖转运蛋白(GLUT)抑制剂细胞松弛素B对其的影响；(3)采用激光扫描共聚焦显微镜检测细胞内ROS，观察高糖诱导系膜细胞ROS产生的情况及不同浓度DHA对其的影响；(4)采用凝胶电泳迁移率法(EMSA)检测活性蛋白1(AP-1)和DNA的结合活性，观察DHA对高糖诱导的系膜细胞内AP-1 活性的影响。结果 (1)AA不能由细胞外进入系膜细胞，而DHA可以进入，并且随着细胞外葡萄糖浓度的增加，其进入速度减慢；细胞松弛素B则完全抑制了DHA进入到系膜细胞。(2)高糖快速诱导系膜细胞ROS产生增多；DHA抑制了高糖的这种作用，并且该抑制作用在≤4 mmol/L的浓度范围内呈浓度依赖性。(3)DHA抑制了高糖诱导的系膜细胞内AP-1 的激活。 结论 (1)系膜细胞是依赖DHA利用Vit C的细胞型；DHA进入该细胞依赖GLUT介导，高糖可抑制其进入细胞。(2)DHA可有效抑制高糖诱导的系膜细胞ROS产生增多，并在一定范围内呈浓度依赖性。(3)DHA在抑制ROS产生的同时，也显著抑制了高糖诱导的AP-1 的激活。     

脱氢抗坏血酸对高糖诱导肾小管上皮细胞产生氧自由基的影响

目的 探讨脱氢抗坏血酸(DHA)对高糖诱导肾小管上皮细胞产生氧自由基的影响｡方法 (1)传代培养肾小管上皮细胞;(2)以Fe3+还原法检测细胞内抗坏血酸(AA)浓度,观察肾小管上皮细胞摄取AA和DHA的情况及葡萄糖和葡萄糖转运蛋白(GLUT)抑制剂细胞松弛素B(cytochalasin B)对其影响;(3)激光扫描共聚焦显微镜检测细胞内氧自由基,观察高糖诱导肾小管上皮细胞氧自由基产生的情况及不同浓度DHA对其影响｡结果 (1)AA不能由细胞外进入肾小管上皮细胞,而DHA可以进入,并且随着细胞外葡萄糖浓度的增加,其进入速度减慢｡细胞松弛素B则完全抑制了DHA进入到肾小管上皮细胞, 而固定葡萄糖浓度(25 mmol/L)时,随着DHA浓度的增加,DHA进入细胞内的速度逐渐增快｡(2)高糖快速诱导了肾小管上皮细胞氧自由基产生增多,DHA抑制了高糖的这种作用,并且该抑制作用在浓度小于､等于4 mmol/L的范围内呈浓度依赖性｡结论 (1)肾小管上皮细胞是依赖DHA利用VitC的细胞型,DHA进入该细胞依赖GLUT介导,高糖可抑制其进入细胞;(2)DHA可有效抑制高糖诱导的肾小管上皮细胞氧自由基产生增多,并在一定范围内呈浓度依赖性｡     

活性氧介导醛固酮诱导的足细胞凋亡

目的 观察醛固酮（ALD）及其受体拮抗剂螺内酯（SPI）对足细胞活性氧（ROS）产生及凋亡的影响,并探讨其可能机制。 方法 体外培养条件的永生化小鼠足细胞系,分为空白对照组、ALD组、SPI组、ALD+SPI组;用荧光分光光度计检测足细胞内ROS水平;间接免疫荧光检测nephrin表达;流式细胞仪检测足细胞凋亡率;RT-PCR、Western 印迹法检测bax、bcl-2 mRNA及蛋白表达。同时观察抗氧化剂N-乙酰半胱氨酸（NAC）对上述效应的阻断作用。 结果 与对照组相比,ALD诱导足细胞ROS产生增多（P < 0.05）,该作用可被SPI阻断（P < 0.05）。ALD可诱导足细胞nephrin表达降低及足细胞凋亡（P < 0.05）,同时伴有bax mRNA、蛋白表达升高及bcl-2 mRNA、蛋白表达降低（P < 0.05）,SPI及NAC可阻断这一变化（P < 0.05）。 结论 ALD通过ROS途径作用于盐皮质激素受体上调促凋亡因子bax表达,下调抑凋亡因子bcl-2表达,进而诱导足细胞凋亡。     

