Role of nitric oxide in ischemia/reperfusion injury and ischemic preconditioning

AIM: To clarify the role of nitric oxide(NO) in ischemic preconditioning(IP) and its effects on apoptosis. METHODS: Seventy-two male Wistar rats were divided into the following six groups:ischemia/reperfusion (IR) group,IP group,IR+L-arg group,IP+L-arg group,IR+L-NAME group and IP+L-NAME group,The following changes were measured:cardiac hemodynamic parameters,infarct size,PMNs counting myocardial MPO activity and TUNEL staining.RESULTS: ①L-arg significantly attenuated ischemia/reperfusion-induced heart injury,reduced PMNs infiltration and cardiomyocyte apoptosis.②L-NAME also significantly reduced infarct size,PMNs infiltration and cardiomyocyte apoptosis compared with IR group,however,L-NAME aggravated ischemia/reperfusions-induced cardiac functional injury.③L-arg or L-NAME did not significantly alter the protective effect of ischemic preconditioning. CONCLUSION: Increased production of endogenous NO before prolonged ischemic period can protect hearts and inhibit apoptosis.L-NAME can inhibit iNOS activity and ONOO^- production in reperfusion period to protect heart.     

The protection of the hepatic ischemic preconditioning is concerned with the NO/ET-1 system

AIM: To study the relationship between the disturbance of nitric oxide/endothelin-1(NO/ET-1) and hepatic ischemia/reperfusion(I/R) injury as well as the regulation of NO/ET-1 system by hepatic ischemic preconditioning(IPC). METHODS: The changes of NO/ET-1 system and their relationship with hepatic I/R injury were compared between I/R group and IPC+I/R group in a rat hepatic I/R model. Two hours after reperfusion, the liver tissues were detected by RT-PCR to see whether there was inducible nitric oxide synthase (iNOS) mRNA expression. RESULTS:In the acute phase of hepatic reperfusion, the ratio of NO/ET-1 was reduced, which was due to a significant reduction of NO₂^-/NO₃^- (the metabolic product of NO) and significant elevation of ET-1 in the blood plasma. The content of ALT, AST, LDH and TNF-α in blood plasma, and of MDA in liver tissue were increased but ATP in liver tissue was reduced, the hepatic damage was deteriorated. The protection of the hepatic IPC was concerned with the elevation of the ratio of NO/ET-1 caused by the elevation of NO₂^-/NO₃^-, and reduction of ET-1 as well. There was no iNOS mRNA detected in the liver tissues.CONCLUSION: Hepatic I/R injury is related to the disturbance of NO/ET-1. The protection of the hepatic IPC in the acute phase might be conducted by its regulation of NO/ET-1 system. The cNOS rather than the iNOS generated the NO in this situation.     

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.     

Cardioprotective effect of sevoflurane preconditioning on ROS production in rat heart with ischemia-reperfusion

AIM: To investigate the effects of sevoflurane preconditioning on the production of reactive oxygen species (ROS) and nitric oxide (NO), the activity of superoxide dismutase(SOD), glutathione peroxide(GPx) and catalase(CAT) in myocardial ischemia-reperfusion injury(IRI). METHODS: Sixty rats were randomly allocated to 8 groups. Following 2% sevoflurane preconditioning for 30 min, the left anterior descending artery was ligated for 30 min and then reperfused for 120 min in vivo. The infarction size of the hearts was measured with the staining of 2,3,5-triphenyltetrazoliumchloride. The myocardial apoptotic index was measured by the method of TUNEL. The ROS fluorescent probe dihydroethidium was used for the measurement of ROS. The myocardium was homogenized for the measurement of NO, SOD, GPx and CAT. To evaluate the effects of ROS and NO on the cardioprotection of sevoflurane preconditioning, ROS scavenger N-(2-mercaptopropionyl) glycine (2-MPG) or NOS inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME) were employed to block their actions. RESULTS: Compared with control group, the production of ROS was induced by sevoflurane preconditioning before ischemia-reperfusion injury (12.0±0.8 vs 2.6±0.5, P<0.05) and decreased after ischemia-reperfusion injury (16.2 ±0.9 vs 24.9±1.3, P<0.05). 2-MPG decreased the elevation of ROS caused by sevoflurane preconditioning before ischemia-reperfusion injury (5.1±0.7 vs 12.0±0.8, P<0.05). No difference of ROS production between treating with 2-MPG+Sevo+IRI and with IRI (24.9±1.4 vs 24.9±1.3, P>0.05) was observed. Compared with control group, sevoflurane preconditioning also induced the generation of NO (34.5±3.2 vs 15.9±1.4, P<0.05) and the activity of SOD(1.5±0.5 vs 0.6±0.2, P<0.05), GPx(22.8±2.5 vs 12.7±2.2, P<0.05) and CAT(15.5±1.8 vs 11.2±1.4, P<0.05). 2-MPG blocked the increase in NO production and inhibited the activity of SOD,GPx,CAT. L-NAME also attenuated the activity of SOD,GPx,CAT. CONCLUSION: Sevoflurane preconditioning protects the rat heart against ischemia-reperfusion injury by reducing the infarction size and apoptosis. Production of ROS at sub-injury dose induced by sevoflurane preconditioning stimulates the myocardium to create SOD,GPx,CAT and NO, thus inhibiting the further formation of ROS and protecting the heart under the condition of ischemia-reperfusion.     

