Mechanism of mesenteric lymph duct ligation against the renal insufficiency in hemorrhagic shock rats

AIM: To observe the effect of mesenteric lymph duct ligation on free radical and inflammatory mediator in serious hemorrhagic shock rats at different periods, and explore the mechanism of intestinal lymphatic pathway on renal insufficiency. METHODS: 78 male Wistar rats were divided into the sham group, shock group, and ligation group. The model of serious hemorrhagic shock was established in shock group, ligation group, and mesenteric lymph was blocked by ligating mesenteric lymph duct in ligation group after resuscitating. All rats were executed and kidneys were taken out for making homogenate of 10 percent to determine levels of MDA, SOD, NO, NOS, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and myeloperoxidase (MPO) at time points after shock 90 min, after transfusion and resuscitate 0 h, 1 h, 3 h, 6 h, 12 h and 24 h. The expression of inducible nitric oxide synthase (iNOS) mRNA in kindey was detected by RT-PCR. RESULTS: The contents of MDA, NO, NOS, TNF-α, IL-6, MPO and iNOS expressions in renal homogenate of shock group were increased after transfusion and resuscitation, and were higher at 6 h and 12 h, and was significantly higher than that in sham group. The acvitity of SOD was significantly lower than that in sham group (P<0.01, P<0.05). The contents of MDA, NO, NOS, TNF-α, IL-6, MPO and iNOS expression in renal homogenate of ligation group after transfusion and resuscitation 6 h, 12 h and 24 h were significantly lower than those in shock group at same points, and the SOD activity was higher (P<0.01, P<0.05). CONCLUSION: The results demonstrate that the ligation of mesenteric lymph duct can antagonise the development of renal failure in serious hemorrhagic shock rats, and its mechanism might relate to reduce the PMN sequestration, decrease the levels of TNF-α and IL-6, inhibit NO production and expression of iNOS mRNA, suppress the release of free radical and consumption of SOD.     

Effect of N-acetyl-L-cystein on blood pressure and endothelial function in aorta of rats exposed to chronic intermittent hypoxia

AIM: To investigate the effect of N-acetyl-L-cystein (NAC) on blood pressure and endothelial function in the aorta of the rats exposed to chronic intermittent hypoxia (CIH). METHODS: Thirty healthy male SD rats were randomly divided into 3 groups: control group, CIH group and CIH+NAC group. The systolic blood pressure (SBP) was measured with tail-cuff me-thod. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of endothelial nitric oxide synthase (eNOS） and endothelin-1 (ET-1) in the thoracic aorta. The protein expression of eNOS in the thoracic aorta was examined by Western blotting. The levels of ET-1 in the thoracic aorta and serum were detected by radioimmunoassay. The serum nitric oxide was determined by nitric acid reduction method.The superoxide dismutase (SOD) activity in peripheral blood plasma was detected by xanthine oxidase method.The serum malondialdehyde content was detected by thiobarbituric acid method, and superoxide anion (O-·2) in thoracic aorta was determined by chemical colorimetric method. RESULTS: Compared with the control animals, CIH exposure was associated with decreased SOD level, and NAC-treated CIH animals showed recovery in SOD level. NAC treatment prevented CIH-induced hypertension as well as CIH-induced increase in MDA. The aorta eNOS mRNA and protein, and serum NO levels in CIH group were lower than those in control group, and those in NAC treatment group were higher than those in CIH group. The increases in ET-1 mRNA,ET-1 protein and O-·2 levels in the aorta, and the elevated circulating ET-1 level were also observed in CIH-exposed animals. Treatment with NAC significantly decreased the mRNA and protein levels of ET-1, the O-·2 content, and the circulating ET-1 level in CIH-exposed animals. CONCLUSION: NAC protects endothelial function and alleviates hypertension by suppressing the oxidant stress in the aorta tissues, indicating that oxidant stress may be involved in the mechanism of endothelial disorder of CIH-induced hypertension.     

