Circulating neutrophils exhibit enhanced apoptosis associated with mitochondrial dysfunctions after surgery under general anaesthesia

BACKGROUND: Evidence suggests that apoptosis plays a main role in the postoperative changes detected in the polymorphonuclear neutrophil (PMN) population. Furthermore, recent studies have demonstrated that mitochondrial alterations constitute critical events of the apoptotic cascade. In this study we investigated whether apoptosis among neutrophils taken from patients undergoing surgical trauma could be associated with perturbation of mitochondrial transmembrane potential (deltapsim) and/or exaggerated production of mitochondrial reactive oxygen species (ROS). METHODS: Twenty-seven patients undergoing elective surgery under general anaesthesia were enrolled in the study. Peripheral blood samples were drawn one day before the operation and at 12 and 24 h after surgery. Apoptosis rate was assessed by staining neutrophils with 7-amino-actinomycin D (7-AAD) and by analysis by a FACScan flow cytometer. In order to evaluate deltapsim, cells were exposed to 3,3-dihexyloxacarbocyanine iodide [DiOC6(3)]; intracellular ROS was measured by means of hydroethidine (HE) and 2,7-diclorofluorescein diacetate (DCFH-DA), followed by analysis on a cytofluorometer. RESULTS: At 12 h following surgery we observed a significantly (P<0.05) increased frequency of apoptotic PMNs compared to that preoperatively (30.79+/-3.68% vs 7.40+/-0.69%). At this same time-point, the rate of neutrophils stained with HE, DCFH-DA and [DiOC6(3)] were significantly (P<0.05) higher compared to baseline (51.05+/-5.44%, 50.58+/-5.84% and 55.31+/-4.33% vs 20.17+/-2.38%, 19.59+/-2.03 and 25.43+/-2.71% respectively). Overall measurements returned to the preoperative values 24 h after surgery. CONCLUSION: These data suggest that surgery under general anaesthesia triggers in the immediate postoperative period pathways of PMN accelerated apoptosis associated with significant alterations in mitochondrial function.     

Effects of semen processing on the generation of reactive oxygen species and mitochondrial membrane potential of human spermatozoa

This study was designed to evaluate the effects of semen processing on the generation of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in spermatozoa, and to develop reliable indexes for the evaluation of sperm quality during sperm preparation. Swim-up and density gradient centrifugation methods were used to separate semen in oligoasthenoteratozoospermia (OAT), leucocytospermia (LC) and normozoospermia groups. Levels of ROS and MMP were measured by flow cytometry. Before preparation, the patients with abnormal semen parameters had a lower MMP and higher ROS, and there was a negative correlation between MMP and ROS. The levels of MMP and ROS increased significantly, especially ROS produced by swim-up. A significant difference was found between the correlation of MMP and total normal motile sperm count after preparation in the OAT group. The level of ROS was associated with the amount of white blood cells in the LC group. The MMP can be used as an objective index to evaluate the sperm quality of OAT patients, and the combination of MMP and ROS can be used to assess the efficiency of sperm preparation in LC patients. These findings can guide selection of the ideal sperm separation technique for different sperm samples.     

人精子体外氧化损伤对线粒体tRNA~(LeuUUR)基因的影响

目的:探讨活性氧(ROS)对人类精子线粒体tRNALeuUUR基因的氧化损伤。方法:采用Percoll梯度离心法筛选具有正常生理功能的精子,作为正常精子模型,并分为损伤组20例和对照组20例,分别加入次黄嘌呤-黄嘌呤氧化酶体系或不予处理,37℃有氧环境中孵育60min。分别提取精子DNA,以Fpg酶切损伤碱基并采用接头介导PCR(LM-PCR)检测线粒体tRNALeuUUR基因的氧化损伤。采用Rhodamine(Rh123)荧光探针标记精子,通过流式细胞仪检测线粒体膜电位,观察精子的功能。结果:与对照组相比,损伤组精子孵育后线粒体膜电位明显降低[(116.27±11.72)%vs(64.00±4.88)%,P<0.05]。Fpg酶切和LM-PCR显示精子线粒体tRNALeuUUR基因损伤。结论:ROS可能通过对精子线粒体tRNALeuUUR基因氧化损伤而影响精子功能(线粒体膜电位明显降低),从而引起不育。     

