Effect of Scutellaria barbata flavonoids on abnormal changes of Bcl-2, Bax,Bcl-xL and Bak protein expression in mitochondrial membrane induced by composite Aβ25-35

AIM：To study the abnormalities of Bcl-2, Bax, Bcl-xL and Bax in the cell mitochondrial membrane of cerebral cortex in the rats injected with β-amyloid beta protein 25-35 (Aβ25-35) in combination with aluminum trichloride (AlCl3) and recombinant human transforming growth factor β1 (rhTGF-β1) (composite Aβ25-35) into lateral cerebral ventricle, and to explore the intervention of Scutellaria Barbata flavonoids (SBF). METHODS：The male SD rats were used to establish the neuronal damaged model by receiving injection of RhTGF-β1 1 μL (10 ng) on day 1 of operation, and then from day 2 of operation, intracerebroventricular injection of Aβ25-35 (4 μg/d, consecutive 14 d) in the morning and 1% AlCl3 in the afternoon (3 μL/d, consecutive 5 d). On the day 49 of operation, the successful model rats were randomly divided into model control and 3 doses of SBF groupss. The rats in SBF groups were daily orally administered with SBF at doses of 35, 70 and 140 mg/kg for 36 d. All the rats were decapitated 60 min after the last administration. The protein expression of Bcl-2, Bax, Bcl-xL and Bak in rat cerebral cortex cell mitochondrial membrane was detected by Western blotting. RESULTS：The composite Aβ25-35 dramatically caused decreases in Bcl-2 and Bcl-xL (P<0.05), and increases in Bax and Bak (P<0.01) in rat cerebral cortex cell mitochondrial membrane. However, oral treatment with SBF for 36 d reversed the above disorders induced by composite Aβ25-35. CONCLUSION：The composite Aβ25-35 induces the expression abnormalities of Bcl-2, Bax, Bcl-xL and Bak in mitochondrial membrane and SBF reverses the above disturbances of apoptotic factors.     

Scutellaria barbata flavonoids inhibits NFT aggregation and regulatory mechanism in rats induced by composited Aβ

AIM: To investigate the effects of Scutellaria barbata flavonoids (SBF) on neurofibrillary tangle (NFT) aggregation, tau protein phosphorylation and the regulated mechanism of glycogen synthase kinase (GSK) 3β and protein phosphatase (PP) 2A in the rats induced by amyloid β protein 25-35 (Aβ_25-35) in combination with AlCl₃ and recombinant human transforming growth factor (RHTGF)-β1(composited Aβ). METHODS: The male SD rats were used to establish the simulated Alzheimer disease (AD) model by intracerebroventricular injection of composited Aβ. The Morris water maze was applied for screening the successful model rats with learning and memory deficits. The successful model rats were daily and orally administrated with SBF at doses of 35, 70 and 140 mg/kg or positive control drug Ginkgo biloba leaves flavonoids (GLF) at 140 mg/kg for 37 d. The silver nitrate staining was used to determine the cortical NFT. The protein levels of total tau, phosphorylated protein of tau at Ser199 and Ser214 sites, GSK3β and PP2A in hippocampus and cortex were determined by Western blot. The mRNA expression of GSK3β and PP2A in the hippocampus and cortex was detected by RT-PCR. RESULTS: Compared with sham group, the cell number of positive NFT with silver nitrate staining in model rat cerebral cortex was significantly increased. The protein levels of phosphorylated tau protein at Ser199 and Ser214 sites, GSK3β in the hippocampus and cerebral cortex in the model rats dramatically elevated, and PP2A was marked decreased as compared with the sham group rats. Meanwhile, the mRNA expression of GSK-3β significantly increased but PP2A was decreased. However, these above abnormalities were differently attenuated by treating with SBF at different doses or GLF at 140 mg/kg for 37 d. CONCLUSION: SBF suppresses the NFT aggregation by inhibition of the regulatory functions of GSK-3β and PP2A, thus reducing the phosphorylation of tau protein.     

