Effects of nitric oxide and inducible nitric oxide synthase on process of atherosclerosis

AIM：To explore the dynamic changes of nitric oxide and inducible nitric oxide synthase during the process of atherosclerosis, and to analyze their influence on the formation of atherosclerosis. METHODS：SD rats (n=60) were randomly divided into control group and atherosclerosis group (30 rats in each group). Atherosclerosis model was induced by feeding high-fat diet and vitamin D3. The values of blood biochemical were analyzed enzymatically using bioMérieux kit. The concentration of serum nitric oxideing was detected by a colorimetric method. The success of atherosclerosis modeling was determined by pathological examination. The protein expression of inducible nitric oxide synthase in atherosclerotic plaque was detected by the method of immunohistochemistry. RESULTS：The atherosclerosis model was successfully established in 90 d. The concentration of serum nitric oxide gradually decreased in atherosclerosis group, and a significant difference among groups was observed. Atherosclerosis index was positively correlated with calcium ion, and negatively correlated with nitric oxide. The protein expression of inducible nitric oxide synthase in the atherosclerotic plaque after 90 d was found. CONCLUSION:The protein expression of inducible nitric oxide synthase in the plaque area of aorta increases and the concentration of serum nitric oxide decreases with the process of atherosclerosis. The anti-atherosclerosis role of nitric oxide is gradually decreased.     

Changes of nitric oxide synthase and nitric oxide in lung of smoking rats

AIM:To observe the changes of iNOS and eNOS in lung tissue and NO in bronchoalveolar lavage fluid (BALF) in smoking rats.METHODS:80 Wistar rats were divided into control, smoking group, L-NIL group and L-NAME group (rats were exposed to smoke and injected (i.p.) with selective iNOS inhibitor L-NIL or NOS inhibitor L-NAME). iNOS and eNOS protein levels in whole lung were detected by immunohistochemical staining, and NOS mRNA was quantified using RT-PCR. In addition, NO₂^-/NO₃^- was determined using Griess assay.RESULTS:The expression of iNOS mRNA and protein in smoking rats increased, the expression of eNOS mRNA and eNOS protein decreased, and the total cell count and the level of NO₂^-/NO₃^-in BALF increased(P＜0.05). In vivo, L-NIL reduced the total cell count and NO₂^-/NO₃^- in BALF (P＜0.05), while L-NAME had no effect on them.CONCLUSION:Cigarette smoke increased expression of iNOS mRNA and protein and decreased expression of eNOS mRNA and protein. The large amount of NO generated by iNOS may amplify inflammation in lung tissue.     

Effects of aspirin on production of nitric oxide and inducible nitric oxide synthase mRNA expression under inflammatory conditions in human vascular endothelial cells

AIM: To explore the effect of aspirin on inducible nitric oxide synthesis and gene expression under inflammation in endothelial cells. METHODS:Using NADPH, Griess methods and RT-PCR, the activity of isozymes of NO synthase (NOS), nitric oxide (NO) level, and iNOS mRNA expression were examined respectively. Also, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content and cell viability were measured. RESULTS: Aspirin (3 mmol/L) reduced inducible NO production and NOS activity(P＜0.05), caused a significant decrease in LDH release rate and MDA content with a further increase in cell viability. Aspirin inhibited inducible NO excretion and alleviated the damage caused by NO in a concentration-dependent manner. However,aspirin had no effect on basal NO levels in the absence of stimulation by inflammatory factor. On the other hand, under middle concentration (＜10 mmol/L), aspirin was able to reduce enzymatic activity of NOS and protein expression by increasing the stability of iNOS mRNA. In contrast, at high concentration (20 mol/L), aspirin could decrease the stability of iNOSmRNA. Sodium salicylate and indomethacin did not inhibit inducible NO production. CONCLUSION:Aspirin could significantly inhibit inducible NO production in vascular endothelial cells during inflammation.     

