Ju Chi Liu

Antihypertensive effects of tannins isolated from traditional Chinese herbs as non-specific inhibitors of angiontensin converting enzyme

The tannins are natural polyphenols, able to precipitate water-soluble alkaloids and possess an inhibitory action on the angiotensin converting enzyme (ACE). We identified 18 polyphenolic compounds (tannins) from Chinese herbs and examined the in vitro effects of these tannins on ACE activity, including determination of the 50% inhibitory concentrations (IC50), specificity and mode of inhibition. We also assessed the in vivo inhibitory effect of the tannins on angiotensin I-induced blood pressure elevation in spontaneously hypertensive rats (SHR). Nine tannins with an IC50 <200 microM for ACE inhibitors were identified belonging to three tannin classes: caffeoylquinates, flavan-3-ols and gallotannins. In vitro, we found caffeoylquinates chelate the ACE zinc cofactor. Two of the flavan-3-ols: epigallocatechin-3-O-gallate and epigallocatechin-3-O-methylgallate, and one of gallotannin: 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose were non-specific inhibitors because also reduced the activity of trypsin and chymotrypsin. The ACE inhibition of 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose was also reduced after addition of bovine serum albumin, suggesting a non-specific mode of action. In vivo, 1,2,3,6-tetra-O-galloyl-beta-D-glucose and epigallocatechin-3-O-methylgallate had a strong dose-dependent hypotensive effect reducing the blood pressure significantly in the SHR with infusion of the angiotensin I. These findings indicate that some of the tannins isolated from herbs inhibit ACE activity non-specifically. The ACE inhibitory effect of these tannins may explain the hypotensive effects of some traditional Chinese herbs.

The effect of stevioside on blood pressure and plasma catecholamines in spontaneously hypertensive rats

Stevioside is a sweet-tasting glycoside, composed of stevia, a diterpenic carboxylic alcohol with three glucose molecules, mainly used as a substitute for non-alcoholic sweetener. It has previously been shown to reduce blood pressure in studies in animals and human. The effect of intravenous stevioside on the blood pressure was studied in spontaneously hypertensive rats (SHR). The hypotensive effect on both systolic and diastolic blood pressure was dose-dependent for intravenous doses of 50, 100 and 200 mg/kg in conscious SHR. The maximum reductions in systolic and diastolic blood pressure were 31.4 +/- 4.2% and 40.8 +/- 5.6% (mean +/- SEM) respectively and the hypotensive effect lasted for more than 60 min with a dose of 200 mg/kg. Serum dopamine, norepinephrine and epinephrine levels were not changed significantly 60 min after intravenous injection of stevioside 100 mg/kg in anesthetized SHR. The present data show that stevioside given intravenously to conscious SHR was effective in blood pressure reduction and there was no change in serum catecholamines in anaesthetized animals with this natural compound.

Role of Reactive Oxygen Species-Sensitive Extracellular Signal-Regulated Kinase Pathway in Angiotensin II-Induced Endothelin-1 Gene Expression in Vascular Endothelial Cells

Background: Circulating angiotensin II (Ang II) increases vascular endothelin-1 (ET-1) tissue levels, which in turn mediate a major part of Ang II-stimulated vascular growth and hypertension in vivo. Ang II also stimulates the generation of reactive oxygen species (ROS) within vascular endothelial cells. However, whether ROS are involved in Ang II-induced ET-1 gene expression, and the related intracellular mechanisms occurring within vascular endothelial cells remain unclear. Methods: Cultured endothelial cells were stimulated with Ang II, and the thus elicited ET-1 gene expression was examined by Northern blotting and a promoter activity assay. Antioxidant pretreatment of endothelial cells was performed prior to Ang II-induced extracellular signal-regulated kinase (ERK) phosphorylation in order to elucidate the redox-sensitive pathway for ET-1 gene expression. Results: The ET-1 gene was induced with Ang II, which was inhibited with Ang II type 1 receptor antagonist (irbesartan). Ang II-enhanced intracellular ROS levels were inhibited by irbesartan and several antioxidants, and antioxidants also suppressed Ang II-induced ET-1 gene expression. Further, Ang II-activated ERK phosphorylation was also significantly inhibited by certain antioxidants. An ERK inhibitor, U0126, inhibited Ang II-induced ET-1 expression completely. Cotransfection of the dominant negative mutant of Ras, Raf and MEK1 (ERK kinase) attenuated the Ang II-enhanced ET-1 promoter activity, suggesting that the Ras/Raf/ERK pathway is required for Ang II-induced ET-1 gene expression. Ang II-induced activator protein-1 (AP-1) reporter activities were inhibited by antioxidants. Moreover, mutational analysis of the ET-1 gene promoter showed that the AP-1 binding site was an important cis element in Ang II-induced ET-1 gene expression. Conclusions: Our data suggest that ROS are involved in Ang II-induced ET-1 gene expression within endothelial cells. The redox-sensitive ERK-mediated AP-1 transcriptional pathway plays an important role in Ang II-induced ET-1 gene expression.

