The authors have declared that no competing interests exist.
Conceived and designed the experiments: XYZ PL YBY MLL. Performed the experiments: XYZ PL YBY. Analyzed the data: XYZ PL. Contributed reagents/materials/analysis tools: XYZ PL YBY. Wrote the paper: XYZ PL.
Xuezhikang is the extract of red yeast rice, which has been widely used for the management of atherosclerotic disease, but the molecular basis of its antiatherosclerotic effects has not yet been fully identified. Here we investigated the changes of eNOS in vascular endothelia and RBCs, eNOS regulatory factor Caveolin-1 in endothelia, and hemorheological parameters in atherosclerotic rats to explore the protective effects of Xuezhikang.
Wistar rats were divided into 4 groups (n = 12/group) group C, controls; group M, high-cholesterol diet (HCD) induced atherosclerotic models; group X, HCD+Xuezhikang; and group L, HCD +Lovastatin. In group X, Xuezhikang inhibited oxidative stress, down-regulated caveolin-1 in aorta wall (
Xuezhikang up-regulated eNOS expression in vascular endothelia and RBCs, increased plasma NOx and improved abnormal hemorheology in high cholesterol diet induced atherosclerotic rats. The elevated eNOS/NO and improved hemorheology may be beneficial to atherosclerotic disease.
Xuezhikang, the extract of red yeast rice, has been widely used as a Chinese traditional medicine for the therapy of patients with cardiovascular diseases. It contains natural Lovastatin and its homologues, as well as unsaturated fatty acids, flavonoids, plant sterols and other biologically active substances
Nitric oxide (NO) is a key regulator of endothelial function and vascular homeostasis
In addition to controlling vascular tone, NO is also a potent regulator of hemorheology mainly due to the improvement of erythrocyte rheological properties
In order to explore the molecular mechanisms of anti-atherosclerotic effects of Xuezhikang, we investigated the changes of eNOS in vascular endothelia and RBCs, NOS regulatory factor Cav-1 in endothelia, and hemorheological parameters after Xuezhikang treatment in high cholesterol diet induced atherosclerotic rats.
Xuezhikang powder was provided by WBL Peking University Biotech Co., Beijing, China. Xuezhikang is the mixture of 13 natural monacolins, such as monacolin K, L, J, M and X. The structure of monacolin K in its lactone form is identical to lovastatin, and lovastatin always be used as the quality standard for Xuezhikang. Xuezhikang contains 0.8% Lovastatin, 8% unsaturated fatty acids (primarily linoleic acid, oleic acid, palmitic acid, stearic acid, etc), as well as essential amino acids, ergosterol and some other components.
Animal study was performed in strict accordance with the Care and Use of Laboratory Animals recommended by National Institute of Health, and was approved by the Animal Research Committee of Peking University Health Sciences. Animal surgery was carried out under sodium pentobarbital anesthesia.
Wistar rats of 200±20 g body weight from the Animal Center of Academy of Military Medical Sciences were randomly assigned into four groups (n = 12/group): (1) controls (group C), (2) fed with high cholesterol diet (HCD), atherosclerotic models (group M), (3) HCD+Xuezhikang (group X), (4) HCD+Lovastatin (group L). Rats in groups M, L and X were intraperitoneally injected with a single dose of vitamin D3 (600,000 U/kg) and fed with the high-cholesterol diet (containing 3% cholesterol, 0.5% cholate, 5% refined sugar, 10% lard and 0.2% propylthiouracil) to induce atherosclerosis. Lovastatin and Xuezhikang were administered by gastric tube with the dose of 2.5 mg·kg−1·d−1 and 300 mg·kg−1·d−1, respectively, based on the doses used in previous studies
At the end of the experiment, rats were anesthetized by sodium pentobarbital (1.25 g/kg) after overnight fasting. Blood was collected by abdominal aorta puncture and anticoagulated with heparin for hemorheological examinations.