Protective effects of total saponins of panax notoginseng on myocardial hypertrophy and fibrosis induced by isoproterenol in rats

AIM: To investigate the protective effects of total saponins of panax notoginseng (PNS) on myocardial hypertrophy and fibrosis induced by isoproterenol (ISO) in rats.METHODS: Myocardial hypertrophy and fibrosis model of rats were induced by injection of ISO (5 mg·kg^-1·d^-1,sc) for 7 days.From day 2,the rats were administered with PNS at dose of 25 and 50 mg·kg^-1·d^-1,ip for 14 days,the control and ISO model group were received saline injection.Then,the heart-weight (HW),left ventricular weight (LVW),the ratio of HW/BW and LVW/BW (LVI) were measured;the hydroxyproline and malondialdehyde (MDA) and angiotensin (AngII) content of left ventricle.The level of nitric oxide (NO),nitric oxide synthase (NOS),superoxide disrnutase (SOD) and glutathione peroxidase (GSH-Px) activities in left ventricle were determined by spectrophotemetry and radioimmunoassay,respectively.RESULTS: Compared with NS control group,the ratio of HW/BW,LVW/BW and the content of hydroxyproline,AngII,MDA and iNOS activity in the left ventricle were significantly increased.The cNOS,SOD,GSH-Px activities and NO content were obriously decreased in the ISO model group.After treatment with PNS,the left ventricular NO content,cNOS,SOD and GSH-Px activities were markedly higher than those in ISO model group.The content of MDA,AngII and iNOS activities and the ratio of HW/BW,LVI were significantly lower than those in ISO model group.CONCLUSION: PNS reverses the myocardial hypertrophy and fibrosis induced by isoproterenol in rats.This effect may be related to eliminating the oxygen free radicals and raising NO level.     

Effects of lipoteichoic acid-induced delayed preconditioning on cardioplegic arrest/reperfusion injury in donor rat heart

AIM: To study the potential effects of lipoteichoic acid (LTA)-induced delayed preconditioning (PC) on cardioplegic arrest/reperfusion injury in donor rat heart. METHODS: The rats were pretreated with LTA (1 mg/kg, ip) 24 h before the experiment, and the isolated hearts were subjected to arrested by cardioplegic solution and stored in Eurocollin's solution for 4 h by the Langendorff method, and to evaluate the changes of cardiac function at the reperfusion for 30 min and 60 min, to measure the amounts of MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH) and total nitric oxide (NO) oxidation products in the coronary effluent, and to detect myocardial apoptosis on tissue samples of left ventricle at the end of reperfusion by TUNEL staining. RESULTS: Pretreated with LTA significantly improved the recovery of cardiac function with a significant increase in coronary flow (CF), left ventricular developed pressure (LVDP), maximal rate of left ventricular developed pressure (+dp/dtmax), and minimal rate of left ventricular decline pressure (-dp/dtmax) at 30 min and 60 min of reperfusion (all P<0.01), reduced CK-MB (P<0.01) and LDH (P<0.01) release and raised the concentrations of NO2-/NO3- in coronary effluent. In addition, LTA pretreatment obviously decreased myocardial apoptosis in left ventricle at the end of reperfusion (P<0.01). The protective effects were abrogated by pretreatment of the rats with L-nitroarginine methyl eater (L-NAME, 10 mg/kg, ip). CONCLUSION: LTA pretreatment significantly improves cardiac function after cardioplegic arrest/reperfusion injury in donor heart of rats, and endogenous NO plays an essential role as an effector of delayed PC induced by LTA.     

iNOS-GC-cGMP signaling activation might be involved in vascular hyporeactivity in endotoxemic rats