Inhibitory effect of resveratrol on angiotensin II-induced cardiomyocyte hypertrophy

Resveratrol is proposed to account in part for the protective effect of red wine on the cardiovascular system. Angiotensin II (Ang II) is a potent hypertrophic stimulus in cardiomyocytes. In this study, we determined the effect of resveratrol on Ang II-induced cardiomyocyte hypertrophy. Cultured neonatal rat cardiomyocytes were stimulated with Ang II, and [3H]leucine incorporation and β-myosin heavy chain (β-MyHC) promoter activity were examined. Intracellular reactive oxygen species (ROS) were measured by a redox-sensitive fluorescent dye, 2’ 7’-dichlorofluorescin diacetate, and the extracellular signal-regulated kinase (ERK) phosphorylation was examined by Western blotting. Resveratrol inhibited Ang II-increased intracellular ROS levels. Furthermore, resveratrol, as well as the antioxidant N-acetyl-cysteine, decreased Ang II- or H2O2-increased protein synthesis, β-MyHC promoter activity, and ERK phosphorylation. In summary, we demonstrate for the first time that resveratrol inhibits Ang II-induced cardiomyocyte hypertrophy via attenuation of ROS generation.

Mechanism of the antihypertensive effect of stevioside in anesthetized dogs

Stevioside is a sweet-tasting glycoside isolated from the leaves of Stevia rebaudiana. It has been used as a noncaloric sugar substitute in Japan and Brazil for decades. Previous studies have shown that it lowered blood pressure in spontaneously hypertensive rats by intravenous injection. This study was designed to evaluate the hypotensive effect of stevioside in dogs and to define the underlying mechanism. After nasogastric administration of stevioside powder (200 mg/kg), the blood pressure of healthy mongrel dogs began to significantly decrease at 60 min and returned to baseline level at 180 min. The reduction of blood pressure was more rapid (at 5–10 min) and effective after intravenous injection. However, no significant change of blood pressure was noted after injection through left vertebral artery, implicating that the hypotensive effect is not related to the central nervous system. Stevioside also showed significant hypotensive effects in renal hypertensive dogs, in a dose-dependent manner. In cultured rat aortic smooth muscle cells (A7r5 cell line), stevioside can dose-dependently inhibit the stimulatory effects of vasopressin and phenylephrine on intracellular Ca2+ in a calcium-containing medium. However, no intracellular Ca2+ inhibitory effect was observed in calcium-free medium, implicating that stevioside may inhibit the Ca2+ influx from extracellular fluid. Our present data show that stevioside did not influence the calcium ionophore (A23187) induced Ca2+ influx, indicating that the antagonistic effect was through Ca2+ channels. This study confirmed that stevioside is an effective antihypertensive natural product, and its hypotensive mechanism may be probably due to inhibition of the Ca2+ influx.

Uric acid activates extracellular signal-regulated kinases and thereafter endothelin-1 expression in rat cardiac fibroblasts

BACKGROUND The association between hyperuricemia and cardiovascular diseases has long been recognized. Elevated levels of uric acid may have a causal role in hypertension and cardiovascular diseases. However, the direct effect of uric acid on cardiac cells remains unclear. Therefore, this study was aimed to examine the effect of uric acid in rat cardiac fibroblasts and to identify the putative underlying signaling pathways. METHODS Cultured rat cardiac fibroblasts were stimulated with uric acid; cell proliferation and endothelin-1 (ET-1) gene expression were examined. The effect of uric acid on NADPH oxidase activity, reactive oxygen species (ROS) formation, and extracellular signal-regulated kinases (ERK) phosphorylation were tested to elucidate the intracellular mechanism of uric acid in ET-1 gene expression. RESULTS Uric acid-increased cell proliferation and ET-1 gene expression. Uric acid also increased NADPH oxidase activity, ROS formation, ERK phosphorylation, and activator protein-1 (AP-1)-mediated reporter activity. Antioxidants suppressed uric acid-induced ET-1 gene expression, and ERK phosphorylation, and AP-1 reporter activities. Mutational analysis of the ET-1 gene promoter showed that AP-1 binding site was an important cis-element in uric acid-induced ET-1 gene expression. CONCLUSIONS These results suggest that uric acid-induced ET-1 gene expression, partially by the activation of ERK pathway via ROS generation in cardiac fibroblasts.

Uric acid stimulates endothelin-1 gene expression associated with NADPH oxidase in human aortic smooth muscle cells

