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GESDAV

Journal of Investigational
Biochemistry

available at www.scopemed.org

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Original Research

Biochemical alterations and endothelial dysfunction in hypercholesterolemic rats: Effects of Bezafibrate

Omayma Ahmed Ragab Abozaid, Mohamed Ragaei Ragab Hasanin, Amal Hanafi

Department of Biochemistry, Faculty of Veterinary Medicine, Moshtohor, Benha University, Egypt

**Received:** January 31, 2013

**Accepted:** March 05, 2013

**Published Online:** March 23, 2013

**DOI:** 10.5455/jib.20130305012038

**Corresponding Author:**
Omayma Ahmed Ragab Abozaid, Department of Biochemistry, Faculty of Veterinary Medicine, Moshtohor, Benha University, Egypt
mohamedsoliman8896@yahoo.com

**Key words:** Hyperlipidemia, bezafibrate, lipid profiles, endothelial dysfunction

**Abstract**

**Background:** Hypercholesterolemia is a serious problem cause biohazards for human health and endothelial dysfunction.

**Objective:** The current study was conducted to test effects of hyperlipidemia alone or in combination with bezafibrate at various doses. Total cholesterol, triacylglycerol high density lipoprotein (HDL), low density lipoprotein (LDL), apolipoprotein B, endothelin-1, histamine, nitric oxide (NO), IL-6, creatinine and alanine amino transferase (ALT) were assayed.

**Methodology:** Rats were randomly divided into four groups (10 rats each). The first group fed a normal diet and represents the control group. The second group fed normal diet enriched with 1% cholesterol and 5% coconut oil (cholesterol-fed group). The third group fed on normal diet and bezafibrate at a dose of 100 mg/kg/day. The fourth group is cholesterol fed and subdivided into A, B, C and supplemented with bezafibrate at various concentration 50, 100, 200 mg/kg/day respectively. Feeding was continued daily for 6 weeks after incidence of hypercholesterolemia.

**Results:** The current study revealed that total cholesterol, triacylglycerols, apolipoprotein E and LDL concentration were significantly elevated in the cholesterol-fed rats compared to control alone and control plus bezafibrate. In addition, plasma endothelin-1, histamine and IL6 concentration were significantly elevated in the cholesterol-fed rats compared to bezafibrate and control group rats. Bezafibrate administration to rats fed high cholesterol diet normalize significantly the changes in all parameters except NO levels compared to hyperlipidemic group. Meanwhile, the plasma nitric oxide in all groups was not significantly different from those of control. However, plasma HDL concentration in the cholesterol fed rats was significantly lowered compared to bezafibrate treated rats.

**Conclusion:** Bezafibrate may acts as a mixed blessing drug to normalize lipid profiles and endothelial dysfunction during hyperlipidemia.

© 2013 GESDAV

INTRODUCTION

The problem of hypercholesterolemia is being of much interest because an elevated concentration of lipoproteins can develop chest pain, heart attack, and accelerate the development of atherosclerosis with its dual sequel of thrombosis and infraction [1]. Moreover, hyperlipidemia, particularly hypercholesterolemia is a major cause of atherosclerosis and atherosclerosis associated conditions including coronary heart disease, ischemic cerebrovascular disease and peripheral vascular disease [2]. In general, monotherapy with a

pharmacologic agent should be attempted first, together with dietary adjustments. Combination treatment may be required for refractory severe hypertriglyceridemia, but should be attempted only with caution and frequent monitoring of serum concentrations of creatine kinase, transaminases and creatinine [3]. Hypercholesterolemia, defined as excessively high plasma cholesterol levels, has emerged as a strong risk factor for cardiovascular disease (CVD). As high total cholesterol levels are considered to be a major independent risk factor for development of coronary artery disease, considerable attention has been directed
toward evaluating the impact and mechanisms of
cholesterol lowering therapies and interventions for
cardiovascular outcomes [4-5].