Overtime expression of plasma membrane and mitochondrial function markers associated with cell death in human spermatozoa exposed to nonphysiological levels of reactive oxygen species

In many cell types, the potential of reactive oxygen species to induce death processes has been largely demonstrated. Studies in spermatozoa have associated the imbalance of reactive oxygen species and phosphatidylserine externalisation as an apoptosis marker. However, the lack of consensus about time effect in the joint expression of these and other death markers has made it difficult to understand the set of mechanisms influenced beyond the concentration effect of reactive oxygen species to stimulate cell death. Here, the plasma membrane permeability and integrity, phosphatidylserine externalisation and mitochondrial membrane potential were jointly evaluated as death markers in human spermatozoa stimulated with H₂O₂. The results showed a profound and sustained effect of dissipation in the mitochondrial membrane potential and an increased phosphatidylserine externalisation in human spermatozoa exposed to 3 mmol⁻¹ of H₂O₂ at 30 min. This was followed by an increased membrane permeability after 45 min. The last observed event was the loss of cell membrane integrity at 60 min. In conclusion, mitochondria are rapidly affected in human spermatozoa exposed to reactive oxygen species, with the barely detectable mitochondrial membrane potential coexisting with the high phosphatidylserine externalisation in cells with normal membrane permeability.     

Apigenin drives the production of reactive oxygen species and initiates a mitochondrial mediated cell death pathway in prostate epithelial cells

BACKGROUND: Phytoestrogens may reduce tumorigenesis in prostate cancer. We screened five phytoestrogens for their effect on cell growth and apoptosis in PWR-1E, LNCaP, PC-3, and DU145 prostate epithelial cells in vitro. METHODS: We assessed cell number, proliferation, and apoptosis using crystal violet assays, flow cytometric analysis, and TUNEL. Focusing specifically on apigenin we assessed the ability of calpain, serine protease, caspase, estrogen receptor, and ceramide synthase inhibitors to block apigenin induced apoptosis. We also analyzed caspase 3, 7, 8, 9, Bcl-2, Bax, Bid, and cytochrome C by Western analysis, and mitochondrial permeability and reactive oxygen species production by flow cytometry using mitosensor(TM) and DCFH-DA, respectively. RESULTS: Apigenin and silybinin significantly reduced cell number, with apigenin inducing apoptosis in PWR-1E, LNCaP, PC-3, and DU145 cells. The PC-3 and DU145 cells were less susceptible to apigenin induced apoptosis then LNCaP and PWR-1E cells. The induction of apoptosis by apigenin was caspase dependent. Apigenin generated reactive oxygen species, a loss of mitochondrial Bcl-2 expression, mitochondrial permeability, cytochrome C release, and the cleavage of caspase 3, 7, 8, and 9 and the concomitant cleavage of the inhibitor of apoptosis protein, cIAP-2. The overexpression of Bcl-2 in LNCaP B10 cells reduced the apoptotic effects of apigenin. CONCLUSIONS: Apigenin induces cell death in prostate epithelial cells using a mitochondrial mediated cell death pathway. Bcl-2 has a role in inhibiting apigenin induced cell death in prostate epithelial cells.     