Differentiation of ejaculates showing reactive oxygen species production by spermatozoa or leukocytes

Summary. Differences between subgroups and correlations between reactive oxygen species (ROS), sperm motility, concentration of leukocytes and viability in semen samples from 143 men were investigated. Patients with azoospermia or leuko-cytospermia were excluded from the study. Spermatozoa were separated by means of glass wool nitration. Reactive oxygen species were determined by means of luminol chemiluminescence before and after sperm separation; thereafter, normozoospermic and oligozoospermic patients were divided into three subgroups using the mean of all patients investigated (17462 count 10⁻⁷ viable spermatozoa) as cut-off value as follows: G1—high reactive oxygen species production in native semen and after glass wool nitration; G2—high production of reactive oxygen species only in native semen; G3—low levels of reactive oxygen species before and after glass wool filtration. In general, reactive oxygen species were significantly higher in oligozoospermic samples than in normozoospermic samples. In men with normal sperm count, ROS production correlated significantly with the number of leukocytes in the ejaculate before glass wool nitration, but not thereafter. Glass wool nitration is useful to distinguish between sperm samples in which reactive oxygen species are generated by leukocytes and those in which reactive oxygen species are excessively generated by the spermatozoa themselves.     

Clinical significance of reactive oxygen species in semen of infertile Indian men

Reactive oxygen species (ROS) levels in semen are believed to play both physiological and pathological roles in male fertility. The study was aimed to find the clinical significance of ROS levels in infertile Indian men. This pilot study included 33 idiopathic infertile men and 18 proven fertile controls. ROS levels in the washed sperm were measured using chemiluminescence assay and expressed as 10⁶ cpm per 20 million spermatozoa. Sperm count, percent sperm motility, and percent normal sperm morphology were found to be significantly ( P  < 0.0001) reduced in infertile men compared with the controls. Median (minimum, maximum range) ROS levels of the infertile group [24.90 (6.89, 44.71)] were found to be significantly ( P  < 0.0001) elevated compared with the fertile controls [0.167(0.15, 2.78)]. No significant correlation was seen between ROS levels and semen parameters. Elevated ROS levels in the idiopathic Indian infertile men may be one of the underlying reasons for impaired fertility. Therefore measurement of seminal ROS levels may be used in Indian infertile men for better understanding of the aetiology and selection of antioxidant regimen in the treatment of male infertility. However, large studies may be urgently warranted to find out the role of antioxidants in ROS elevated Indian infertile men through randomised, controlled clinical study.     

Analysis of the relationship between reactive oxygen species production and leucocyte infiltration in fractions of human semen separated on Percoll gradients

We have shown previously that the reactive oxygen species generated by washed human ejaculates originate from cells which can be isolated in the low density region of discontinuous Percoll gradients. In this study we have used a simplified two-step (40/80%) Percoll gradient to separate human ejaculates (n = 109) into two populations of spermatozoa, exhibiting either a high or a low capacity for reactive oxygen species generation. We have then examined the relationships between this activity and other properties of the isolated fractions, with particular emphasis on the presence of leucocytes, which we have quantified using a monoclonal antibody directed against the common leucocyte antigen. The low-density cells recovered from the 40%/80% interface of the Percoll gradients, differed from the high-density fraction in exhibiting significantly reduced sperm motility, poorer sperm morphology, and a considerably enhanced capacity for reactive oxygen species production (P less than 0.001). In six cases the elevated levels of reactive oxygen species generation were associated with leucocyte concentrations in excess of 1 x 10(6) per 10(7) sperm, suggesting that leucocytes enter the seminal compartment in an activated, oxygen-radical generating, state. However, in the majority of cases exhibiting high levels of reactive oxygen species production, leucocyte numbers were low or absent and the semen profiles were unremarkable, except that seminal sperm concentrations tended to be low. These results suggest that the oxidative stress responsible for defective sperm function involves reactive oxygen species originating from two sources; the sperm and infiltrating leucocytes.     