Protection of the limb ischemic preconditioning on the injury of gut is concerned with NO/ET-1 system in rats

AIM: To study the relationship between disturbance of nitric oxide/endothelin-1 (NO/ET-1) and the injury of gut following limb ischemia-reperfusion (I/R) in rats as well as the regulation of NO/ET-1 system by limb I/R preconditioning (IPC). METHODS: A limb ischemia-reperfusion injury model in rats was established. The animals were randomly divided into three groups: control group, IR group and IPC group. The contents of diamide oxidase(DAO), nitric oxide (NO), endothelin-1 (ET-1) and ratio of nitric oxide/endothelin-1 (NO/ET-1) in the plasma and the gut were measured. The leavels of myeloperoxidase, ratio of DNA chain (%), total nitric oxide synthase (tNOS), inducible nitric oxide synthase (iNOS) and constitutive nitric oxide synthase (cNOS) in the gut were determined. The expression of iNOS and endothelial NOS (eNOS) were detected by the immunohistochemical method. RESULTS: It was found that the levels of NO, ET-1 in the plasma and the gut tissue all increased after reperfusion, while the values of NO/ ET-1 decreased. The values of DAO in the plasma and MPO in the gut increased, while the contents of DAO and the ratio of DNA chain (%) in the gut decreased. The expression of iNOS elevated, cNOS (mainly eNOS) reduced and total NOS increased. The protection of the limb IPC attenuated the disturbance of NO/ET-1. CONCLUSION: The intestinal injury following limb I/R is related to the disturbance of NO/ET-1. The protection of the limb IPC might be conducted by its regulating NO/ET-1 system. The endothelial NOS increases and non-endothelial NOS decreases in this situation.     

Effect of nitric oxide on adiponectin-induced inhibition of platelet aggregation in hyperlipidemia rats

AIM: To investigate the mechanism that adiponectin inhibits platelet aggregation via nitric oxide (NO) signaling pathway. METHODS: Adult rats were fed with normal or high-fat diet for 14 weeks. Their platelets were immediately isolated and treated with or without recombinant full-length adiponectin (rAPN). The platelet aggregation, NO and superoxide production, endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) expression, and antioxidant capacity were determined. RESULTS: Treatment with rAPN inhibited platelet aggregation induced by hyperlipidemia (P<0.05). Interestingly, total NO, a crucial molecule depressing platelet aggregate and thrombus formation, was significantly reduced, rather than increased in rAPN-treated platelets. Treatment with rAPN significantly decreased superoxide production by 62% (P<0.05) and increased antioxidant capacity by 38% (P<0.05) in hyperlipidemic platelets. Importantly, hyperlipidemia-induced reduction of eNOS phosphorylation and increase in iNOS expression were markedly reversed by rAPN treatment (P<0.05 and P<0.01, respectively). CONCLUSION: Adiponectin is an adipokine that inhibits platelet aggregation by enhancing eNOS activation and attenuating oxidative/nitrative stress including blockage of iNOS expression and superoxide production.     

Effect of altitude hypoxia on glutamate,aspartate and NOS in the rat hypothalamus

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. The 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 ligating 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 endothelial nitric oxide synthase gene transfection on cytokines and cAMP production in human phagocytic cells

AIM and METHOD: Human endothelial nitric oxide synthase (eNOS) gene was transfected into human phagocytic cell U937 and the effects of gene transfer on cytokines and cAMP production were observed. RESULTS: A functional eNOS was stably expressed in transfected U937 cells, but NO release was undetectable in intact transfectants. However, eNOS gene expression upregulated tumor necrosis factor-α release and downregulated interleukin-10 and cAMP production in either presence or absence of NOS inhibitor N^ω-monomethyl-L-arginine. CONCLUSION: The function of tranfected eNOS gene product showed cellular speciality. The effector molecule that changed the produced pattern of cytokines and cAMP in phagocytic cells seems not likely the nitric oxide.     