As known, Fibric acid derivatives such as bezafibrate
are the mainstay in hypertriglyceridemia treatment [6].
These fibrates can reduce plasma triglyceride levels by
up to 50% and raise plasma HDL-C concentrations as
much as 20% [7]. The complex mechanism of action of
fibrates includes modulation of the activity of
peroxisome proliferator-activated receptor-α in the
liver, with reduced hepatic secretion of VLDL and
increased lipolysis of plasma triglycerides [7]. Fibrates
reduce the quantity of small, dense LDL particles and
increase HDL-C [8]. So, it is better to treat
hyperlipidemia with safe and less side effect drugs.
Bezafibrate is widely used as anti-hyperlipidemic drug
in human therapy. Based on these facts our study was
designed to evaluate the hypolipidemic effect of
bezafibrate at various doses on endothelium derived
substances and hyperlipidemia related changes as total
cholesterol, triacylglycerol, high density lipoprotein-
cholesterol (HDL-C), low density lipoprotein-
cholesterol (LDL-C), apolipoprotein B, endothelin-1,
histamine, nitric oxide (NO), interleukin-6 (IL-6),
creatinine and alanine amino transferase (ALT).

MATERIALS AND METHODS

Animals and chemicals

Male white albino rats, 6 weeks age and weighting (140
– 150g) were used in the experiment. Rats were housed
in separate metal cage with free access to water. Rats
were kept under constant and nutritional environmental
condition throughout the experiment. Rats were left for
a week before beginning of experiment for
acclimatization. Cholesterol and coconut oil were
purchased from El-Goumhouria Co. for Trading
Chemicals, Egypt. Hyperlipidemia induced by
continuous supplementation of high fat (coconut oil 2%
wt/wt) and high cholesterol (1% wt/wt) diet.
Bezafibrate was obtained from EIPICO Pharmaceutical
Co., Egypt. Bezafibrate was dissolved in 20% ethanol
to be used at doses 50, 100 and 100 mg/kg.bw/day
orally. All measured kits were purchased from Sigma
Aldrich Co. USA.

Experimental design and sampling

Rats were divided into 3 groups (10 per each). Group 1
was fed on normal diet and served as control group.
Group 2 was fed on high fat diet for 14 weeks. Group 3
was fed on normal diet and bezafibrate at a dose of 100
mg/kg/day. Another 4th group (30 rats) were
hyperlipidemic and were subdivided into 3 groups (10
rats each). Group 3A is hyperlipidemic and bezafibrate
50 (fed high fat diet plus bezafibrate at a dose 50

mg/kg/day). Group3B is hyperlipidemic and
bezafibrate 100 (fed high fat diet plus bezafibrate at a
dose 100 mg/kg/day). Group3C is hyperlipidemic and
bezafibrate 200 (fed high fat diet plus bezafibrate at a
dose 200 mg/kg/day). At the end of experimental
procedures heparinized blood samples were collected
from animals after 14 weeks (time of
hypercholesterolemia) and at 2, 4 and 6 weeks from the
beginning of bezafibrate administration. The samples
were collected in the morning after overnight fasting.
Plasma was separated by centrifugation at 3000 r.p.m
for 10 minutes. The clear obtained plasma was kept in
deep freezer at -20 °C until assayed.

Biochemical Assays

Kits for total cholesterol, triacylglycerol (TG), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), apolipoprotein B, endothelin-1, histamine, nitric oxide (NO), IL-6, creatinine and ALT were assayed spectrophotometrically.

Statistical analysis

Statistical analysis was done using SPSS software
version 15. The inter-group variation was measured by
one way analysis of variance (ANOVA) followed by
Post Hoc LSD test. Results were expressed as means ±
SEM. The mean difference is significant at the P<0.05
level.