Aortic expression of HSP22, TNF-α and eNOS in rats with hyperlipidemia and effects of atorvastatin

AIM:To establish a rat hyperlipidemia model for studying the aortic expression of heat shock protein 22 (HSP22), tumor necrosis factor alpha (TNF-α) and endothelial nitric oxide synthase (eNOS) and the effect of atorvastatin intervention. METHODS:Hyperlipidemia model was established in SD rats. Afterwards, the rats were divided into normal control group, high fat group and high fat+atorvastatin intervention group. The expression of HSP22 and TNF-α in the rat aortas was detected by immunohistochemical assay and the expression of eNOS was assessed by Western blotting. RESULTS:No detectable expression of HSP22 and TNF-α in the normal control group was observed. However, the expression of HSP22 and TNF-α was positive in the high fat group and the atorvastatin intervention group. The mean densities of HSP22 and TNF-α positive particles were significant lower in the atorvastatin intervention group as compared with high fat group (both P＜0.05). The expression of eNOS protein in the high fat group and atorvastatin intervention group was significantly lower than that in normal control group (P<0.01). However, no marked difference of eNOS protein expression between high fat group and atorvastatins intervention group was observed. CONCLUSION: The expression of HSP22 and TNF-α in the rat aortas is increased in the hyperlipidemia rat model. This effect can be restored by atorvastatin treatment. The expression of eNOS in the rat aortas is decreased in the hyperlipidemia rat model, but this tendency could not be attenuated by atorvastatin.     

Liver protection by Sini decoction in hemorrhagic shock and its mechanism relating to oxygen free radical and nitric oxide

AIM:The work was designed to explore protective effects of a traditional Chinese medicine-sini decoction (SD) on liver in hemorrhagic shock and its mechanism relating to oxygen free radical and nitric oxide.METHODS:Anesthetized Wistar rats were subjected to a hemorrhagic shock protocol for 60 min followed by intravenous injection with normal sodium chloride solution or SD solution. Superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (NO) in liver were examined. The inducible nitric oxide synthase (iNOS) was determined immunohistochemically. RT-polymerase chain reaction (RT-PCR)was used to assay the mRNA, which were corresponding to eNOS (endothelial nitric oxide synthase) and iNOS.RESULTS:The activity of SOD decreased, while the concentration of MDA increased in liver during hemorrhagic shock. SD enhanced SOD activity and inhibited a increase in MDA level in liver (P＜0.01). The NO concentrations in liver in SD group increased at three hours after resuscitation (P<0.01). In addition, it was found that the expression of iNOS was upregulated in sodium chloride-treated group, while SD upregulated the expression of eNOS.CONCLUSION:SD reduces the liver injury caused by oxygen free radicals during hemorrhagic shock. The increasing NO concentration by SD is through upregulation of endothelial NOS expression.     

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.     

Effect of caveolin-1 expression on high-salt diet-induced endothelial dysfunction in type 1 diabetic rats

AIM: To investigate the underlying mechanisms responsible for endothelial dysfunction of type 1 diabetes mellitus (DM) rats fed with high-salt diet. METHODS: Type 1 DM was induced by intraperitoneal injection of streptozotocin (70 mg/kg). Normal and diabetic rats were fed high-salt food (HS, 8% NaCl) and standard food for 6 weeks, respectively. Isometric tension of the mesenteric arteries were measured. The expression of Akt, endothelial nitric oxide synthase (eNOS) and caveolin-1 (Cav-1) was examined by Western blot. RESULTS: The rats in DM+HS group exhibited more pronounced impairment of vasorelaxation to acetylcholine and insulin compared with either DM group or HS group (P<0.01). Akt and eNOS phosphorylation levels, and nitric oxide (NO) concentration in DM+HS group were significantly lower than those in DM group (P<0.01). The level of Cav-1 in DM+HS group was significantly higher than that in DM group and HS group. CONCLUSION: Impaired endothelial Akt activation, increased Cav-1 expression and resultant decreased eNOS activation contribute to aggravate high-salt diet-induced endothelial dysfunction and hypertension in DM rats.     

