Carbon monoxide decreases the level of iNOS protein and active dimer in IL-1β-stimulated hepatocytes

There is evidence that NO can regulate CO production, however less is known about CO regulation of NO synthesis. Our studies were undertaken to define how CO regulates iNOS in cultured hepatocytes. CO (250 ppm) exposure resulted in a significant decrease in iNOS protein, nitrite production, level of active iNOS dimer and cytosolic iNOS activity in cells stimulated with cytokines (IL-1β) or transfected with the human iNOS gene. However, IL-1β-stimulated iNOS mRNA expression was unaffected by CO. These effects of CO on iNOS protein levels were inhibited when CO was scavenged using hemoglobin. HO-1 induction with an adenoviral vector carrying HO-1 showed a decrease in total iNOS protein, nitrite production, and iNOS dimer level from cells stimulated by IL-1β. iNOS protein level was significantly higher in lung endothelial cells isolated from HO-1 knockout mice compared to wild type cultures stimulated with cytokines mixture. CO was found to increase p38 phosphorylation and p38 inhibition using SB203580 increased iNOS protein levels in response to IL-1β. Interestingly, proteasome inhibitors (MG132 and Lactacystin) and an autophagy inhibitor (3-methyladenine) reversed CO influence iNOS levels. Our results imply that CO exposure decreases NO production by suppressing dimer formation and increasing iNOS degradation through a process involving p38 activation.     

Increased HO-1 expression and decreased iNOS expression in the hippocampus from adult spontaneously hypertensive rats

Brain expression of heme oxygenase (HO) and nitric oxide synthase (NOS) in hypertension may participate in the pathogenesis of hypertension-related neuronal disorders, such as vascular dementia. In the present study, expression levels of HO and NOS in spontaneously hypertensive rats (SHR) were investigated using Western immunoblotting assay. Expression level of inducible HO-1 in hippocampus of 4-wk prehypertensive SHR was about twofold of that in age-matched Sprague-Dawley (SD) rats (p<0.01). In 23-wk SHR with fully developed hypertension, hippocampal HO-1 level was significantly greater than that of age-matched SD rats (p<0.05), but not different from 4-wk SHR. There was no difference in expression levels of hippocampal HO-2 between SHR and SD rats at different ages. Total enzymatic activity of hippocampal HO was significantly greater in 23-wk SHR than in age-matched SD rats or 4-wk SD/SHR (p<0.01). Although hippocampal expression of nNOS protein was relatively unchanged, iNOS expression in 23-wk SHR was about fourfold lower than that in age-matched SD rats and 4-wk SD/SHR (p<0.01). Total enzymatic activity of hippocampal NOS was significantly lower in 23-wk SHR than in age-matched SD rats or 4-wk SD/SHR (p<0.01). Significantly suppressed Morris water maze performance was found in 23-wk SHR in comparison with age-matched SD rats. Because SHR has been used as a model of vascular dementia and hippocampus is essential for spatial learning and memory, understanding of altered HO/CO and NOS/NO systems in the hippocampus of adult SHR may shed light on the pathogenic development of memory deficits associated with vascular dementia.     

醒脑静联合古拉定对急性酒精中毒患者血清H2O2、SOD、MDA及iNOS水平的影响

目的:探讨醒脑静联合古拉定治疗急性酒精中毒的疗效及对患者血清过氧化氢(H_2O_2)、超氧化物歧化酶(SOD)、丙二醛(MDA)和诱导型一氧化氮合酶(i NOS)水平的影响。方法:选取我院2015年4月至2017年4月收治的80例急性酒精中毒患者,采用随机数字表法分为两组。对照组40例,采用醒脑静治疗;观察组40例,采用醒脑静联合古拉定治疗。记录比较两组症状改善时间,治疗前后肝功能及血清学指标变化,临床疗效与不良反应的发生情况。结果:观察组血压与心率恢复正常及清醒时间均较对照组显著缩短(P0.01)。与治疗前相比,两组治疗后血清谷酰转肽酶(GGT)、谷草转氨酶(AST)和谷丙转氨酶(ALT)水平均显著下降(P0.01),且观察组以上指标均显著低于对照组(P0.01)。治疗4、8 h后,两组血清H_2O_2、MDA、iNOS水平均较治疗前显著下降(P0.01),血清SOD水平较治疗前显著升高(P0.01),且观察组以上指标改善较对照组更显著(P0.01)。观察组治疗后总有效率达97.50%,较对照组(90.00%)略高,但差异无统计学意义(P0.05)。观察组苏醒后不适率为27.50%,较对照组显著降低(67.50%,P0.01)。结论:采用醒脑静及古拉定治疗急性酒精中毒患者可更快速、有效消除临床症状,改善肝功能,减少患者不适感,疗效更显著,可能与其有效降低血清MDA、i NOS水平并升高血清SOD水平有关。     

Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression

In order to elucidate the mechanism of anti-inflammatory effect of 1-o-acetylbritannilatone (ABL) isolated from Inula Britannica-F, we investigated ABL for its ability to inhibit the inflammatory factor production in RAW 264.7 macrophages. The studies showed that ABL not only inhibited LPS/IFN-gamma-mediated nitric oxide (NO) production and inducible nitric synthase (iNOS) expression, but also decreased LPS/IFN-gamma-induced prostaglandin E2 (PGE2) production and cyclo-oxygenase-2 (COX-2) expression in a concentration-dependent manner. EMSA demonstrated that ABL inhibited effectively the association of NF-kappaB, which is necessary for the expression of iNOS and COX-2, with its binding motif in the promoter of target genes. These data suggest that ABL suppress NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression, respectively. The anti-inflammatory effect of ABL involves blocking the binding of NF-kappaB to the promoter in the target genes and inhibiting the expression of iNOS and COX-2.     

Synthesis and biological evaluation of 4,5-dihydro-1H-pyrazole derivatives as potential nNOS/iNOS selective inhibitors. Part 2: Influence of diverse substituents in both the phenyl moiety and the acyl group

In a preliminary article, we reported a series of 4,5-dihydro-1H-pyrazole derivatives as neuronal nitric oxide synthase (nNOS) inhibitors. Here we present the data about the inhibition of inducible nitric oxide synthase (iNOS) of these compounds. In general, we can confirm that these pyrazoles are nNOS selective inhibitors. In addition, taking these compounds as a reference, we have designed and synthesized a series of new derivatives by modification of the heterocycle in 1-position, and by introduction of electron-donating or electron-withdrawing substituents in the aromatic ring. These derivatives have been evaluated as nNOS and iNOS inhibitors in order to identify new compounds with improved activity and selectivity. Compound 3r, with three methoxy electron-donating groups in the phenyl moiety, is the most potent nNOS inhibitor, showing good selectivity nNOS/iNOS.     

Antiarrhythmic effect of lithium in rats after myocardial infarction by activation of Nrf2/HO-1 signaling

Glycogen synthase kinase-3 (GSK-3) signaling has been shown to play a role in the regulation of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of antioxidant genes, including heme oxygenase-1 (HO-1). We assessed whether lithium, a GSK-3 inhibitor, attenuates cardiac sympathetic reinnervation after myocardial infarction, a status of high reactive oxygen species (ROS), by attenuating nerve growth factor (NGF) expression and whether Nrf2/HO-1 signaling is involved in the protection. Twenty-four hours after ligation of the left anterior descending artery, male Wistar rats were treated for 4 weeks. The postinfarction period was associated with increased oxidative–nitrosative stress, as measured by myocardial superoxide, nitrotyrosine, and dihydroethidium fluorescent staining. In concert, myocardial norepinephrine levels and immunohistochemical analysis of sympathetic nerve revealed a significant increase in innervation in vehicle-treated rats compared with sham-operated rats. Arrhythmic scores during programmed stimulation in the vehicle-treated rats were significantly higher than those in sham. This was paralleled by a significant upregulation of NGF protein and mRNA in the vehicle-treated rats, which was reduced after administration of LiCl. LiCl stimulated the nuclear translocation of Nrf2 and the transactivation of the Nrf2 target gene HO-1. Inhibition of phosphoinositide 3-kinase by wortmannin reduced the increase in Nrf2 nucleus translocation and HO-1 expression compared with lithium alone. In addition, the lithium-attenuated NGF levels were reversed in the presence of the Nrf2 inhibitor trigonelline, HO-1 inhibitor SnPP, and peroxynitrite generator SIN-1, indicating the role of Nrf2/HO-1/ROS. In conclusion, lithium protects against ventricular arrhythmias by attenuating NGF-induced sympathetic innervation via antioxidant activation of the Nrf2/HO-1 axis.     