RESULTS

Effect of high cholesterol diet on lipid profiles in rats

The results in table 1, 2 and 3 revealed that rats fed
hyperlipidemic diet showed significant increase in
plasma lipid profiles as total cholesterol, triglyceride,
LDL and apolipoprotein B (APOB), and significant
decrease in HDL compared with control group at 2, 4,
and 6 weeks after feeding. Moreover, no significant
difference between control fed rats and control fed rats
plus bezafibrate (100 mg/kg.bw) was observed.

Effect of bezafibrate on hyperlipidemic fed rats

To examine the effect of bezafibrate on changes
induced by feeding rats high cholesterol diet,
bezafibrate was given in 3 different doses 50, 100 and
200 mg/kg.bw/day. As seen in table 1, 2 and 3,
bezafibrate administration significantly decreased
plasma levels of total cholesterol, triglycerides, LDL
and apolipoprotein B with significant increase in high
density lipoprotein after 2, 4 and 6 weeks compared to
hyperlipidemic non treated group.

Effect of bezafibrate on endothelial derived
substances in hyperlipidemic rats.

As seen in table 1, 2 and 3, hyperlipidemic diet showed
a significant decrease of nitric oxide (NO) and a significant increase endothelin-1 when compared with control group. Bezafibrate administration caused significant decrease in endothelin-1 after 2, 4 and 6 weeks of administration, and partial normalization in nitric oxide concentration after when compared to hyperlipidemic non treated group.

Effect of bezafibrate on IL-6, histamine, alanine transaminases and creatinine in hyperlipidemic rats

Table 1, 2 and 3 revealed that rats fed on hyperlipidemic diet showed a significant increase in IL-6, histamine, ALT and creatinine when compared to control group. Moreover, bezafibrate administration at different doses (table 1, 2 and 3) induced, a significant normalization in IL-6, histamine, ALT and creatinine levels at 2, 4 and 6 weeks when compared to hyperlipidemic non treated group in dose dependent manner.

