Hui-Ping Wang

Apigenin protects endothelium-dependent relaxation of rat aorta against oxidative stress

Apigenin is shown to have cardiovascular effects, but the effects of apigenin on aortas injured by exogenous oxidants are unknown. The objective of this study was to investigate the effect of apigenin on endothelium-dependent vasorelaxation in isolated rat aortic rings exposed to superoxide anion produced by pyrogallol, and its mechanism. The male Sprague-Dawley rat thoracic aorta was rapidly dissected out and the effect of apigenin on tension of aortic rings pretreated with 500 microM pyrogallol, inducing oxidative stress injury, was measured. The activity of nitric oxide synthase (NOS), the level of nitric oxide (NO) and the inhibition of superoxide anion in aortic tissues were measured. We found that pretreatment with pyrogallol concentration-dependently decreased acetylcholine-induced endothelium-dependent vasorelaxation. Apigenin (0.5-72.0 microM) evoked a concentration-dependent relaxation in aortas (pD(2): 5.304+/-0.049), which was weakened by L-NAME (the maximal relaxation fell from 87.6+/-6.7% to 37.1+/-8.8%, P<0.01), but not by aminoguanidine and indomethacin. Apigenin markedly attenuated the inhibition of vasorelaxation induced by pyrogallol (the maximal relaxation elevated from 55.8%+/-6.6% to 69.5%+/-6.4%, and the pD(2) increased from 6.559+/-0.119 to 7.057+/-0.145, P<0.01) and increased the inhibition of superoxide anion (from 94.6% to 74.5%), the NO level (from 77.1% to 94.4%), and the constitutive NOS activity (from 35.1% to 62.5%). These results indicate that pyrogallol decreased endothelium-dependent vasorelaxation in rat aortas via oxidative stress, which was markedly attenuated by apigenin. This may be mediated by weakening the oxidative stress and the NO reduction.

Luteolin reduces high glucose-mediated impairment of endothelium-dependent relaxation in rat aorta by reducing oxidative stress

While luteolin, a flavone rich in many plants, has some cardiovascular activity, it is not clear whether luteolin has beneficial effects on the vascular endothelial impairment in hyperglycemia/high glucose. Here, we reveal the protective effect of luteolin on endothelium-dependent relaxation in isolated rat aortic rings exposed to high glucose. The thoracic aorta of male Sprague-Dawley rats was rapidly dissected out and the effect of luteolin on the tension of aortic rings pretreated with high glucose (44mM) for 4h was measured in an organ bath system. The levels of nitric oxide (NO), hydroxy radical (OH(-)) and reactive oxygen species (ROS), and the activity of superoxide dismutase (SOD) and nitric oxide synthase (NOS) were measured in aortas. The vasorelaxation after treatment with luteolin for 8 weeks in aortic rings from diabetic rats was also determined. We found that exposure to high glucose decreased acetylcholine-induced endothelium-dependent relaxation. However, high mannitol had no effect on vasorelaxation. Luteolin evoked a concentration-dependent relaxation in aortic rings previously contracted by phenylephrine, and the pD(2) value was 5.24+/-0.04. The EC(50) of luteolin markedly attenuated the inhibition of relaxation induced by high glucose, which was significantly weakened by pretreatment with l-NAME (0.1mM), but not by indomethacin (0.01mM). Luteolin significantly inhibited the increase of ROS level and OH(-) formation, and the decrease of NO level, NOS and SOD activity caused by high glucose. The improving effect of luteolin on endothelium-dependent vasorelaxation in diabetic rat aortic rings was reversed by pretreatment with l-NAME or methylene blue. The results indicate that the decrease of endothelium-dependent relaxation in rat aortic rings exposed to high glucose is markedly attenuated by luteolin, which may be mediated by reducing oxidative stress and enhancing activity in the NOS-NO pathway.

Betulinic acid ameliorates endothelium-dependent relaxation in L-NAME-induced hypertensive rats by reducing oxidative stress.