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.     

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.     

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.     

Nitric oxide production,NOS activity and expression in pulmonary arterioles of rats with chronic hypoxic hepercapnic pulmonary hypertension

AIM: To clarify the role of nitric oxide (NO) system in development of chronic hypoxic hypercapnic pulmonary hepertension. METHODS: Male Sprague-Dawley rats were randomly divided into control group and hypoxic hypercapnic group. NO content of plasma was determined, constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) were examined using the technique of immunohistochemistry, expression of cNOS mRNA and iNOS mRNA of arteriole were detected by in situ hybridization. RESULTS: Plasma NO concentration, cNOS activity and cNOS mRNA expression in arteriole of chronic hypoxic hypecapnic group were significantly lower than that of control group (P<0.01); activity of iNOS and expression of iNOS mRNA in arteriole showed significantly higher compared with control. CONCLUSION: The disturbance of NO production and NOS expression in arteriole are involved in hypoxic hypercapnic pulmonary hepertension.     

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

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

Puerarin pretreatment protects human umbilical vein endothelial cells against hypoxia/reoxygenation injury via increasing protein expression of endothelial nitric oxide synthase

AIM: To investigate the protective effects of puerarin (PUE) pretreatment on hypoxia/reoxygenation (H/R) injury in human umbilical vein endothelial cells (HUVECs), as well as its possible mechanism and the signal transduction pathways involved. METHODS: HUVECs were randomly divided into normal control group, H/R group, PUE pretreatment group and PUE+H/R group (1.0×10^-3 mol/L, PUE pretreated the cells for 24 h before H/R). The protein expression of endothelial nitric oxide synthase (eNOS) was measured by Western blot. The activity of constitutive NOS (cNOS) was determined via chemical colorimetric methods. Apoptosis of HUVECs was detected by TUNEL assay. In addition, the cells were treated with ERK inhibitor U0126 (1.0×10^-5 mol/L) or PKB/Akt inhibitor LY294002 (5.0×10^-5 mol/L) for 1 h before PUE pretreatment, and then H/R was performed.RESULTS: Compared with control group, H/R decreased the protein expression of eNOS (P<0.05), and PUE pretreatment up-regulated it (P<0.05). This effect of PUE was inhibited by U0126 or LY294002 (P<0.05). Compared with control group, the activity of cNOS decreased in H/R group (P<0.05), while it increased after PUE pretreatment (P<0.05). Compared with control group, the apoptotic index significantly increased in H/R group (P<0.01). PUE pretreatment reduced the apoptotic index (P<0.01). CONCLUSION: H/R decreases the protein expression and enzyme activity of eNOS in HUVECs, and induces apoptosis of HUVECs. PUE pretreatment up-regulates the protein expression and enzyme activity of eNOS, and reduces the apoptosis of HUVECs with H/R injury. The protective effect of PUE might be through increasing eNOS protein expression via ERK1/2 and PKB/Akt signaling pathways.     

Roles of protein phosphatase 4 in down-regulation of eNOS Ser633 phosphorylation induced by palmitic acid in human umbilical vein endotheli?al cells