<table><caption>Table 1. Effect of 2 weeks administration of bezafibrate on some biochemical blood parameters of hyperlipidemic rats</caption><thead><tr><th></th><th>Chol.<br>(mg/dl)</th><th>TG<br>(mg/dl)</th><th>HDL-C<br>(mg/dl)</th><th>LDL-C<br>(mg/dl)</th><th>Apo(B)<br>(mg/dl)</th><th>NO<br>(nmol/l)</th><th>Endothelin-1<br>(ng/dl)</th><th>Histamine<br>(ng/dl)</th><th>IL-6<br>(pg/mL)</th><th>ALT<br>(IU/mL)</th><th>Creatinine<br>(mg/dl)</th></tr></thead><tbody><tr><td>Control</td><td>147.8<sup>c</sup><br>± 0.012</td><td>82.7<sup>a</sup><br>± 0.002</td><td>40.1<sup>a</sup><br>± 0.0009</td><td>133.3<sup>c</sup><br>± 0.003</td><td>105.5<sup>d</sup><br>± 0.003</td><td>0.2<sup>a</sup><br>± 0.008</td><td>1.86<sup>d</sup><br>± 0.006</td><td>0.6<sup>d</sup><br>± 0.001</td><td>9.33<sup>c</sup><br>± 0.005</td><td>11.66<sup>b</sup><br>± 0.001</td><td>0.85<sup>d</sup><br>± 0.02</td></tr><tr><td>Hyperlipidemic</td><td>218.4<sup>a</sup><br>± 0.006</td><td>198.5<sup>a</sup><br>± 0.003</td><td>23.5<sup>b</sup><br>± 0.009</td><td>207.4<sup>a</sup><br>± 0.003</td><td>204.3<sup>a</sup><br>± 0.004</td><td>0.17<sup>a</sup><br>± 0.008</td><td>2.3<sup>d</sup><br>± 0.001</td><td>2.2<sup>a</sup><br>± 0.004</td><td>18.64<sup>a</sup><br>± 0.002</td><td>20.00<sup>a</sup><br>± 0.005</td><td>2.06<sup>a</sup><br>± 0.08</td></tr><tr><td>Control + Beza100</td><td>150.7<sup>c</sup><br>± 0.009</td><td>100.75.3<sup>a</sup><br>± 0.002</td><td>35.2<sup>b</sup><br>± 0.0006</td><td>124.6<sup>c</sup><br>± 0.005</td><td>108.3<sup>d</sup><br>± 0.004</td><td>0.19<sup>a</sup><br>± 0.005</td><td>1.38<sup>a</sup><br>± 0.001</td><td>0.58<sup>d</sup><br>± 0.001</td><td>9.37<sup>c</sup><br>± 0.003</td><td>10.0<sup>b</sup><br>± 0.005</td><td>0.85<sup>d</sup><br>± 0.02</td></tr><tr><td>Hyperlipidemic +<br>Beza 50 (mg/kg/day)</td><td>194.8<sup>b</sup><br>± 0.005</td><td>182.4<sup>a</sup><br>± 0.002</td><td>23.1<sup>d</sup><br>± 0.0008</td><td>155.2<sup>c</sup><br>± 0.005</td><td>167.2<sup>b</sup><br>± 0.001</td><td>0.17<sup>a</sup><br>± 0.005</td><td>1.01<sup>b</sup><br>± 0.002</td><td>2.0<sup>b</sup><br>± 0.003</td><td>18.2<sup>a</sup><br>± 0.005</td><td>19.66<sup>a</sup><br>± 0.002</td><td>1.93<sup>a</sup><br>± 0.02</td></tr><tr><td>Hyperlipidemic +<br>Beza 100<br>(mg/kg/day)</td><td>196.2<sup>b</sup><br>± 0.005</td><td>135.8<sup>a</sup><br>± 0.003</td><td>26.1<sup>c</sup><br>± 0.0005</td><td>158.2<sup>c,b</sup><br>± 0.002</td><td>149.5<sup>c</sup><br>± 0.002</td><td>0.18<sup>a</sup><br>± 0.002</td><td>1.01<sup>b</sup><br>± 0.006</td><td>2.0<sup>b</sup><br>± 0.001</td><td>15.30<sup>b,a</sup><br>± 0.005</td><td>18.33<sup>a</sup><br>± 0.001</td><td>1.7<sup>b</sup><br>± 0.05</td></tr><tr><td>Hyperlipidemic +<br>Beza 200<br>(mg/kg/day)</td><td>182.4<sup>a,b</sup><br>± 0.002</td><td>157.3<sup>a</sup><br>± 0.002</td><td>33.1<sup>b</sup><br>± 0.0007</td><td>144<sup>a,c</sup><br>± 0.003</td><td>111<sup>d</sup><br>± 0.002</td><td>0.19<sup>a</sup><br>± 0.002</td><td>1.01<sup>c</sup><br>± 0.009</td><td>1.7<sup>c</sup><br>± 0.005</td><td>14.25<sup>b</sup><br>± 0.001</td><td>16.00<sup>a</sup><br>± 0.002</td><td>1.4<sup>c</sup><br>± 0.09</td></tr></tbody></table>

Data are presented as (mean ± S.E.). S.E = Standard error for 10 rats per each group. Mean values with different superscript letters in the same column are significantly different at (P<0.05).