Zizyphi Spinosi semen (ZSS) is one of the most widely used traditional Chinese herbs with protective effects on the cardiovascular system. It is not clear whether betulinic acid (BA), the key active constituent of ZSS, has beneficial cardiovascular effects on N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertensive rats. The objective of this study was to investigate the effect of BA on endothelium-dependent vasorelaxation in isolated aortic rings from L-NAME-induced hypertensive rats and its underlying mechanisms. Male Sprague-Dawley rats were injected with L-NAME (15 mg/kg/d, i.p.) for 4 weeks to induce hypertension. After treatment with L-NAME for 2 weeks, rats with mean blood pressure >120 mm Hg measured by tail-cuff method were considered hypertensive and then injected with BA (0.8, 4, 20 mg/kg/d, i.p.) for the last 2 weeks. The effect of BA on the tension of rat thoracic aortic rings was measured in an organ bath system. The levels of nitric oxide (NO), reactive oxygen species (ROS), and the activity of superoxide dismutase (SOD) and nitric oxide synthase (NOS) in aortas were assayed. We found that BA (0.1-100 μM) evoked a concentration-dependent vasorelaxation in endothelium-intact normal rat aortic rings, which was significantly attenuated by pretreatment with L-NAME (100 μM) or methylene blue (MB, 10 μM), but not by indomethacin (10 μM). Pretreatment with EC(50) (1.67 μM) concentration of BA enhanced the acetylcholine (ACh)-induced vasorelaxation, which was also markedly reversed by both L-NAME and MB. The blood pressure in hypertensive rats increased to 135.22±5.38 mm Hg (P<0.01 vs. control group), which was markedly attenuated by high dose of BA. The ACh-induced vasorelaxation in hypertensive rat aortic rings was impaired, which was markedly improved by chronic treatment with BA (20 mg/kg/d) for 2 weeks. The increase of ROS level and the decrease of NO level, SOD and eNOS activities in hypertensive rat aortas were all markedly inhibited by BA. These results indicate that BA decreased blood pressure and improved ACh-induced endothelium-dependent vasorelaxation in L-NAME-induced hypertension rats, which may be mediated by reducing oxidative stress and retaining the bioavailability of NO in the cardiovascular system.

Inhibition of superoxide anion-mediated impairment of endothelium by treatment with luteolin and apigenin in rat mesenteric artery

This study was designed (i) to test the hypothesis that the endothelium-derived hyperpolarizing factor (EDHF) component of ACh-induced vasorelaxation and hyperpolarization of smooth muscle cells (SMCs) are impaired following exposure to superoxide anion, and (ii) to further investigate whether luteolin and apigenin induce vasoprotection at the vasoactive concentrations in rat mesenteric artery. Rat mesenteric arterial rings were isolated for isometric force recording and electrophysiological studies. Perfusion pressure of mesenteric arterial bed was measured and visualization of superoxide production was detected with fluorescent dye. 300 microM pyrogallol significantly decreased the relaxation and hyperpolarization to ACh. Luteolin and apigenin both induced vasoprotection against loss of the EDHF component of ACh-induced relaxation and attenuated the impairment of hyperpolarization to ACh. Oxidative fluorescent microtopography showed that either luteolin or apigenin significantly reduced the superoxide levels. The results suggest that superoxide anion impairs ACh-induced relaxation and hyperpolarization of SMC in resistance arteries through the impairment of EDHF mediated responses. Luteolin and apigenin protect resistance arteries from injury, implying that they may be effective in therapy for vascular diseases associated with oxidative stress.

Mitochondrial nitric oxide synthase participates in septic shock myocardial depression by nitric oxide overproduction and mitochondrial permeability transition pore opening.

ABSTRACT The aim of this study was to determine whether mitochondrial nitric oxide (NO) synthase (NOS) is involved in septic shock myocardial depression. The cecal ligation and puncture (CLP) method was used to induce septic shock. There was a significant depression of hemodynamic parameters recorded in the septic shock stage. After using nonselective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME), inducible NOS inhibitor aminoguanidine (AMG), and neuronal NOS inhibitor 7-nitroindazole (7-NI), depression of the parameters was partly attenuated. Nitric oxide production in isolated cardiac mitochondria increased obviously in the CLP–septic shock stage, L-NAME and 7-NI both decreased NO production significantly. Nitrite/nitrate (NOx-) production in the septic shock stage was much greater than those in the corresponding sham groups, and NOx- production in the cytosol by inducible NOS was greater. Treatment with AMG suppressed NOx- production in the cytosol by iNOS, whereas treatment with 7-NI decreased NOx- production in the mitochondria. Mitochondrial NOS expression increased significantly in the septic shock stage, and its overexpression was attenuated using 7-NI. There was no significant decrease in the mitochondrial permeability transition pore measurement in the CLP–septic shock group, whereas a significant decrease was observed in those treated with L-NAME or 7-NI. These results indicate that overexpression of mitochondrial NOS is involved in myocardial depression.

