Analysis of the nitric oxide-cyclic guanosine monophosphate pathway in experimental liver cirrhosis suggests phosphodiesterase-5 as potential target to treat portal hypertension

AIM To investigate the potential effect of inhibitors of phosphodiesterase-5(PDE-5) for therapy of portal hypertension in liver cirrhosis.METHODS In the rat model of thioacetamide-induced liver fibrosis/cirrhosis the nitric oxide-cyclic guanosine monophosphate(NO-cGMP) pathway was investigated. Expression and localization of PDE-5, the enzyme that converts vasodilating cGMP into inactive 5'-GMP, was in the focus of the study. Hepatic gene expression of key components of the NO-cGMP pathway was determined by qRT-PCR: Endothelial NO synthase(eNOS), inducible NO synthase(iNOS), soluble guanylate cyclase subunits α1 and β1(sGCa1, sGCb1), and PDE-5. Hepatic PDE-5 protein expression and localization were detected by immunohistochemistry. Serum cGMP concentrations were measured using ELISA. Acute effects of the PDE-5 inhibitor Sildenafil(0.1 mg/kg or 1.0 mg/kg) on portal and systemic hemodynamics were investigated using pressure transducers.RESULTS Hepatic gene expression of eNOS(2.2-fold; P = 0.003), sGCa1(1.7-fold; P = 0.003), sGCb1(3.0-fold; P = 0.003), and PDE-5(11-fold; P = 0.003) was increased in cirrhotic livers compared to healthy livers. Overexpression of PDE-5(7.7-fold; P = 0.006) was less pronounced in fibrotic livers. iNOS expression was only detected in fibrotic and cirrhotic livers. In healthy liver, PDE-5 protein was localized primarily in zone 3 hepatocytes and to a lesser extent in perisinusoidal cells. This zonation was disturbed in cirrhosis: PDE-5 protein expression in perisinusoidal cells was induced approximately 8-fold. In addition, PDE-5-expressing cells were also found in fibrous septa. Serum cGMP concentrations were reduced in rats with cirrhotic livers by approximately 40%. Inhibition of PDE-5 by Sildenafil caused a significant increase in serum cGMP concentrations [+ 64% in healthy rats(P = 0.024), + 85% in cirrhotic rats(P = 0.018)]. Concomitantly, the portal venous pressure was reduced by 19% in rats with liver cirrhosis. CONCLUSION Overexpression and abrogated zonation of PDE-5 likely contribute to the pathogenesis of cirrhotic portal hypertension. PDE-5 inhibition may therefore be a reasonable therapeutic approach for portal hypertension.

Studies on Relationship between Serum Nitric Oxide and Plasma Cyclic Guanosine Monophosphate and Prolonged Bleeding after Medical Abortion as well as Prophylaxis and Treatment of Bleeding with Traditional Chinese Medicine

Objectives To study the relationship between serum nitric oxide (NO and plasma cyclic guanosine monophosphate (cGMP) and prolonged bleeding after medical abortion. Methods A total of 120 women having received medical abortions at random were recruited and divided into two groups: the one (Group A,n=60) taking 'Gong Fu Mixture(Uterus Recovering Mixture)' and the other (Group B,n=60) not taking it after abortion. On d 10, 20 and 30 after medical abortion, serum NO and plasma cGMP were tested before and after mifepristone administration and 10 d later by Gresis reaction method and radioimmunoassay respectively. Results NO concentration in serum and cGMP concentration in plasma decreased significantly after taking mifepristone given (P<0.05). Ten days later, the number of those with bleeding discontinuation in the group A was significantly greater than that in the group B (P<0.05). Serum NO level and plasma cGMP level in the group A decreased more significantly than those in the group B (P<0.05). Conclusion The slow decrease of serum NO and plasma cGMP is closely related to prolonged bleeding after medical abortion. “Gong Fu Mixture (uterus recovering mixture)” is effective in prevention and treatment of prolonged bleeding.