<table><caption>Table 2. Effect of 4 weeks administration of bezafibrate on some biochemical blood parameters of hyperlipidemic rats</caption><thead><tr><th></th><th>Chol.<br>(mg/dl)</th><th>TG<br>(mg/dl)</th><th>HDL-C<br>(mg/dl)</th><th>LDL-C<br>(mg/dl)</th><th>Apo(B)<br>(mg/dl)</th><th>NO<br>(nmol/l)</th><th>Endothelin-1<br>(ng/dl)</th><th>Histamine<br>(ng/dl)</th><th>IL-6<br>(pg/mL)</th><th>ALT<br>(IU/ML)</th><th>Creatinine<br>(mg/dl)</th></tr></thead><tbody><tr><td>Control</td><td>152.4<sup>c</sup><br>± 0.013</td><td>89.8<sup>a</sup><br>± 0.003</td><td>43.3<sup>a</sup><br>± SE<br>0.001</td><td>133.2<sup>d,c</sup><br>± 0.002</td><td>102.2<sup>c</sup><br>± 0.001</td><td>0.2<sup>a</sup><br>± 0.006</td><td>1.3<sup>d</sup><br>± 0.006</td><td>0.6<sup>e</sup><br>± 0.02</td><td>9.85<sup>d</sup><br>± 0.003</td><td>11.6<sup>d</sup><br>± 0.001</td><td>0.85<sup>d</sup><br>± 0.02</td></tr><tr><td>Hyperlipidemic</td><td>246.8<sup>a</sup><br>± 0.06</td><td>207.8<sup>a</sup><br>± 0.005</td><td>19.8<sup>e</sup><br>± 0.006</td><td>21.6<sup>b</sup><br>± 0.006</td><td>207.4<sup>a</sup><br>± 0.004</td><td>0.17<sup>d</sup><br>± 0.009</td><td>2.9<sup>a</sup><br>± 0.008</td><td>2.3<sup>a</sup><br>± 0.07</td><td>20.61<sup>a</sup><br>± 0.002</td><td>25.66<sup>b,a</sup><br>± 0.001</td><td>2.27<sup>a</sup><br>± 0.006</td></tr><tr><td>Control +<br>Beza100</td><td>148<sup>c</sup><br>± 0.07</td><td>71.3<sup>a</sup><br>± 0.003</td><td>40.5<sup>b</sup><br>± 0.0006</td><td>126.9<sup>d</sup><br>± 0.005</td><td>106.1<sup>c</sup><br>± 0.002</td><td>0.24<sup>a</sup><br>± 0.01</td><td>1.2<sup>d</sup><br>± 0.008</td><td>0.5<sup>e</sup><br>± 0.01</td><td>08.65<sup>d</sup><br>± 0.002</td><td>19.00<sup>b,c</sup><br>± 0.005</td><td>0.84<sup>d</sup><br>± 0.06</td></tr><tr><td>Hyperlipidemic +<br>Beza 50<br>mg/kg/day</td><td>186.8<sup>b</sup><br>± 0.005</td><td>168.2<sup>a</sup><br>± 0.003</td><td>28.1<sup>d</sup><br>± 0.001</td><td>153<sup>b</sup><br>± 0.007</td><td>139.5<sup>b</sup><br>± 0.003</td><td>0.18<sup>d,c</sup><br>± 0.004</td><td>1.0<sup>b</sup><br>± 0.003</td><td>2.0<sup>b</sup><br>± 0.01</td><td>15.3<sup>b</sup><br>± 0.005</td><td>20.00<sup>c</sup><br>± 0.001</td><td>1.8<sup>b</sup><br>± 0.06</td></tr><tr><td>Hyperlipidemic +<br>Beza 100<br>mg/kg/day</td><td>183<sup>b</sup><br>± 0.007</td><td>168.2<sup>a</sup><br>± 0.003</td><td>30.2<sup>d</sup><br>± 0.002</td><td>143.4<sup>d,c</sup><br>± 0.001</td><td>134.3<sup>b</sup><br>± 0.004</td><td>0.19<sup>c</sup><br>± 0.0008</td><td>1.8<sup>c</sup><br>± 0.001</td><td>1.6<sup>c</sup><br>± 0.07</td><td>12.25<sup>b,c</sup><br>± 0.001</td><td>17.66<sup>b,c</sup><br>± 0.001</td><td>1.5<sup>c</sup><br>± 0.009</td></tr><tr><td>Hyperlipidemic +<br>Beza 200<br>mg/kg/day</td><td>167<sup>c,b</sup><br>± 0.003</td><td>137.5<sup>a</sup><br>± 0.002</td><td>34.9<sup>c</sup><br>± 0.0006</td><td>133.5<sup>d,c</sup><br>± 0.004</td><td>113.6<sup>c</sup><br>± 0.006</td><td>0.23<sup>b</sup><br>± 0.003</td><td>1.2<sup>c</sup><br>± 0.009</td><td>1.5<sup>d</sup><br>± 0.01</td><td>11.96<sup>c</sup><br>± 0.006</td><td>15.33<sup>a</sup><br>± 0.008</td><td>1.3<sup>c</sup><br>± 0.09</td></tr></tbody></table>