Decreased GLT-1 and increased SOD1 and HO-1 expression in astrocytes contribute to lumbar spinal cord vulnerability of SOD1-G93A transgenic mice

The SOD1-G93A transgenic mouse is a widely used ALS model, but the death of lower motor neurons is the hallmark. Here, we show that the SOD1-G93A transgene and HO-1 are preferentially over-expressed in the lumbar spinal cord, particularly in the activated astrocytes of the transgenic mice. We also show down-regulation of GLT-1 in spite of the proliferating astrocytes. However, GLT-1, SOD1-G93A transgene and HO-1 expression were not obviously changed in the motor cortex. Our data link spinal cord vulnerability to relatively decreased expression of GLT-1, and high expression of the transgene and HO-1 in astrocytes in SOD1-G93A transgenic mice.     

Tranilast, an orally active anti-allergic drug, up-regulates the anti-inflammatory heme oxygenase-1 expression but down-regulates the pro-inflammatory cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW264.7 macrophages

Tranilast (N-[3′,4′-dimethoxycinnamonyl] anthranilic acid), an orally active anti-allergic drug, is reported to exert the anti-inflammatory effects, but the underlying mechanisms that could explain the anti-inflammatory actions of tranilast remain largely unknown. Here, we found that tranilast induces heme oxygenase-1 (HO-1) expression through the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway in RAW264.7 macrophages. Tranilast suppressed cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS) expression, and thereby reduced COX-2-derived prostaglandin E₂ (PGE₂) and iNOS-derived NO production in lipopolysaccharide (LPS)-stimulated macrophages. Similarly, tranilast diminished tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production. Interestingly, the effects of tranilast on LPS-induced PGE₂, NO, TNF-α, and IL-1β production were partially reversed by the HO-1 inhibitor tin protoporphyrin, suggesting that tranilast-induced HO-1 expression is at least partly responsible for the resulting anti-inflammatory effects of the drug. Thus, HO-1 expression via ERK1/2 activation may be at least one of the possible mechanisms explaining the anti-inflammatory actions of tranilast.     

乳腺癌HPV16感染对iNOS、P53蛋白表达的影响

目的研究人乳头状瘤病毒（HPV16）在乳腺癌中的表达及作用机制,检测HPV16、诱导一氧化氮合酶（iNOS）及P53蛋白在乳腺癌的表达及其间的相关性。方法应用免疫组化SP法,共检测了52例乳腺癌和16例乳腺良性瘤HPV16、iNOS和P53蛋白的表达。结果HPV16和P53蛋白在乳腺癌组的阳性表达率均显著高于乳腺良性瘤组。统计分析表明,iNOS和P53蛋白表达阳性率与HPV感染率密切相关（P〈0．05）。结论HPV16感染参与了乳腺癌的发生,其致癌机制可能是通过诱导iNOS表达增加,产生NO,诱导p53基因突变,从而导致乳腺癌的发生。     