Effects of aminoguanidine on nitric oxide production induced by inflammatory cytokines and endotoxin in cultured rat hepatocytes

AIM: To study the effects of aminoguanidine (AG) and two L-arginine analogues N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(omega)-nitro-L-arginine (L-NNA) on nitric oxide (NO) production induced by cytokines (TNF-alpha, IL-1 beta, and IFN-gamma) and bacterial lipopolysaccharide (LPS) mixture (CM) in the cultured rat hepatocytes, and examine their mechanisms action. METHODS: Rat hepatocytes were incubated with AG, L-NAME, L-NNA, Actinomycin D (ActD) and dexamethasone in a medium containing CM (LPS plus TNF-alpha, IL-1 beta, and IFN-gamma) for 24h. NO production in the cultured supernatant was measured with the Griess reaction. Intracellular cGMP level was detected with radioimmunoassy. RESULTS: NO production was markedly blocked by AG and L-NAME in a dose-dependent manner under inflammatory stimuli condition triggered by CM in vitro. The rate of the maximum inhibitory effects of L-NAME (38.9%) was less potent than that obtained with AG(53.7%, P 【 0.05). There was no significant difference between the inhibitory effects of AG and two L-arginine analogues on intracellular cGMP accumulation in rat cultured hepatocytes. Non-specific NOS expression inhibitor dexamethasone (DEX)and iNOS mRNA transcriptional inhibitor ActD also significantly inhibited CM-induced NO production. AG(0.1 mmol x L(-1)) and ActD (0.2 ng x L(-1)) were equipotent in decreasing NO production induced by inflammatory stimuli in vitro, and both effects were more potent than that induced by non-selectivity NOS activity inhibitor L-NAME (0.1 mmol x L(-1)) under similar stimuli conditions (P【0.01). CONCLUSION: AG is a potent selective inhibitor of inducible isoform of NOS,and the mechanism of action may be not only competitive inhibition in the substrate level, but also the gene expression level in rat hepatocytes.

Analytical Solutions of Nonlinear Differential Equations in the Mathematical Model for Inactivation of Nitric Oxide by Rat Cerebellar Slices

A mathematical model for the inactivation of nitric oxide by rat cerebellar slices under non-steady state condition has been analyzed. This diffusion-inactivation model was used to estimate the kinetics of NO consumption by the rat cerebellar slices. He’s Homotopy perturbation method is used to solve the first order nonlinear differential equations which describe the concentrations given by net of diffusion and inactivation by the slices. Analytical expressions for the concentration of nitric oxide have been derived for all values of parameters. The obtained analytical results are compared with the simulation results (Matlab/Scilab program) and are found to be in good agreement.

Nitric oxide promotes survival of cerebellar granule neurons cultured in vitro through the Akt pathway

In this study, cerebellar granule neurons were used to examine the role of nitric oxide on cell survival. The N-methyI-D-aspartic acid receptor antagonist, MK-801, and the soluble guanylate cyclase antagonist, 1H-[1, 2, 4]oxadiazolo-[4, 3-a] quinoxalin-1 -one, decreased cell viability, induced caspase-3, and decreased phosphorylated-Akt levels, suggesting that blockade of nitric oxide production promotes apoptosis of differentiating cerebellar granule neurons. After administration of sodium nitroprusside, an endogenous nitric oxide donor, cell viability recovered, caspase-3 expression was decreased, and phosphorylated-Akt levels increased. This study provides direct evidence that nitric oxide can sustain the survival of developing cerebellar granule neurons in vitro through the nitric oxide-Akt pathway. Moreover, endogenous nitric oxide exerts these effects in a cyclic guanosine monophosphate-dependent manner while exogenous nitric oxide does so in a cyclic guanosine monophosphate-independent manner.

Changes of plasma nitric oxide in patients with pregnancy induced hypertension

Objective: To explore the role of nitric oxide (NO), an internal vasodilative factor, in occurrence ofpregnancy induced hypertension (PIH). Methods: The antepartum and postpartum levels of NO2-/NO3-, the stable metabolic end product of NO, and those of cyclic guanosine monophosphate (cGMP) in 30 patients with PIHand 30 healthy women in their late pregnancy were measured with greiss reagent. ResultS: ① The plasma levels ofNO2-/NO3- and cGMP were significantly decreased in patients with PIH as compared with the healthy women (P<0. 05). ②In patients with PlH, the antepartum level of NO2-/NO3- was markedly lower than the postpartum one(P<O. ol ). ③There was a negative correlation between the level of plasma NO2- /NO3- and systolic blood pressurein patients with PIH (P<0. 01). ④ A positive correlation was found between the level of plasma NO2-/NO3- andthat of cGMP in patients with PIH (P<0. 01). Conclusion: The decrease of NO synthesis may play an importantrole in occurrence of PIH.