AIMTo investigate the roles of protein phosphatase 4 (PP4) in down-regulation of endothelial nitric oxide synthase (eNOS) Ser633 phosphorylation induced by palmitic acid (PA). METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with PA at 25 μmol/L, 50 μmol/L, 100 μmol/L and 200μmol/L for 36 h, or treated with PA at 100 μmol/L for 12 h, 24 h, 36 h and 48 h. Protein phosphatase 2A (PP2A) family inhibitor fostriecin (FST, 20 nmol/L) or okadaic acid (OA, 5 nmol/L) was selected to pretreat the HUVECs for 30 min. Protein phosphatase 4 catalytic subunit (PP4c) siRNA or protein phosphatase 2A catalytic subunit (PP2Ac) siRNA was transfected into the HUVECs. The protein expression levels of of eNOS, PP4c and PP2Ac, as well as the level of eNOS Ser633 phosphorylation, were detected by Western blot. The intracellular nitric oxide (NO) content was measured by DAF-FM DA. RESULTS(1) Compared with control group, the levels of eNOS Ser633 phosphorylation were decreased in PA groups in which the HUVECs were treated with 25 μmol/L, 50 μmol/L, 100 μmol/L and 200 μmol/L PA for 36 h (P<0.05) and 100 μmol/L PA for 24 h, 36 h and 48 h (P<0.05). No significant difference in the level of total eNOS protein expression among all the groups was observed. (2) Compared with control group, both FST and OA pretreatment reversed the reduction of eNOS Ser633 phosphorylation (P<0.05) and the decrease in intracellular NO content (P<0.05) induced by PA. No significant difference in the level of total eNOS protein expression among all the groups was observed. (3) Compared with si-Control group, the PP4c protein expression was significantly reduced (P<0.05), while the level of eNOS Ser633 phosphorylation was significantly increased in si-PP4c group (P<0.05). Although the levels of PP2Ac protein expression declined significantly (P<0.05), the level of eNOS Ser633 phosphorylation remained unchanged in si-PP2Ac group. No significant differencein the level of total eNOS protein expression among all the groups was found. CONCLUSION PA significantly reduces the level of eNOS Ser633 phosphorylation and the content of NO in the HUVECs, which may be due to PA inducing the activation of the PP2A family member PP4 rather than PP2A.     

Role of P2Y1 receptor in activation of astrocytes induced by Aβ25-35

AIM: To study the role of P2Y₁ receptor in the activation of astrocytes induced by Aβ_25-35.METHODS: Astrocytes were isolated and cultured from newborn Wistar rats and divided into control group, Aβ_25-35 group, MRS2179(P2Y₁receptor inhibitor)+Aβ_25-35 group and MRS2179 group by treating the cells with the corresponding reagents. The expression levels of GFAP and P2Y₁ were determined by the methods of immunohistochemistry, immunofluorescence and Western blotting.RESULTS: No significant change of the astrocyte numbers in all groups was observed. Compared with the control cells, the fluorescence intensity of GFAP significantly increased in Aβ_25-35 group and decreased in both MRS2179+Aβ_25-35 group and MRS2179 group. The expression level of GFAP determined by Western blotting and immunofluorescence showed the similar trend of change in each group. Compared with control group, the expression of P2Y₁ in Aβ_25-35 group was significantly increased (P<0.05), and no significant change between MRS2179+Aβ_25-35 group and MRS2179 group was found (P>0.05).CONCLUSION: Aβ_25-35 activates astrocytes by activation of P2Y₁ receptor.     

Astragalus polysaccharides protects against free fatty acid-induced human vascular endothelial cell dysfunction via AMPK-eNOS pathway