AIM: To evaluate the activation of inducible nitric oxide synthase (iNOS)-guanylate cyclase(GC)-cyclic guanosine monophosphate(cGMP) signaling on vascular hyporeactivity in endotoxemic rats. METHODS: Twenty-four SD rats were randomly divided into 4 groups as follows: sham operation group (sham group), lipopolysaccharide group(LPS group), LPS+polymyxin B group (LPS+PMX-B group) and polymyxin B group (PMX-B group). Cannulation of the carotid artery was performed to record mean arterial blood pressure (MABP). The levels of plasma NO, iNOS and TNF-α were detected. The tension of the thoracic aortic rings was measured by a biological analytical system. RESULTS: Compared with sham group, MABP in LPS group was significantly lower (P<0.01), whereas MABP in LPS+PMX-B group was significantly higher than that in LPS group (P<0.05), and no statistical difference of MABP between PMX-B group and sham group was observed (P>0.05). The plasma levels of NO and iNOS in LPS group were significantly higher than those in sham group and LPS+PMX-B group (P<0.01). The contraction of isolated thoracic aortic rings stimulated by phenylephrine and the relaxation response by acetylcholine in LPS group were significantly lower than those in sham group (P<0.01), whereas those in LPS+PMX-B group were significantly improved (P<0.01). The vascular hyporeactivity to vasoconstrictors was completely reversed by pretreatment either with aminoguanidine, a selective iNOS inhibitor, or with methylene blue, an inhibitor of NO-sensitive GC. CONCLUSION: The iNOS-GC-cGMP signaling activation might be involved in vascular hyporeactivity in LPS-induced endotoxemic rats. Polymyxin B partly reverses the vascular hyporeactivity to vasoconstrictors by reducing the level of serum TNF-α, which may be mediated by the iNOS-GC-cGMP signal pathways to attenuate the overexpression of iNOS and NO production.     

Effects of lipopolysaccharide and interleukin 1 receptor antagonist on proliferation and nitric oxide synthesis of mesangial cells

AIM: To investigate the effects of lipopolysaccharide (LPS) and interleukin-1 receptor antagonist (IL-1ra) on mesangial cells proliferation and nitric oxide synthesis. METHODS: Glomerular mesangial cells from SD rats were cultured. The first and second passages of cultured cells were used for the experiment. LPS and LPS plus IL-1ra were added in cell cultures, respectively. By using chemical method the nitrite in supernatants was measured, [³H]-TdR incorporation was determined to evaluate the GMC proliferation. Northern and slot hybridizations were performed to detect the expression of iNOS mRNA. RESULTS: There were expression of iNOS mRNA, more production of nitrite (0.64±0.25 vs 0.12±0.06 nmol/10⁴ cell) in supernatants and GMC proliferation (3735±1177.9 vs 1785±280.6) in LPS group compared to the control. While compared with LPS group, in LPS+IL-1ra GMC group, expression of iNOS mRNA decreased by 40%, nitrite increased (3.28±0.33nmol/10⁴ cell), proliferation of GMC decreased (818±77.27). CONCLUSION: LPS could activate the GMC to express iNOS mRNA and produce more nitrite. IL-1ra could partially inhibit the effects of LPS on the expression of iNOS mRNA in GMC, but not nitrite. There is no synchronous correlation between NO production and GMC proliferation.     

Resveratrol regulates iNOS to inhibit atherosclerosis in C57BL/6J mice

AIM: To investigate the effect of resveratrol on the lipids(CHOL, TG, LDL-C and HDL-C), nitric oxide(NO), peroxynitrite anion(ONOO^-) and the expression of inducible nitric oxide synthase(iNOS) in the artery of the mice with ovariotomy(OVX).METHODS: The lipid levels and NO level in the serum were measured. The changes of atherosclerosis were evaluated with Oil Red O staining. The expression of iNOS was measured by DAB staining and Western blot. The ONOO^- production was measured by DAB staining.RESULTS: Compared with sham group, the levels of the lipids and NO production in OVX+ high fat(HF) group were increased(P<0.05). Compared with OVX+HF group, the levels of the lipids and NO production in resveratrol group were decreased(P<0.05). Fourteen weeks later, the atherosclerosis model was successfully established. Compared with OVX+HF group, the iNOS expression and the ONOO^- production in resveratrol group were decreased(P<0.05), while those in sham group were increased(P<0.05).CONCLUSION: Resveratrol prevents and treats atherosclerosis by inhibiting the iNOS expression in C57BL/6J mice.     

