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.     

Effects of atorvastatin on expression of cytochrome P450 hydroaylase in spontaneously hypertensive rats

AIM: To investigate the effects of atorvastatin on blood pressure and expression of cytochrome P450 hydroaylase (CYP) 4A1 in spontaneously hypertensive rats (SHR). METHODS: SHRs (n=18) were randomly divided into three groups: SHR control group, 50 mg atorvastatin (HATV) group and 10 mg (LATV) group. Six male Wistar-Kyoto rats were selected as normal control group (WKY group). All rats were given vehicle once a day by gavage for 10 weeks. Systolic blood pressure (SBP) was measured before and after treatment every 2 weeks. The expressions of CYP 4A1 mRNA and protein in heart, liver, kidney, and aorta were detected by RT-PCR and Western blotting analysis, respectively. Plasma lipids were also measured.RESULTS: SBP in all SHR groups was much higher than that in WKY group before experiment (P<0.01). Compared with SHR control group, SBP significantly decreased in HATV group at 6, 8, 10 weeks and in LATV group merely at 10 weeks (P<0.01 or P<0.05). The expressions of CYP 4A1 mRNA and protein in heart, liver, kidney and aorta tissues of SHR control group were significantly higher than those in WKY group (P<0.01 or P<0.05). After 10 weeks, the levels of CYP 4A1 mRNA, protein and plasma lipids in HATV and LATV groups were markedly lower than those in SHR control group (P<0.01 or P<0.05).CONCLUSION: Atorvastatin down-regulates the expressions of CYP 4A1 mRNA and protein in SHR, which may be the mechanism of the favorable effects of statins on regulation of hypertension.     

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.     

Folic acid reduces monocyte chemoattractant protein-1 expession in aorta of rats with hyperhomocysteinemia

AIM: To investigate the effects of folic acid on the expression of monocyte chemoattractant protein-1 (MCP-1) in aorta of rats with hyperhomocysteinemia induced by ingestion of execess methionine (Met). METHODS: Thirty male SD rats ［(200±20） g］ were divided into 3 groups (n=10 for each group): control group (fed with normal diet), high Met group (fed with normal diet enriched by 1.7% Met) and Met plus folate group (fed with normal diet plus 1.7% Met and 0.006% folate). The animals were fed with different regiments for 45 days. Levels of total plasma homocysteine (Hcy) were detected. The expression of MCP-1 protein in aorta was detected by immunohistochemistry and Western blotting. RESULTS: The high-methionine diet resulted in a significant increase in plasma Hcy levels (P<0.01). Serum Hcy levels were significantly lower in rats fed with high-methionine plus folate-rich diet than that in rats fed with high-methionine diet (P<0.01). The expression of MCP-1 were higher in aorta of rats fed with high-methionine diet than those in control rats (46.41±4.23 vs 15.73±2.74, P<0.05). The expression of MCP-1 was significantly reduced in aorta of rats fed with high-methionine plus folate-rich diet compared with rats fed with high-methionine diet (23.12±4.40 vs 46.41±4.23, P<0.05). CONCLUSION: High methionine diet for 45 days is sufficient to induce hyperhomocysteinemia. Folate supplementation to the rats fed with the high-methenine diet prevents elevation of Hcy levels in blood, and reduces the expression of MCP-1 in aorta of rats with hyperhomocysteinemia.     

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 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.     

PP2A activation in mesenteric arteries of angiotensin Ⅱ-induced hypertensive rats