Akt-eNOS-NO signal pathway mediates regulatory effect of Ang-1 and Ang-2 on vascular hyperreactivity in early hemorrhagic shock rats

AIM:To observe the role of endothelial nitric oxide synthase(eNOS) in the regulatory effect of angiopoietin-1(Ang-1) and angiopoietin-2(Ang-2) on the biphasic change of vascular reactivity after hemorrhagic shock in rats. METHODS:The protein expression of eNOS was measured in the superior mesenteric artery(SMA) after hemorrhagic shock by Western blotting. The effect of eNOS inhibitor on the vascular reactivity of SMA treated with Ang-1 and Ang-2 in the early(hyperreactivity) and late(hyporeactivity) periods of hypoxia were observed via an isolated organ perfusion system. The protein levels of eNOS in the hypoxic mixture of vascular endothelial cells(VECs) and vascular smooth muscle cells(VSMCs), and the concentration of nitric oxide(NO) in the medium supernatant of the mixture cells treated with Ang-1, Ang-2 and the inhibitors of Tie-2, Akt, p38 MAPK and ERK were measured. RESULTS:The protein expression of eNOS in SMA was low in normal control group, and increased significantly after hemorrhagic shock, which was 1.84, 3.55, 4.75, 5.96 and 6.33 folds of the normal control level in shock 10 min, 30 min, 1 h, 2 h and 4 h groups, respectively(P<0.01). Inhibitor of eNOS decreased the vascular hyperreactivity in hypoxia 10 min group, in which the E_max of norepinephrine(NE) was decreased from 13.479 mN to 9.043 mN(P<0.05). It also repressed the maintenance effect of Ang-1 on vascular reactivity in hypoxia 10 min group, in wihich the E_max of NE was decreased from 15.283 mN to 11.219 mN(P<0.01). The effect of Ang-2 on the vascular hyperreactivity in hypoxia 10 min group, the vascular hyporeactivity in hypoxia 4 h group, or the effect of Ang-1 or Ang-2 on the vascular reactivity in hypoxia 4 h group did not change. The protein expression of eNOS was increased 10 min after hypoxia as compared with the normal control, which was decreased by Ang-2 and the inhibitors of Tie-2 and Akt(P<0.01), but was not decreased by p38 MAPK and ERK inhibitors. The concentration of NO in the medium supernatant was increased 10 min after hypoxia, and was significantly decreased by Ang-2 and the inhibitors of Tie-2, Akt and eNOS, while the inhibitors of p38 MAPK and ERK had no influence on it. CONCLUSION:Ang-1 and Ang-2 regulate the vascular hyperreactivity in the early hemorrhagic shock rats through Akt-eNOS-NO pathway.     

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 cholecystokinin octapeptide on cerebral cortex injury during endotoxic shock

AIM: To study the effects of cholecystokinin octapeptide (CCK-8) on cerebral cortex injury during endotoxic shock (ES) and its mechanisms. METHODS: Rabbits were injected intravenously with lipopolysaccharide (LPS, 8 mg/kg) to establish ES model. Rabbits (n=32, 8 in each group) were randomly assigned to receive one of four treatments intravenously: normal saline (as control), LPS, CCK-8 pretreatment 30 min before LPS, proglumide (Pro, nonspecific antagonist of CCK receptors) pretreatment 30 min before LPS. The changes of mean arterial pressure (MAP) were measured. The morphologic changes in cerebral cortex were observed through light microscope (LM) and transmission electron microscope (TEM). The alterations of activities of nitric oxide synthase (NOS) and superoxide dismutase (SOD), contents of nitric oxide (NO) and malondialdehyde (MDA) in cerebral cortex were assayed. The expressions of protein of inducible NOS (iNOS) and neuronal NOS (nNOS) were detected by immunohistochemistry staining in cerebral cortex in 4 groups of Sprague-Dawley (SD) rats (n=12, 3 in each group) which were grouped as that of the rabbits. RESULTS: LPS administration resulted in lower MAP than that in control group (P<0.01). Hydropic degeneration of neurons and severe injuries to capillaries were observed in cerebral cortex of ES rabbits. LPS administration induced the expression of iNOS protein in the cytoplasm of neurocytes, and lead to stronger positive signals of nNOS than that in control group. NOS activity, NO2ˉ/NO3ˉ level and MDA content were higher (P<0.05, P<0.01 and P<0.01), while SOD activity was lower in cerebral cortex of ES rabbits than those in control group (P<0.01). CCK-8 pre-administration alleviated the changes induced by LPS, while Pro pre-administration aggravated those alterations. CONCLUSION: CCK-8 protects brain tissues against the injury induced by LPS, which may be associated with its effects of suppressing the overproduction of NO and free radicals.     