Testicular hypofunction caused by activating p53 expression induced by reactive oxygen species in varicocele rats

We investigated the mechanism of the testicular hypofunction induced by hypoxia in varicocele rats. Varicocele was induced by partial ligation of the left renal vein. Seven weeks later, left testis mass index was measured. The sperm counts and motility were tested by CASA. The change of seminiferous tubule tissue was observed by HE staining. The reactive oxygen species (ROS) level in left testicular tissue was measured by ELISA, and the expressions of HIF‐1α and p53 were detected by immunohistochemistry and Western blot. The left testis mass index and the sperm motility were significantly lower in surgery group. By HE staining, the left seminiferous epithelial cell arrangement was incompact, disordered and vacuolated in surgery group. The ROS level in surgery group was significantly higher than the other groups. The results of immunohistochemistry and Western blot indicated that the expressions of HIF‐1α and p53 increased significantly in surgery group. Our study demonstrated that varicocele caused hypoxia that could cause the rise of ROS level to induce the increase of p53 expression, leading to the decrease of testis mass index and changes of seminiferous tubules, which would reduce sperm motility and result in male infertility eventually.     

氧自由基在异氟醚诱导小鼠前脑细胞凋亡中的作用

目的 观察小鼠暴露于异氟醚后前脑Caspase-3蛋白和氧自由基的变化,以了解氧自由基(reactive oxygen spceies,ROS)在异氟醚神经损伤中的作用.方法 56只雄性C57BL/6J小鼠按随机数字表法分为异氟醚组(Iso组,n=20)、二甲基硫脲±异氟醚组(DMTU±Iso组,n=8)、二甲基硫脲组...     

氧自由基在异氟醚诱导小鼠前脑细胞凋亡中的作用

Objective By observing the changes of Caspase-3 protein and reactive oxygen species (ROS) in the forebrain of mice induced by isoflurane inhalation to study the role of ROS in neural damages of isoflurane. Methods Fifty-six male C57BL/6Jmice were randomly divided into isoflurane group(Iso, n=20), dimethylthiourea group(DMTU,n=8), imethylthiourea plus isoflurane group(DMTU+lso, n=8), and control group(Con,n=20). Mice in Iso and DMTU+Iso groups were exposed to three segments of 1.4%isoflurane, 2 h for each segment. Thirty min before oxygen inhalation in DMTU group and first segment of isoflurane inhalation in DMTU+Iso group, 50 mg/kg of radical scavenger DMTU were intraperitoneally injected to the mice in the two groups. The changes of caspase-3 expression in prefrontal cortex were observed by immunocytochemical staining. And the activities of superoxide dismutase (SOD) and the amount of MDA were detected. Results Compared with Con group, the number of caspase-3 positive cells in Iso group increased significantly (3.38 times, P＜0.05); but in DMTU+Iso group( 1.43 times of Con group) and DMTU group(0.74 times of Con group),it did not change obviously (P＞0.05). There was no significant difference in the activities of SOD between Iso group [(25±10) U/mgprot] and Con group [(20±6) U/mgprot](P＞0.05), but lipid peroxidation malondialdehyde (MDA) content in Iso group [(13.7±6.1 ) nmol/mgprot] was significantly higher than that in Con group [(5.7±2.7) nmol/mgprot](P＜0.05). Conclusion 1.4% isoflurane exposure significantly increase the expression of caspase-3 in the forebrain of mice and its molecular mechanism may be related to the increase of free radicals.     

