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.     

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.     

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.     

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.     

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.     

Alterations of nitric oxide synthase in cardiac sarcoplasmic reticulum from rats with myocardial calcification

AIM: To study alterations of nitric oxide synthase (NOS) in cardiac sarcoplasmic reticulum from rats with myocardial calcification, and to explore the mechanism of inhibition of SR function in the rats with myocardial calcification. METHODS: Compared with control, myocardial calcium content in the 6 weeks increased by 408%（Ｐ＜0.01）, the NO production, NOS activity and NOS protein expression in the SR with myocardial calcification increased versus control（Ｐ＜0.01）.Myocardial calcium content was not alterations significantly, but the NOS/NO pathway in the SR was up-regulated slightly in the 2 weeks. RESULTS: Compared with control, myocardial calcium content in the 6 weeks increased by 408%(P＜0.01), the NO production, NOS activity and NOS protein expression in the SR with myocardial calcification increased versus control(P＜0.01).Myocardial calcium content was not alterations significantly, but the NOS/NO pathway in the SR was up-regulated slightly in the 2 weeks. CONCLUSION: The up-regulated NOS/NO system in the SR with myocardial calcification is the important mechanism of function inhibition of the SR.     

Effects of nitric oxide synthase inhibitor in two-week oral treatment on hyperdynamic circulatory statein cirrhotic rats

AIM:To investigate the ef ects of low dosage of nitric oxide synthase(NOS)inhibitor N^G-nitro-L-arginine methyl ester(L-NAME)in two-week treatment on the hyperdynamic circulatory state in rats with cirrhosis.METHODS:Cirrhosis model was induced in male SD rats by injection of 60%CCl4 oily solution subcutaneously.Cirrhotic rats were treated with L-NAME(0.5 mg kg^-1d^-1)by gavage for two weeks.Mean arterial pressure(MAP), portal pressure(PP), cardiac output(CO), cardiac index(CI), splanchnic vascular resistance(SVR), splanchnic blood flow(SBF)and serum nitrite levels were determined in L-NAME-treated, L-NAME-untreated cirrhotic rats and controls by using 57Co-labled microsphere technique and a fluorometric assay, respectively.RESULTS:Untreated cirrhotic rats had significantly lower MAP, SVR and higher PP, CO, CI, SBF and nitrite concentration than those of the controls(all, P<0 01).In treated cirrhotic rats, L-NAME significantly at enuated the increase of CO, CI, SBF, nitrite concentration and the decrease of MAP and SVR.In treated cirrhotic rats, L-NAME induced a marked decrease of nitrite concentration than untreated cirrhotic rats[(1.471 0.907)mol/L vs(4.204 1.253)mol/L, P<0 01].CONCLUSION:The endogenous NO may play an important role in the changes of hemodynamics pat ern in cirrhosis, andhyperdynamic circulatory state in rats with cirrhosis can be ameliorated by oral two-week administration of lower dose of L-NAME.     

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.     

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 exogenous salicylic acid and nitric oxide on lipid peroxidation and antioxidant enzyme activities in leaves of Brassica napus L. under nickel stress

The effects of nickel in combination with salicylic acid (SA) and sodium nitroprusside (SNP), a donor of nitric oxide (NO) on 21-day-old canola plants were evaluated. Exposure to 0.5 mM NiCl₂·6H₂O for 10 days resulted in toxicity symptoms such as chlorosis and necrosis at leaves. Addition of 0.2 mM SA or 0.2 mM SNP slightly reduced the toxic effects of nickel. After application of both SA and NO, these symptoms considerably decreased. Treatment with Ni resulted in a decrease in dry weight of roots and shoots and chlorophyll content of leaves. In Ni-treated plants, level of lipoxygenase activity and malondialdehyde (MDA), H₂O₂ and proline contents significantly increased, while the activities of the antioxidant enzymes such as catalase, guaiacol peroxidase and ascorbate peroxidase decreased in leaves. The results indicated that Ni caused an oxidative stress in canola plants. The Ni-stressed plants exposed to SA or NO, especially to SA + NO, exhibited an improved growth as compared to Ni-treated plants. SA or NO, especially both together considerably reduced root-to-shoot translocation of Ni and increased the activities of the antioxidant enzymes in leaves of Ni-stressed plants. Interaction of SA and NO improved the chlorophyll content and decreased the level of lipid peroxidation, H₂O₂ and proline accumulation in leaves. These results suggest that SA or NO in particular their combination counteract the negative effects of Ni on canola plants.     

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.     

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.     

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.     