AIM: To investigate the mechanism of protein phosphatase 2A (PP2A) activation in mesenteric arteries of angiotensinⅡ(AngⅡ)-induced hypertensive rats. METHODS: Adult male Sprague-Dawley (SD) rats were subjected to AngⅡinfusion (500 ng·kg^-1·min^-1) using osmotic minipump up to 14 d to established the hypertension model. The rats (n=40) were randomly divided into 4 groups:control group (n=10), AngⅡgroup (n=10), candesartan (CAN; AngⅡtype 1 receptor blocker)+AngⅡgroup (n=10) and CAN group (n=10). The rats in CAN+AngⅡgroup and CAN group were administered with candesartan ester at the dose of 10 mg·kg^-1·d^-1 by gavage on the first day after implantation of osmotic minipump. The rats were sacrificed on the 15th day after minipump implantation. Serum and mesenteric arteries were collected. Systolic blood pressure was measured by tail-cuff method. The serum levels of AngⅡ were measured by ELISA. The protein levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (Ser1177), PP2A catalytic subunit (PP2Ac), phosphorylated PP2Ac (Tyr307) and PP2A inhibitor 2 (I₂^PP2A) in the mesenteric arteries were determined by Western blot. The activity of PP2A in the arteries was detected using PP2A activity assay kit. RESULTS: Compared with control group, the systolic blood pressure in AngⅡgroup was significantly increased(P<0.05), while those in CAN+AngⅡgroup and CAN group were significantly decreased (P<0.05). The serum levels of AngⅡ in AngⅡ group and CAN+AngⅡ group were significantly higher than that in control group (P<0.05). Compared with control group, the phosphorylation levels of eNOS Ser1177 were decreased in AngⅡgroup (P<0.05), but the activity of PP2A was significantly increased (P<0.05), and Pearson correlation analyses showed a negative correlation between PP2A activity and eNOS S1177 phosphorylation (r=-0.842, P<0.05). Compared with AngⅡgroup, the phosphorylation levels of eNOS Ser1177 in CAN+AngⅡgroup were significantly increased (P<0.05), but the activity of PP2A was reduced (P<0.05). Compared with control group, the protein levels of phosphorylated PP2Ac (Tyr307) and I₂^PP2A in the mesenteric arteries were decreased in AngⅡgroup (P<0.05), but increased in CAN+AngⅡgroup (P<0.05). No significant difference in all above-mentioned measures between control group and CAN group, nor in the levels of total eNOS and PP2Ac protein expression among all the groups was observed. CONCLUSION: AngⅡmay reduce the protein levels of phosphorylated PP2Ac (Tyr307) and I₂^PP2A in the mesenteric arteries of AngⅡ-induced hypertensive rats through AngⅡ/AngⅡ type 1 receptor-mediated signaling pathway, resulting in the activation of PP2A, then leading to down-regulation of eNOS S1177 phosphorylation, which ultimately mediates the occurrence of vascular endothelial dysfunction.     

Retarding effect of simvastatin on artery remodeling induced by high-salt and high-fat diet in rats

AIM: To investigate the effect of simvastatin intervention on the changes of blood pressure, serum lipid fluctuation and aortic configuration induced by high-sodium and high-fat diet in rats. METHODS: Sixty adult male SD rats were randomly divided into 5 groups (n=12): control (N)group, high salt (S)group, high fat (F) group, high salt+ high fat (SF) group and high salt+high fat + simvastatin (T) group. After fed for 16 weeks, the rats were subject to determine blood pressures and serum concentrations of triglycerides (TG),total cholesterol(TC) and soluble CD40 ligand (sCD40L). The expression of CD40/CD40L in the root of ascending aorta was detected by immunohistochemical method. The thickness of intima media in the ascending aorta as well as the ratio of lumen area/total vascular area were measured and calculated after HE staining. RESULTS: In S group, F group and SF group, systolic blood pressure was significantly higher than that in N group (P<0.01). Systolic blood pressure in T group were slightly higher than that in N group with statistical significance and significantly lower than that in SF group. The serum concentrations of TG and TC in F group and SF group were significantly higher than those in N group and T group (P<0.01), and no significant difference among S group, N group and T group was observed. In S group, F group and SF group, the serum concentrations of sCD40L were higher than that in N group and T group (P<0.05), meanwhile that in SF group was also higher than that in S group and F group (P<0.05). However, no significant difference of sCD40L concentration between S group and F group as well as N group and T group was observed. The expression of CD40/CD40L in the ascending aorta in S group, F group and SF group was higher than that in N group and T group (P<0.05), and that in SF group was also higher than that in S group and F group (P<0.05).No significant difference of CD40/CD40L expression between S group and F group as well as N group and T group was observed. The thickness of intima media in S group, F group and SF group was significantly thicker than that in N group (P<0.01), and no significant difference of the intima media thickness between T group and N group was observed. The ratio of lumen area/total vascular area in S group, F group and SF group was smaller than that in N group (P<0.05), and no significant difference of the ratio between T group and N group was found. CONCLUSION: Feeding high-fat and high-salt diet leads to blood pressure elevation, induces atherosclerosis, increases serum concentration of sCD40L and enhances the expression of CD40/CD40L in arterial tissues. The combination of the stimuli has stronger effect than a single factor. Statins protect the arterial tissues against atherosclerosis by decreasing the level of serum sCD40L and inhibiting the arterial expression of CD40/CD40L.     