Modulation of CYP3A4 expression by ceramide in human colon carcinoma HT-29 cells

Cytochrome P450 3A4 (CYP3A4) enzyme is responsible for the metabolic activation and inactivation of the majority of clinically used drugs in human liver and intestines. Recent studies have increasingly implicated various inflammatory stimuli to cause changes in the activities and expression levels of CYPs. However, the underlying mechanisms are largely unknown. In the present study, our studies investigated the effects of ceramide on CYP3A4 expression in human colon carcinoma HT-29 cells. Treatment with the cell-permeable ceramide analog C(6)-ceramide to the cells significantly decreased the expression of CYP3A4. By contrast, C(6)-dihydroceramide, a biologically inactive analog of C(6)-ceramide, did not affect CYP3A4 expression. We found that bacterial sphingomyelinase (SMase) and tumor necrosis factor-alpha (TNF), which are known to increase intracellular ceramide levels, also markedly suppressed the synthesis of CYP3A4. To elucidate whether nitric oxide (NO) participates in suppression of CYP3A4 expression by ceramide, the effects of NO modulators were determined. Treatment with N(G)-monomethyl-L-arginine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), was able to protect ceramide-dependent CYP3A4 suppression. In contrast, the addition of S-nitroso-N-acetylpenicillamine, a NO donor, to HT-29 cells reduced CYP3A4 expression. The addition of iNOS antisense oligonucleotide prevented ceramide-mediated induction of iNOS expression and restored CYP3A4 expression. Wortmannin which is known to inhibit phosphatidylinositol 3-kinase (PI3-K) blocked CYP3A4 suppression by ceramide. Taken together, our results demonstrate that ceramide-mediated suppression of CYP3A4 is due to production of NO, which might result from activation of PI3-K.     

Behaviour of two-dimensional arrays of gold nanoparticles under H2S: agglomeration and regeneration

The use of an inorganic link, H₂S, leads to two-dimensional assemblies of the Au quantum dots via a gas phase procedure. The morphology of the networks created in this way and the behaviour of the Au nanoparticles under H₂S have been studied by scanning electron microscopy (SEM) and the absorption spectroscopy in the visible region.     

脓毒症患者血清H2S、GDF-15、PTX-3水平与凝血功能、炎症指标及病情评分的相关性分析

摘要 目的：探讨脓毒症患者血清硫化氢（H₂S）、生长分化因子-15（GDF-15）、穿透素-3（PTX-3）水平与其凝血功能、炎症指标及病情评分的相关性。方法：随机选取我院2018年2月～2020年2月收治的脓毒症患者52例作为脓毒症组,另选取我院同期收治的脓毒症休克患者46例作为休克组以及同期于我院进行体检的健康者50例作为对照组。检测三组血清H₂S、GDF-15、PTX-3水平以及凝血功能、炎症指标,其中凝血功能指标包括血小板计数（PLT）、部分凝血活酶时间（APTT）、凝血酶原时间（PT）、纤维蛋白原（FIB）。炎症指标包括降钙素原（PCT）、C反应蛋白（CRP）。采用急性生理学和慢性健康状况评分系统Ⅱ（APACHEⅡ）、快速序贯器官功能（qSOFA）评分对脓毒症组、休克组患者病情进行评分。分析血清H2S、GDF-15、PTX-3与患者凝血功能、炎症指标及病情评分的相关性。结果：脓毒症组、休克组的血清H₂S、PLT均低于对照组,且休克组低于脓毒症组（P＜0.05）。脓毒症组、休克组的血清GDF-15、PTX-3、APTT、PT、FIB、PCT、CRP均高于对照组,且休克组高于脓毒症组（P＜0.05）。脓毒症组APACHEⅡ、qSOFA评分均显著低于休克组（P＜0.05）。血清H2S与PLT呈正相关（P＜0.05）,与APTT、PT、FIB、PCT、CRP、APACHEⅡ评分、qSOFA评分呈负相关（P＜0.05）。血清GDF-15、PTX-3与PLT呈负相关（P＜0.05）,与APTT、PT、FIB、PCT、CRP、APACHEⅡ评分、qSOFA评分呈正相关（P＜0.05）。结论：脓毒症患者的血清H₂S明显下降,而血清GDF-15、PTX-3增高,三者与凝血功能、炎症以及病情评分均存在密切关联,这可能是影响脓毒症进展的重要原因之一。     

Inducible nitric oxide synthase and cyclooxgenase-2 mediate protection of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress in PC12 cells