Effect of dietary black rice outer layer fraction on inducible nitric oxide synthase expression in apoE-deficient mice

AIM: To explore the effect of dietary black rice outlayer fraction (BRF) on inducible nitric oxide synthase (iNOS) expression, and elucidate the possible mechanism of BRF anti-atherogenesis in apoE-deficient mice. METHODS: After 16 weeks intervention by 5% BRF, aortic iNOS activity in different groups was determined by RIA. iNOS mRNA expression in aorta were analyzed by RT-PCR. RESULTS: Mice in BRF group showed weaker expression of iNOS mRNA and lower iNOS activity than those in positive and WRF group (P＜0.05). No significant difference was observed between positive group and WRF group (P＞0.05). CONCLUSION:The supplementation of BRF has dra- matically reduced aortic sinus atherosclerotic plaque areas compared to WRF in apoE-deficient mice and its action of ant-atherogenesis may be attributed to its inhibition of iNOS activity and iNOS mRNA expression.     

Effects of hypoxia on levels of nitric oxide,endothelin-1 and inducible nitric oxide synthase mRNA expression in cultured human umbilical vein endothelial cells

AIM: To observe the effects of hypoxia on the levels of nitric oxide (NO), endothelin (ET-1) and the expression of inducible nitric oxide synthase (iNOS) mRNA in human umbilical vein endothelial cells (HUVECs), and further investigate the mechanism of hypoxic pulmonary hypertension. METHODS: On the basis of the HUVECs culture model, the methods of nitrate reductase and radioimmunoassay were used to determine the changes of NO and ET-1 in the medium secreted by HUVECs, and the expression of iNOS mRNA was analyzed by semi quantitative RT-PCR after exposure to hypoxia (3% O2) for 6, 12 or 24 h. RESULTS: The contents of NO2-/NO3- and ET-1 in hypoxia group in the medium was significantly higher than that in control group at different time points (P<0.05). Also, iNOS mRNA expression increased significantly (P<0.05). CONCLUSION: Hypoxia stimulates the release of NO and ET-1 from HUVECs, also induces iNOS-mRNA expression. The change of NO may be the result of iNOS mRNA upregulation induced by hypoxia.     

The augmentative effect of inducible nitric oxide synthase on pulmonary fibrosis progression

AIM: To study the up-regulation of inducible nitric oxide synthase (iNOS) in lung of pulmonary fibrosis and its relationship with fibrosis. METHODS: The changes of amount of iNOS positive stain cells and type Ⅰ?Ⅲ collagen were examined on the day 7, 14 and 30 after intratracheal administration of bleomycin A₅. The contents of NO₂^-/NO₃^- (nitrite/nitrate) in out-flowing pulmonary blood (OPB), hydroxyproline in lung and the histological changes were detected after iNOS was blocked by aminoguanidine (AG). RESULTS: (1) The number of iNOS-positive stain cells increased significantly in BLMA₅ 7 d, 14 d and 30 d groups compared with that in control group (P<0.01). Furthermore, the increment of the number of iNOS-positive stain cells in BLMA₅ 7 d, 14 d groups was more than that in BLMA₅ 30 d group. There was an increment of collagen in BLMA₅ 14 d group and in BLMA₅ 30 d group , with an increase in type Ⅲ collagen in BLMA₅ 14 d group and an increase in type Ⅰcollagen in BLMA₅ 30 d group. (2) The high level of NO₂^-/NO₃^- in OPB and hydroxyproline level in lung could be reversed by AG, a selective inhibitor of iNOS. Large amount of fibroblasts and macrophages were also abated by AG. CONCLUSION: In the development of pulmonary fibrosis, the expression of iNOS is up-regulated, which induces nitric oxide (NO) production and promotes propagation of pulmonary fibrosis.     

Changes of nuclear factor-κB and inducible nitric oxide synthase in hypoxic pulmonary hypertension

AIM: To observe the changes of nuclear factor-κB (NF-κB) activity and inducible nitric oxide synthase (iNOS) expression in hypoxic pulmonary hypertension (HPH). METHODS :The rat model of HPH was used. The NF-κB activity and iNOS expression in lung tissue were determined by immunohistochemistry (IHC), in situ hybridization (ISH), RT-PCR and Western blot. RESULTS: ISH showed that iNOS mRNA expression in intraacinar pulmonary arteriole (IAPA) in H28d group (hypoxic treatment for 28 days) was stronger than that in normal group, H5d group and H14d group. RT-PCR showed that the iNOS mRNA in H28 group was 2.1 times, 1.9 times and 1.8 times higher than that in normal group, H5d group and H14d group, respectively. The nucleic anti-NF-κB stain was observed in H28d group, which was significantly stronger, but the I-κB amount was 2.8 times, 2.7 times and 2.5 times lower than that in normal group, H5d group and H14d group, respectively. CONCLUSION: The activity of NF-κB was correlated with the hypoxic pulmonary vessel structural remodeling and iNOS expression.     

