Cardiovascular Journal of Africa: Vol 28 No 5 (September/October 2017)

CARDIOVASCULAR JOURNAL OF AFRICA • Volume 28, No 5, September/October 2017

300

AFRICA

Plasma levels of oxLDL were measured by ELISA using a

specific ELISA kit (MBS729489, My BioSource Ltd, USA). The

coefficient of variation for the assay was 4.4 to 5.6. The coated

monoclonal antibody (mAb) was against a conformational

epitope in the apolipoprotein B-100 (apoB-100) moiety of LDL.

All procedures were carried out according to the manufacture’s

instruction.

Briefly, the mAb-coated wells were loaded with 50 µl standards

or samples. In order to determine quantitative amounts of

oxLDL in the sample, polyclonal antibody (conjugated to

horseradish peroxidase) specific for oxLDL, was added to

each well. The wells underwent washing three times with PBS

following incubation for one hour at room temperature. TMB

substrate solution was then added and allowed to react for 20

minutes. The chromogenic reaction was halted by adding the

stop solution. The absorbance of the wells was read using a

spectrophotometer (Awareness, USA) at 450 nm. The amount

of oxLDL concentration in each sample was determined using a

standard curve (

concentration and

optical density).

Statistical analysis

The required sample sizes were obtained based on a previous

article so no statistical methods were used to determine sample

sizes.

Resultant data are presented as means

standard

deviation (SD). The means of the variables and the differences in

mean values between the groups were evaluated using the Mann–

Whitney

-test. A

value of

0.05 was considered significant.

Results

We intended to determine perturbations in the plasma lipid

profile of the rats in each group to confirm the induction of

hypercholesterolaemia after 20 weeks of treatment. As shown in

Fig. 1, the serum total cholesterol levels of the HD group were

approximately three-fold higher than those of the ND group

[229.35

13.26 vs 67.89

5.14 mg/dl (5.94

0.34 vs 1.76

0.13

mmol/l);

0.01]. The serum levels of LDL-C in the HD group

were more than 10-fold higher than those in the ND group

[177.39

10.38 vs 15.20

2.34 mg/dl (4.59

0.27 vs 0.39

0.06

mmol/l);

0.05]. A slight but non-significant decrease was also

observed in HDL-C levels [31.27

4.69 vs 33.66

2.90 mg/dl

(0.81

0.12 vs 0.87

0.08 mmol/l);

ns].

The cholesterol-lowering activity of AV was also evaluated.

As shown in Fig. 1, AV displayed a strong cholesterol-lowering

activity at a dose of 20 mg/kg. Serum levels of total cholesterol in

the HD

AV group decreased approximately 50% in comparison

with those of the HD group [119.00

9.187 vs 229.35

13.26

mg/dl (3.08

0.24 vs 5.94

0.34 mmol/l);

0.01]. The serum

levels of LDL-C in the HD

AV group decreased 75% in

comparison to those of the HD group [44.284

6.905 vs 177.398

10.386 mg/dl (1.15

0.18 vs 4.59

0.27 mmol/l);

0.01]. The

plasma levels of oxLDL were significantly increased in the HD

versus the ND group [214.42

17.46 vs 69.13

9.92 mg/dl (5.55

0.45 vs 1.79

0.26 mmol/l);

0.01] but AV diminished the

plasma levels of oxLDL in the HD

AV group compared to the

AV group [126.52

9.46 vs 214.42

17.46 mg/dl (3.28

0.25 vs

5.55

0.45 mmol/l)] (Fig. 2).

The protein expression of cleaved caspase-3 and bcl-2, and

phosphorylation of p38 were determined to ascertain whether

cholesterol induced apoptosis. The protein expression levels of

cleaved caspase-3 were measured by Western blotting analysis.

Based on the results, cholesterol increased the cleavage of caspase-3

HD + AV

mg/dl

250

200

150

100

Total serum cholesterol (mg/dl)

LDL-cholesterol (mg/dl)

HDL-cholesterol (mg/dl)

HDL/LDL

VLDL (mg/dl)

## #

Fig. 1.

Bar graph showing the attenuating effect of adminis-

tration of oral atorvastatin (20 mg/kg for eight weeks)

on cholesterol-induced serum lipid profiles of triglyc-

erides (TG), total cholesterol (TC), HDL-C and LDL-C

in hypercholesterolaemic rats. Data are presented as

mean

SD;

10. *

0.05, **

0.001 compared

with normal diet group;

0.05,

0.001 compared

with hypercholesterolaemic rats using the ANOVA test.

normal diet, HD

high-cholesterol (2%) diet, HD

high-cholesterol diet plus AV (20 mg/kg), and

AV control group.

HD + AV

Serum-ox (ng/dl)

250

200

150

100

Fig. 2.

Box plot showing the reducing effect of admin-

istration of oral atorvastatin (20 mg/kg for eight

weeks) on cholesterol-induced plasma oxLDL levels.

Data are presented as mean

SD;

10. **

0.001 compared with normal diet group;

0.001

compared with hypercholesterolaemic rats using the

ANOVA test. ND

normal diet, HD

high-cholesterol

(2%) diet, HD

high-cholesterol diet plus AV (20

mg/kg ) and AV

AV control group.