The induction of inducible nitric oxide synthase (iNOS) in response to different stress is associated with simultaneous induction of cyclooxygenase-2 (COX-2) in various cell types. Both iNOS and COX-2 have been reported to mediate the late phase of cardioprotection induced by different preconditioning. However, whether both iNOS and COX-2 are mediators in the neuroprotection induced by preconditioning with hydrogen peroxide (H(2)O(2)) at low concentration is unknown. In this study, using the neurosecretory cell line-PC12 cells to set up the model of neuroprotection of preconditioning with H(2)O(2) against apoptosis, we first investigate what changes in expression of iNOS and COX-2 appear during H(2)O(2) preconditioning, then determine if both iNOS inhibitor and COX-2 inhibitor interfere with the neuroprotection elicited by preconditioning with H(2)O(2). We found that preconditioning with H(2)O(2) at 10 microM significantly protected PC12 cells against apoptosis induced by lethal H(2)O(2) (50 microM) and increased the expression of iNOS and COX-2 and that selective iNOS inhibitor, aminoguanidine (AG) and COX-2 inhibitor, NS-398 obviously blocked the protective effects induced by preconditioning with 10 microM H(2)O(2). The results of this study suggest that both iNOS and COX-2 are mediators of the neuroprotection induced by preconditioning with oxidative stress (H(2)O(2) at low concentration) in PC12 cells.     

硫化氢对1型糖尿病大鼠膈肌NO含量和iNOS活性的影响

目的：观察硫化氢（H2S）对1型糖尿病大鼠膈肌一氧化氮（NO）含量和诱导型一氧化氮合酶（iNOS）活性的影响。方法：将32只雄性SD大鼠随机分为4组：正常组（NC组）、糖尿病组（DM组）、糖尿病治疗组（DM + NaHS组）和NaHS对照组（NaHS组）（n=8）。采用一次性腹腔注射链脲佐菌素55 mg/kg制备1型糖尿病大鼠模型,造模成功后第4周起,DM + NaHS组和NaHS组大鼠腹腔注射NaHS溶液14μmol/kg干预治疗。连续注射5周后,测大鼠空腹血糖值（FBG）和膈肌重量/体重量比（DW/BW）;HE染色观察膈肌显微结构变化;利用NOS分型测试盒测膈肌组织iNOS活性;硝酸还原法测定膈肌组织NO含量;利用RT-PCR和Western blot分别检测膈肌组织iNOS mRNA和蛋白表达。结果：与NC组比较,DM组大鼠FBG显著升高,膈肌显微结构损伤明显,DW/BW下降,膈肌组织iNOS活性和NO含量显著增加,iNOS mRNA和蛋白表达明显增高,NaHS组各项指标差异无统计学意义。与DM组比较,DM + NaHS组膈肌显微结构明显改善,DW/BW增高,膈肌组织iNOS活性和NO含量明显下降,iNOS mRNA和蛋白表达显著降低。结论：外源性补充H2S可能通过下调膈肌组织iNOS活性和蛋白表达,降低NO含量,进而保护糖尿病大鼠膈肌的功能。     