Cardioprotective effects of luteolin on ischemia/reperfusion injury in diabetic rats are modulated by eNOS and the mitochondrial permeability transition pathway.

Abstract: Myocardial ischemia/reperfusion (I/R) injury in diabetes is associated with oxidative stress, endothelial nitric oxide synthase (eNOS) dysfunction, and mitochondrial collapse, whereas luteolin is known to protect the cardiovascular system against diabetes and I/R injury. Here, we investigated whether luteolin pretreatment diminishes myocardial I/R injury in diabetic rats by affecting eNOS and the mitochondrial permeability transition pore (mPTP). After diabetic rats were produced by streptozotocin treatment (65 mg/kg) for 3 weeks, luteolin (100 mg·kg−1·d−1) or L-NAME (25 mg·kg−1·d−1) was administered intragastrically for 2 weeks. Hearts were then isolated and subjected to 30 minutes of global ischemia followed by 120 minutes of reperfusion. Pretreatment with luteolin significantly improved left ventricular function and coronary flow throughout reperfusion, increased cardiac tissue viability and manganese superoxide dismutase (MnSOD) activity, and reduced coronary lactate dehydrogenase release, and the myocardial malonaldehyde level in diabetic I/R rat hearts. All these improving effects of luteolin were significantly attenuated by L-NAME. Luteolin also significantly upregulated eNOS expression in diabetic rat hearts after I/R. Ca2+-induced mPTP opening and mitochondrial inner membrane potential reduction were significantly inhibited in ventricular myocytes isolated from luteolin-treated diabetic rats, and this effect was attenuated by L-NAME. These findings indicate that luteolin protects the diabetic heart against I/R injury by upregulating the myocardial eNOS pathway, and downstream effects include the enhancement of MnSOD and inhibition of mPTP.

Carvedilol protected diabetic rat hearts via reducing oxidative stress

Oxidative stress plays a dominant role in the pathogenesis of diabetes mellitus. Bcl-2 gene has close connection with antioxidant stress destruction in many diseases including diabetes. Carvedilol, an adrenoceptor blocker, also has antioxidant properties. To study the effect of carvedilol on the antioxidant status in diabetic hearts, we investigated carvedilol-administrated healthy and streptozotocin-induced diabetic rats. After small and large dosage carvedilol-administered for 5 weeks, hemodynamic parameters, the levels of malondialdehyde, activities of antioxidant enzymes and expression of Bcl-2 mRNA in the cardiac tissues were measured. The diabetic rats not only had cardiac disfunction, weaker activities of antioxidant enzymes, but also showed lower expression of Bcl-2. Carvedilol treatment increased activities of antioxidant enzymes and expression of Bcl-2 in healthy rats as well as diabetic rats. These results indicated that carvedilol partly improves cardiac function via its antioxidant properties in diabetic rats.

Reducing the oxidative stress mediates the cardioprotection of bicyclol against ischemia-reperfusion injury in rats

ObjectiveTo investigate the beneficial effect of bicyclol on rat hearts subjected to ischemia-reperfusion (IR) injuries and its possible mechanism.MethodsMale Sprague-Dawley rats were intragastrically administered with bicyclol (25, 50 or 100 mg/(kg·d)) for 3 d. Myocardial IR was produced by occlusion of the coronary artery for 1 h and reperfusion for 3 h. Left ventricular hemodynamics was continuously monitored. At the end of reperfusion, myocardial infarct was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and serum lactate dehydrogenase (LDH) level and myocardial superoxide dismutase (SOD) activity were determined by spectrophotometry. Isolated ventricular myocytes from adult rats were exposed to 60 min anoxia and 30 min reoxygenation to simulate IR injuries. After reperfusion, cell viability was determined with trypan blue; reactive oxygen species (ROS) and mitochondrial membrane potential of the cardiomyocytes were measured with the fluorescent probe. The mitochondrial permeability transition pore (mPTP) opening induced by Ca2+ (200 μmol/L) was measured with the absorbance at 520 nm in the isolated myocardial mitochondria.ResultsLow dose of bicyclol (25 mg/(kg·d)) had no significant improving effect on all cardiac parameters, whereas pretreatment with high bicyclol markedly reduced the myocardial infarct and improved the left ventricular contractility in the myocardium exposed to IR (P<0.05). Medium dose of bicyclol (50 mg/(kg·d)) markedly improved the myocardial contractility, left ventricular myocyte viability, and SOD activity, as well decreased infarct size, serum LDH level, ROS production, and mitochondrial membrane potential in rat myocardium exposed to IR. The reduction of ventricular myocyte viability in IR group was inhibited by pretreatment with 50 and 100 mg/(kg·d) bicyclol (P<0.05 vs. IR), but not by 25 mg/(kg·d) bicyclol. The opening of mPTP evoked by Ca2+ was significantly inhibited by medium bicyclol.ConclusionsBicyclol exerts cardioprotection against IR injury, at least, via reducing oxidative stress and its subsequent mPTP opening.