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 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.     

Hydrogen sulfide attenuates human umbilical vein endothelial cell senescence via modulation of Sirt1/eNOS pathway

AIM: To explore the role of Sirt1/eNOS signalling pathway in the protective effect of hydrogen sulphide (H₂S) against endothelial cell senescence induced by high glucose.METHODS: High glucose (33 mmol/L) was applied to induce senescence in primary human umbilical vein endothelial cells (HUVECs). The cell viability, the proportion of senescence-associated β-galactosidase (SA-β-Gal) positive cells and the plasminogen activator inhibitor 1 (PAI-1) expression were detected to assess the senescence model. Mean while, Sirt1 siRNA was used to examine the effect of Sirt1 on eNOS expression and the senescence-related parameters.RESULTS: Treatment of HUVECs with high glucose decreased the cell viability slowly with a larger proportion of the cells stained with SA-β-Gal, and the protein expression of PAI-1 was dramatically increased. The increased cell viability, reduced SA-β-Gal positive cells and decreased protein expression of PAI-1 were detected after sodium hydrosulfide (NaHS, 100 μmol/L) treatment. Furthermore, NaHS treatment upregulated the protein expression of Sirt1 and eNOS, and eventually increased the production of nitric oxide (NO).CONCLUSION: Exogenous H₂S modulates Sirt1/eNOS/NO pathway to prevent HUVECs against high glucose-induced senescence.     

Effect of cilazapril on pulmonary vascular endothelial dysfunction in hypoxic rats

AIM:To study the effect of cilazapril on pulmonary vascular endothelial dysfunction in hypoxic rats. METHODS:The structure and function of endothelium in hypoxic rats were studied by biochemical analysis, radioimmunoassay, transmission electron microscope and correlated with hemodynamic. RESULTS:1) The change and damage of ultrastructure in endothelial cell (EC) were obsevered in hypoxic rats. 2) The contents of plasma nitric oxide (NO) and superoxide dismutase (SOD) activity in blood as well as endothelial nitric oxide synthase (eNOS) activity in the lung tissue were significantly lower in the hypoxic rat than those in contral animals. The concentrations of plasma endothelin-1(ET-1) and angiotensin converting enzyme(ACE) as well as malondialdehyde(MDA) were significantly higher in the hypoxic rat than these in contral animals. The relaxing and contracting factors had a significant positive/negative correlation with mean pulmonary artery pressure (mPAP). 3) Cilazapril significantly decreased the level of ET-1 and ACE and significantly increased the level of NO and activity of eNOS and SOD. At the same time, cilazapril extenuated hypoxia-induced injuries of EC. CONCLUSION:The results indicate that damaging structure and dysfunction of EC existes in hypoxic rats. The cilazapril effectively preventes and treates the chronic hypoxic PH by relieving the injury and improving secretion in EC.     