Effect of inhaled nitric oxide on aquaporin expression in newborn rat lung in acute hyperoxic lung injury

AIM:To investigate the effect of inhaled nitric oxide on aquaporin expression and alveolar epithelial fluid transport in newborn rats with acute hyperoxic lung injury. METHODS:32 newborn SD rats were randomized to breathe for 48 h room air (C), >95%O₂ (O), >95%O₂+10^-5 NO (NO only in the first 24 h, ONO), room air + NO (CN). Then, the rats were killed, the lung wet-to-dry weight ratio (Q_W/Q_D), the histology, and AQP1, AQP5, α₁-NKA, α-ENaC mRNA expressions in the lungs were measured. RESULTS:Compared with C group, the Q_W/Q_D in O group significantly increased (P<0.01), and AQP1 mRNA expression decreased significantly (P<0.01). Compared with O group, ONO group had a lower level of Q_W/Q_D (P<0.05), and AQP1 mRNA expression increased (P<0.05). AQP5 mRNA expression in all groups remained unchanged. CONCLUSION:In newborn rats with acute hyperoxic lung injury, inhaled 10-5 nitric oxide for 24 h may attenuate lung edema and increase AQP1 mRNA expression, suggesting that inhaled 10^-5 nitric oxide for 24 h may promote the AQP1 expression in lung in this model of acute lung injury.     

Effect of ischemic preconditioning on expression of nitric oxide synthase in rat small-for-size liver graft

AIM:To investigate the effect of ischemic preconditioning (IPC) on expression of nitric oxide synthase (NOS) in rat small-for-size liver graft and its significance. METHODS:Sixty SD rats were randomly divided into 3 groups (n=10 pairs/group):nonwarm ischemia group (NWI);warm ischemic group (WI);and ischemic preconditioning group (IPC). The models of rat small-for-size liver transplantation were set up by two-cuff technique. Expression of eNOS mRNA and iNOS mRNA in hepatic tissue were detected by fluorescence-quantitating-PCR. RESULTS:Heptic expression of eNOS mRNA post-IPC was higher than that pre-IPC (P<0.05). Heptic expression of eNOS mRNA in each group at 0.5, 1, 2 and 3 h post-reperfusion was higher than that pre-operation (P<0.05). It was not different significantly between NWI and WI group (P>0.05). It was higher in IPC group than that in NWI and WI group (P<0.05 or P<0.01). Hepatic expression of iNOS mRNA was detected 1 h after reperfusion of liver graft. It was lower in IPC group than that in WI group (P<0.05 or P<0.01) and lower in NWI group than that in IPC group (P<0.05 or P<0.01) 2 h and 3 h post-reperfusion. CONCLUSION:IPC might protect liver graft by increasing the expression of eNOS mRNA at early stage after reperfusion and decreasing the expression of iNOS mRNA at later stage after reperfusion.     

Hydrogen sulfide regulates the expression of nitric oxide/nitric oxide synthase system during recurrent febrile seizures

AIM: To explore the effect of hydrogen sulfide (H2S) on nitric oxide (NO)/nitric oxide synthase (NOS) system during recurrent febrile seizures (FS). METHODS: Sprague-Dawley rats aged 21 days were randomly divided into four groups: control group (37.0 ℃ water, n=8); FS group (45.2 ℃ water, n=8); FS+NaHS group (45.2 ℃ water, n=8), FS+HA (hydroxylamine) group (45.2 ℃ water, n=8). FS in rats were induced ten times in a bath of warm water, once every 2 days. The plasma level of H2S and NO was detected by the spectrophotometer method. The expression of NOS mRNA was examined by in situ hybridization. The expression of nNOS protein was observed by immunohistochemistry. RESULTS: The plasma level of NO decreased significantly in FS+NaHS group while elevated obviously in FS+HA group compared with that in FS group. At the same time, the expression of nNOS down-regulated in FS+NaHS group while up-regulated in FS+HA group compared with that in FS group. CONCLUSION: H2S down-regulated the expression of NO/NOS system during recurrent FS.     