Effects of oxidative stress on high-fat,high-salt diet and sucrose solution-induced metabolic syndrome in nephropathy rats

AIM: To establish a suitable animal model of nephropathy associated with metabolic syndrome (MS) induced by abnormal diet, and to investigate the effects of oxidative stress on renal damage in MS rats. METHODS: Normal 7-week-old male SD rats were randomly divided into 2 groups.The animals were fed with normal chow (control group, n=10) or high-fat and high-salt diet plus 20% sucrose solution (MS model group, n=10) for 20 weeks. Systolic blood pressure (SBP) was measured monthly. The levels of blood glucose, serum and urinary creatinine (Cr), total cholesterol (TC), triglycerides (TG), fasting insulin (FIns), urinary protein, urinary albumin and urinary sodium were determined. Insulin resistance (HOMA-IR), creatinine clearance (Ccr), urinary protein excretion (UPE), urinary albumin excretion (UAE) and urinary sodium excretion (USE) were calculated. Renal total-antioxidant capacity (T-AOC), inhibiting superoxide anion capacity (ISAC), malondialdehyde (MDA) content, and antioxidant enzyme activity were measured. Renal protein expression of Cu/Zn-SOD, NADPH oxidase subunit p47^phox and p22^phox was detected by Western blotting. In addition, pathological changes of the kidney were observed with PAS and Masson staining,and degree of glomerulosclerosis (GS) and tubulointerstitial injury was evaluated. RESULTS: Compared with control rats, SBP, TC, TG, FIns, USE and UAE were increased in MS rats. Furthermore, the MS rats showed a significant elevation of renal MDA content, p47^phox protein expression and GS score, and reduction of T-AOC, ISAC, SOD activity, and Cu/Zn-SOD protein expression in the kidney. CONCLUSION: SD rats fed with abnormal diet produce a suitable animal model of MS nephropathy that mimics the major features of human MS. Oxidative stress caused by up-regulation of NADPH oxidase expression and down-regulation of SOD expression may be one of the mechanisms leading to MS renal damage.     

Role of TGF-β1/Smads and ERK expression in vascular remodeling induced by high-salt diet in Wistar rats

AIM: To investigate the role of transforming growth factor β₁ (TGF-β₁)/Smads and extracellular signal-regulated kinase(ERK) expression in vascular remodeling induced by high-salt diet in Wistar rats. METHODS: Wistar rats were randomly divided into 3 groups: normal control group (n=13), high salt (8%) model group and high salt+telmisartan group (n=13). Tail-cuff arterial pressure was determined every 2 weeks. After 24 weeks, the rats in high salt model group were divided into model animals with hypertension group (MH, n=12) and model animals without hypertension group (MN, n=12). The remodeling of aorta and mesenteric artery was observed by HE and Masson staining. In addition, the techniques of immunohistochemistry and real-time PCR were applied to detect the expression of proliferating cell nuclear antigen (PCNA), TGF-β₁, p-Smad2/3, p-ERK1/2 and Smad7 at both protein and mRNA levels. RESULTS: Compared with normal control group, blood pressure in MH group was much higher, and media thickness (MT) and collagen volume fraction (CVF) of arteries in MH and MN groups were higher.The mRNA expression of TGF-β₁, Smad2 and Smad7 in the aorta was significantly increased, and the protein levels of PCNA, p-ERK1/2, TGF-β₁ and p-Smad2/3 in the aorta and mesenteric artery media were elevated, but Smad7 decreased. After telmisartan treatment, MT and CVF were much lower,and the protein levels of PCNA, TGF-β₁, p-Smad2/3 and p-ERK1/2 were significantly reduced, whereas Smad7 was increased. CONCLUSION: The abnormal expression of TGF-β₁/Smads and ERK may be involved in the mechanism of remodeling of aorta and mesenteric artery induced by high-salt diet. Telmisartan prevents the vascular remodeling via regulating TGF-β₁/Smads and ERK signal pathways mediated by angiotensinⅡ type 1 (AT₁) receptor, at least in part.     