L-Arginine Supplementation and Nitric Oxide Production:No Additional Effect When Associated to Exercise

L-arginine is an amino acid semiessencial considered a precursor of nitric oxide, a gas mainly produced in endothelial cells. Nutritional supplements based on the amino acid L-arginine have been broadly marketed in order to increase vasodilation and the blood supply to muscle in order to optimize metabolic responses induced by exercise. The main objective of this study was to evaluate the effect of L-arginine supplementation on nitric oxide production in response to exercise. Furthermore, the biochemical parameters of muscle fatigue were assessed. Fourteen trained runners were divided in two groups, supplemented with L-arginine (ARG) and placebo (PLA). Blood samples were collected before supplementation (T0), immediately after the first exercise session (T1), immediately after the second exercise session (T2), and after 20 minutes of rest (T3). Plasma cyclic guanosine monophosphate was assessed as a marker of nitric oxide production. The biochemical parameters of muscle fatigue analyzed were plasma lactate and ammonia. There was significant increase in plasma cyclic guanosine monophosphate in both groups in response to exercise: ARG (T0: 3.6 ± 1.4;T1: 17.9 ± 5.8;T2: 15.9 ± 5.3;T3: 7.3 ± 2.5 pmol/mL) and PLA (T0: 4.1 ± 1.1;T1: 18.8 ± 9.9;T2: 16.1 ± 3.5;T3: 9.3 ± 3.7 pmol/mL). A significant reduction in plasma lactate and ammonia were observed in the recovery period after exercise (T3). However, no significant difference was observed between groups in the variables studied. Therefore, L-arginine supplementation was unable to increase the effects of exercise on nitric oxide production and did not improve the metabolic responses to exercise in runners.

The mechanistic study on Wa medicine Niang-Mu-Liang medicinal liquor mitigating diabetes mellitus erectile dysfunction in rats by inhibiting ferroptosis

Background:This study aims to investigate the therapeutic effect of Wa medicine Niang-Mu-Liang medicinal liquor(NML)on rats with diabetes mellitus erectile dysfunction(DMED)and its impact on the ferroptosis signaling pathway.Methods:Thirty Sprague-Dawley rats were randomly divided into three groups:Control,DMED,and NML.After establishing the DMED model,treatments were administered for 8 weeks.After the administration,apomorphine hydrochloride tests were conducted to measure the mass and organ index of testes and epididymides,sperm concentration and viability in each group.Penile corpus cavernosum tissues were stained with hematoxylin and eosin.Nitric oxide and cyclic guanosine monophosphate levels in the penile corpus cavernosum tissues were determined using biochemical kits and enzyme-linked immunosorbent assay,while the expression of proteins related to the ferroptosis signaling pathway was measured by Western blot.Results:Compared to the DMED group,the DMED rats treated with NML showed significantly increased erection frequency,testicular and epididymal mass and index,sperm count and viability,along with noticeable improvement in the pathological morphology of penile corpus cavernosum.The content of nitric oxide and cyclic guanosine monophosphate,and the expression of ferritin heavy chain,ferritin light chain,and glutathione peroxidase 4 proteins in penile corpus cavernosum tissue were elevated,while the expression of transferrin and STEAP3 proteins was reduced.Conclusion:NML can improve erectile function in DMED rats by inhibiting the ferroptosis signaling pathway.

Cell-specific expression and immunolocalization of nitric oxide synthase isoforms and the related nitric oxide/cyclic GMP signaling pathway in the ovaries of neonatal and immature rats

Objective: The present study is designed to investigate the cellular expressions and immunolocalizations of three different nitric oxide synthase (NOS) isoforms and the related nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway in the ovaries of neonatal and immature rats.Methods: The ovaries were obtained from ICR (Institute for Cancer Research) female Sprague-Dawley rats at postnatal days 1,5,7,10,and 19.Then we carried out the histologic examination,immunohistochemistry,measurement of NOS activity,and modifications within the NO/cGMP pathway.Results: During postnatal days 1,5,7,10,and 19,all three isoforms of NOS were mainly localized to the oocytes and expressed as a gradual increase in granulosa cells and theca cells within the growing follicle.The ovarian total NOS activities and NO levels were increased at postnatal days 7 and 10 compared with other days.Conclusions: Our findings suggest that the locally produced NO and the NO/NOS signaling systems are involved in the follicular development to puberty.