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 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.     

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.     

Role of hydrogen sulfide in acute lung injury induced by ischemia-reperfusion of hind limbs in rats

AIM: To explore the role of endogenous and exogenous hydrogen sulfide (H₂S) in acute lung injury (ALI) induced by ischmia-reperfusion (IR) of hind limbs in rats.METHODS: A Sprague-Dawley rat model of acute lung injury was induced by ischemia of the hind limbs for 4 h and reperfusion for another 4 h. The rats (n=120) were randomly divided into 4 groups: control, IR, NaHS (H₂S donor)+IR, and propargylglycine +IR. The animals were sacrificed after reperfusion. Lung weight/body weight ratio (LW/BW) was measured and calculated. Morphological changes of the lung tissues were observed. The concentrations of H₂S, nitric oxide (NO) and carbon monoxide (CO) in plasma were tested. The content of malondialdehyde (MDA), the activity of CSE, inducible nitric oxide synthase (iNOS) and hemeoxygenase (HO) in the lungs were determined. The polymorpho-nuclear neutrophils(PMN) and protein content in bronchoalveolar lavage fluid(BALF) were also measured. The correlation of H₂S content with the above indices was analyzed.RESULTS: Compared with control group, severe injuries of the lung tissues, raised LW/BW, MDA concentration, PMN and protein contents in BALF were observed in IR group. Limb IR also made a drop in the concentration of plasma H₂S and the activity of lung CSE, while the activity of iNOS and HO in the lung tissues and the levels of plasma NO and CO increased. Administration of NaHS before IR attenuated the changes induced by IR, while pre-administration of PPG exacerbated the IR injuries and increased the plasma NO level and lung iNOS activity. The H₂S content was positively correlated with CSE activity, CO content and HO-1 activity (P<0.01), and negatively correlated with the other indices (P<0.01).CONCLUSION: Down-regulation of H₂S/CSE is involved in the pathogenesis of acute lung injury induced by IR. Endogenous and exogenous H₂S protects against lung injuries. The anti-injury effects of H₂S are related with its anti-oxidative activity to attenuate the inflammatory over-reactions in the lung induced by PMN. Down-regulation of NO/iNOS system and up-regulation of CO/HO-1 system by H₂S are also involved in the process of anti-injury to ALI.     

Effects of external counterpulsation on nitric oxide system in myocardial infarction canines

AIM:To investigate the effects of external counterpulsation(ECP)on nitric oxide(NO)and nitric oxide synthase(NOS)and the expression of NOS gene in myocardial infarction canines.METHODS:Nineteen healthy dogs were randomly divided into three groups ie.controls, ischemia group, ischemia and ECP group.Serum NO concentrations and myocardium NO levels and NOS specific activity were determined by modified nitrate reductase method.T he protein synthesis of sub-type NOS including inducible NOS(iNOS)and endothelial NOS(eNOS)of myocardial tissue were also determined by immunohistochemical method.The constitutive NOS(cNOS)mRNA was measured via in situ hybridization.RESULTS:120 and 180 minutes after the ligat ing of LAD, serum NO concentration in ECP groups were higher than those in ischemic groups(P<0.05).The NO levels and NOS specific activity in myocardium of ischemic dogs were lower than those in controls and ECP group(P<0.05).Protein synthesis of iNOS increased and that of eNOS decreased in ischemic myocardium.But ECP could control the protein synthesis of iNOS, and increase that of eNOS.Further studies showed that the expression of cNOS mRNA decreased in ischemic myocardial tissue, ECP might promote the expression of it and regulate NOS in the gene level.CONCLUSION:The results suggested that it was one of the most important mechanisms through raising the NO levels to protect ischemic myocardium in ECP.     

Effects of nitric oxide on hepatic encephalopathy in cirrhotic rats induced by LPS

AIM: To investigate the effects of nitric oxide (NO) on hepatic encephalopathy in cirrhotic rats induced by LPS. METHODS: The cirrhotic model of rats was established by complex pathogeny. Since the end of the 8 th week, the rats were intragastrically-infused with 0.9% salt, L-arginine(L-arg) and LNNA respectively for 2 weeks.The hepatic encephalopathy in cirrhotic rats were induced by 3 mg/kg LPS (ip) 4 hours before the rats were sacrificed. RESULTS: The normal behaviors and electroencephalograph were appeared in L-arg group. LNNA group showed hepatic encephalopathy. The content of NO₂^-/NO₃^- of brain tissue was markedly higher in L-arg group than LNNA group(P<0.05), but the content of histamine in brain tissue was lower in L-arg group than LNNA group(P<0.05). There was a negative correlation between the content of histamine in brain tissue and the content of NO₂^-/NO₃^- of brain tissue. CONCLUSION: NO can prevent hepatic encephalopathy in cirrhotic rats induced by LPS.     