Decreased expression of mitochondrial aldehyde dehydrogenase‐2 induces liver injury via activation of the mitogen‐activated protein kinase pathway

The aim of this study was to determine the role of ALDH2 in the injury of liver from brain‐dead donors. Using brain‐dead rabbit model and hypoxia model, levels of ALDH2 and apoptosis in tissues and cell lines were determined by Western blot, flow cytometry (FCM), and transferase (TdT)‐mediated biotin‐16‐dUTP nick‐end labeling (TUNEL) assays. After the expression of ALDH2 during hypoxia had been inhibited or activated, the accumulations of 4‐hydroxynonenal (4‐HNE) and molecules involved in mitogen‐activated protein kinase (MAPK) signaling pathway were analyzed using ELISA kit and Western blot. The low expression of phosphorylated ALDH2 in liver was time‐dependent in the brain‐dead rabbit model. Immunohistochemistry showed ALDH2 was primarily located in endothelial, and the rates of cell apoptosis in the donation after brain‐death (DBD) rabbit groups significantly increased with time. Following the treatment of inhibitor of ALDH2, daidzein, in combination with hypoxia for 8 h, the apoptosis rate and the levels of 4‐HNE, P‐JNK, and cleaved caspase‐3 significantly increased in contrast to that in hypoxic HUVECs; however, they all decreased after treatment with Alda‐1 and hypoxia compared with that in hypoxic HUVECs (P < 0.05). Instead, the levels of P‐P38, P‐ERK, P‐JNK, and cleaved caspase‐3 decreased and the ratio of bcl‐2/bax increased with ad‐ALDH2 (10⁶pfu/ml) in combination with hypoxia for 8 h, which significantly alleviated in contrast to that in hypoxic HUVECs. We found low expression of ALDH2 and high rates of apoptosis in the livers of brain‐dead donor rabbits. Furthermore, decreased ALDH2 led to apoptosis in HUVECs through MAPK pathway.     

The role of reactive oxygen species and apoptosis in the pathogenesis of varicocele in a rat model and efficiency of vitamin E treatment

We investigated role of reactive oxygen species (ROS) and apoptosis in the pathogenesis of infertility in experimental model of varicocele. The protective effect of vitamin E was also examined. Three groups of rats were constructed as the first group had sham operation, experimental varicoceles were established by partial ligation of the left renal vein in later two groups. Third group had received vitamin E. Production of ROS was determined by chemiluminescence assay (CL). The in situ end labelling technique was utilized to investigate apoptosis. Tissue vitamin E levels were measured by high performance liquid chromatography. The differences between luminol enhanced CL levels of groups were not statistically significant. However, the difference between CL levels of lucigenin probe in left testicles of sham and varicocele groups were statistically significant ( p = 0.0007). Similarly, the results of the third group receiving vitamin E significantly differed from the varicocele group ( p = 0.0025). The difference of apoptotic index was also statistically significant between sham and varicocele groups ( p = 0.0038). Although the values of apoptotic index detected in the vitamin E group were lower compared with the varicocele group, the difference was not significant. This study proposes that ROS production and apoptosis in the testicles were induced with experimental varicocele. Vitamin E had a protective role. An increased rate of apoptosis with experimental varicocele suggests a molecular alteration, which may involve ROS overproduction as the triggering mechanism. Consequently, this study indicates an association between varicocele and infertility at molecular level through stimulation of ROS and apoptosis.     

High glucose‐induced excessive reactive oxygen species promote apoptosis through mitochondrial damage in rat cartilage endplate cells