AIM: To study the protective effect of Astragalus polysaccharides (APS) on free fatty acid-induced injury in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs were divided into control group, APS group [APS (200 mg/L) treated for 24 h], free fatty acid group [free fatty acid (0.25 mmol/L) treated for 24 h], free fatty acid plus APS group [free fatty acid (0.25 mmol/L) and APS (200 mg/L) treated for 24 h], and compound C group [free fatty acid (0.25 mmol/L) and APS (200 mg/L) and AMPK inhibitor compound C (10 μmol/L) treated for 24 h]. The cell viability was detected by MTT assay. Nitric oxide (NO) content in the medium was determined by nitrate reductase assay. The protein levels of total adenosine monophosphate-activated protein kinase (AMPK), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), endothelial nitric oxide synthase (eNOS) and phosphorylated endothelial nitric oxide synthase (p-eNOS) were measured by Western blot. RESULTS: No significant difference of all indexes between APS group and control group was observed. The cell viability in free fatty acid group decreased significantly compared with control group. The cell viability in free fatty acid plus APS group was significantly improved as compared with free fatty acid group. The cell viability in compound C group was almost the same as that in free fatty acid group. The No content and protein levels of p-AMPK and p-eNOS in free fatty acid group decreased obviously as compared with control group, while the NO content and protein levels of p-AMPK and p-eNOS in free fatty acid plus APS group increased obviously compared with free fatty acid group. No significant difference of the p-AMPK and p-eNOS protein levels between free fatty acid plus APS group and free fatty acid group was observed. No significant difference of the AMPK and eNOS protein levels in all groups was found. CONCLUSION: APS attenuates the free fatty acid-induced injury, and its mechanism is related to the AMPK-eNOS signal pathway.     

PPARs ligands antagonizes Ang-induced decrease in endothelial NO production by upregulating eNOS expression

AIM: We hypothesized that PPARγ ligands stimulate endothelial-derived nitric oxide (NO) release to protect the vascular wall. Thus, the purpose of this study is to investigate the effects of ciglitazone (Cig) and fenofibrate (Fen) on angiotensin Ⅱ (AngⅡ)-induced decrease in endothelial NO synthase (eNOS) expression and NO production in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were preincubated for 24 h with Cig (10-7, 10-6, 10-5, 10-4 mol/L) or Fen (10-5 and 10-4 mol/L), then incubated for 12 h with 10-7 mol/L AngⅡ. Total RNA was extracted, and the expression of mRNA and protein of eNOS was assessed by RT-PCR and Western blotting. NO production was measured by Griees method. RESULTS: In the presence of 10-7 mol/L AngⅡ for 12 h, NO production in cultured HUVECs was decreased (P<0.01). Cig and Fen pretreatments enhanced NO production (P<0.01) and antagonized Ang-induced decrease in eNOS mRNA and protein levels in HUVECs. CONCLUSION: PPARγ activator, ciglitazone, and PPARα activator, fenofibrate, antagonize Ang-induced decrease in endothelial NO production by directly upregulating eNOS expression.     

Eeffect of HMGB1 on expression of NF-κB in BV-2 cells stimulated with Aβ25-35

AIM: To investigate the effect of high mobility group box-1 protein (HMGB1) on the expression of nuclear factor-κB (NF-κB) in BV-2 cells stimulated with amyloid β-protein (Aβ)_25-35. METHODS: Cultured BV-2 cells in logarithmic growth phase were divided into 4 groups:normal cell group (without any treatment), model group (treated with Aβ_25-35 at 40 μmol/L), RNA interference (RNAi) group (conducted with HMGB1-siRNA followed by Aβ_25-35 stimulation) and solvent control group (treated with 0.1% DMSO). After treatment with Aβ_25-35 for 24 h, the protein levels of HMGB1 and NF-κB in BV-2 cells were determined by Western blot. RESULTS: Aβ_25-35 at 40 μmol/L was used to stimulate BV-2 cells. The GFP fluorescence-tagged HMGB1-siRNA (30 nmol/L) was used to transfect BV-2 cells and its transfection efficiency was about 80%~90%. The results of Western blot showed that the protein level of HMGB1 was significantly decreased after the interference of siRNA fragment (P<0.05). The protein levels of HMGB1 and nucleic NF-κB p65 were dramatically increased in BV-2 cells stimulated with Aβ_25-35 (P<0.05). After RNA interference with HMGB1, the expression of HMGB1 and nucleic NF-κB p65 were significantly decreased in BV-2 cells stimulated with Aβ_25-35 (P<0.05). CONCLUSION: RNA interference with HMGB1 reduces the expression of nucleic NF-κB in BV-2 cells stimulated with Aβ_25-35.     

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.