Data are presented as (mean ± S.E.). S.E = Standard error for 10 rats per each group. Mean values with different superscript letters in the same column are significantly different at (P<0.05).
<table><caption>**Table 3.** Effect of 6 weeks administration of bezafibrate on some biochemical blood parameters of hyperlipidemic rats</caption><thead><tr><th></th><th>Chol.<br>(mg/dl)</th><th>TG<br>(mg/dl)</th><th>HDL-C<br>(mg/dl)</th><th>LDL-C<br>(mg/dl)</th><th>Apo(B)<br>(mg/dl)</th><th>NO<br>(nmol/l)</th><th>Endothelin-1<br>(ng/dl)</th><th>Histamine<br>(ng/dl)</th><th>IL-6<br>(pg/mL)</th><th>ALT<br>(IU/ML)</th><th>Creat<br>(mg/dl)</th></tr></thead><tbody><tr><td>Control</td><td>145<sup>d,c</sup><br>± 0.01</td><td>84.7<sup>d</sup><br>± 0.003</td><td>41.3<sup>a</sup><br>0.0005</td><td>127.3<sup>c</sup><br>± 0.003</td><td>101.1<sup>d</sup><br>± 0.0005</td><td>0.2<sup>b</sup><br>± 0.006</td><td>1.4<sup>a</sup><br>± 0.004</td><td>0.6<sup>e</sup><br>± 0.02</td><td>9.85<sup>c,d</sup><br>± 0.3</td><td>11.66<sup>c</sup><br>± 0.001</td><td>0.85<sup>e</sup><br>± 0.02</td></tr><tr><td>Hyperlipidemic</td><td>260.8<sup>a</sup><br>± 0.008</td><td>230.3<sup>a</sup><br>± 0.006</td><td>18.2<sup>a</sup><br>± 0.0007</td><td>224.8<sup>a</sup><br>± 0.006</td><td>209.2<sup>a</sup><br>± 0.005</td><td>0.14<sup>c</sup><br>± 0.004</td><td>2.2<sup>c</sup><br>± 0.003</td><td>2.4<sup>a</sup><br>± 0.09</td><td>22.34<sup>a</sup><br>± 0.17</td><td>25.66<sup>b</sup><br>± 0.001</td><td>2.27<sup>a</sup><br>± 0.001</td></tr><tr><td>Control + Beza100</td><td>124<sup>d</sup><br>± 0.005</td><td>65.7<sup>e</sup><br>± 0.002</td><td>42.3<sup>a</sup><br>± 0.0007</td><td>125.9<sup>c</sup><br>± 0.003</td><td>105.1<sup>c</sup><br>± 0.004</td><td>0.24<sup>a</sup><br>± 0.01</td><td>2.0<sup>c</sup><br>± 0.001</td><td>0.6<sup>e</sup><br>± 0.01</td><td>8.7<sup>d</sup><br>± 0.24</td><td>13.66<sup>c</sup><br>± 0.008</td><td>0.846<sup>e</sup><br>± 0.02</td></tr><tr><td>Hyperlipidemic +<br>Beza 50 mg/kg/day</td><td>173.8<sup>b</sup><br>± 0.006</td><td>155.3<sup>b</sup><br>± 0.003</td><td>33.2<sup>a</sup><br>± 0.0009</td><td>143.4<sup>b</sup><br>± 0.001</td><td>136.3<sup>b</sup><br>± 0.0006</td><td>0.19<sup>b</sup><br>± 0.005</td><td>1.4<sup>a</sup><br>± 0.01</td><td>1.5<sup>b</sup><br>± 0.04</td><td>14.25<sup>b</sup><br>± 0.11</td><td>16.66<sup>b</sup><br>± 0.001</td><td>1.4<sup>b</sup><br>± 0.09</td></tr><tr><td>Hyperlipidemic +<br>Beza 100 mg/kg/day</td><td>161.6<sup>c,b</sup><br>± 0.005</td><td>148.1<sup>b</sup><br>± 0.02</td><td>35.6<sup>a</sup><br>± 0.001</td><td>133.5<sup>c,b</sup><br>± 0.004</td><td>128.6<sup>c</sup><br>± 0.006</td><td>0.2<sup>b</sup><br>± 0.0008</td><td>1.6<sup>c,b</sup><br>± 0.002</td><td>1.3<sup>c</sup><br>± 0.01</td><td>11.96<sup>b,c</sup><br>± 0.06</td><td>13.33<sup>a</sup><br>± 0.008</td><td>1.23<sup>c</sup><br>± 0.003</td></tr><tr><td>Hyperlipidemic +<br>Beza 200 mg/kg/day</td><td>142.4<sup>d,c</sup><br>± 0.004</td><td>127<sup>c</sup><br>± 0.003</td><td>37.9<sup>a</sup><br>± 0.007</td><td>124.6<sup>c</sup><br>± 0.005</td><td>115.3<sup>d</sup><br>± 0.005</td><td>0.23<br>± 0.004</td><td>1.2<sup>c</sup><br>± 0.001</td><td>1.1<sup>d</sup><br>± 0.02</td><td>10.13<sup>c,d</sup><br>± 0.013</td><td>11.33<sup>c</sup><br>± 0.008</td><td>1.1<sup>d</sup><br>± 0.008</td></tr></tbody></table>