Role of nitric oxide during early phase myocardial ischemic preconditioning in rats

Background To date, there have been no reports on altered nitric oxide （NO） content in ischemia/reperfusion with regard to in vivo preconditioning procedures. These studies are important for understanding the mechanisms of NO during early myocardial ischemic preconditioning. The aim of the present study was to investigate the mechanisms of NO during early myocardial ischemic preconditioning by measuring levels of NO and cyclic guanosine monophosphate （cGMP）, as well as activity of nitric oxide synthase （NOS） in ischemia/reperfusion with respect to preconditioning in rats. Methods Sixty-six female Sprague-Dawley rats were randomly divided into four groups： ischemic preconditioning group （IP）, ischemia/reperfusion group （I/R）, control group （CON）, and preconditioning procedure group （PC）. In the PC group, rats were further divided into PC1-, PC1＋, PC2-, PC2＋, PC3-, and PC3＋ subgroups. Rats underwent left coronary artery occlusion and reperfusion, and subsequently, NOS activity and levels were assessed with spectrophotometric analysis. cGMP contents were measured with radioimmunoassay. Results The level of NO and cGMP, as well as the activity of NOS, were significantly higher in the IP group compared to the I/R and CON groups （P 〈0.05）. During preconditioning prior to prolonged ischemia, NO and cGMP levels varied markedly with ischemia and reperfusion. The levels of NO repeatedly increased when the heart was exposed to three episodes of 5-minute ischemia, and were almost completely reversed during each reperfusion period. NO and cGMP levels were significantly different between the 5-minute period of ischemia and the same period of reperfusion during preconditioning. Conclusions NO plays an important role during early phase myocardial ischemic preconditioning in rats. NO and cGMP could be triggers and mediators of early phase myocardial ischemic preconditioning. Altered NOS activity following ischemic stress could be the primary inducer of higher NO levels detected. NO and cGMP fluctuations might be the trigger for protection during early phase myocardial ischemic preconditioning.

鸟苷酸环化酶激活剂维利西呱在心力衰竭治疗中的研究进展

随着对心力衰竭(心衰)发病机制及治疗方法的研究不断深入,近年来许多新型药物取代以往“强心、利尿、扩血管”的心衰传统治疗理念,采用神经内分泌调控为理论基础的治疗药物。维利西呱是一种新型的可溶性鸟苷酸环化酶激活剂,它也可以通过一氧化氮(NO)-可溶性鸟苷酸环化酶(sGC)-环磷鸟嘌呤核苷(cGMP)通路,最终产生增加心肌细胞内cGMP浓度的效应,从而发挥治疗慢性心衰的功效,同时维利西呱对于心衰合并症例如慢性肾功能不全(CKD)的治疗也有所帮助。目前维利西呱正逐渐成为治疗心衰的热点药物。本文对于维利西呱的相关研究进展及其作用机制和临床应用展望进行综述。

大鼠脑缺血再灌流脑区一氧化氮变化的研究

目的研究大鼠脑缺血及再灌流后脑部一氧化氮的变化。方法采用荧光法和放射免疫法测定４个脑区一氧化氮（ＮＯ）代谢产物ＮＯ２和环磷酸鸟苷（ｃＧＭＰ）。结果脑缺血１０ｍｉｎ，各脑区ＮＯ２和ｃＧＭＰ含量明显增高；脑缺血３０ｍｉｎ，各脑区ＮＯ２和ｃＧＭＰ含量开始下降。缺血１０ｍｉｎ再灌流１５ｍｉｎ以及缺血３０ｍｉｎ再灌流１５ｍｉｎ，各脑区ＮＯ２和ｃＧＭＰ含量再次增加，与单纯脑缺血组相比，有显著差异性（Ｐ＜０．０５或Ｐ＜０．０１）。

