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 ischemic preconditioning on hepatocyte apoptosis induced by hepatic ischemia-reperfusion in cirrhotic rats

AIM:To investigate the protective effect of ischemic preconditioning (IPC) on hepatic ischemia-reperfusion(I/R) injury in cirrhotic rats and its possible mechanism. METHODS:Hepatic I/R was induced by Pringle maneuver. The cirrhotic rats were randomized into three groups: Group A: before 30 min of ischemia, a short period of 5 min ischemia and 5 min reperfusion were given; Group B: before 30 min of ischemia, a short period of 10 min ischemia and 10 min of reperfusion were given; Group C: 30 min ischemia only. The serum alanine transferase (ALT), hepatic Fas-mRNA, caspase-3 activity and hepatocyte apoptosis were analyzed. RESULTS:The 7-day survival rate in the group A and B were 100%, respectively. However, it was only 62.5% in the group C. After 6 h of reperfusion, the ALT levels in both group A and B were significantly lower than that of in group C, P＜0.01. The ALT level of group A was also lower than that of group B, P＜0.01. The hepatic Fas-mRNA expression, caspase-3 activity and apoptotic hepatocyte in group A were significantly lower than those of in group C, P＜0.01. CONCLUSIONS:IPC has significant protective effect against hepatic I/R injury. An IPC with 5 min of ischemia and 5 min of reperfusion has the maximal protective effect. The protective mechanism of IPC against hepatic I/R injury is via down-regulation of Fas-mRNA expression, inhibiting caspase-3 activity and subsequently inhibiting hepatocyte apoptosis.     

Effects of nitric oxide and endothelin on relaxation and contraction of isolated splanchnic vascular strips in cirrhotic rats

AIM: To investigate the different vasoactive effects of nitric oxide (NO) and endothelin (ET) on splanchnic arterial and venous vessels in cirrhotic rats. METHODS: Cirrhosis was induced in Wistar rats by subcutaneously administration of carbon tetrachloride. Maximal relaxation (Rmax) and contraction (Cmax) to NO and ET were determined in vitro using isolated vascular strips prepared from portal vein (PV) and mesenteric artery (MA) of both cirrhotic and normal rats, and EC50 was calculated for effects of NO and ET, respectively. RESULTS: Rmax of PV and MA to sodium nitroprusside (SNP) (releasing NO) were significantly higher in cirrhotic rats (n=8) than those in normal rats (n=7), and EC50 of NO were dramatically lower in cirrhotic rats than those in control (P<0.05,P<0.01). Cmax of PV and MA to ET were significantly decreased in cirrhotics compared with control, and EC50 of ET were obviously increased in cirrhotic rats compared with normal rats (P<0.05,P<0.01). Furthermore, there were significant differences in Rmax, Cmax and EC50 to NO and ET between PV and MA in both of cirrhotic and normal rats, but these differences in cirrhotics were greater than those in control (P<0.05 or P<0.01). CONCLUSION: There are significant different vasoactive effects of NO and ET on splanchnic arterial and venous vessels in cirrhotic rats, and it may play a crucial role in the pathogenesis of portal hypertension.     

Study on the relationship between melatonin and hepatic encephalopathy

AIM:To explore the relationship between change of serum melatonin (MT) and pathogenesis of hepatic encephalopathy (HE). METHODS: Changes of MT level in sera of cirrhosic patients with HE and without HE were determined by ELISA, normal serum served as control. The change of serum MT level in exacerbation and remission in HE was also determined.RESULTS:MT level in patients with HE was higher than that withour HE (P＜0.01). MT levels of both groups were higher than that of normal group (P＜0.01). They were (308.53±59.07) ng/L, (139.85±34.59)ng/L,(77.73±28.41)ng/L, respectively. Serum MT level in exacerbation was higher than that in remission (P＜0.01), they were (301.52±66.42)ng/L and (147.81±23.31) ng/L, respectively. CONCLUSION: The elevation of MT content in sera may be closely related to the onset of hepatic coma.     

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.     

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.     