Role of inducible nitric oxide synthase in the metabotropic glutamate receptor2/3 mediated-brain ischemic tolerance

目的：阐明诱导型一氧化氮（NO）信号通路在代谢型谷氨酸受体2/3（mGluR2/3）参与海马脑缺血预处理（CIP）保护机制中的作用。方法： 本实验采用免疫组化,观察脑室应用mGluR2/3阻断剂α-甲基-(4-四唑基-苯)甘氨酸 (MTPG) 对CIP诱导的iNOS表达的影响。36只永久凝闭椎动脉的SD大鼠随机分为sham、CIP、损伤性缺血、CIP+损伤性缺血、MTPG+CIP和MTPG+CIP+损伤性缺血6组,每组6只。各组大鼠均在术后或末次缺血再灌后,常温饲养5 d取材观察。结果： Sham组胞浆有较弱的iNOS表达。CIP组和CIP+损伤性缺血组 NOS表达与sham组相比明显增加。在CIP前20 min 脑室注射mGluR2/3阻断剂MTPG,可阻断CIP引起的iNOS表达增加,但对神经元的形态无影响。而MTPG+CIP+损伤性缺血组中,iNOS的表达较MTPG+CIP组明显增加,同时锥体神经元的轮廓、形态出现明显的损伤性改变。结论： CIP能够引起一定程度的iNOS表达增加,而MTPG能阻断CIP引起的iNOS的表达增加,同时又能阻断CIP抗后续损伤性缺血的作用,提示iNOS信号通路在mGluR2/3介导的脑缺血耐受过程中发挥重要作用。     

Progress in mitochondrial nitric oxide synthase

Nitric oxide (NO) is an intra- and intercellular messenger with a broad spectrumof activities in the central nervous system, cardiovascular and immune systems. Mitochondrial nitric oxide synthase (mt-NOS), which might be a new form of NOS in mitochondria, has been discovered to be active in the regulation ofmitochondrial respiration, energy metabolism and many pathophysiological processes.In this review, the location, properties, physiological and pathophysiological significance of mtNOS were summarized.     

Inducible nitric oxide synthase in diseases

A Review Nitric oxide (NO) is a biologically active molecule in vivo,it has been found to play an important role not only as a cytotoxic effector but also an immune regulatory mediator and a signal molecule of message transmission.The inducible NO synthase (iNOS) produces NO after cells were activated and play a rather complicated pathophysiological action in a variety of human diseases or disorders. The molecular biological character of iNOS,its expression in human diseases and probable significance were reviewed.     

Construction of human neuronal nitric oxide synthase expression system in Escherichia coli

AIM: To construct a high-level expression system of recombinant human neuronal nitric oxide synthase (hnNOS) full-length enzyme in Escherichia coli. METHODS: The coding sequence of hnNOS full-length was firstly amplified by PCR, and then ligated into the expression vector pCWori+. The recombinant plasmid was transformed into Escherichia coli BL21 for high-level expression. After having been checked with Western blot, the enzyme was used for large-scale culture and purification. Finally, the property of the enzyme was determined by spectrophotometric method. RESULTS: The constructed expression system could give a yielding of 3 mg/L initial culture. CONCLUSION: The expression system constructed is fully sufficient to express the active human neuronal nitric oxide synthase.     

Fenofibrate increases endothelial nitric oxide synthase expression in bovine aortic endothelial cells