Effects of taurine-zinc on learning and memory abilities of vascular dementia mice

AIM:To observe the effects of taurine-zinc (TZC) on the learning and memory abilities of vascular dementia (VD) mice and to investigate the related mechanism. METHODS:The mice were randomly divided into model group, sham group, and TZC at 50 mg/kg, 100 mg/kg and 200 mg/kg groups. The mice in drug groups were given TZC by gavage at 10 mL/kg once daily. The mice in sham group and model group were given equal volume of distilled water. VD mice were established by intercepting both common carotid arteries and bleeding at caudal vein after 14 d of gavage. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISA. The levels of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) were measured via spectrophotometer. Step-down test and Morris water maze test were used to examine the abilities of learning and memory in the mice. RESULTS:TZC at 50 mg/kg, 100 mg/kg and 200 mg/kg reduced the levels of TNF-α, IL-1β, iNOS and NO in the brain tissues. In the water maze test, TZC at 100 mg/kg and 200 mg/kg significantly decreased the error times and latency compared with model group. In the step-down test, the escape latency was prolonged and error times were lowered significantly by treatment with TZC at 50 mg/kg, 100 mg/kg and 200 mg/kg as compared with model group. CONCLUSION:TZC improves the abilities of learning and memory, which might be related to the reduction of TNF-α, IL-1β, iNOS and NO levels in VD mice.     

Reversion of the decline of cardiac function in endotoxin shock rats by cholecystokinin octapeptide

AIM: To verify the effect of cholecystokinin octapeptide(CCK-8) on cardiac function in endotoxin shock (ES) rats. METHODS: The rats were divided into four groups:control,lipopolysaccharide(LPS),CCK-8 and CCK-8+LPS. The left ventricle pressure(LVP),the maximal/minimum rate of LVP,heart rate (HR) and mean arterial pressure (MAP) were measured. The activity of superoxide dismutase (SOD),the contents of malondialdehyde (MDA) and nitric oxide (NO) in both serum and myocardium were also measured,respectively. RESULTS: CCK-8 (40 μg·kg^-1, iv) elicited bradycardia in short time and gently increase MAP,LVP and ±LVdp/dt_max. Lipopolysaccharide(LPS, 8 mg·kg^-1, iv) caused a variation in heart rate (HR)(a bradycardia following a tachycardia) and rapid decreases in MAP,LVP and ±LVdp/dt_max. The rapid variation of HR and the decline of MAP,LVP and ±LVdp/dt_max were reversed by pretreatment with CCK-8 in ES rats, but didn't restore to normal. The activity of SOD was increased and the contents of MDA and NO were decreased by pretreatment with CCK-8 in ES rats. CONCLUSION: The decline of cardiac function in ES rats could be reversed by pre-administration of CCK-8 and the decrease in NO production may be one of the mechanisms.     

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.     

Association among COX2, iNOS and p38MAPK in late phase of preconditioning in rat cardiomyocytes

AIM:To explore the association among COX₂ , iNOS and p38MAPK in late phase of preconditioning. METHODS: The expression of COX₂ , iNOS and p38MAPK activity were determined by western blotting and the injury of cardiomyocyte was assessed by LDH release and trypan blue exclusion in four groups: control group, group IPC (ischemic preconditioning), group SMT (iNOS inhibitor), group NS₃₉₈ (COX₂ inhibitor) and group SB (p38MAPK inhibitor).RESULTS:The expression of COX₂ in group IPC increased markedly in comparision with group SMT and group SB.The expression of iNOS in group SB was lower than that in group IPC and group NS₃₉₈.The difference of the amount of iNOS was not significant between group IPC and group NS₃₉₈.The difference of the amount of phospho-p38MAPK was not significant among group IPC, group SMT and group NS₃₉₈(P>0.05).The LDH was lower, and cell viability was higher in group IPC than those in control group.The LDH was higher, and cell viability was lower in group SMT, group NS₃₉₈ and group SB than those in group IPC. CONCLUSIONS:In late phase of preconditioning , the p38MAPK activity and expression of iNOS and COX₂ increase significantly in rat cardiomyocytes, activited p38MAPK mediates iNOS, then promotes COX₂ expression.     

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.