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 eplerenone on expression and activity of aortic endothelial nitric oxide synthase in hypertensive rats induced by high-salt intake

AIM: To explore the effects of eplerenone on the expression and activity of aortic endothelial nitric oxide synthase(eNOS) in high salt-induced hypertensive rats.METHODS: Male Wistar rats(4 week old, weighting 50～60 g) were randomly divided into control group, high-salt diet group and eplerenone group. The rats in control group were fed with ordinary rodent animal diet, the rats in high-salt group and eplerenone group were exposed to 5% salt diet for 16 weeks and administrated with the same dosage of saline or eplerenone(40 mg·kg^-1·d^-1) by gavage for 4 weeks, respectively. Systolic blood pressure(SBP) was measured by tail-cuff every 2 weeks. The rats were sacrificed after 16 weeks and the thoracic aorta was collected. The aldosterone content in the aorta was measured by ELISA. The protein levels of mineralocorticoid receptor(MR) and eNOS were determined by Western blot. The activitie of constitutive NOS(cNOS) was measured by chemocolorimetry. The protein localization of eNOS, neuronal nitric oxide synthase(nNOS) and MR was observed by immunohistochemistry.RESULTS: A process of 8-week high-salt diet increased SBP gradually. SBP in the rats exposure to high salt for 16 weeks was significantly higher than that in control group(P<0.05). After 4 weeks of eplerenone treatment, SBP in the rats was significantly lower than that before treatment(P<0.05). Compared with control group, the aldosterone content in the aorta were significantly increased in high-salt diet group and eplerenone group(P<0.05), the expression level of MR also increased significantly(P<0.05). Compared with control group, both eNOS protein expression(P<0.05) and cNOS activity in high-salt diet group were significantly decreased(P<0.05). The protein expression of eNOS as well as cNOS activity in aorta increased significantly in eplerenone group compared with high-salt diet group(P<0.05).CONCLUSION: Aldosterone content in aorta of high-salt-induced hypertensive rats increases significantly. Aldosterone attenuates the protein expression of eNOS and reduces the enzyme activity through the activation of mineralocorticoid receptor. The selective mineralocorticoid receptor antagonist eplerenone enhances the protein expression of eNOS and its activity, thereby improves eNOS function.     

Role of endogenous nitric oxide synthase inhibitor in erectile dysfunction of diabetic rats

AIM: To investigate the role of nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in erectile dysfunction of diabetic rats.METHODS: Type 2 diabetic rat model was established by 4 weeks of high-fat diet plus a single intraperitoneal injection of streptozotocin and continued high-fat diet feeding for 8 weeks. Corpus cavernosum was isolated from the rats under anesthetization, and the endothelium-dependent relaxation response to acetylcholine (ACh) was tested in an organ chamber to reflect erectile function. The level of ADMA in serum was detected. The NOS activity, nitric oxide (NO) content and cyclic guanosine monophosphate (cGMP) content in corpus cavernosum were measured. The protein expression of ADMA-NOS-NO pathway-related molecules and phosphodiesteras 5 (PDE5) in the corpus cavernosum was detected by Western blot. Superoxide dismutase activity and malondialdehyde content were analyzed to evaluate oxidative stress.RESULTS: Elevated blood glucose and lowered insulin sensitivity were observed in the diabe-tic rats, indicating that type 2 diabetic rat model was successfully established. Compared with control group, the relaxation response to ACh of corpus cavernosum from diabetic rats was significantly decreased, which was accompanied with the elevation of serum ADMA level and reduction of NOS activity, NO content and cGMP content in the corpus cavernosum. The protein expression of ADMA-generating enzyme protein arginine methyltransferase 1 was up-regulated, while ADMA-metabolic enzymes dimethylarginine dimethylaminohydrolases 1 and 2, and ADMA-targeting enzymes endothelial NOS and neuronal NOS were down-regulated. The protein expression of PDE5 was up-regulated, accompanied with an increase in oxidative stress in the corpus cavernosum of diabetic rats. Incubation of isolated corpus cavernosum from normal rats with NOS inhibitor ADMA induced the similar relaxation dysfunction of corpus cavernosum response to ACh and decreased NO and cGMP contents in diabetic rats.CONCLUSION: Elevated endogenous NOS inhibitor ADMA plays an important role in erectile dysfunction of diabetic rats. The underlying mechanism may be related to the reduction of NO production and the increase in oxidative stress.     

Production of nitric oxide and change of nitric oxide synthase activity in brains mitochondria of the rats with focal cerebral ischemia/reperfusion

AIM and METHOD:To determine the production of nitric oxide(NO) and change of NO synthase(NOS) activity in mitochondria isolated from the rat brains of the ischemia/reperfusion rat model produced by transient occlusion of middle cerebral artery on the following time points:2 h after occlusion of artery and 30 min,2 h, 4h after reperfusion.RESULTS:After the occlusion of middle cerebral artery,the respiratory control rate(RCR) of mitochondria significantly decreased and slightly increased at 4h after reperfusion.Meantime,the production of NO in mitochondria increased significantly.But with the increase of perfusion, production of NO gradually decreased and reached normal level as in the control group.It also shows that cerebral ischemia increased NOS's activity significantly in the mitochondria and still kept a higher level than the control group although it decreased gradually after reperfusion.But the iNOS's activity did not show obvious change.The change of total NOS's activity depends on the change of cNOS's activity.CONCLUSION: The activation of NO/NOS system in the mitochondria might play an important role in the reperfusion injury during reperfusion of ischemic brain.