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.     

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.     

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.     

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 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.     

Effects of folic acid on antioxidant enzyme,nitric oxide synthase and nitric oxide in ovariectomized rats

AIM: To observe the effects of folic acid (FA) on antioxidant enzyme, nitric oxide synthase (NOS) and nitric oxide (NO) in ovariectomized (OVX) rats.METHODS: Forty three-month-old female SD rats were randomly divided into 5 groups: sham group, OVX group, diethylstilbestrol group (0.03 mg·kg^-1·d^-1), low-dose FA group (5 mg·kg^-1·d^-1) and high-dose FA group (20 mg·kg^-1·d^-1). Gastric gavage started 1 week after operation and lasted for 10 weeks. The rats in sham group and OVX group were given distilled water instead of FA as controls. At the end of the 10th week, the L₅ vertebra and right femur were removed for determination of bone mineral density (BMD). The bone homogenates were made using the L₃ and L₄ vertebrae. The levels of the total antioxidant capacity (TAC), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), NOS and NO were detected in plasma and bone homogenates.RESULTS: Compared with sham group, the BMD levels in L₅ vertebra and right femur and the levels of GSH-Px and NO in the plasma were all decreased. The levels of TAC, GSH-Px, NOS and NO in the bone homogenates were also decreased, while the MDA concentration was increased in OVX group (all P < 0.01). Compared with OVX group, the levels of TAC, GSH-Px, NOS, NO and BMD of the L₅ vertebra and right femur were all increased, while the MDA concentration was decreased in high-dose FA group (all P < 0.01). CONCLUSION: In female SD rats, ovariectomy leads to a significant reduction of antioxidant enzyme, NOS and NO levels. Oxidative stress is possibly involved in the development of osteoporosis. Protection against osteoporosis by high-dose FA may be linked to improvement of antioxidant enzyme activity, the levels of NOS and NO as well as a reduction of oxidative stress in ovariectomized rats.     

Effect of advanced glycosylation end products on level of nitric oxide and activity of nitric oxide synthase in cultured macrophages

AIM:To study the mechanism and effect of advanced glycosylation end products (AGEs) on NO pathway in cultured macrophages.METHODS:The level of NO and NOS activity were measured by NO and NOS kits in cultured macrophages. RESULTS:The results showed that AGEs induced decreases in NO level and NOS activity in a time and dose-related manner in interleukin-1 (IL-1)-stimulated macrophages. VitE can significantly inhibited effects of AGEs on IL-1-stimulated macrophages. CONCLUSION:AGEs can decrease NO production via inhibiting NOS activity in IL-1-stimulated macrophages. VitE can protect the cells from AGEs injury. It is an important theoretical basis for preventing chronic complication in diabetes mellitus.     

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.     

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.     

Effects of nitric oxide inhalation on nitric oxide synthase and endothelin-1 in patients with hypoxic pulmonary hypertension

AIM: To investigate the effects of nitric oxide (NO) inhalation on nitric oxide synthase (NOS) and endothelin-1 (ET-l) of patients with hypoxic pulmonary hypertension. METHODS: Examined 13 pulmonic blood samples to determine the concentration of NOS in leukocyte and ET-1 in plasma before NO inhalation, 30 minutes after inhalation, 2 and 12 hours after stopping of inhalation respectiviy. RESULTS: The values taken before inhalation was NOS (0.70 ± 0.21 )mol/min·mg^-1, ET-1 (78.89 ± 46.59) Pmol/L; 30 minutes after inhalation (0.74±0.14)mol/min·mg^-1, ET-1 (88.27 ± 45.41 )pmol/L; 2 hours after stopping of inhalation NOS (0.64 ± 0.22)mol/min·mg-1, ET-1 (80.76±42.66)pmol/L; and 12 hours after stopping of inhalation NOS (0. 63± 0. 17)mol/min.mg-1, ET-1(61.07±29.44)pmol/L. NO significant difference was found in the values of NOS and ET- 1 before and after inhalation, P＞ 0.05. CONCLUSION: The effects of NO inhalation on NOS and ET-l in patients with hypoxic pulmonary hypertension are not significant according to the above investigation.     