Data are presented as (mean ± S.E.). S.E. = Standard error for 10 rats per each group. Mean values with different superscript letters in the same column are significantly different at (P<0.05).

DISCUSSION

The findings of the present study showed that hypercholesterolemia induced alteration in lipid profiles (VLDL, LDL and TG). Many reviews like that of Selvin and Erlinger [5], Ros [7] and Huijgen [9] discussed the dangerous effects of hypercholesterolemia on cardiovascular system but no direction for the treatment. Unlike our study, we focused on the problem and gave attention for the treatment through the usage of bezafibrate at various doses. Saturated fats mostly increase the concentration of cholesterol, LDL, and to a less extent VLDL levels. The increase in LDL levels produced by saturated fats seems to be related mainly to the decrease in catabolic rate. Moreover, Csont [1] demonstrated that elevated levels of LDL, appearing in the circulation upon feeding the hyperlipidemic diet, is mainly derived from LDL is up taken by the receptor in liver and extra hepatic tissue. The production of LDL exceeds the capacity of LDL receptors i.e. efflux of cholesterol from the liver is more than influx [1]. This could be explaining the elevated serum LDL levels observed in our study. The lipid-lowering effects of bezafibrate and reduction of the risk of cardiovascular events in dyslipidemic patients with coronary artery disease is the target of many physicians. The molecular mechanism underlying the triglyceride-reducing effect of bezafibrate is due to in part to the induction of lipoprotein lipase activity mediated by the activation of peroxisome proliferators-activated receptor α (PPAR-α) [10]. Furthermore, Pennacchio et al., [12], demonstrated that, the activation of PPAR-β by bezafibrate induces lipoprotein lipase in the liver, which plays a key role in triglyceride-rich lipoprotein catabolism. It also affects the binding and clearance in liver of remnant lipoprotein particles by LDL-related receptors. The fibrate-induced increased lipoprotein catabolism may also be related to a PPAR mediated

