Mechanisms of cardiovascular effects of oleanolic acid and related synthetic oleanane derivatives : an experimental study.
Madlala, Hlengiwe Pretty.
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Introduction Despite the various conventional treatments that are available to treat hypertension, this disease continues to be globally responsible for approximately 9.4 million deaths each year. The high mortality can partly be attributed to side effects of available drugs or to the inaccessibility of current synthetic drugs to communities from poor socioeconomic background because of their relative high cost. This problem has resulted in a growing interest in the use of medicinal plant products because they are considered to be cheap, believed to possess few side effects and are easily accessible to the general population in developing countries. Although traditional herbal remedies are widely used in Africa for the management of various disorders including cardiovascular diseases, very little reliable data is available on their therapeutic and pharmacological effects. In search for plants with therapeutic properties for the treatment of hypertension and complications, our laboratory has scientifically evaluated several plant species. In particular, we have isolated Syzygium spp-derived triterpenes and focused on the therapeutic effects of oleanolic acid (OA) and maslinic acid (MA). In the present study, we investigated the effects of Syzygium aromaticum-derived OA and related synthetic derivatives on arterial pressure and evaluated the underlying mechanisms in Wistar, spontaneously hypertensive rats (SHR) and Dahl salt-sensitive (DSS) animals. Materials and methods OA was extracted from dried flower buds of Syzygium aromaticum using a previously validated protocol in our laboratory. Oleanolic acid methyl ester (Me-OA) and a brominated derivative of OA (Br-OA) were synthesised according to methods described by Fu and Gribble. The structures of extracted OA and synthesised derivatives were confirmed using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and were comparable to previously reported data. Acute renal clearance studies investigated the influence of OA and derivatives on mean arterial pressure (MAP) and Na+ handling in the proximal tubule of anaesthetised Wistar rats using lithium clearance. Animals were given water with lithium (12 mmol L-1) for 48 hours following which they were anaesthetised and cannulated using a previously validated standard protocol that has been reported from our laboratories. After a 3.5 h equilibration, animals were challenged with hypotonic saline for 4 h of 1 h control, 1.5 h treatment and 1.5 h recovery periods. OA, Me-OA and Br-OA were added to the infusate during the treatment period followed by measurements of arterial pressure, fluid and electrolyte handling. Sub-chronic study experiments were restricted to OA because of the low amounts of synthetic derivatives obtained during the synthetic studies to prepare the derivatives. Various doses of OA (30, 60, 120 mg kg-1, p.o.) were administered to separate groups of male Wistar, SHR and DSS rats twice (8 h apart) every third day for nine weeks. Rats given dimethyl sulphoxide (DMSO)-saline (3 mL kg-1, p.o.) acted as untreated controls. Mean arterial pressure (MAP) was monitored every third consecutive day using non-invasive tail cuff method with photoelectric sensors. Measurements of body weight, food and water intake, Na+, K+, Cl-, urea and creatinine were taken 24 h after dosing. At the end of this 9 week study, the cardiac, renal and hepatic tissues harvested from hypertensive animals were evaluated for oxidative status by measuring malonyldialdehyde (MDA, lipid peroxidation marker) and antioxidant enzymes; superoxide dismutase (SOD) and glutathione peroxidase (GPx). In addition, we evaluated the effects of OA and derivatives on aldosterone and arginine vasopressin (AVP) secretion on plasma samples from both sub-chronic and acute experimental settings. Additional Ex vivo studies to unravel the mechanisms of action of OA and derivatives were carried out in isolated ventricular cardiomyocytes and arteries. Measurements of cell shortening and Ca2+ currents were done in cells isolated from Wistar and DSS animals using edge detection and whole cell patch clamp techniques, respectively. Isometric tension measurements were done in endothelium-intact and denuded aortic rings and mesenteric arteries of Wistar and DSS rats. The influence of indomethacin (INDO), N-nitro-L-arginine methyl ester (L-NAME), glibenclamide (Gli) and 4-aminopyridine (AP) on the effects of OA and derivatives was investigated. Results The purity of the plant-derived OA was approximately 98% and the percentage yield varied from 0.79% to 1.72% of the dry plant material. The percentage yield of the synthetic derivatives, Me-OA and Br-OA was 65% and 30%, respectively, from the starting materials. Our results show that OA decreased MAP in both acute and sub-chronic experimental settings, and that the MAP lowering effect was more marked in hypertensive animals compared to normotensive rats. Similarly, the two OA derivatives, used in acute settings, also decreased MAP. OA increased urinary Na+ excretion rate under acute and sub-chronic conditions. A similar but quantitatively more marked increase was obtained with Br-OA derivative under acute conditions. Untreated hypertensive animals had elevated levels of plasma aldosterone and AVP in comparison to Wistar rats. OA treatment significantly reduced aldosterone secretion in these animals but had no influence on plasma concentrations of AVP. Compared with respective control rats, OA-treated