Role of reactive oxygen species in inhibition of endothelial cell migration by oxidized low-density lipoprotein

OBJECTIVE: Endothelial cell migration is inhibited by oxidized low-density lipoprotein (oxLDL) and lysophosphatidylcholine (lysoPC). The purpose of this study was to explore the mechanism of this inhibition, specifically the role of reactive oxygen species. METHODS: The ability of oxLDL, lysoPC, and known superoxide generators to stimulate endothelial cell production of reactive oxygen species and inhibit endothelial cell migration under the same conditions was assessed. Reactive oxygen species production was assessed with dichlorofluorescein. Migration was studied with a razor scrape assay and measured after 24 hours. In addition, the ability of various antioxidants, added before initiation of the scrape assay, to restore endothelial cell migration in oxLDL was determined. RESULTS: OxLDL and lysoPC, at concentrations that stimulated reactive oxygen species production, also inhibited endothelial cell migration. Other agents that generated superoxide also inhibited endothelial cell migration, but hydrogen peroxide did not. Of a variety of antioxidants assessed for their ability to preserve endothelial cell migration in the presence of oxLDL, only superoxide dismutase and reduced nicotinamide adenine dinucleotide (phosphate) oxidase inhibitors (diphenyleneiodonium, quinacrine, hydralazine) preserved endothelial cell migration. CONCLUSIONS: These data suggest that oxLDL inhibits endothelial cell migration through a superoxide-dependent mechanism and that reduced nicotinamide adenine dinucleotide (phosphate) oxidase is the cellular source of the superoxide. CLINICAL RELEVANCE: OxLDL inhibits endothelial cell migration, and may impair healing of arterial injuries. The mechanism of oxidized LDL inhibition is not known. Our in vitro studies show that the inhibitory properties are related to production of reactive oxygen species. Superoxide dismutase or inhibitors of reduced nicotinamide adenine dinucleotide phosphate oxidase can preserve endothelial migration in the presence of oxLDL. This might improve the healing of endothelial injuries at sites of arterial repair or angioplasty, especially in lipid-laden arterial walls.     

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.     

活性氧簇调控破骨细胞分化的研究进展

破骨细胞是影响骨重塑的关键细胞,其分化过程受包括活性氧簇(reactive oxygen species, ROS)在内的多种因子调控。ROS是氧气转化为水的过程中氧化还原反应的中间产物,可通过激活RANKL/RANK信号通路下游的NF-κB、MAPK和AKT等多条信号通路来促进破骨细胞分化。近年来,众多新型抗氧化剂显现出对破骨细胞分化成熟的靶向抑制作用,有望为骨质疏松症等溶骨亢进型疾病提供治疗新思路。本文对破骨细胞中ROS的产生与清除、ROS对于破骨细胞分化的调控作用和抗氧化剂治疗溶骨性疾病的研究进展进行综述,探讨ROS作为溶骨性疾病治疗靶点的巨大潜力。     

丙泊酚对大鼠小肠缺血-再灌注后肺损伤氧自由基变化的影响

目的 探讨不同临床剂量丙泊酚对大鼠小肠缺血-再灌注后肺损伤氧自由基变化的影响及其保护作用。方法 雄性SD大鼠40只,随机分为假手术对照组(C组)、小肠缺血-再灌注组(R组)及分别给予丙泊酚4mg·kg~(-1)·h~(-1)、8mg·kg~(-1)·h~(-1)、10mg·kg~(-1)·h~(-1)(P4、P8、P10)五组;制作小肠缺血-再灌注模型。实验结束即刻留取血样和肺组织,待测脂质过氧化物和肺组织形态学变化。结果 R、P4组血中脂质过氧化物明显升高,与C、P8、P10组相比均有非常显著差异(P<0.01),P8、P10组与C组相比无统计学差异;肺组织脂质过氧化物各实验组与对照组相比差异非常显著(P<0.01),以R组改变最为明显;各用药组指标均得以改善,与R组相比有显著差异(P<0.01),P4与P8、P10组相比亦差异显著(P<0.01)。假手术组肺组织结构正常,R组可见肺泡压闭实变,腔内有大量渗出,炎性细胞浸润,毛细血管扩张,支气管壁增厚,各用药组肺部改变较R组明显减轻,P8、P10两组除了有少量改变外,肺组织结构基本接近正常。结论 氧自由基在大鼠小肠缺血-再灌注后肺损伤中有重要作用,临床剂量丙泊酚不同程度地抑制脂质过氧化反应,对小肠缺血-再灌注后氧自由基介导的肺损伤产生保护作用。     