Effects of short-chain acyl-CoA dehydrogenase on hypertensive vascular remodeling

AIM: To investigate the changes of short-chain acyl-CoA dehydrogenase (SCAD) in hypertensive vascular remodeling and to explore the relationship between SCAD and vascular remodeling in hypertension.METHODS: The spontaneously hypertensive rats (SHR; 24 weeks old) and Wistar rats (24 weeks old) were used as experimental control groups. The SHR and Wistar rats of 16 weeks old were trained by swimming as experimental groups. The systolic pressure was measured periodically. The thickness of vascular wall and the diameter of the vascular lumen were measured. The contents of ROS and ATP, the enzyme activity of SCAD, and the expression of SCAD at mRNA and protein levels in the aorta were determined. The free fatty acid in the serum and aorta was also measured.RESULTS: Compared with Wistar group, the diameter of vascular lumen decreased in SHR group. The thickness of vascular wall, the ratio of vascular wall and the diameter of vascular lumen, and the blood pressure in SHR group were increased significantly (P<0.05). Compared with SHR group, the diameter of vascular lumen increased in SHR+swim group. The thickness of vascular wall, the ratio of vascular wall and the diameter of vascular lumen, and the blood pressure in SHR+swim group were decreased significantly. Compared with control group, the expression of SCAD at mRNA and protein levels, the enzyme activity of SCAD, and the content of ATP were decreased in SHR group. However, the free fatty acid in the serum and aorta, and the content of ROS in the aorta were increased in SHR group. The expression of SCAD at mRNA and protein levels, the enzyme activity of SCAD, the content of ATP were increased in Wistar+swim group and SHR+swim group. However, the free fatty acid in serum and aorta, and the content of ROS in the aorta were decreased in Wistar+swim group and SHR+swim group.CONCLUSION: Decrease in SCAD expression may be associated with hypertensive vascular remodeling. Swimming training can reverse hypertensive vascular remodeling by increasing the expression of SCAD in the aorta.     

Effect of atorvastatin on HMG-CoA reductase expression in spontaneously hypertensive rats

AIM: To evaluate the effect of atorvastatin on HMG-CoA reductase (HMGR) expression level in spontaneously hypertensive rats (SHR). METHODS: Twelve eight-week-old SHR was randomized into distilled water group (SHR_DW group, n=6) and atorvastatin-treated group (SHR_ATV group, n=6). The age-matched Wistar-Kyoto rats (WKY) were used as control (WKY group, n=6). RT-PCR and Western blotting were used to detect HMGR mRNA and protein expression levels, respectively. Meanwhile, systolic blood pressure (SBP) and serum lipid levels were examined. RESULTS: Ten weeks a treatment, SBP in SHR_ATV group was markedly decreased compared with before treatment and SHR_DW group (P<0.05). The levels of TC, TG, LDL-C and HDL-C in SHR_ATV group were also markedly lower than those in SHR_DW group and WKYgroup (P<0.05). 10 weeks later, HMGR mRNA levels decreased markedly in SHR_ATV group compared to WKY group and SHR_DW group (P<0.05). The similar results were found in HMGR protein expression levels. CONCLUSIONS: Atorvastatin downregulates mRNA and protein expression levels of HMGR in SHR, which not only decreases the serum lipids levels, but also impacts blood pressure to a certain extent.     

Effects of phytosterol ester on aortic aging and expression of related genes in rats