氯胺酮对大鼠吗啡戒断症状的影响及其作用机理

目的 研究氯胺酮对大鼠吗啡戒断症状的影响及其可能的机理。方法 建立大鼠吗啡依赖模型 ,在用纳洛酮催瘾前 2min给予不同剂量的氯胺酮 ,观察其戒断症状的改变 ;用分光光度法测定戒断时大鼠一氧化氮 (NO)含量、一氧化氮合酶 (NOS)活性 ,用放射免疫法测定环鸟苷酸 (cGMP)含量。结果 3个剂量的氯胺酮 (5、10和 2 0mg·kg- 1)均可缓解吗啡戒断时探究、扭体、湿狗样抖动、跳跃等运动反应 ,减少活动次数 ,抑制植物神经系统症状。10、2 0mg·kg- 1氯胺酮可显著减轻吗啡戒断所致的体重下降 ,小剂量氯胺酮 (5mg·kg- 1)可抑制吗啡依赖大鼠前额叶皮质、小脑的NOS活性和NO、cGMP含量的增高。结论 氯胺酮可缓解大鼠吗啡戒断症状 ,其作用机理可能与减弱大鼠吗啡戒断时NMDA NO cGMP通路效应有关。

γ-羟基丁酸受体在大鼠局灶性脑缺血再灌注损伤中的作用

研究γ-羟基丁酸(gamma-hydroxybutyric acid,GHB)受体在大鼠局灶性脑缺血再灌注损伤中的作用及其机制。选用GHB受体选择性激动剂NCS-356和特异性拮抗剂NCS-382作为工具药,采用改良的Longa法制备大鼠大脑中动脉栓塞(MCAO)模型。缺血2 h再灌注2 h后,动物进行Longa法行为功能评分;再灌注24 h后,部分动物用TTC染色法测定大鼠脑梗死体积;部分动物应用流式细胞仪测定神经细胞内游离钙离子浓度;分光光度法测定缺血侧大脑皮质中总一氧化氮合酶(tNOS)、诱导型一氧化氮合酶(iNOS)活性和一氧化氮(NO)含量;放射免疫法测定大鼠缺血侧大脑皮层环磷酸鸟苷(cGMP)含量。Isc/R组大鼠行为功能评分、脑梗死体积、神经细胞内游离Ca2+浓度、cGMP和NO含量、tNOS及iNOS活性均显著高于假手术组;NCS-356 160μg.kg-1(N1)、NCS-356 320μg.kg-1(N2)、NCS-356 640μg.kg-1(N3)和尼莫地平600μg.kg-1(Nim)组的上述各指标均不同程度地低于Isc/R组,而NCS-382 640μg.kg-1+NCS-356 640μg.kg-1(NCS-382+N3)组则能显著对抗N3组的作用。激动GHB受体对大鼠局灶性脑缺血再灌注损伤具有一定的保护作用,其作用机制可能与降低神经细胞内游离Ca2+浓度,减少NO及cGMP含量有关。

异丙酚对大鼠脑NO/cGMP信号转导系统的影响

目的 :了解异丙酚对大鼠脑一氧化氮 (NO) /环鸟苷酸 (cGMP)信号转导系统的影响。方法 :32只SD大鼠 ,随机分为对照组和异丙酚组 ,分别腹腔注射 (ip)生理盐水 10ml/kg或异丙酚 10 0mg/kg。用分光光度法测定脑组织一氧化氮合酶 (NOS)活性和NO产量 ,放射免疫法测定脑组织cGMP含量。结果 :与对照组比较 ,大鼠ip异丙酚 10 0mg/kg不但明显抑制小脑、海马和大脑皮层的NOS活性 ,而且显著减少上述脑区NO产量和cGMP含量 (P <0 0 5或P <0 0 1)。结论

脂多糖对大鼠血清及内脏组织一氧化氮产物水平的影响

作者观察了LPS(脂多糖)对大鼠血清及内脏组织一氧化氮(NO)代谢产物NO_(2-)/NO_(3-)水平的影响。结果表明给于LPS后,血清 NO_2^-/NO_3^-水平急剧升高,24h达峰值,为对照组的20倍。心、肺组织的NO_2^-/NO_3^-含量也显著增加;但肝脏和肾脏组织NO_2^-/NO_3^-仅在短时期内增加。同时血清和内脏组织cGMP水平明显升高。给于NO合成酶(NOS)抑制剂L-NMMA,能降低血清和心、肺组织NO_2^-/NO_3^-升高幅度,并能减轻组织水肿程度。提示LPS能通过诱导NOS活性而增加体内NO水平,过多的NO以及cGMP可能是内毒素性组织的重要因素之一。

