Methods
Chemicals
Asiatic acid (AA; ³ 98% purity by HPLC analysis) was purchased from Leap Labchem Co., Ltd. (Hangzhou, China). Cadmium chloride (CdCl2), glutathione, ethylenediamine tetraacetic acid (EDTA), thiobarbituric acid (TBA), sodium dodecylsulfate (SDS), 1, 1, 3, 3-tetraethoxypropane, metaphosphoric acid (MPA), 2,4-dinitrophenylhydrazine (DNPH), bovine serum albumin fraction V (BSA), N-(1-nepthyl)ethylenediamine dihydrochloride (NED), sodium nitrite (NaNO2) and sodium nitrate (NaNO3) were from Sigma-Aldrich Pte. Ltd. (Singapore). Acetylcholine chloride and sodium nitropusside were obtained from Fluka Chemika Co. Ltd. (Buchs, Switzerland). All other chemicals used were of analytical grade quality.
Animals
Male Sprague-Dawley rats weighing 180200 g were obtained from the National Laboratory Animal Center, Nakhon Pathom, Thailand. The rats were housed in the Heating, Ventilation and Air-Conditioning (HVAC) System with 12 h dark/light cycle in the Northeast Laboratory Animal Center, Khon Kaen University, Thailand, and were fed with a standard chow diet (Chareon Pokapan Co. Ltd., Samutprakarn, Thailand). The study protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Khon Kaen University (AEKKU-NELAC 17/2558). All surgical procedures were performed under standard anesthesia, and all efforts were made to minimize suffering. After one week of acclimatization, rats were randomly divided into five groups (n = 8/group): group I-control, group II-control + AA 30 mg/kg b.w., group III-Cd alone, group IV-Cd + AA 15 mg/kg b.w., and group V-Cd + AA 30 mg/kg b.w. The control groups received deionized water (DI) as drinking water whereas Cd-treated groups received drinking water containing CdCl2 (10 ppm or 10 mg/L) continuously for 16 weeks. AA (15 or 30 mg/kg) was orally administered once daily for the last 4 weeks of the experiment.
Indirect blood pressure measurement
To monitor the changes in arterial blood pressure of rats during exposure to Cd, blood pressure was assessed by the indirect tail cuff method. Briefly, a conscious rat was placed in restrainer and allowed to rest inside the cage for 15 min prior to blood pressure measurement. The rat tail was placed inside the tail cuff, an automatically inflated and released. Systolic blood pressure (SBP) values of 3-4 consecutive readings were recorded by using a rat tail sphygmomanometer (IITC model 179 blood pressure analyzer, Woodland Hills, CA, USA).
Measurement of hemodynamic status and vascular responsiveness
After 16 weeks, rats were anaesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg). A tracheotomy was performed for spontaneous breathing, and left femoral artery was cannulated with polyethylene catheter connected to a pressure transducer for continuous monitoring of blood pressure (BP) using the Acqknowledge data acquisition analysis software (BIOPAC Systems Inc., California, USA). The left femoral vein was cannulated with polyethylene tubing for infusion of vasoactive drugs. Baseline BP values were monitored in animals for 10 min. After obtaining stable baseline measurements, an endothelium-dependent vasodilator, acetylcholine (ACh; 3, 10, 30 nmol/kg) and an endothelium-independent vasodilator, sodium nitroprusside (SNP; 1, 3, 10 nmol/kg), were randomly infused intravenously, while blood pressure was continuously monitored. Thereafter, hindlimb blood flow (HBF) was measured by placing electromagnetic flow probes around the abdominal aorta connected to an electromagnetic flowmeter (Carolina Medical Electronics, North Carolina, USA). Hindlimb vascular resistance (HVR) was calculated from the mean arterial pressure (MAP) divided by HBF. At the end of experiment, rats were sacrificed by overdose of the anesthetic drug. Blood samples were withdrawn from abdominal aorta for assays of oxidative stress markers. The aortas were rapidly excised from the animals and used for analysis of superoxide anion (O2 -) production and Cd content.
Assay of oxidative stress markers and NO metabolites
O2 - production in vascular tissues was measured using lucigenin-enhanced chemiluminescence method 10. Lipid peroxidation, as measured by malondialdehyde (MDA) level in plasma, was estimated using thiobarbituric acid (TBA) as described previously 10. The antioxidant glutathione (GSH) in whole blood was assayed by a previously described method 11. Nitrite and nitrate, the end products of nitric oxide (NO) metabolism, were used as indicator of NO production. Plasma nitrate/nitrite was determined by an enzymatic conversion method with the Griess reaction as previously described 12.
Assay of Cd contents
Cd content in whole blood and aortic tissues were determined by using inductively coupled plasma mass spectrometry (ICP-MS) method (Agilent 7500 ICP-MS model, Santa Clara, CA, USA) according to the manufacturers recommendation as previously described 12. The Cd contents were expressed in mg/L and mg/g of tissue wet weight.