Anticoagulated blood was centrifuged 3,000 rpm for 10 min at 4°C to separate plasma and erythrocytes. Plasma samples were used for the measurements of Triglyceride (TG), low density lipoprotein cholesterol (LDL-C), NOx (total nitrite and nitrate), MDA, SOD and T-AOC. Packed erythrocytes were washed twice with heparin-phosphate-buffered saline (PBS), and added to five volumes of hypotonic buffer (10 mmol/L Tris-HCl, pH 7.4) to make hemolysis. After centrifugation, the supernatant was collected as erythrocyte cytoplasm for cGMP determination. The pellet was washed and centrifuged three times to obtain erythrocyte membrane sample. The erythrocyte membrane sample was lysed in sample buffer (20 mmol/L Tris pH 7.4, 2.5 mmol/L EDTA, 1% Triton X-100, 1% deoxycholic acid, 0.1% sodium dodecyl sulfate, 100 mmol/L NaCl, 10 mmol/L NaF, and protease inhibitor cocktail) for eNOS determination by western blotting.
Aorta between the aortic valve cusps and bifurcation at iliac arteries was isolated and taken off the gross adventitial tissue. The ascending aorta was quickly fixed in 4% formaldehyde for histological studies. Aortic arch was homogenized in RIPA buffer and centrifuged at 12,000 rpm for 20 minutes at 4°C. Protein lysates of aortic arch were used for the measurements of eNOS, p-eNOS and caveolin-1 by western blotting, the measurement of cGMP by ELISA, and immunoprecipitation. Protein concentration in aortic arch lysate and erythrocyte membrane sample was assayed by a BCA protein quantification kit (KeyGen Biotechnology, China).
Rat blood samples were measured for erythrocyte deformation index (EDI), whole blood viscosity (WBV), plasma viscosity (PV), and hematocrit (Hct). EDI was determined at various fluid shear stresses by using an ektacytometer (Model LBY-BX2, Precil Co., China). Erythrocytes were re-suspended in 15% polyvinylpyrrolidone (PVP, MW 30 kDa) buffer (61 mM NaCl, 0.8 mM Na2HPO4, 0.2 mM KH2PO4, pH 7.4, 290 mOsm/kg, viscosity 15 mPa·s) and adjusted to 2×107 cells/ml to measure EDI. The EDI was calculated for shear rates from 50 to 1000 s−1. Whole blood viscosity was measured by using a cone-plate viscometer (Model LBY-N6A, Precil Co., China). Plasma viscosity was measured with a capillary viscometer (Model LBY-NM2, Precil Co., China). Hematocrit was measured in a capillary tube after centrifugation (15,000 rpm for 3 min) by a microhematocrit centrifuge. All measurements were carried out at 37°C according to the International Guidelines for the measurements of hemorheologic parameters.
NO is chemically active and rapidly converted into nitrate (NO3−) and nitrite (NO2−) in vivo. NO2− is further converted into NO3−. In this study, specific nitrate reductase method and Griess reaction were performed to detect NO metabolites (nitrite and nitrate).
Malondialdehyde (MDA) was analyzed by TBA assay. SOD was measured by degree of inhibition on nitroblue tetrazolium (NBT) produced by superoxide radicals generated from the xanthine/xanthine oxidase system. Total antioxidant capacity (T-AOC) in plasma was measured based on the level of Fe3+ reduced to Fe2+. We followed the instruction of the kit (Nanjing Jiancheng Biology Engineering, China), and measured using an ultraviolet/visible scanning spectrophotometer.