animals exhibited significantly lower MDA levels and increased activity of the antioxidant enzymes, SOD and GPx in hepatic, cardiac and renal tissues. OA and derivatives had a positive inotropic effect on isolated ventricular cardiomyocytes in Wistar rats and had no influence on the contraction of cells from hypertensive animals. OA and derivatives caused relaxation in aortic rings and mesenteric arteries of both Wistar and DSS animals. This effect was partly inhibited by INDO and K+ channel blockers when used independently. Addition of L-NAME did not further inhibit the cyclooxygenase (COX)-resistant relaxation. The combination of INDO, Gli and AP completely abolished OA or derivatives-evoked relaxation in endothelium-intact arteries indicating involvement of both endothelium-dependent and independent mechanisms. Discussion The present study investigated the effects of OA and related synthetic derivatives (Me-OA and Br-OA) on blood pressure and thereafter examined the possible underlying mechanisms. The absolute stereo-structure of S. aromaticum-derived OA and synthetic derivatives elucidated from the spectra using 1H- and 13C-NMR was comparable to previously reported data. Our results confirm the previously reported antihypertensive properties of OA in experimental models of hypertension. However, the present study demonstrates a more marked action in SHR and DSS by comparison with non-hypertensive animals, suggesting a specifically enhanced action in disease conditions. We noticed that urinary Na+ excretion in control, untreated animals tended to spontaneously increase with time during the following 9 weeks post weanling. In both hypertensive models, no such increase with time was obtained, instead, urinary Na+ excretion tended to decrease with time after weanling in the DSS model supporting Na+ retention previously reported in these animals. To further support this theory we found increased aldosterone levels in the plasma of non-treated hypertensive rats. Our data show that OA decreased aldosterone secretion and increased urine Na+ excretion, relatively larger increases were obtained in the hypertensive models. This suggests that treatment with the drugs was accompanied by alleviation of Na+ retention in these animals. This increase in urinary Na+ output is, at least in part, mediated via inhibition of proximal tubular Na+ reabsorptions as indicated by increased Li+ clearance. Indeed, we found a positive correlation between the increase in urinary Na+ excretion rate and the decrease in MAP. However, despite the potent natriuretic effects of these triterpenes mediated by decreased aldosterone levels, the urine flow rate was not changed as supported by unchanged levels of AVP. Our experiments indicated that OA induces vasorelaxation of aortic rings and small mesenteric arteries in both normotensive and hypertensive animals. The maximum relaxation evoked by the derivatives, particularly Br-OA, was significantly larger in the mesenteric vessels although these differences were not observed in the aorta. Glibenclamide and 4-aminopyridine used allowed a complete blockade of the relaxation to OA, Br-OA and Me-OA suggesting that ATP-dependent and voltage-activated K+ channels opening mediate the endothelium-independent relaxation. Our experiments using isolated cardiomyocytes showed that OA and derivatives do not decrease but rather tend to increase myocyte shortening and had no influence on Ca2+ currents in Wistar rats. Increase cardiomyocyte contractility implies an increase in the force of cardiac tissue contraction and increase cardiac output which may be ideal for a drug which causes natriuresis. The results showed that OA-treated DSS animals had normal levels of MDA, SOD and GPx in comparison to untreated hypertensive rats. Therefore, we suggest that OA-evoked decreased reactive oxygen species (ROS) and increased antioxidant enzymes may have enhanced the production of prostanoids, thereby improving vasodilation. Hence, we speculate that antioxidant properties of OA could play a role in hypotensive mechanisms of this triterpene. Conclusion The results of this study introduce the first evidence that OA and its oleanane derivatives induce similar effects. These involve 1) increased urinary Na+ output mediated by inhibition of proximal and distal tubular Na+ reabsorption as indicated by increased Li+ clearance and decreased aldosterone levels, respectively. 2) Modulation of oxidative status in cardiac, renal and hepatic tissues in hypertensive animals. As well as 3) decreased vascular resistance via endothelium-dependent COX/prostanoids pathway and endothelium-independent opening of ATP-dependent and voltage-activated K+ channels. The results of this study are novel and clinically relevant because OA and related triterpene derivatives exert multiple blood pressure lowering mechanisms while increasing the force of cardiac contraction hence balancing the fluid volume in the circulatory system so as to avoid a state of hypotension. Limitations and direction for future studies Due to low amounts obtained for the synthetic derivatives, we were unable to study their sub-chronic effects in conscious animals, therefore this should be explored in future. OA effects on renal function seem to mimic oxytocin-like activities i.e. increase Na+ output without changing the urine flow rate, hence future studies should explore whether this hormone can have synergistic effects with OA and derivatives. This study did not evaluate the effects of these triterpenes on nitric oxide production or expression of eNOS and phosphorylation of K+ channels.
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