Statistical analysis
Data are presented as means ± S.E.M. Statistical differences were evaluated by one-way analysis of variance (ANOVA) and followed by Student NewmanKeul's test to show specific group differences. All analysis was performed using SigmaStat software version 3.1. Statistical significance was determined at a level of p < 0.05.
Results
Effect of AA on hemodynamics and vascular responsiveness
The baseline data of systolic blood pressure (SBP) in all studied groups obtained from indirect blood pressure measured by using a rat tail sphygmomanometer are displayed in Figure 1. There were no differences in SBP among all groups at the beginning of experiments. SBP increased progressively in Cd-treated rats during experimental periods for 16 weeks (p < 0.05, Fig. 1), indicating the development of hypertension in rats exposed to Cd. A significant reduction in SBP was found in rats exposed to Cd treated with AA (15 or 30 mg/kg) for 4 weeks when compared with those treated with Cd alone (p < 0.05, Fig. 1). The effect of AA was found in a dose-dependent manner.
Figure 1 Effect of asiatic acid on systolic blood pressures monitoring throughout 16 weeks of experiments.
Data are expressed as mean ± S.E.M. (n = 6-8/group). *p<0.05 compared to control group, †p<0.05 compared to Cd-treated group, $p<0.05 compared to Cd+AA15 mg/kg treated group.
A significant increase in SBP, diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) was found in Cd-treated rats (p < 0.05, Table 1), which is consistent with the data obtained from indirect blood pressure measurement. The elevation of arterial blood pressure was accompanied by decreasing HBF and increasing HVR (p < 0.05, Table 1). Rats supplemented with AA during Cd exposure showed a significant decrease in blood pressure and HVR whereas HBF increased (p < 0.05, Table 1). The improvement of hemodynamic status in Cd-exposed rats treated with AA was found in a dose-dependent manner. The data indicate that AA supplementation reduced arterial blood pressure and total peripheral resistance, which leads to prevent the development of hypertension during Cd exposure. There was no alterations in all hemodynamic parameters in control rats-treated with AA, indicating that AA does not change the normal physiological condition (Table 1).
Table 1 Effect of asiatic acid on hemodynamics of rats in all experimental groups
|
Parameters |
Control |
Control+AA30 |
Cd |
Cd+AA15 |
Cd+AA30 |
|
SBP (mmHg) |
118 ± 4 |
119 ± 3 |
158 ± 4* |
139 ± 4*† |
131 ± 4*†$ |
|
DBP (mmHg) |
84 ± 4 |
85 ± 3 |
102 ± 4* |
98 ± 4*† |
92 ± 3*†$ |
|
MAP (mmHg) |
95 ± 4 |
96 ± 3 |
121 ± 4* |
111 ± 4*† |
105 ± 4*†$ |
|
HR (beats/min) |
354 ± 41 |
359 ± 44 |
421 ± 30* |
410 ± 25*† |
388 ± 31*†$ |
|
HBF (ml/min/
100 g tissue) |
8.7 ± 0.5 |
8.8 ± 0.6 |
5.5 ± 0.6* |
6.8 ± 0.3*† |
7.4 ± 0.4*†$ |
|
HVR (mmHg/ml/
min/100 g tissue) |
10.9 ± 1.1 |
11.0± 1.2 |
21.9 ± 3.2* |
16.4 ± 1.3*† |
14.2 ± 1.3 *†$ |
SBP, systolic blood pressure; DBP, diastolic blood pressure, MAP, mean arterial pressure; HR, heart rate; HBF, hindlimb blood flow; HVR, hindlimb vascular resistance.
Data are expressed as mean ± S.E.M. (n = 6-8/group). *p<0.05 compared to control group, †p<0.05 compared to Cd-treated group, $p<0.05 compared to Cd+AA15 mg/kg treated group.
A marked reduction in vasodilation to ACh was found in rats with long-term exposure to Cd (p < 0.05, Fig. 2A), suggesting the endothelial dysfunction was present in animals exposed to Cd. AA, especially at high dose improved endothelial function by increasing the vasodilating responses to ACh (Fig. 2A). It is found that the vasodilating responses to SNP were not different in all groups (Fig. 2B).
Figure 2 Effect of asiatic acid on mean arterial pressure in responses to acetylcholine and sodium nitroprusside at various concentrations. Data are expressed as mean ± S.E.M. (n = 6-8/group). *p<0.05 compared to control group, †p<0.05 compared to Cd-treated group, $p<0.05 compared to Cd+AA15 mg/kg treated group.