AIM: We hypothesize that peroxisome proliferator-activated receptor α(PPARα) agonists act directly on nitric oxide (NO) production in vascular endothelium. Thus, the purpose of this study is to investigate the effects of fenofibrate on endothelial NO synthase(eNOS) activity and its expression in cultured vascular endothelial cells. METHODS: Bovine aortic endothelial cells (BAECs) were treated with the PPARα activator fenofibrate. The eNOS activity and the expression of eNOS protein and its mRNA were determined. RESULTS: Our data show that fenofibrate increased eNOS activity in a dose-and time-dependent manner. At the concentration of 10 μmol/L or more, fenofibrate treatment caused a significant increase in eNOS activity. The maximal increase in eNOS activity(2.32±0.47 fold of the control) was observed with 50 μmol/L fenofibrate treatment for 48 h. Fenofibrate failed to increase eNOS activity at 1 and 12 h. RT-PCR analysis demonstrated that eNOS mRNA relative to β-actin mRNA significantly increased at concentrations of 5 μmol/L or more. It reached 2.08±0.33 fold of the control with 50 μmol/L fenofibrate. Significant increase in eNOS mRNA levels was observed after 6 h, and lasted for 48 h. The peak increase in eNOS mRNA levels(2.13±0.30 fold of the control,P<0.01) was observed with 50 μmol/L fenofibrate treatment for 12 h. Longer incubation of cells with 50 μmol/L fenofibrate caused no further increase. The treatment of BAECs with fenofibrate for 48 h demonstrated a concentration-dependent increase in eNOS protein levels as measured by Western blot analysis. Densitometric analysis indicated that there was a significant increase in eNOS to β-actin ratios after fenofibrate treatment at concentrations of 10,50 and 100 μmol/L(1.80±0.45, 2.70±0.42 and 2.20±0.32 fold of the control, respectively, P<0.01). The significant increase in eNOS protein levels was observed 12 h after treatment and lasted for 48 h. CONCLUSION: PPARα activator fenofibrate, enhances endothelial NO production by directly upregulating eNOS expression and activity.     

Changes of serum levels of nitric oxide and nitric oxide synthase in patients during liver transplantation

AIM: To study the changes of serum levels of nitric oxide (NO) and nitric oxide synthase (NOS) in patients during liver transplantation. METHODS: Samples were obtained from 30 patients in end liver disease at five time points during liver transplantation. NO level and NOS activity were measured by radioimmunoassay and colorimetry, respectively. Arterial and mixed venous blood samples used for blood gas analysis were taken at the same time. Intrapulmonary shunt (Qs/Qt) was calculated according to the standard formula. The hemodynamics parameters including continuous cardic output (CO), HR, MABP, CVP, SVR were measured during liver transplantation. RESULTS: (1) NO2-/NO3- level at 10 min before anhepatic period was significantly higher than the baseline level. Compared with NO2-/NO3- level at 10 min before anhepatic period, NO2-/NO3- level at 30 min after anhepatic period was significantly decreased. NO2-/NO3- level at 30 min after neohepatic period was significantly higher than the baseline level and at 30 min after anhepatic period. (2) No significant change of tNOS activity was observed. Compared with the baseline activity of inducible nitric oxide synthase (iNOS), the activity at 10 min before anhepatic period and at 30 min after neohepatic period was significantly increased. The activity at 30 min after neohepatic period was significantly higher than that at 30 min after anhepatic period. (3) MABP decreased significantly when opening the inferior vena cava. CO and CVP decreased in the anhepatic stage and increased in the reperfusion stage. SVR increased during anhepatic stage and decreased significantly during neohepatic period. (4) Qs/Qt decreased significantly during anhepatic stage and increased significantly at 30 min after neohepatic period. CONCLUSIONS: Serum level of NO and NOS activity are significantly changed during liver transplantation. High level of NO may result in low systemic vascular resistance and increasing in intrapulmonary shunt.     

Effects of hypoxia on endothelial nitric oxide synthase expression in cerebral artery endothelial cells

AIM: To investigate the molecular mechanism by which hypoxia affect the endothelial nitric oxide synthase (eNOS) expression in cerebral artery endothelial cells (CAECs). METHODS: Primary cultured porcine CAECs were exposed to hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h. The eNOS mRNA level was determined by RT-PCR. The level of eNOS protein was detected by Western blotting. After specific PKC inhibitors BIM Ⅰ(1 μmol/L) and G6983 (1 μmol/L) were added, CAECs were exposed to hypoxia for 24 h. The effect of hypoxia on eNOS mRNA stability was analyzed after actinomycin D was added. RESULTS: After exposure to hypoxia for 2 h, the levels of eNOS mRNA and protein in CAECs were increased. The levels of eNOS mRNA and protein reached peak after 12 h of hypoxia (about 2.5 fold and 2.0 fold, respectively, compared to control), and remained at higher level even after 48 h of hypoxia. Moreover, hypoxia did not change the stability of eNOS mRNA. The specific PKC inhibitors BIM Ⅰ and G6983 attenuated significantly the effects of hypoxia on eNOS gene expression. CONCLUSION: These results suggest that hypoxia may enhance the expression of eNOS gene in CAECs through PKC signaling pathway, which might be one of the mechanisms of cerebral artery dilation and neuroprotection during cerebral hypoxia.