Diabetes mellitus (DM) is an important factor in intervertebral disc degeneration (IDD). Apoptosis of cartilage endplate (CEP) cells is one of the initiators of IDD. However, the effects of high glucose on CEP cells are still unknown. Therefore, we conducted the present study to evaluate the effects of high glucose on CEP cells and to identify the mechanisms of those effects. Rat CEP cells were isolated and cultured in 10% foetal bovine serum (FBS, normal control) or high‐glucose medium (10% FBS + 0.1 M glucose or 10% FBS + 0.2 M glucose, experimental conditions) for 1 or 3 days. In addition, CEP cells were treated with 0.2 M glucose for 3 days in the presence or absence of alpha‐lipoic acid (ALA, 0.15 M). Flow cytometry was performed to identify and quantify the degree of apoptosis. The expression of reactive oxygen species (ROS) was assessed by flow cytometry, and mitochondrial damage (mitochondrial membrane potential) was assessed by fluorescence microscopy. Furthermore, the expression levels of cleaved caspase‐3, cleaved caspase‐9, Bcl‐2, Bax, and cytochrome c were evaluated by Western blotting. High glucose significantly increased apoptosis and ROS accumulation in CEP cells in a dose‐ and time‐dependent manner. Meanwhile, a disrupted mitochondrial membrane potential was detected in rat CEP cells cultured in the two high glucose concentrations. Incubating in high glucose enhanced the expression levels of cleaved caspase‐3, cleaved caspase‐9, Bax, and cytochrome c but decreased the level of the anti‐apoptotic protein Bcl‐2. ALA inhibited the expression of cleaved caspase‐3, cleaved caspase‐9, Bax, and cytochrome c but enhanced the expression of Bcl‐2. ALA also prevented disruption of the mitochondrial membrane potential in CEP cells. This study demonstrates that high glucose‐induced excessive reactive oxygen species promote mitochondrial damage, thus causing apoptosis in rat CEP cells in a dose‐ and time‐dependent manner. ALA could prevent mitochondrial damage and apoptosis caused by high glucose in CEP cells. The results suggest that appropriate blood glucose control may be the key to preventing IDD in diabetic patients. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2476–2483, 2018.

     

Typha capensis (Rohrb.)N.E.Br. (bulrush) extract scavenges free radicals, inhibits collagenase activity and affects human sperm motility and mitochondrial membrane potential in vitro: a pilot study

The biodiversity in South Africa provides more than 30,000 higher plants, of which more than 3000 are used by traditional healers to treat diseases. Typha capensis (bulrush) is one of the medicinal plants used in South Africa to treat male fertility problems. Considering that South African traditional healers have been recognised by Law and the health benefits of T. capensis have not been scientifically investigated yet, this study aimed at investigating the in vitro effects of aqueous extracts from this plant on male reproductive functions. Both leaves and rhizomes of T. capensis were dried, infused with distilled water and freeze-dried. Motile sperm from 50 men were isolated by swim-up and incubated with 1 μg ml(-1) aqueous extract of Typha rhizome for 1 h at 37 °C. Vitality, motility, sperm production of reactive oxygen species and mitochondrial membrane potential were analysed in the test sample, a control and in the pellet from the swim-up. Results showed that the rhizome extract had significant (P < 0.0001) negative effects on all parameters. The extracts from the leaves and rhizomes revealed dose-dependent inhibitory activity for collagenase and free radical formation. No inhibitory activity for elastase was found. The inhibitory activity for collagenase might indicate possible anti-cancer effects.     

The essential role of the mitochondria and reactive oxygen species in Cisplatin-mediated enhancement of fas ligand-induced apoptosis in malignant pleural mesothelioma

Cytotoxic chemotherapeutic drugs such as cisplatin (CDDP) synergistically interact with soluble Fas ligand (sFasL) to mediate profound induction of apoptosis in cancer cells, particularly those refractory to this death-inducing ligand. The goal of this study was to evaluate the roles of the mitochondria-dependent apoptotic cascade and the CDDP-generated reactive oxygen species (ROS) in mediating the supra-additive enhancement of cytotoxicity and apoptosis in combination-treated malignant pleural mesothelioma (MPM) cells. MPM cells were treated with sequential CDDP/sFasL in vitro. Cell viability and apoptosis were determined by MTT and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays. Stable transfectants expressing high levels of Bcl2 were created by retroviral gene transfer. Specific proteolytic activity of caspases 3, 8, and 9 were measured using fluorescent substrates. Pretreating MPM cells with CDDP increased their susceptibility to sFasL by 2- to more than 20-fold. Overexpression of either Bcl-2, the selective caspase 9 inhibitor z-LEHD-fmk, or the antioxidant N-acetylcysteine significantly abrogated combination-induced cytotoxicity and apoptosis. Moreover, the robust activation of caspase 8 in combination-treated cells was completely suppressed by Bcl-2 overexpression, thus implicating a mitochondria-mediated amplification feedback loop. As an in vivo correlate, sequential intraperitoneal administration of CDDP and sFasL significantly inhibited the growth of intraperitoneal MPM human xenografts in nude mice. Our data indicate that the mitochondria-dependent feedback loop of the caspase activation cascade and the generation of ROS are both essential in mediating profound cytotoxicity and apoptosis of MPM cells treated with CDDP and sFasL. This mechanistic study establishes a the translational framework for the clinical application of sequential CDDP/sFasL in the treatment of MPM.     