MECHANISMS UNDERLYING BIOCHANIN A‐INDUCED RELAXATION OF THE AORTA DIFFER BETWEEN NORMOTENSIVE AND HYPERTENSIVE RATS

1 The aim of the present study was to investigate the mechanism underlying biochanin A‐induced relaxation of the aorta in spontaneously hypertensive rats (SHR). 2 The tension in isolated ring preparations of thoracic aortas from normotensive (Wistar‐Kyoto (WKY) rats) and SHR at 5 and 10 weeks of age was measured isometrically. 3 Biochanin A (10‐7 to 10‐4 mol/L) induced a concentration‐dependent relaxation in aortic rings from both strains at the age of 5 and 10 weeks and the relaxation was greater in rings from 10‐week‐old SHR compared with age‐matched WKY rats. The vasorelaxation induced by biochanin A was significantly reduced by denudation of the endothelium in aortic rings from SHR, but not WKY rats. Treatment with either indomethacin, a cyclo‐oxygenase inhibitor, or Nw‐nitro‐l‐arginine methyl ester, a nitric oxide synthase inhibitor, had little effect on the relaxation induced by biochanin A in aortic rings from either strain. Glibenclamide, a selective inhibitor of ATP‐sensitive potassium channels, significantly attenuated the relaxation induced by biochanin A in aortic rings from both strains, although the extent of reduction was greater in WKY rats than SHR. Conversely, treatment with 4‐aminopyridine, a selective inhibitor of voltage‐dependent potassium channels, or tetraethylammonium, an inhibitor of calcium‐activated potassium channels, significantly reduced the vasorelaxation induced by biochanin A in rings from SHR but not WKY rats. 4 The greater vasorelaxation produced by biochanin A in aortic rings from 10‐week‐old SHR is endothelium dependent. Different mechanisms underlie the relaxant effects of biochanin A in aorta from SHR and WKY rats. The mechanisms of biochanin A‐induced vasorelaxation in thoracic aortas from both normotensive and hypertensive rats involve ATP‐sensitive potassium channels and, in addition, in rings from the hypertensive strain at 10 weeks of age, an endothelium‐derived activation of smooth muscle cell potassium channels contributes to the vasorelaxation observed.

Endothelium-dependent and -independent vasorelaxant actions and mechanisms induced by total flavonoids of Elsholtzia splendens in rat aortas.

Elsholtzia splendens (ES) is, rich in flavonoids, used to repair copper contaminated soil in China, which has been reported to benefit cardiovascular systems as folk medicine. However, few direct evidences have been found to clarify the vasorelaxation effect of total flavonoids of ES (TFES). The vasoactive effect of TFES and its underlying mechanisms in rat thoracic aortas were investigated using the organ bath system. TFES (5-200mg/L) caused a concentration-dependent vasorelaxation in endothelium-intact rings, which was not abolished but significantly reduced by the removal of endothelium. The nitric oxide synthase (NOS) inhibitor N(ω)-nitro-l-arginine methyl ester (100μM) and the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (30μM) significantly blocked the endothelium-dependent vasorelaxation of TFES. Meanwhile, NOS activity in endothelium-intact aortas was concentration-dependently elevated by TFES. However, indomethacin (10μM) did not affect TFES-induced vasorelaxation. Endothelium-independent vasorelaxation of TFES was significantly attenuated by KATP channel blocker glibenclamide. The accumulative Ca(2+)-induced contraction in endothelium-denuded aortic rings primed with KCl or phenylephrine was markedly weakened by TFES. These results revealed that the NOS/NO/cGMP pathway is likely involved in the endothelium-dependent vasorelaxation induced by TFES, while activating KATP channel, inhibiting intracellular Ca(2+) release, blocking Ca(2+) channels and decreasing Ca(2+) influx into vascular smooth muscle cells might contribute to the endothelium-independent vasorelaxation conferred by TFES.