The kinetic alteration of nitric oxide formation in the lungs in the development of pulmonary fibrosis of rats

AIM: To observe the kinetic alteration of nitric oxide formation in the lungs in the development of pulmonary fibrosis in the rat. METHODS: The contents of hydroxyproline in the lungs, NO₂^-/NO₃^- (nitrite/nitrate) in out-flowing and in-flowing pulmonary blood (OPB, IPB) were assayed on the day 7, 14, 21, 30 and 70 after intratracheal administration of bleomycin A₅ . The content of NO₂^-/NO₃^- in supernatants of culture of the alveolar macrophages (AMs) and the amount of iNOS positive stain cells in lung tissue section were also observed in the rat on 14th day after-bleomycin A₅ administration. RESULTS: The content of lung hydroxyproline had no change on the 7th day, increased on the 14th day (P＜0.05), increased significantly on the 21th day, 30th day and 70th day post-bleomycin A₅ compared with control rats. On the 7th day and 14th day, the content of NO^- ₂ /NO₃^- increased in OPB and decreased in IPB (P＜0.01). On the 21th day, the content of NO₂^-/NO₃^- abated in OPB (P＞0.05) but still decreased in IPB (P＜0.01). On the 30th day and the 70th day, the NO₂^-/NO₃^- level recovered both in OPB and IPB. AMs from rats on the 14th day post-bleomycin A₅ showed significant elevation (P＜0.01) in NO₂^-/NO₃^- level. The amount of iNOS positive stain cells increased in rats on the 14th day post-bleomycin A₅. CONCLUSION: The amount of NO in the lungs was high in the initial phase of fibroproliferative reaction induced by bleomycin A₅ ,and these might be associated with the enhanced ability of AMs to release NO and the increased amount of iNOS.     

Effect of L-arginine on nitric oxide and brain edema after subarachnoid hemorrhage in rats

AIM:To investigate the role of nitric oxide in the development of brain edema after subarachnoid hemorrhage(SAH), and the influence of L-arginine on them. METHODS:Noncraniotomy models of SAH in Wistar rats were used and animals were divided into sham-operated group, SAH group and SAH plus L-arginine group. Dynamic changes of regional cerebral blood flow within 24 hours were measured. Serum nitric oxide level, brain water and sodium content at different time points within 24 hours were also detected. RESULTS:Regional cerebral blood flow and serum nitric oxide level at every time point after operation in SAH group were lower than those in sham-operated group, while brain water content and sodium content in the former group were higher than those in the latter group. Above pathological alterations in SAH plus L-arginine group were not so obvious as in SAH group.CONCLUSION:Decrease in serum nitric oxide plays a role in the development of brain edema after SAH, which may be partly reversed by administration of L-arginine.     

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.     

Effect of hyperlipidemia on nitric oxide levels of blood and myocardial tissues in rats

AIM: To study the impact of hyperlipidemia on vasoactive substances and the mechanism of gemfibrozil in regulating the endothelial function. METHODS: The hyperlipidemic model was established with rats. The serum levels of lipid, NO, angiotensin Ⅱ (Ang Ⅱ), vascular endothelial growth factor(VEGF) and levels of NO, AngⅡ in myocardial tissues were measured. RESULTS: Hyperlipidemic rats had lower level of NO and higher level of VEGF than control subjects. Significant correlation had been shown between serum levels of lipid and NO. Administration of gemfibrozil significantly elevated serum VEGF, NO and myocardial tissue NO levels. CONCLUSIONS: Hyperlipidemia not only reduced serum NO level but also reduced myocardial NO content, which could cause endothelial dysfunction. Gemfibrozil reversed the above changes induced by hyperlipidemia, which might have relevance to VEGF.     