Preliminary study on overproduction of reactive oxygen species by neutrophils in diabetes mellitus

AIM:To assess the amount and pattern of reactive oxygen species(ROS)production in diabetic patientderived neutrophils.METHODS:Blood samples from type 2 diabetes mellitus(DM)patients and volunteers(controls)were subjected to neutrophil isolation and the assessment of neutrophil oxidative burst using chemiluminescence assay.Neutrophils were activated by using phorbol myristate acetate(PMA)and neutrophils without activation were kept as a negative control.The chemilu-minescence readings were obtained by transferring cell suspension into a 1.5 m L Eppendorf tube,with PMA and luminol.Reaction mixtures were gently vortexed and placed inside luminometer for a duration of 5 min.RESULTS:Our results showed that in the resting condition,the secretion of ROS in normal non-diabetic individuals was relatively low compared to diabetic patients.However,the time scale observation revealed that the secreted ROS declined accordingly with time in non-diabetic individuals,yet such a reduction was not detected in diabetic patients where at all the time points,the secretion of ROS was maintained at similar magnitudes.This preliminary study demonstrated that ROS production was significantly higher in patients with DM compared to non-diabetic subjects in both resting and activated conditions.CONCLUSION:The respiratory burst activity of neutrophils could be affected by DM and the elevation of ROS production might be an aggravating factor in diabetic-related complications.     

The efficacy of antioxidants in sperm parameters and production of reactive oxygen species levels during the freeze-thaw process: A systematic review and meta-analysis

To investigate the impact of antioxidants in sperm parameters and reduction in reactive oxygen species production during the freeze-thaw process. PubMed, Scopus, Web of Science, Embase and Cochrane central library were systematically searched. Of the 1583 articles, 23 studies were selected for data extraction. Our results show that antioxidants improved sperm progressive motility (standardised mean difference (SMD) = 1; 95% CI: 0.62, 1.38; p < .001) and viability (SMD = 1.20; 95% CI: 0.50, 1.91; p = .001) and reduced sperm DNA fragmentation (SDF) and hydrogen peroxide (H₂O₂) production, but there was no significant improvement in total sperm motility after thawing. Acetyl-l-carnitine/l-carnitine, melatonin and catalase had a significant positive impact on progressive motility. The role of tempol and melatonin in improving viability was significant compared to other antioxidants. Moreover, a significant reduction in SDF was observed after addition of butylated hydroxytoluene, tempol and vitamin E. However, the prevention of H₂O₂ production was significant only after the addition of tempol. Our overall results displayed the positive impact of antioxidants on progressive sperm motility, viability and reduction in SDF and H₂O₂ production, but no significant impact of antioxidants on total sperm motility was seen during the freeze-thaw process.     

Proteomic analysis of sperm proteins in infertile men with high levels of reactive oxygen species

Oxidative stress is a significant risk factor for male infertility. A pro‐oxidant testicular environment may alter the expression profile of functional sperm proteins and result in poor sperm quality. Patients and donors were divided into ROS (‐) and ROS (+) groups. Using computational studies, and data mining of available literature on spermatozoa, oxidative stress and proteomics, we identified three core regulatory proteins angiotensin‐converting enzyme (ACE), heat‐shock protein (Hsp70) family A member 2 (HSPA2) and ribosomal protein subunit 27A (RPS27A) and seven interlink proteins NOS2, SUMO2, UBL4A, FBXO25, MAP3K3, APP and UBC. HSPA2 was validated by Western Blot, while the localisation of ACE, RPS27A, MAP3K3 and APP was identified by immunocytochemistry. The obtained results showed that HSPA2 was 1.2 (ROS+) and 2.1 (ROS?) fold downregulated in spermatozoa from patients with high levels of reactive oxygen species (ROS). ACE and APP were localised in the post‐acrosomal region of spermatozoa, whereas RPS27A and MAP3K3 were localised either in the tail or sperm neck area. Our data show that these proteins may play a role in ROS‐induced male infertility.