Aqueous leaf extract of Moringa oleifera reduced intracellular ROS production,DNA fragmentation and acrosome reaction in Human spermatozoa in vitro

The effects of aqueous leaf extract of Moringa oleifera (MO) on human sperm functions and integrity was studied in vitro. Semen was obtained by masturbation after 3–5 days' abstinence from 34 healthy donors in Western Cape, South Africa. Liquefied semen was washed in human tubular fluid supplemented with 1% bovine serum albumin (HTF-BSA;1:5) with 10 min centrifugation at 300 g. Sperm suspensions were subsequently incubated with MO extract (0.625, 6.25, 62.5 and 625 µg/ml) for 1 hr, where HTF-BSA served as control. Sperm motility, vitality, DNA fragmentation, reactive oxygen species production, mitochondrial membrane potential, capacitation and acrosome reaction were assessed. Sperm motility, vitality, mitochondrial membrane potential and capacitation remained unchanged (p > .05). A dose-dependent decrease in sperm reactive oxygen species production (p < .0001), DNA fragmentation (p < .0001) and acrosome reaction (p < .001) was observed. An increase in the percentage of non-capacitated sperm (p < .01) was noted at 625 µg/ml. The antioxidant properties of MO actively maintained basic sperm functions, inhibited excess sperm free superoxide production and preserved acrosome reaction and DNA integrity. Further studies are needed to confirm the effect of aqueous MO leaf extract on fertility potential.     

Mitochondrial permeability transition and cytochrome c release in ischemia-reperfusion injury of the rat liver

BACKGROUND: We investigated whether ischemia-reperfusion causes activation of caspases and whether this activation is related to cytochrome c release from the mitochondria into the cytosol as a result of the mitochondrial inner membrane permeability transition. MATERIALS AND METHODS: Rats were subjected to 30 min to 120 min of hepatic ischemia followed by 6 h of reperfusion. Cyclosporin A or ruthenium red (inhibitors of the mitochondrial inner membrane permeability transition) was given intravenously at 60 and 30 min before ischemia, respectively. RESULTS: Reperfusion after ischemia caused the release of liver enzymes accompanied by mitochondrial membrane depolarization, DNA fragmentation, and translocation of cytochrome c from the mitochondria into the cytosol. Accumulation of cytochrome c in the cytosol and activation of caspase-3-like protease was already detected during ischemia and before reperfusion. Pretreatment with cyclosporin A or ruthenium red significantly ameliorated the loss of the mitochondrial membrane potential, the increase of plasma membrane permeability, the cytosolic accumulation of cytochrome c, DNA fragmentation, and caspase-3-like protease activation. CONCLUSIONS: The mitochondrial inner membrane permeability transition occurs during ischemia and/or after reperfusion, resulting in translocation of cytochrome c and activation of caspases.     

Small interference RNA targeting heat-shock protein 27 inhibits the growth of prostatic cell lines and induces apoptosis via caspase-3 activation in vitro