Effect of AA on oxidative stress
Increased oxidative stress was found in rats-exposed to Cd, as shown by increasing vascular O2 - production and elevating plasma MDA (p < 0.05, Fig. 3A and B), whereas the blood GSH was markedly decreased (p < 0.05, Fig. 3C). Decrease in plasma nitrate/nitrite was found in Cd-treated rats compared to untreated controls (p < 0.05, Fig. 3D). Treatment with AA, particularly at high dose significantly alleviated oxidative stress, increased antioxidant GSH and increased plasma nitrate/nitrite in Cd-exposed rats (p < 0.05, Fig. 3). It appeared that alleviation of oxidative stress after AA treatment was associated with a restoration of hemodynamics.
Figure 3 Effect of asiatic acid on vascular superoxide production (A), plasma malondialdehyde (MDA) (B), glutathione (GSH) level in whole blood (C) and plasma nitrate/nitrite (D) in all experimental groups. Data are expressed as mean ± S.E.M. (n = 6-8/group). *p<0.05 compared to control group, †p<0.05 compared to Cd-treated group, $p<0.05 compared to Cd+AA15 mg/kg treated group.
Effect of AA on Cd contents
Data in Figure 4 demonstrate the levels of Cd contents in blood and vascular tissues of all experimental groups. A marked increase in Cd concentrations was found in the blood and vascular tissues of rats after Cd exposure for 16 weeks (p < 0.05, Fig. 4A and B). Meanwhile, the level of Cd contents in blood and tissues were very low in controls (Fig. 4). AA supplementation in a dose-dependent manner significantly reduced Cd contents in both blood and tissues (p < 0.05, Fig. 4).
Figure 4 Effect of asiatic acid on cadmium content in blood (A) and vascular tissues (B) in all experimental groups. Data are expressed as mean ± S.E.M. (n = 6-8/group). *p<0.05 compared to control group, †p<0.05 compared to Cd-treated group, $p<0.05 compared to Cd+AA15 mg/kg treated group.
Discussion
In this study, AA can restore the hemodynamics of rats exposed to Cd by lowering blood pressure, increasing hindlimb blood flow, reducing peripheral vascular resistance and decreasing heart rate. The improvement of hemodynamic status are associated with the increase in endothelial function, decrease in oxidative stress and reduction in Cd accumulations in the blood and tissues. It has been demonstrated that Cd decreased the functional availability of the potent vasodilator NO, which leads to increase blood pressure 13. In consistent with our results, the increase in blood pressure of Cd exposed rats was accompanied by a blunted responses to endothelial-dependent vasodilator, ACh. Yoopan et al. suggested that the impairment of endothelial function after Cd exposure might be due to a dysfunction of muscarinic cholinergic and NO-dependent pathway in the vascular endothelium14. Moreover, L-arginine-NO pathway has been proposed to play an important role in Cd-induced endothelial dysfunction and vascular inflammation 15. In agreement with this, we have found that endothelial NO synthase (eNOS) was decreased while inducible NO synthase (iNOS) was increased in the vascular tissues after Cd exposure 11, and tetrahydrocurcumin could modulate the eNOS/iNOS pathway. However, the effect of AA on eNOS/iNOS pathway is needed for further exploration.
It is evident that oxidative stress plays a key role in endothelial dysfunction and also exerts its effects in various pathophysiological conditions 16. It is found that Cd interacts with reactive thiols in the mitochondrial membrane and causes mitochondrial permeability transition, which inhibits the respiratory chain reaction, thereby generates reactive oxygen species (ROS) 17. O2 - is one of the major species of ROS that react with NO to form peroxynitrite, which causes NOS uncoupling in endothelial cells and leads to low NO bioavailability. As shown in our results, there was increased in O2 - production and reduced plasma nitrate/nitrite in Cd exposed rats, and these alterations were ameliorated after treatment with AA. The plausible mechanism involved with this effect is the antioxidant property of AA, which might be through the L-arginine-NO pathway since AA significantly stimulates the NO production. Moreover, AA also alleviated oxidative stress, increased antioxidant GSH and reduced Cd accumulation in the animal body after Cd exposure. Therefore, AA may be used as an antioxidant and metal chelator for detoxification.
Conclusion
The present study provides evidence about the beneficial effects of AA on reduced hypertension, improved endothelial function and alleviated oxidative stress in rats after exposure to Cd for 16 weeks. The ameliorating effects of AA against Cd toxicity may be mediated by its antioxidant and chelating properties. The overall findings support the idea of using Bua-Bok which contains high amount of asiatic acid as a food supplement to prevent hypertension and reduce oxidative stress in Cd-intoxication.
Acknowledgements
This work was supported by the Invitation Research Fund (IN61217), Faculty of Medicine and Joint Funding (591H217) under the Program for Supporting Lecturer to Admit High Potential Student to Study and Research on His Expert Program from the Graduate School, Khon Kaen University, Thailand.
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