Shear stress regulates NO production in vascular endothelial cells through Pim1/eNOS signaling pathway

AIM: To determine whether laminar shear stress regulates nitric oxide (NO) production in vascular endothelial cells through Pim1/endothelial nitric oxide synthase (eNOS) signaling pathway. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to laminar shear stress using a parallel-plate flow system. NO production is evaluated by NO assay kit. Pim1 protein expression and eNOS phosphorylation were determined by Western blot. A specific small interfering RNA was used to knock down Pim1 gene expression, and then the changes of above indicators were detected. RESULTS: After 15-min exposure of HUVECs to laminar shear stress (15 dyn/cm²), rapid increases in Pim1 protein expression and NO production were observed (P < 0.05). Shear stress also caused time-dependent stimulation of eNOS phosphorylation (P < 0.05). The shear-induced Pim1 expression and NO production were abrogated in the HUVECs transfected with siPim1 (P < 0.05). Pim1 silencing also prevented shear-induced rise of eNOS-Ser1177 phosphorylation (P < 0.05). CONCLUSION: Pim1 may account for shear-induced NO production in endothelial cells due to phosphorylation activation of eNOS.     

Effect of gingko biloba extract on liver from experimental type 2 diabetic rats

AIM:To study the protective effect of the ginkgo biloba (EGB) extract on liver from experimental type 2 diabetic rats and to explore its possible mechanism. METHODS:Thirty-nine male Sprague-Dawley rats were divided randomly into four groups: normal control group, high-fat group, diabetic group and EGB-treated group. After fed with high-fat diet for 4 weeks, the later two groups were injected with streptozotocin intraperitoneally to induce type 2 diabetes mellitus. EGB-treated group was injected intraperitoneally with EGB at a dose of 8 mg·kg^-1·d^-1, and the other three groups were treated with normal saline of the same volume. After 8 weeks, the morphologic change of hepatic tissue was observed under transmission electron microscope (TEM) and light microscope (LM), respectively. In addition, the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), total nitric oxide synthase (TNOS), inducable nitric oxide synthase (iNOS) and the content of malondialdehyde (MDA), nitric oxide (NO) in liver homogenate were detected biochemically. RESULTS:Obvious liver fatty degeneration, apparent decrease of glycogen granules in cytoplasm of hepatocytes under light microscope and hepatocytes pyknosis, lots of lipid deposits in cytoplasm of hepatocytes, proliferation of hepatic stellate cells and collagen under TEM were observed in diabetic group. The activity of SOD, CAT, GSH-PX decreased but the activity of tNOS, iNOS and the content of MDA, NO^-₂/NO^-₃ increased in diabetic group compared with normal control group. The pathological change was relieved in EGB-treated group. The activity of SOD, CAT, GSH-PX increased, the activity of tNOS, iNOS and the content of MDA, NO^-₂/NO^-₃ decreased in the liver of rats in EGB-treated group compared with diabetic group. CONCLUSION:EGB exerts a beneficial effect on liver in experimental type 2 diabetic rats. Anti-lipid peroxidation and suppression of NO production may be involved in this process.     

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

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

Role of PI3K/Akt/eNOS signaling pathway in inhibitory effects of puerarin on ox-LDL-induced TF expression in vascular endothelial cells

AIM:To explore the role of phosphatidylinositiol 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathways in the inhibitory effects of puerarin on oxidized low-density lipoprotein (ox-LDL)-induced tissue factor (TF) expression in vascular endothelial cells.METHODS:The mRNA expression of TF was detected by real-time fluorescent quantitative PCR.The protein levels of TF and Akt was determined by Western blot.The content of the nitric oxide (NO) was measured by nitrate reduction method.RESULTS:Compared with control group,incubating endothelial cells with ox-LDL significantly induced TF expression at mRNA and protein levels and the dephosphorylation of Akt protein,and decreased NO production.Incubation of the endothelial cells with puerarin for 1 h and then treatment of the cells with ox-LDL decreased the TF expression at mRNA and protein levels,increased Akt protein phosphorylation and intracellular NO content.Co-incubation of the endothelial cells with PI3K inhibitor LY294002 and puerarin for 1 h and then treatment of the cells with ox-LDL augmented the TF expression at mRNA and protein levels and the Akt protein dephosphorylation,and decreased NO production.Co-incubation of the endothelial cells with eNOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) and puerarin significantly decreased the inhibitory effect of puerarin on ox-LDL-induced TF expression at mRNA and protein levels in the endothelial cells,and reduced Akt protein phosphorylation and NO production.CONCLUSION:Puerarin inhibits ox-LDL-induced TF expression at mRNA and protein levels in the human umbilical vein endothelial cells via activation of PI3K/Akt/eNOS signaling pathway.     