AbstractAim:Recent experimental and human studies have shown that hyperuricemia is associated with hypertension and cardiovascular diseases. Elevated levels of endothelin-1 (ET-1) has been regarded as one of the most powerful independent predictors of cardiovascular diseases. For investigating whether uric acid-induced vascular diseases are related to ET-1, the uric acid-induced ET-1 expression in human aortic smooth muscle cells (HASMC) was examined.Methods:Cultured HASMC treated with uric acid, cell proliferation and ET-1 expression were examined. Antioxidant pretreatments on uric acid-induced extracellular signal-regulated kinases (ERK) phosphorylation were carried out to elucidate the redox-sensitive pathway in proliferation and ET-1 gene expression.Results:Uric acid was found to increase HASMC proliferation, ET-1 expression and reactive oxygen species production. The ability of both N-acetylcysteine and apo-cynin (l-[4-hydroxy-3-methoxyphenyl]ethanone, a NADPH oxidase inhibitor) to inhibit uric acid-induced ET-1 secretion and cell proliferation suggested the involvement of intracellular redox pathways. Furthermore, apocynin, and p47phox small interfering RNA knockdown inhibited ET-1 secretion and cell proliferation induced by uric acid. Inhibition of ERK by U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) significantly suppressed uric acid-induced ET-1 expression, implicating this pathway in the response to uric acid. In addition, uric acid increased the transcription factor activator protein-1 (AP-1) mediated reporter activity, as well as the ERK phosphorylation. Mutational analysis of the ET-1 gene promoter showed that the AP-1 binding site was an important cis-element in uric acid-induced ET-1 gene expression.Conclusion:This is the first observation of ET-1 regulation by uric acid in HASMC, which implicates the important role of uric acid in the vascular changes associated with hypertension and vascular diseases.

Effect of trilinolein on the activity and gene expression of superoxide dismutase in cultured rat brain astrocytes

Stroke is one of the major causes of morbidity and mortality in recent. Oxygen free radicals produced during cerebral infarction increases the damage to neurons. Superoxide dismutase (SOD) is the endogenous antioxidant enzyme that can effectively scavenge superoxide radicals. Catechin is a hydrophilic antioxidant usually existed in tea, fruits and vegetables. In the cultured rat brain astrocytes (RBA), the activity of SOD (both Cu, Zn-SOD and Mn-SOD subtypes) was markedly increased by incubation with catechin at low concentration (0.1 microM) for 2 days (short-term) and 7 days (long-term). This stimulatory effect of catechin was not related to the incubating concentration. Similar changes were also observed in the gene expression of SOD in RBA. The increase in quantity of SOD-messenger RNA was remarkable and seemed to be more obvious than the other antioxidants such as vitamin E. This result shows that catechin is an effective antioxidant to increase the activity of SOD in RBA which would be beneficial to neurons subjected to oxygen free radical damage.

Involvement of reactive oxygen species in urotensin II-induced proliferation of cardiac fibroblasts

Urotensin II, a cyclic dodecapeptide, has recently been demonstrated to play an important role in cardiac remodeling and fibrosis. Cardiac fibroblast is the cell type known to proliferate during cardiac fibrosis and to produce the excess matrix proteins characteristic of cardiac remodeling. However, the effect of urotensin II on cardiac fibroblast proliferation and the intracellular mechanisms remain to be clarified. Cultured neonatal rat cardiac fibroblasts were stimulated with urotensin II, cell proliferation and the reactive oxygen species generation were examined. We also examined the effects of antioxidant pretreatment on urotensin II-induced cell proliferation, extracellular signal-regulated kinase phosphorylation, and the tyrosine phosphorylation of epidermal growth factor receptor, to elucidate the redox-sensitive pathway in urotensin II-induced cell proliferation. Urotensin II-increased cell proliferation and intracellular reactive oxygen species levels which were inhibited by antioxidants N-acetylcysteine, and the flavin inhibitor diphenyleneiodonium. Urotensin II potently activated the tyrosine phosphorylation of epidermal growth factor receptors and extracellular signal-regulated kinase. Pretreatment of cells with U0126, an inhibitor of the upstream activator of mitogen-activated protein kinase kinase, or with AG1478, a selective epidermal growth factor receptor kinase inhibitor, reduced the urotensin II-increased extracellular signal-regulated kinase phosphorylation. Antioxidants, U0126, and AG1478, all significantly inhibited urotensin II-increased cell proliferation in cardiac fibroblasts. Our data suggest that the redox-sensitive intracellular signaling pathway plays a role in urotensin II-induced proliferation in rat cardiac fibroblasts.

Antiproliferative Effect of Isosteviol on Angiotensin-II-Treated Rat Aortic Smooth Muscle Cells

Isosteviol is a derivative of stevioside, a constituent of Stevia rebaudiana, which is commonly used as a noncaloric sugar substitute in Japan and Brazil. The aims of this study were to examine whether isosteviol alters angiotensin-II-induced cell proliferation in rat aortic smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with isosteviol, then stimulated with angiotensin II, after which [3H]thymidine incorporation and endothelin-1 secretion were examined. Isosteviol (1–100 µmol/l) inhibits angiotensin-II-induced DNA synthesis and endothelin-1 secretion. Measurements of 2′7′-dichlorofluorescin diacetate, a redox-sensitive fluorescent dye, showed an isosteviol-mediated inhibition of intracellular reactive oxygen species generated by the effects of angiotensin II. The inductive properties of angiotensin II on extracellular signal-regulated kinase (ERK) phosphorylation were found reversed with isosteviol and antioxidants such as N-acetylcysteine. In summary, we speculate that isosteviol inhibits angiotensin-II-induced cell proliferation and endothelin-1 secretion via attenuation of reactive oxygen species generation. Thus, this study provides important insights that may contribute to the effects of isosteviol on the cardiovascular system.