Effects of nitric oxide on mitochondrial damage caused by exogenous calcium

AIM: To study the effects of nitric oxide (NO) on mitochondrial damage caused by exogenous calcium. METHODS: Normal myocardial mitochondria were divided into three groups; L-arginine control group (CG), Ca^2＋-damaged group (DG) and L-NAME-preserved group (PG). Mitochondria of all groups were incubated at 30 ℃ with reaction medium containing 20 μmol/L EDTA, 100 μmol/L CaCl₂ and 1 μmol/L L-NAME with 100 μmol/L CaCl₂ respectively. Then the NO₂^-/NO₃^- contents, mitochondrial viability and membrane potential were investigated. RESULTS: The NO₂^-/NO₃^- contents of DG was obviously higher than that of CG and PG, meanwhile, there was no obvious difference between CG and PG. Mitochondrial viability and membrane potential of DG were significantly lower than that of CG and PG, and negatively related to NO^-₂/NO^-₃ contents (r=-0.5297, P＜0.01; r=-0.6041, P＜0.01). But, the mitochondrial viability and membrane potential of PG were still lower than that of CG. CONCLUSION: Exogenous calcium could activate mitochondrial nitric oxide synthase resulting in NO production and the latter play an important role in decreasing mitochondrial viability and membrane potential.     

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 nitric oxide and peroxynitrite on lung injury induced by ischemia-reperfusion of hind limbs in rats

AIM:To observe the changes in nitric oxide(NO) and peroxynitrite anion (ONOO^- ) in the injuried lung following the ischemia-reperfusion of hind limbs and evaluate the contribution of NO and ONOO^- to tissue injury.METHODS:A model of hind limbs ischemia was made by clamping infrarenal aorta with a microvascular clip and lung injury occurring after reperfusion.Lung t issue was obtained from the animals received sham operation(group 1),4 hours ischemia without reperfusion(group2),1 hour reperfusion following 4 hours ischemia(group3)and 4 hours reperfusion fol owing 4 hours ischemia(group4).The contents of MDA,NO₂^-/NO₃^- and the activities of SOD in the lung were examined.Immunohistochemical echnique was used to determine the immunoreactivity to iNOS and nitrotyrosine(NT)-a specific "footprint" of peroxynitrite.RESULTS:Compared with group1 and group2,the contents of MDA and NO₂^-/NO₃^- increased significantly (P<0.05) and the activities of SOD decreased markedly(P＜0.05) in group3 and group4.Immunohistochemical examination demonstrated intense staining for iNOS and NT throughout the lung in group3 and group4.CONCLUSION:NO and ONOO^- are involved in oxidant-mediated lung injury following reperfusion of ischemic hind limbs.     

Effect of NG-nitro-L-arginine on mitochondria in acute lung injury induced by LPS in rats

AIM: To examine the effect of nonselective nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NA), on mitochondria from acute lung injury induced by lipopolysaccharides(LPS) in rats. METHODS: The rats were randomly divided into control group, LPS injury group and L-NA treatment group. The model of acute lung injury was prepared with injection of LPS in rats. L-NA was respectively administrated through intraperitoneal injection at 3 h after injury induced by LPS. The rats were killed and the mitochondria in lung tissues were isolated by differential centrifugation. The activities of T-NOS, iNOS, ATPase, SOD and GSH-Px, and the contents of NO and MDA from mitochondria were respectively measured. The changes of ultrastructure in lung mitochondria were examined by electronic microscope after injury and L-NA treatment. RESULTS: The activities of T-NOS and iNOS were significantly increased, the activities of ATPase, SOD and GSH-Px were significantly decreased, the contents of NO and MDA were increased after acute lung injury. L-NA significantly enhanced the activities of ATPase, SOD and GSH-Px, and markedly decreased the contents of NO and MDA and the activities of T-NOS and iNOS. CONCLUSION: L-NA inhibits the activity of NOS in mitochondria, decreases the production of NO, improves mitochondria energy pump, ameliorates oxidative injury, and effectively protects lung tissue against acute lung injury induced by LPS.     