Renoprotective effects of sesamin on renal hypertensive and hyperlipidemic rats

AIM: To investigate the effects of sesamin on progression of renal injury in renal hypertensive and hyperlipidemic rats (RHHR). METHODS: RHHR was induced by 2K1C and high lipid baitvessel. After 7 weeks of intragastric administration with sesamin, the contents of serum creatinine (Scr), blood urea nitrogen (BUN), 24 h urinary protein excretion (UPE) were measured. In addition, the activity of total antioxidative capacity (T-AOC), superoxide dismutase (SOD), the concentrations of malondialdehyde (MDA), hydrogen peroxide (H2O2), and the nitric oxide synthase (NOS) and nitric oxide (NO) levels in renal homogenate were measured. RESULTS: Compared with the model group, seasamin (in 100 mg·kg-1 and 33 mg·kg-1 groups) evidently decreased the contents of Scr, BUN, UP and the concentration of MDA, iNOS, H2O2 in renal tissure. It also improved the levels of NO, cNOS and activity of SOD, T-AOC in renal tissure. CONCLUSION: Sesamin ameliorates hypertensive and hyperlipidemic-induced renal injury, probably by enhancing antioxidative activity, scavenging hydroxyl radical and restraining iNOS level.     

Erythropoietin inhibits the proliferation of neonatal rat cardiac fibroblasts induced by angiotensin II via PI3-K/Akt signaling pathway

AIM: To investigate the effects of erythropoietin (EPO) on the proliferation of rat cardiac fibroblasts induced by angiotensin Ⅱ(Ang Ⅱ) and to identify the roles of phosphatidylinositol-3-kinase/Akt (PI3-K/Akt) signaling pathway and nitric oxide synthase (NOS) in this process. METHODS: Neonatal rat cardiac fibroblasts (CFs) were isolated by collgenase, trypsinase and technique of differential attachment. EPO, Ang Ⅱ, LY294002 (an inhibitor of PI3-K), and L-NAME (an inhibitor of NOS) were added in related group respectively. Growth curves of CFs were established by cell counting and methyl thiazolyl tetrazolium (MTT). The levels of nitric oxide (NO), and the activities of NOS and its isoforms were measured by chemical enzymic method. The expressions of Akt, p-Akt, endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) were detected by Western blotting. RESULTS: Ang Ⅱ markedly enhanced the proliferation of CFs. The NO level in CFs culture fluid was increased and the proliferation of CFs induced by Ang Ⅱ was suppressed by EPO in a dose dependent manner. After 4 d of administrations, the proliferation ratio of CFs was suppressed 24.4%, 41.5% and 50.5% by EPO at doses of 5×103 U/L, 1×104 U/L and 2×104 U/L respectively. The expressions of phosphated Akt, p-Akt, and eNOS were all up-regulated by EPO. The effect of EPO on NO was blocked by LY294002 and L-NAME, and the suppression of CFs proliferation induced by Ang Ⅱ was diminished similarly. However, LY294002 also down-regulated the expression of eNOS but the L-NAME had no effect on it. CONCLUSION: EPO suppresses the proliferation of neonatal rat CFs induced by Ang Ⅱ in dose dependent manner. The suppressive effects may be due to up-regulating the expression of eNOS and enhancing the production of NO via activating the PI3-K/Akt signaling pathway.