OBJECTIVES: To evaluate synthetic small interference RNA (siRNA) compounds targeting heat-shock protein 27 (Hsp27) as an alternative approach to Hsp27 'knockdown' in prostate cancer cells, as Hsp27 expression is highly up-regulated in prostate cancer cells after androgen withdrawal or chemotherapy, to become uniformly highly expressed in androgen-independent (AI) prostate cancer. MATERIALS AND METHODS: We recently showed that targeting Hsp27 by a 2'-methoxyethyl modified phosphorothioate antisense oligonucleotide, OGX-427, inhibits Hsp27 expression and enhances hormone- and chemotherapy in prostate cancer xenograft models. In the present study, a 'gene walk' screening different siRNAs was initially used in PC-3 and LNCaP cells to determine the most potent sequence to down-regulate Hsp27 mRNA and protein levels. The effects of Hsp27 silencing on in vitro growth rates were studied by tetrazolium-blue and crystal violet assays. Apoptosis was determined by single-stranded DNA nuclear and cleaved caspase-3 immunostaining, as well as flow cytometry. Spotted microarrays with 14,000 human oligonucleotides were used to examine changes in gene expression. RESULTS: Low concentrations of 1 nm siRNA decreased Hsp27 mRNA levels by 19-fold and suppressed protein expression to undetectable levels. Silencing of Hsp27 in prostate cancer cells by siRNA # 2 increased apoptotic rates 2.4-4 fold and caused 40-76% inhibition of cell growth in LNCaP and PC-3 cells. Characteristic cleavage of caspase-3 occurred after treatment with Hsp27 siRNA (1 nm). cDNA microarray analysis from LNCaP and PC-3 cell lines revealed differential gene expression profiles after Hsp27 down-regulation that could be used to identify various survival pathways involved in androgen-dependent and AI growth. CONCLUSIONS: These findings illustrate the potential utility of Hsp27-silencing therapy and highlight Hsp27 siRNA strategies as a novel and highly effective tool, with the potential for future targeted therapy in enhancing the efficacy of chemotherapy in advanced prostate cancer.     

Hyperbaric oxygen treatment prevents nitric oxide‐induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70

Heat shock proteins (HSPs), inflammatory cytokines, nitric oxide (NO), and localized hypoxia‐induced apoptosis are thought to be correlated to the degree of cartilage injury. We investigated the effect of hyperbaric oxygen (HBO) on (1) interleukin‐1β (IL‐1β)‐induced NO production and apoptosis of rabbit chondrocytes and (2) healing of articular cartilage defects. For the in vitro study, RT‐PCR and Western blotting were performed to detect mRNA and protein expressions of HSP70, inducible NO synthase (iNOS), and caspase 3 in IL‐1β‐treated chondrocytes. To clarify that the HSP70 was necessary for anti‐iNOS and anti‐apoptotic activity by HBO, we treated the cells with an HSP70 inhibitor, KNK437. For the in vivo study, cartilage defects were created in rabbits. The HBO group was exposed to 100% oxygen at 2.5 ATA for 1.5 h a day for 10 weeks. The control group was exposed to normal air. After sacrifice, specimen sections were sent for examination using a scoring system. Immunohistochemical analyses were performed to detect the expressions of iNOS, HSP70, and caspase 3. Our results suggested that HBO upregulated the mRNA and protein expressions of HSP70 and suppressed those of iNOS and caspase 3 in chondrocytes. KNK437 inhibited the HBO‐induced downregulation of iNOS and casapase 3 activities. The histological scores showed that HBO markedly enhanced cartilage repair. Immunohistostaining showed that HBO enhanced HSP70 expression and suppressed iNOS and caspase 3 expressions in chondrocytes. Accordingly, HBO treatment prevents NO‐induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 376–384, 2013     

Reactive oxygen species mediate detrusor overactivity via sensitization of afferent pathway in the bladder of anaesthetized rats

OBJECTIVES

To investigate the effects of reactive oxygen species (ROS) on the micturition reflex in vivo, especially in bladder afferent signalling in rats, as several pathophysiological conditions in the urinary bladder (e.g. ischaemia/reperfusion and inflammation) are characterized by the formation of ROS.