Changes in L-arginine/nitric oxide pathway of the aorta in septic shock rats

AIM:To observe the change of nitric oxide (NO) generation system in the vascular adventitia, media and intima in septic shock rats.METHODS:The septic shock model was made in rats by caecal ligation and puncture. The intima, media and adventitia of the rat aorta were separated. NO production (NO₂^-), nitric oxide synthase(NOS) activity and L-arginine (L-Arg) transport were measured, separately. Inducible NOS (iNOS) distribution was detected by immunohistochemistry.RESULTS:Both in early and late stage of septic shock, NO₂^- from the intima was decreased by 66.1% and 78.9%(P<0.01), while NO₂^- from the media was increased by 1.1 and 2.2 folds(P＜0.01), and the adventitia 9.6 and 18.6-fold (P＜0.01), as compared with the sham group, respectively. The changes of NOS activity and the L-arginine transport in the intima, the media layer and the adventitia of the aorta in the septic shock rat paralleled with that of NO₂^- in these tissues. The results of iNOS immunohistochemistry showed that there were obviously positive staining in the media layer and adventitia, especially the adventitia of the rat aortas in septic shock, as compared with that in the sham control.CONCLUSIONS:During septic shock, NO production in the aortic intima was progressively suppressed. However, it was progressively increased in the aortic medial layer and adventitia, especially the adventitia with shock processes. These changes result from different changes of L-arginine transport, NOS activity and its expression in three layers of the aorta from the septic shock rat.     

Phenytoin inhibited changes in nitric oxide of rat hippocampus induced by stress

AIM: To investigate the changes in nNOS and iNOS expression of hippocampal CA3 pyramidal neurons and NO₂^-/NO₃^- level of hippocampal homogenate of rats induced by stress, and to explore the effect of phenytoin on them. METHODS: Rats were subjected to forced-swimming stress, phenytoin was administered(ip) at 30 min before stress. Using the immunohistochemistry and the computerized image technique, the expression levels of nNOS and iNOS of rat hippocampal CA3 pyramidal neurons were assayed quantitatively, and the NO₂^-/NO₃^- level of hippocampal homogenate was also measured using nitric acid deoxidize enzyme method. RESULTS: The nNOS average grey degree of hippocampal CA3 pyramidal neurons was significantly lower in stress group (155.42±3.77)than that in control group(164.54±4.62)and in stress plus phenytoin group(164.27±2.55)(P<0.01); The iNOS relative sectional area proportion of hippocampal CA3 pyramidal neuron was significantly larger in stress group(5.87%±2.90%) than that in control group (0.90％±0.89%) and in strers plus phenytoin groups (0.90%±0.88%)(P<0.01); The NO₂^-/NO₃^- level of hippocampal homogenate was significantly higher in stress group(42.75 umol/L±14.49 umol/L)than that in control group(21.23 umol/L±6.99 umol/L)and in stress plus phenytoin group(18.40 umol/L±8.11 umol/L)(P<0.01). CONCLUSION: It is suggested that the stress could induce nNOS and iNOS expression in CA3 pyramidal neurons and excessive production of NO in hippocampus of rats, which could be inhibited by phenytoin.     

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.     

The cytotoxicity of nitric oxide induced by inflammatory cytokine in combination with LPS in endothelial cells

AIM: To explore the mechanism underlying inducible nitric oxide (NO) caused injury of endothelial cells during inflammation. METHODS:The activity of iso-enzymes of NO synthase (NOS), NO level and iNOS expression were examined using NADPH method, Griess reaction and RT-PCR, respectively. Furthermore, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content were also measured. RESULTS:Co-administration of cytokines (TNF-α 5×10⁵ U/L, IL-1β 2×10⁵ U/L, INF-γ 2×10⁵ U/L) and LPS (10 mg/L) caused an obvious increase in NOS activity, NO levels (about two-fold) and a significant injury of the cells. At the same time, a significant increase in iNOS mRNA was also detected. Wheareas, treatment of the cells separately with cytokines or LPS for 24 h had no significant effect on NOS activity and NO level in cell lysates, however, it caused a significant increase in LDH release and MDA content. Also, the effect of cytokines and LPS on cell viability was concentration-and time-dependent. L-NMMA, a inhibitor of NOS, can suppress inducible NO production and protect cells against NO induced injury. CONCLUSION:Co-administration of cytokines (TNF-α, IL-1β and INF-γ) and LPS significant activated iNOS and NO production which, in turn, induced oxidative reaction in endothelial cells.