AIM: To explore the protective effect of phytosterol ester (PSE) on aortic aging in rats. METHODS: The female SD rats (12 months old, n=42) were randomly divided into control group, model group and PSE group. During the experiment, the rats in control group, model group and PSE group were treated with basic feed, high-fat diet (HFD) and HFD with 2% PSE (W/W) for 6 months, respectively. The morphological changes of the aorta were observed by HE staining and Masson staining, and the absolute area of smooth muscle cells and collagen fiber in the vascular wall were measured by image analysis. The levels of advanced glycosylation end products (AGEs), malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) in the plasma were detected. The expression of silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor γ (PPARγ) at mRNA and protein levels in the vascular tissue was determined by real time PCR and Western blot, respectively. RESULTS: PSE significantly lowered plasma TC and LDL-C, and increased plasma HDL-C level (P<0.05), but had no effect on plasma TG level. PSE significantly attenuated the thickening of intima and media of aging aortic, and decreased the migration of vascular smooth muscle cells (VSMC) and the amount of VSMC and collagen fiber in the aorta (P<0.05). PSE significantly reduced the contents of AGEs and MDA (P<0.05), but had no effect on the activity of SOD and CAT in the plasma. PSE also down-regulated the expression of PPARγ and up-regulated the expression of SIRT1 (P<0.05). CONCLUSION: PSE is able to attenuate the senescence process in the aorta by reducing the production of reactive oxygen species in plasma, and activating SIRT1, or inhibiting the expression of PPARγ in vascular tissues.     

Effect of salvianolic acid B on vasodilatory function,NF-κB activation and inflammatory cytokine expression in diabetic rats

AIM: To investigate the ameliorative effect of salvianolic acid B on vasodilatory function in diabetic rats and the possible mechanisms. METHODS: SD rats (n=40) were fed on high-sugar and high-fat diet for 4 weeks, followed by a single intraperitoneal injection of streptozotocin (40 mg/kg). The rats with random blood glucose level over 16.7 mmol/L were considered diabetic and randomly allocated to 3 groups, namely model group, low dose (80 mg·kg^-1·d^-1) of salvianolic acid B group and high dose (160 mg·kg^-1·d^-1) of salvianolic acid B group. The rats in salvianolic acid B groups were intragastrically administered with corresponding doses of salvianolic acid B for 6 weeks. Vasodilatory function was measured as endothelium-dependent and-independent vasodilation of the aortic rings. The primary histopathological changes of aorta were observed by HE staining. Serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP) were measured by ELISA. The levels of total antioxidant capacity, malondialdehyde (MDA) and nitric oxide (NO) in aortic tissues were evaluated by colorimetric assays. The protein levels of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein-1 (MCP-1), and the activation of nuclear factor-κB (NF-κB) were determined by Western blot. RESULTS: Treatment with salvianolic acid B evidently ameliorated endothelium-dependent diastolic function and pathological changes of aorta in diabetic rats (P<0.05 or P<0.01). Supplementation with salvianolic acid B resulted in significant increases in NO content and total antioxidant capacity in aortic tissues, accompanied by marked decreases in the level of MDA in aorta tissues and the serum levels of IL-6, TNF-α and CRP (P<0.05 or P<0.01). Salvianolic acid B markedly down-regulated NF-κB p65 nuclear translocation and protein expression of ICAM-1 and MCP-1 in aorta tissues (P<0.05 or P<0.01). CONCLUSION: Salvianolic acid B effectively ameliorates endothelium-dependent diastolic function of aorta in diabetic rats, which might be attributed to suppression of NF-κB activation and subsequent expression of inflammatory cytokines. The beneficial effect of salvianolic acid B on vascular endothelium might be derived from its antioxidant capacity.     

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.     

Endogenous nitric oxide synthase inhibitor increase skeletal muscle contractility and mitochondria biosynthesis in 4-week running rats

AIM: To observe the effect of endogenous nitric oxide synthase(NOS) inhibitor asymmetric dimethylarginine(ADMA) and its signaling pathways on NO levels and skeletal muscle contractility in 4-week running rats. METHODS: The 4 weeks running rat model was established. The twitch tension, tetanic tension and the fatigue test of soleus muscle induced by electrical stimulation ex vivo were detected. The ATP content, mitochondrial DNA levels and the mRNA expression of peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α), nuclear respiratory factor(NRF) were measured to reflect the mitochondrial function and biosynthesis in the skeletal muscle. Serum ADMA concentration was detected by high performance liquid chromatography. The endogenous ADMA enzymes PRMT1 and 2 subtypes of ADMA metabolism enzyme DDAH, 3 subtypes of NOS protein expression in the skeletal muscle were determined by Western blot. NOS activity and nitric oxide(NO) content were analyzed by colorimetric method. RESULTS: Compared with normal control group, the twitch tension, tetanic tension and the anti-fatigue capability of soleus muscle in running group were significantly enhanced, ATP content, mitochondrial DNA content and the mRNA expression of PGC-1α and NRF were significantly increased(P<0.01). In addition, the protein expression of constitute type NOS(cNOS) and NOS activity were significantly increased(P<0.01), but the increase in NO content was relatively smaller in soleus muscle in exercise group(P<0.05). Moreover, serum ADMA concentration in running group was increased, while the DDAH2 expression in skeletal muscle was decreased.CONCLUSION: Short-term endurance exercise enhances the twitch tension, tetanic tension and fatigue resistance of soleus muscle. The mechanism may be that increased cNOS expression feedbacks to increase ADMA concentration, thus maintaining the increase in NO synthesis at a relatively low level, and resulting in promoting skeletal muscle mitochondria biosynthesis and mitochondrial function.     