MATERIALS AND METHODS

Adult female Sprague‐Dawley rats under urethane anaesthesia (normal or pretreated with 125 mg/kg capsaicin, subcutaneously, 4 days before) were assessed by continuous cystometrography (CMG) with or without the intravesical administration of H₂O₂ (0.003–3%) to stimulate ROS damage. To investigate the mechanism of H₂O₂, catalase (a H₂O₂ scavenger) was applied intravesically (2000 IU/mL), or rats were given intravenous injections with superoxide dismutase (SOD, 20 000 IU/kg, a superoxide anion scavenger), dimethylthiourea (DMTU, 100 mg/kg, a hydroxyl radical scavenger), deferoxamine (20 mg/kg, an iron‐chelator that prevents the formation of hydroxyl radical), indomethacin (3 mg/kg, a cyclooxygenase inhibitor) or ketoprofen (1 mg/kg, a cyclooxygenase inhibitor) just before or during the intravesical administration of H₂O₂. Prostaglandin (PG) levels (PGE₂ and 6‐keto‐PGF_1α) were measured in the bladder of rats treated with intravesical 0.3% H₂O₂ for 30 min with or without indomethacin.

RESULTS

Intravesical administration of H₂O₂ induced detrusor overactivity, as shown by a reduction in the mean (sem ) intercontraction interval (ICI), in a dose‐dependent manner in normal rats (0.3% H₂O_2, P < 0.01, 36.2 (4.7)% of the control ICI). H₂O₂‐induced detrusor overactivity was almost abolished by catalase and significantly suppressed by DMTU, deferoxamine, capsaicin pretreatment, indomethacin or ketoprofen but not by SOD. The level of PGs was significantly increased by H₂O₂ instillation, and indomethacin significantly inhibited the increase in PGs.

CONCLUSION

These results indicate that oxidative stress induced by H₂O₂ activates capsaicin‐sensitive C‐fibre afferent pathways, at least in part, mediated via stimulation of the cyclooxygenase pathway, thereby inducing detrusor overactivity. Thus, rats treated with intravesical H₂O₂ appear to be a suitable model for the study of detrusor overactivity.     

长时受精过程中活性氧及精子的DNA损伤对体外受精结局的影响

目的通过检测精液处理后不同时间的精子DNA损伤程度与活性氧的变化,分析对比长时受精与短时受精两种精卵孵育时间的实验室参数及临床结局,旨在探讨长时受精对体外受精-胚胎移植治疗中的不利影响.方法接受常规IVF受精的夫妇146例,共取卵146个周期,移植113个周期.在取卵当日采集精液,经处理后将精子浓度调整为10×106/ml,留取标本3份,各1.0ml.第一份标本定为加精后0h(即在CO2培养箱内培养至该患者卵子加精时检测),第二份标本定为加精后5h(即在CO2培养箱内培养至该患者卵子加精后5h后检测),第三份标本定为加精后20h(即在CO2培养箱内培养至该患者卵子加精后20h后检测),每份标本均用于检测H2O2、CTA和精子DNA损伤.精子DNA损伤采用精子吖啶橙染色方法测定,H2O2及CTA含量测定采用分光光度比色测定法.在胚胎移植日,除外取消移植的患者33名,其余的113名患者随机分成两组,A组57人选择短时受精的胚胎进行移植,B组56人选择长时受精的胚胎进行移植.结果精子DNA损伤、H2O2水平在加精后20h均明显高于授精时及授精后5h(P<0.05),CTA水平明显低于授精时及授精后5h(P<0.05).精子DNA损伤率在授精后三个时间均与H2O2浓度呈正相关(r值分别为0.688、0.532和0.491, P<0.05);精子DNA损伤率与授精后20h的CTA浓度呈负相关(r=-0.347, P<0.05).B组的多精受精率明显高于A组(P<0.05),A组的优胚率明显高于B组(P<0.05),两组间的受精率、卵裂率、植入率、临床妊娠率无统计学差异.结论长时受精时,精子可以产生过量的活性氧并且导致DNA损伤的精子增多,对卵子及胚胎有不利影响.短时受精在不影响受精率及卵裂率的前提下,可以降低多精受精率,提高优胚率.