支气管哮喘患儿血清硫化氢、嗜酸性粒细胞趋化因子水平与炎症因子及肺功能的关系研究

摘要 目的：探讨支气管哮喘患儿血清硫化氢（H2S）、嗜酸性粒细胞趋化因子（Eotaxin）水平与炎症因子及肺功能的关系。方法：选取我院收治的95例支气管哮喘患儿,按照《儿童支气管哮喘诊断及防治指南(2016年版)》将支气管哮喘患儿分为急性发作期组43例和缓解期组52例。检测血清中H2S 水平;检测Eotaxin、肿瘤坏死因子-α（TNF-α）,白介素6（IL-6）和白介素13（IL-13）的水平;对患儿进行肺功能测定,采用Pearson检验分析血清H₂S、Eotaxin水平与血清炎症因子及肺功能之间的相关性。ROC曲线分析血清H2S、Eotaxin对支气管哮喘患儿病情的预测价值。结果：急性发作期组患儿血清H₂S水平、第一秒用力呼气容积占预计值百分比（FEV₁%pred）、最大呼气流量占预计值百分比（PEF%pred）和FEV₁/用力肺活量（FVC）低于缓解期组患儿,Eotaxin、TNF-α、IL-6和IL-13水平高于缓解期组患儿（P<0.05）;支气管哮喘患儿血清H₂S水平与TNF-α、IL-6和IL-13水平均呈负相关（P<0.05）,与FEV₁%pred、FEV₁/FVC和PEF%pred均呈正相关（P<0.05）;支气管哮喘患儿血清Eotaxin水平与TNF-α、IL-6和IL-13水平均呈正相关（P<0.05）,与FEV₁%pred、FEV₁/FVC和PEF%pred均呈负相关（P<0.05）。血清H₂S预测支气管哮喘患儿病情的曲线下面积为0.854,灵敏度和特异度分别为77.89%和81.05%,Youden指数为0.5894;血清Eotaxin预测支气管哮喘患儿病情的曲线下面积为0.924,灵敏度和特异度分别为92.71%和84.38%,Youden指数为0.7709。结论：支气管哮喘急性发作期患儿与缓解期患儿相比,其血清H2S水平较低,Eotaxin水平较高,血清H₂S和Eotaxin水平与机体炎症反应及肺功能密切相关,有助于辅助评估支气管哮喘患儿的病情。     

Heme oxygenase-1 and heme oxygenase-2 have distinct roles in the proliferation and survival of olfactory receptor neurons mediated by cGMP and bilirubin,respectively

Heme oxygenase (HO) is implicated in protection against oxidative stress, proliferation and apoptosis in many cell types, including neurons. We utilized olfactory receptor neurons (ORNs) as a model to define the roles of HO-1 and HO-2 in neuronal development and survival, and to determine the mediators of these effects. The olfactory system is a useful model as ORNs display neurogenesis post-natally and do not contain nitric oxide synthase (NOS) activity, which could confound results. HO isoforms were expressed in ORNs during embryogenesis and post-natally. Mice null for either HO-1 or HO-2 displayed decreased proliferation of neuronal precursors. However, apoptosis was increased only in HO-2 null mice. Cyclic GMP immunostaining was reduced in ORNs in both genotypes, providing direct evidence that HO mediates cGMP production in vivo. Bilirubin immunostaining was reduced only in HO-2 null mice. These roles for HO-1 and HO-2 were confirmed using detergent ablation of the epithelium to observe increased neurogenesis of ORNs after target disruption in HO null mice. Primary cultures of ORNs revealed that proliferative and survival effects of HO were mediated through cGMP and bilirubin, respectively. These results support a role for HO, the CO-cGMP signaling system and bilirubin in neurodevelopment and in response to injury.     

Involvement of Bcl-2 Family and Caspase-3-Like Protease in NO-Mediated Neuronal Apoptosis

Abstract: To clarify mechanisms of neuronal death in the postischemic brain, we examined whether astrocytes exposed to hypoxia/reoxygenation exert a neurotoxic effect, using a coculture system. Neurons cocultured with astrocytes subjected to hypoxia/reoxygenation underwent apoptotic cell death, the effect enhanced by a combination of interleukin-1β with hypoxia. The synergistic neurotoxic activity of hypoxia and interleukin-1β was dependent on de novo expression of inducible nitric oxide synthase (iNOS) and on nitric oxide (NO) production in astrocytes. Further analysis to determine the neurotoxic mechanism revealed decreased Bcl-2 and increased Bax expression together with caspase-3 activation in cortical neurons cocultured with NO-producing astrocytes. Inhibition of NO production in astrocytes by N ^G-monomethyl- l -arginine, an inhibitor of NOS, significantly inhibited neuronal death together with changes in Bcl-2 and Bax protein levels and in caspase-3-like activity. Moreover, treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by an NO donor, sodium nitroprusside. These data suggest that NO produced by astrocytes after hypoxic insult induces apoptotic death of neurons through mechanisms involving the caspase-3 activation after down-regulation of BCl-2 and up-regulation of Bax protein levels.