lower hepatic Apo CIII synthesis [11]. It is well known that Apo CIII delays the catabolism of triglyceride rich lipoproteins, since it inhibits their binding to the endothelial surface and lipolysis by lipoprotein lipase and interferes with Apo E-mediated receptor clearance of remnant particles from plasma [12]. Finally, a new additional mechanism contributing to the fibrate-induced reduction in triglyceride rich-lipoproteins was also proposed is a recently discovered lipoprotein that influences plasma triglyceride levels [13]. Moreover, Fruchart et al [14] demonstrated that fibrates increase HDL-C may be related to accelerate triglyceride-rich lipoprotein catabolism leading to an increase in pre-HDL, which is the key acceptor of cholesterol for peripheral cells during reverse cholesterol transport.

The importance of the endothelium in vessel reactivity and subsequent abnormalities of arterial responses has fostered the use of the term “endothelial dysfunction”. Importantly, endothelial dysfunction includes alterations in any of the functional roles. The endothelium plays role in maintenance of normal tone, limiting thrombosis, and protecting against leukocyte adhesion [15]. Reactivity of the arterial wall is controlled in part by biomechanical inputs through blood flow and blood pressure. Endothelin-1 (ET-1) promotes blood vessel constriction, as evident with the decrease in blood flow after endothelial ET-1 release [16]. ET-1 also induces smooth muscle cells proliferation. These ET-1 responses are countered by endothelial release of nitric oxide (NO), which stimulates vasodilatation, thus increasing blood flow [17-18]. Endothelial nitric oxide synthase (eNOS) is the key enzymatic step in producing NO, a secondary messenger produced by EC that can inhibit NF-κB activation and attenuate endothelial inflammatory responses, including adhesion molecule expression. The increase in ET-1 production and decrees in NO production, results in vasoconstriction, abnormal
vasomotor responses and promotion of atherosclerosis
[19].

The results of this study are parallel with that of Stokes
et al., [20] where hypercholesterolemia leads to an
inflammatory response within the microvasculature that
reflected by endothelial cell activation, leukocyte
recruitment, rolling and adherence, as well as platelet
activation and adhesion. Moreover, monocyte chemo
tactic protein-1 (MCP-1), IL-6 and histamine are
important in hypercholesterolemic patients, acting to
increase monocyte recruitment and adherence which
leads to wall remodeling. Also, Stapleton et al. stated
that macrophages derived from monocytes, begin to
accumulate LDL and oxidized LDL (ox-LDL) which
develop into foam cells between the basal lamina of the
endothelium and the smooth muscle layer [21]. These
foam cells lead to the production of numerous
inflammatory and oxidative stress markers, cytokines,
chemokines, and growth factors which aggravate the
balance of endothelial equilibrium leading to vascular
dysfunction. The increase in IL-6 and histamine is
interesting point as Gervois and his colleagues [22]
reported that bezafibrate reduced IL-6 and histamine
possibly through the activation of the peroxisome
proliferator-activated receptor (PPAR)-α with a
consequent reduction of NF-κB activation. These
discrepancies may be due to the differences in
inflammatory status at baseline, duration of the study
period, or the lack of control group reported in their
study. The hyperlipidemic diet induced a significant
increase ALT activity and creatinine compared with
control group. Those findings may be attributed to the
increase in LDL levels which is regulated by LDL
receptors [23]. The elevation of aminotransferases
levels has been frequently observed after the
administration of bezafibrate and this phenomenon is
considered to be non-pathological because bezafibrate
activates the gene expression of the aminotransferases.
Recently, fibrate has been used not only for
hypercholesterolemia but also for primary biliary
cirrhosis [24-25]. In conclusion, these results indicate
that bezafibrate normalized the biochemical changes
induced during hyperlipidemia and its accompanied
endothelial dysfunction in rats and suggested that other
studies are needed in order to directly translate
bezafibrate effects in humans.

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