Role of miRNA-24 in regulation of endothelial nitric oxide synthase expression and vascular endothelial cell proliferation

AIM：To investigate whether miRNA-24 is involved in the regulation of endothelial nitric oxide synthase (eNOS) expression and vascular endothelial cell proliferation. METHODS：A plasmid that highly expressed miRNA-24 was constructed, and was transfected into the human umbilical vein endothelial cells (HUVECs) by liposome. The cell proliferation was detected by MTT assay. The expression of eNOS and Sp1 at mRNA and protein levels was exa-mined by real-time PCR, immunohistochemistry and Western blotting.RESULTS：Compared with control group, the proliferation of endothelial cells in miRNA-24 group was significantly decreased by 41.97 % (0.47±0.04 vs 0.81±0.03, P<0.01), and the expression of eNOS at mRNA and protein levels was decreased by 44.8% (0.48±0.01 vs 0.87±0.03, P<0.05) and 71.92% (0.16±0.06 vs 0.57±0.08, P<0.05), respectively. Meanwhile, the mRNA and protein levels of Sp1 were significantly decreased by 53.00% (0.45±0.02 vs 0.93±0.01, P<0.05) and by 62.31% (0.13±0.07 vs 0.31±0.09, P<0.05), respectively. In miRNA-24 inhibitor group, the above indexes were decreased compared with control group, but significantly increased compared with miRNA-24 group. CONCLUSION：miRNA-24 significantly inhibits the proliferation of HUVECs and the eNOS expression. Sp1 possibly acts as one of the important factors in the regulation of eNOS expression by miRNA-24.     

Meconium induces formation of nitrotyrosine and expression of inducible nitric oxide synthase in the rat lung

AIM: To evaluate the contribution of inducible nitric oxide synthase (iNOS) and nitrotyrosine to acute lung injury (ALI) in rats with meconium aspiration. METHODS: 16 health male Sprage-Dawley rats were randomized to control group and meconium group, followed by intratracheally administration of 1 mL/kg saline or 1 mL/kg 20% human newborn meconium suspension. The animals were killed after 24 h of treatment. The measurements included bronchoalveolar lavage fluid (BALF) cell count, pulmonary myoloperoxidase (MPO) activity and nitric oxide (NO) level. Western bloting was used to determine the expression of pulmonary nitrotyrosine-a specific “footprint” of peroxynitrite and iNOS. RESULTS: Compared to control group, the rats in the meconium group had increased BALF cell counts ［(4.04±1.01)×109cells/L vs （0.53±0.19)×109cells/L］, pulmonary MPO activity ［(1.49±0.22）U/g wet lung tissue vs (0.62±0.16) U/g wet lung tissue］, NO level ［(12.77±5.00） mmol/g protein vs （4.89±1.32) mmol/g protein］, increased expression of nitrotyrosine and iNOS (0.46±0.19 and 1.49±0.60 vs 0.15±0.04 and 0.09±0.04, respectively), all P<0.01. CONCLUSIONS: Meconium results in an increase in expression of pulmonary iNOS, leading to over production of NO and nitrotyrosine, which may be of pathogenic importance in the ALI with meconium aspiration.     

Expression of human inducible nitric oxide synthase in fibroblast cell line V79 and effects of H4Bon iNOS activity

AIM: To explore the feasibility of human iNOS transfected into V₇₉ cells by gene transfer and investigate the effects of H₄B on iNOS activity. METHODS: Human iNOS was transfected into V₇₉ cells with the karyocyte expressive vector. The cloned cells were selected by G₄₁₈. The expression of iNOS mRNA was quantified by RT-PCR and iNOS expression was observed by immunofluorescence. NO product in cells was determined by measuring nitrite (NO^-₂) release using the Griess reaction. RESULTS: V₇₉ cells infected human iNOS was proved to have iNOS mRNA at 462 bp by RT-PCR, and iNOS protein in the cytochylema by immunofluorescence. When the cells were incubated without H₄B, the content of NO in pcDNA₃ cells was minimal, with NO^-₂ production (82.32±13.08) just above the normal group (74 38±9 80, P>0.05, n=6) There was no significant difference between pcDNA₃ cells incubated with or without H₄B, (P>0.05, n=6) NO^-₂ production by pcDNA₃-iNOS cells without H₄B was higher (105 58±13 33) (n=6, P<0.01vs the normal cells or pcDNA cells). However, in pcDNA₃-iNOS cells incubated with H₄B, NO^-₂ production was much higher (236 57±3183) (n=6, P<0.01vs the all former groups). CONCLUSION: iNOS activity was increased by adding H₄B in pcDNA₃-iNOS cells, and the fibroblast can be a target cell of iNOS gene transfer.     