Equal amount of proteins were separated by SDS-polyacrylamide gel (SDS-PAGE) electrophoresis at 4°C, and the proteins were transferred onto a nitrocellulose membrane. After blocking nonspecific sites, the membrane was incubated with anti-eNOS antibody (1∶1000 sc-376751, Santa Cruz Biotech), anti-eNOS p-ser 1177 antibody (1∶500, Santa Cruz Biotech), anti-tubulin antibody (1∶1500, Santa Cruz Biotech), anti-caveolin-1 antibody (1∶1000, Santa Cruz Biotech), or anti-β-actin antibody (1∶1500, Santa Cruz Biotech) overnight. Blotted antibodies were detected by a horseradish peroxidase-labeled second antibody (1∶8000, Santa Cruz Biotech) and enhanced chemiluminescent technique. Density of the bands was measured by an image analyzer.
Protein lysate of aorta wall was pre-cleared by incubation with 50 µl of 50% (V/V) protein A sefinose (Sangon Biotech Co., China) for 30 min to pre-clean the lysate. 5 µl of anti-caveolin-1 antibody (Santa Cruz Biotech, CA, USA) and 50 µl of 50% (V/V) protein A sefinose were added to the pre-cleaned supernatant and the mixture was incubate at 4°C for 5 hours on a shaker. The resin was collected by centrifugation at 2,500 rpm at 4°C for 1 min, washed with 1 ml PBS for 3 times, and re-suspended in 80 µl loading buffer. After heated at 100°C for 5 min and centrifugation, the samples were used for western blotting to detect eNOS by the anti-eNOS antibody.
Longitudinal sections of ascending aorta were used for the examinations. The slides were heated in a microwave oven for 15 min in 10 mM sodium citrate buffer pH 6.0 and then incubated in the buffer at room temperature for 20 min to retrieve antigens. After washing, the slides were incubated in 0.3% H2O2 in 100% methanol for 30 min to inactivate endogenous biotin. After washing with PBS and blocking in 1.5% normal goat serum, the slides were then incubated with anti-eNOS monoclonal antibody (1∶100) or anti-caveolin-1 polyclonal antibody (1∶100) overnight at 4°C. After washing with PBS, the slides were sequentially incubated with biotinylated goat anti-mouse IgG or goat anti-rabbit IgG antibody, HRP-conjugated streptavidin and development reagent. A high power microscope with digital imaging system was used for photograph after counterstaining the slides with hematoxylin.
Hemoglobin in erythrocyte cytoplasm was removed by an ultrafilter (cutoff 50-kDa, Millipore) as described previously
Results were presented as means±standard deviations. Statistical differences were evaluated by one-way analysis of variance (ANOVA) with Tukey or Dunnett post hoc analysis. A value of
In group M, plasma LDL-C was significantly increased as compared with that in group C (
Group (n = 12/group) | TG(mmol/L) | LDL-C(mmol/L) | HDL-C(mmol/L) |
M | 0.67±0.19 | 6.43±1.93 |
1.12±0.32 |
L | 0.31±0.11 | 3.69±1.08 | 0.96±0.33 |
X | 0.29±0.16 |
3.32±0.29 |
0.94±0.31 |
C | 0.64±0.06 | 0.28±.012 | 0.64±0.06 |
M: atherosclerosis model group, rats were fed with high cholesterol diet (HCD); L: Lovastatin group, rats were fed with HCD+Lovastatin; X: Xuezhikang group, rats were fed with HCD+Xuezhikang. C: control group, rats were fed with regular diet.
In group M, plasma MDA increased and SOD and T-AOC decreased as compared with those of group C (
Group (n = 12/group) | MDA (nmol/ml) | SOD (U/ml) | T-AOC (U/ml) |
M | 11.52±1.98 |
62.39±12.08 |
6.64±2.31 |
L | 9.62±0.48 |
72.13±4.75 |
8.05±1.03 |
X | 8.68±0.40 |
95.57±4.15 |
8.57±0.96 |
C | 6.23±0.63 | 107.7±6.07 | 9.08±0.54 |
Microscopic examination of the ascending artery revealed no pathological changes in group C. Rats in group M developed typical plagues with macrophage infiltration and thickened intima. In group X, the morphology of aorta was comparable to that of group C (
(H-E staining, 20x).