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.     

Qishen-Yiqi dripping pills attenuate atherosclerosis by regulating Ca2+ homeostasis via TRPC1/STIM1 pathway

AIM:To explore the therapeutic effect of Qishen-Yiqi dripping pills (QS) on atherosclerosis (AS) and the mechanism. METHODS:AS rat model was established by high-fat diet, and SD rats were randomly divided into normal control group, AS model group, low-dose, middle-dose and high-dose QS groups, and positive group (n=6 each). After administration for 12 weeks, serum samples were collected to detect the serum lipid and Ca²⁺ levels. HE staining was used evaluated the histopathological changes of arterial tissue. The serum levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were measured by ELISA. The nitric oxide (NO) level was detected by nitrate reductase method. The protein levels of transient receptor potential channel protein 1 (TRPC1), stromal interaction molecule 1 (STIM1) and endothelial NO synthase (eNOS) were determined by Western blot. RESULTS:QS significantly reduced the arterial damage via inhibiting the formation of atherosclerotic plaque and attenuated intimal thickening and vascular stenosis. Compared with AS group, the serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) were decreased significantly and the levels of high-density lipoprotein cholesterol (HDL-C) were increased significantly in high-dose QS group (P<0.05). The serum levels of IL-1β, IL-6 and TNF-α in high-dose QS group were lower than those in AS group (P<0.05). Compared with AS group, the serum Ca²⁺ level was lowered and the arterial tissue NO level was elevated in QS groups (P<0.05). Compared with AS rats, the protein levels of TRPC1 and STIM1 were decreased significantly and the protein level of eNOS was increased significantly in the rats treated with QS (P<0.05). CONCLUSION:QS regulate calcium homeostasis via TRPC1/STIM1 pathway, increase the production of NO and inhibit the inflammatory responses, thus exerting anti-AS effect.     

Alleviation of aortic vascular dysfunction in STZ/HFD-induced type 2 diabetic rats by nFGF1

AIM: To investigate the protective effect of non-mitogenic fibroblast growth factor 1 (nFGF1) on the aortic vascular function in streptozotocin (STZ)/high-fat diet (HFD)-induced type 2 diabetic rats and its underlying mechanisms. METHODS: Five-week-old male SD rats (n=30) were randomly divided into 3 groups (n=10 in each group), including normal control group, type 2 diabetic group and nFGF1 treatment group (type 2 diabetic rats were intraperitoneally injected with 0.5 mg/kg nFGF1 every other day for 4 weeks). After the rats were sacrificed, blood glucose, cholesterol and triglyceride levels, aorta diastolic function and superoxide dismutase (SOD) level in the aorta of each group were measured. Besides, the protein levels of cyclooxygenase-2 (COX-2), phosphorylated extracellular signal-regulated kinase (p-ERK) and endothelial nitric oxide synthase (eNOS) in the aorta were determined by Western blot. RESULTS: nFGF1 markedly lowered blood glucose, cholesterol and triglyceride levels, enhanced aorta SOD activity and upregulated protein level of eNOS in the type 2 diabetic rats. Furthermore, the increased protein levels of COX-2 and p-ERK in the type 2 diabetic rats were largely abrogated by nFGF1. CONCLUSION: nFGF1 effectively attenuates aortic vascular dysfunction in the type 2 diabetic rats, which may be associated with decreasing blood glucose, cholesterol and triglyceride levels, reducing inflammation and oxidative stress response, and activating eNOS signaling pathway.