Effects of nitric oxide synthase inhibitors on focal cerebral ischemic injury in rats

AIM:To observe the effects of nitric oxide and different isoforms of nitric oxide synthase inhibitors on the focal cerebral ischemic injury in rats. METHODS:After the rat model of focal cerebral ischemia were established with middle cerebral artery occlusion (MCAO), aminoguanidine(AG)and N^G-nitro-L-arginine(L-NA )were administrated and the cerebral infarct size, NO production,MDA content, nitric oxide synthase(NOS) and SOD activities in the focal ischemic brain tissues were examined. RESULTS:AG could significantly attenuate the focal cerebral ischemic injury, and L-NA had a protective effect when it was administrated at 1 h,6 h but not at 3 h after surgery.CONCLUSION:Cerebral ischemic injury could be attenuated by both selective and nonselective inhibition of NOS.     

Effect of diltiazem on heme oxygenase-1 and nitric oxide synthase in rats with pulmonary hypertension

AIM: To investigate the action of diltiazem (a calcium antagonist) on the expression of heme oxygenase (HO) -1 and nitric oxide synthase (NOS) in the small pulmonary arteries (SPA) of rat in chronic hypoxia. METHODS: Chronic pulmonary arterial hypertension models were established by treating the rats in hypoxic environment for 6 weeks. After 2 weeks of hypoxia, rats were treated with diltiazem (15 mg/kg/day). Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were measured. Pathological changes in the lungs were observed under the light microscope and transmission electron microscope. The expression and distribution of heme oxygenase (HO) -1, endothelial NOS (eNOS) and inducible NOS (iNOS) were tested by immunohistochemistry and Western blot. Guanosine-3', 5'-cyclic monophosphate (cGMP) of lung tissues were detected with radioimmunoassay. RESULTS: Diltiazem significantly decreased abnormal RVSP, and RVHI in model rats, attenuated the SPA media thickeness, and recovered abnormal eNOS and iNOS expression in SPA. Whereas diltiazem had little effect on the increased HO-1 expression in SPA caused by hypoxia and ultrastructure injury in endothelium. cGMP levels were corresponded with HO-1. CONCLUSION: Diltiazem has a significant effect on inhibiting hypoxic pulmonary hypertension structural remodeling. These effects might be partly attributed to the suppression of iNOS, promotion of eNOS, and not attenuation HO-1 expression in the lung of hypoxic rats.     

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.     

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.     

Expression of nitric oxide synthase and its clinical significance in hepatic cellular carcinoma

AIM: To investigate the expression of nitric oxide synthase (NOS) and its clinical significance in hepatic cellular carcinoma (HCC). METHODS: The NOS1, NOS2 and NOS3 of 51 cases of HCC and 46 cases of liver tissue beside carcinoma (LTBC) were detected by immunohistochemistry. RESULTS: The expressive rates of NOS1, NOS2 and NOS3 in LTBC were significantly higher than those in HCC (P<0.01). The expressive rates of NOS1 in the recurrent group was significantly higher than that in the non-recurrent group (P<0.01). The expressive rate of NOS2 in the group without carcinoma embolus was significantly higher than that in the group with carcinoma embolus (P<0.05). The expressive rate of NOS3 in the recurrent group was significantly higher than that in the non-recurrent group (P<0.01). CONCLUSION: The expression of NOS1, NOS2 and NOS3 are closely related with the biological behaviors of HCC.