In group M, erythrocyte deformation index (EDI) decreased and whole blood viscosity (WBV) and plasma viscosity (PV) increased as compared with those in group C (
WBV (mPa·s) | EDI (max) | Pv (mPa·s) | HCT | |||
Group (n = 12/group) | 50/S−1 | 100/S−1 | 150/S−1 | |||
M | 5.47±0.41 |
4.91±0.39 |
4.58±0.33 |
0.31±0.02 |
1.81±0.16 |
0.41±0.01 |
L | 4.66±0.59 | 4.17±0.45 | 3.97±0.39 | 0.32±0.01 | 1.68±0.16 | 0.41±0.01 |
X | 4.50±0.26 |
4.04±0.19 |
3.87±0.20 |
0.35±0.01 |
1.54±0.20 |
0.41±0.02 |
C | 4.72±0.68 | 4.34±0.64 | 3.99±0.48 | 0.36±0.01 | 1.56±0.15 | 0.42±0.01 |
Plasma NO metabolites (nitrite and nitrate; NOx) were measured to estimate NO production. Plasma NOx increased significantly in groups X and L as compared with that in group M (
(n = 12/group). *:
In group M, cGMP level in aorta wall and erythrocyte cytoplasm decreased as compared with that in group C (
Hemoglobin in erythrocyte cytoplasm had been removed by ultrafiltration before the assay. *:
eNOS was expressed on rat erythrocyte membrane (see
About a 134 kDa band was identified by anti-eNOS antibody. β-tubulin was used as an internal reference. *:
In group M, eNOS and p-eNOS in aorta wall decreased as compared with that of group C. In group X, eNOS and p-eNOS was significantly higher than those in group C and group L (
(A) Protein lysate of aorta wall was used for the detection of eNOS, eNOS p-ser1177 by western blotting (n = 6/group). *:
Caveolin-1 expression changed reversely with eNOS expression in aorta wall among the four groups, i.e., caveolin-1 increased in group M, decreased in groups L and X, and the decrease was more significant in group X than in group L (
(A) Protein lysate of aorta wall was used for the detection of caveolin-1 (n = 6/group). *:
Protein lysates of aorta wall were incubated with anti-caveolin-1 antibody and protein A resin, and the immuno-precipitated proteins were blotted by anti-eNOS antibody by western blotting. Negative control sample was taken from the mixed supernatant from groups M, L, X and C, and was subjected to immunoprecipitation without the addition of anti-caveolin-1 antibody.
There is no plasma cholesterylester transfer protein (CETP) in rats, and 80% of total plasma cholesterol exists in HDL particles. Therefore, rats are considered to be resistant to atherogenesis and require higher cholesterol diet to form arterial plagues
In this study, we found that Xuezhikang increased eNOS expression in vascular endothelia and erythrocytes, and decreased the expression of caveolin-1 in aorta. These changes are expected to increase NO production, which was confirmed by the increase of NOx (nitrate and nitrite) in plasma and cGMP in aorta wall. Besides, Xuezhikang treatment ameliorated the hemorheological abnormalities and oxidative stress, lowered the higher serum lipids, and improved the pathology of atherosclerosis.
Previous studies showed that the cholesterol synthesis inhibitor rosuvastatin can increase eNOS activity in endothelial cells
RBCs also contain functional eNOS
Hemorheological status is determined by many factors including erythrocyte deformability, blood volume, plasma viscosity, platelet activation, etc. Hemorheological abnormalities play an important role in the pathogenesis and development of atherosclerosis
We conclude that Xuezhikang up-regulated eNOS expression in vascular endothelia and RBCs, increased plasma NOx and improved abnormal hemorheology in high cholesterol diet induced atherosclerotic rats. The elevated eNOS/NO and improved hemorheology may be beneficial to atherosclerotic disease.
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