支气管哮喘与一氧化氮及cGMP关系的初步研究

研究目的小儿哮喘时一氧化氮的作用机制及临床意义。研究方法哮喘患儿３３例，正常健康儿童２０例为对照组。测定一氧化氮（ＮＯ）水平及ｃＧＭＰ水平，并对测定结果进行统计学分析。研究结果所有哮喘患儿血浆ＮＯ及ｃＧＭＰ水平在治疗前均明显高于正常儿童（Ｐ均＜０．００１），其中９例重度哮喘患儿血浆ＮＯ水平较２４例轻中度哮喘患儿更高（Ｐ＜０．００１）；治疗后血浆ＮＯ及ｃＧＭＰ水平均恢复正常。结论哮喘发作时一氧化氮产生过多，发挥其细胞及组织毒性作用，使肺上皮细胞等损伤，从而引起和加重哮喘发病，且血浆ＮＯ水平与哮喘病情密切相关。

杏仁核中一氧化氮对睡眠-觉醒的影响

目的研究杏仁核中一氧化氮（ＮＯ）对大鼠睡眠觉醒的影响，并分析其作用机制。方法多导睡眠描记和杏仁核微量注射。结果一氧化氮合酶抑制剂Ｌ硝基精氨酸（ＬＮＮＡ）可增加慢波睡眠（ＳＷＳ）和减少觉醒（Ｗ），而一氧化氮（ＮＯ）供体硝普钠（ＳＮＰ）可增加Ｗ、减少ＳＷＳ，并可对抗ＬＮＮＡ的促睡眠效应；ＮＯ前体Ｌ精氨酸（ＬＡｒｇ）对睡眠觉醒无直接影响，但可对抗ＬＮＮＡ的促睡眠效应。环磷酸鸟苷（ｃＧＭＰ）具有明显的增加Ｗ和减少ＳＷＳ效应，而鸟苷酸环化酶抑制剂亚甲蓝（ＭＢ）增加睡眠、减少觉醒，并可阻断ＳＮＰ的促睡眠效应。结论杏仁核参与睡眠觉醒调节，杏仁核中ＮＯ具有促进Ｗ、抑制ＳＷＳ效应，这一作用是通过激活鸟苷酸环化酶使ｃＧＭＰ增多实现的。

急性一氧化碳中毒对大鼠脑循环的影响

目的 探讨急性一氧化碳中毒后脑内血液流变学的变化 ,以进一步弄清迟发性脑病的发病机制 ,为解决其有效防治提供新的线索。方法 体重 2 40～ 2 80g雄性SD大鼠腹腔间断注射染毒 ,分别于染毒后 1、 3、 7、 14、 2 1d颈静脉取血监测血液流变学特性 ,同时检测血浆Fib、ET 1、MDA、NO及cGMP含量。结果 染毒后脑循环血液流变学特性发生明显改变 ,粘度增加 ,且见血管内皮严重受损 ,缩血管物质增加 ,NO cGMP系统受到抑制。结论 急性一氧化碳中毒对大鼠脑循环存在持续性影响 。

外源性一氧化碳对大鼠脑内源性气体信使系统的影响

目的 探讨外源性一氧化碳对脑内气体信使系统的影响 ,为迟发性脑病机制的研究提供新思路。方法 体重 2 40～ 2 80g雄性SD大鼠腹腔单次注射染毒 ,分别于染毒前及染毒后 0 5、 1、 2、 4、 8h ,颈静脉取血测定血浆NO及cGMP含量 ;取双侧脑海马组织 ,测定HO、NOS活性和cGMP含量。结果 染毒后脑循环中NO cGMP通路一过性上调 ,随后抑制 ;脑海马组织中HO、NOS活性下降和cGMP含量降低。结论 急性一氧化碳中毒可引起体内气体信使系统功能失衡 。