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Introduction
Atherosclerosis can lead to the development of coronary heart disease (CHD), cerebrovascular disease, and other
peripheral vascular diseases. CHD, which causes more than twice as many deaths as do all forms of malignancy in USA, is
currently the leading cause of death in many countries. As lipid deposition is a characteristic feature of athero-sclerosis,
patients suffering from atherosclerosis often have plasma total cholesterol (TC) and/or triglyceride
(TG) levels higher than those in non-affected
populations[1-3]. In general, the mean plasma TC level is low in undeveloped/developing countries
whose incidences of CHD are much lower than those in the developed countries, where the mean plasma TC level in the
population is higher. Although many studies have shown that the pathogenesis of CHD involves multiple factors, high
blood lipid levels frequently correlate with higher incidences of CHD. In recent years, much attention has been paid to the
development of drugs used for the prevention and treatment of hyperlipidemia. In this regard, several animal models of
hyperlipidemia, including diet-
induced and transgenic models, have been developed for
screening drug candidates that can lower blood TC/TG
level[4-8].
Treatment with schisandrin B (Sch B), a dibenzocyclo-octadiene derivative isolated from Fructus Schisandrae, at high
oral doses caused apparent increases in serum lipid levels in
mice[9]. This observation has led
to further investigations with an attempt to establish an animal model of hyperlipoproteinemia using Sch
B[10]. Bifendate (biphenyl-dicarboxylate), a
synthetic intermediate of schisandrin C (also a dibenzocyclooctadiene derivative) was found to protect against drug-induced
liver injury and hepatitis in animal
models[11,12], with the mode of action similar to that of Sch B and schisandrin C. Bifendate
is now used clinically for the treatment of hepatitis. In the present study, the effects of bifendate treatment on serum and
hepatic lipid levels as well as apolipoprotein levels are examined in rabbits and mice.
Materials and methods
Chemicals and reagents Bifendate (powdered pill suspended in 0.5% CMC) was purchased from Beijing Xiehe
Pharmaceutical Factory (Beijing, China). Cholesterol (CHO) and bile salt were obtained from Beijing Chemical Reagent Company
(Beijing, China). Inositol nicotinate and fenofibrate were bought from Beijing Yongkang Medical Company and Yimin
Medical Company (Beijing, China), respectively. Both sodium carboxymethylcellulose (CMC) and polyethylene glycol 6000
(PEG) were obtained from Beijing Xudong Chemical Plant (Beijing, China). Assay kits for triglyceride (TG), total cholesterol
(TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), apolipo-protein (Apo) A,
and Apo B were bought from Zhong-sheng Beikong Bio-technology and Science Inc (Beijing, China).
Animal treatment Adult male ICR mice (26-28 g) [Grade II, Certificate
No SCXK(jing)2002-0003] and Japanese male white
rabbits (2.5-2.7 kg) were supplied from Vital River Lab Animal Company
(Beijing, China). Animals were maintained under
standard laboratory conditions (12-h light-dark cycle, 20-21
oC and a relative humidity of 50%-55%) and had access to food
and water ad libitum. Eight mice were housed per cage. Mice were allowed a 4-day habituation period in the laboratory
where the experiments were carried out. Rabbits were housed individually and allowed to acclimatize for 10 d prior to the
experiments. Both mice and rabbits were orally administered with bifendate (powdered pill suspended in 0.5% CMC). Control
animals received the vehicle only (ie, 0.5% CMC) at 20 mL/kg. Positive control groups were administered CHO (2 g/kg) plus
bile salt (0.5 g/kg). Blood and liver tissue samples were obtained from animals that were fasted for 12 h and subjected to
biochemical analyses. All experimental protocols were approved by the University Committee on Research Practice at the
Beijing University of Chinese Medicine.
Determination of lipid and apolipoprotein levels
Serum samples were prepared by centrifuging the whole blood (obtained
from the orbital vein in mice or ear vein in rabbits) for 8 min at
2000×g and then frozen at -20
oC until assay within 5 d. Liver tissue sample was homogenized in 9 volumes of 0.9%
(w/v) NaCl solution by two 10-s bursts of a tissue disintegrator at 13 500
r/min, and the homogenate was then centrifuged at
2000×g for 15 min to obtain the supernatants. Hepatic supernatants 10 µL
and 40 µL were used to determine the TG and TC concentrations, respectively. Both serum and hepatic TG and TC levels were
artificially measured using 722 spectrophotometer (made in China), but serum LDL-C, HDL-C, Apo A-I, and Apo B were
automatically determined using Olympus An 400 biochemical analysis apparatus (made in Japan).
Statistical analysis Data were analyzed using one-way ANOVA and expressed as mean±SEM. Significant difference
between groups was detected by Duncan’s multiple range test using SPSS 12.0 software.
Student’s t-test was used for comparison between two groups. The inter-group difference was considered as significant
when P<0.05.
Results
Effects of a single dose of bifendate on serum TG and TC levels in rabbits
Prior to drug administration, a blood sample was obtained and serum TG and TC levels were measured in the rabbits. The values were used as baselines for comparison.
Bifendate treatment (0.3 g/kg, ig) caused a time-dependent and biphasic change in serum TG level, with the extent of increase
ranging from 39%-200% and the value reaching a maximum between 24-36 h post-dosing when compared with the baseline
value. Serum TC levels in bifendate-treated rabbits was reduced (by 11%-15%) when compared with the baseline value, but
the difference was not statistically significant (Figure 1).
Effects of a single dose of bifendate on serum and hepatic TG levels in mice
Bifendate treatment (0.25-1 g/kg) increased serum TG levels in a dose-dependent manner (39%-76% and 14%-39%, respectively) at 24 and 48 h post-dosing. Bifendate
treatment (0.25-1 g/kg) also dose-dependently increased the hepatic TG level at 6 h post-dosing. However, the time-course
of bifendate-induced change in hepatic TG level varied with doses, with the lower dose achieving a maximum increase in later
time, and vice versa (Figure 2). CHO/bile salt treatment did not produce any detectable changes on serum and hepatic TG
levels in mice. To exclude the possibility that the hypertriglyceridemic effect of the bifendate pill may be attributed to PEG
(the main excipient present in the formulation), the effect of PEG treatment (10
g/kg, suspended in 0.5% CMC, ig) was examined along with mice treated with the vehicle (0.5% CMC, 20 mL/kg) and bifendate
pill (1 g/kg, in 0.5% CMC). The result indicated that, while bifendate pill produced a significant increase in serum TG level at
24 h post-dosing when compared with the vehicle control, PEG treatment did not cause any detectable change (data not
shown).
Effects of bifendate on serum and hepatic TC levels in mice
Treating mice with bifendate at 1 g/kg decreased serum TC
(13%) and hepatic TC (11%) levels at 24 h post-dosing. But the difference in hepatic TC was not statistically significant.
When mice were treated with bifendate at daily doses of 0.25 and 1 g/kg for 4 d, hepatic TC level was slightly but significantly
reduced (by 9%-10%) at 24 h after the last dosing. Single or multiple doses of CHO/bile salt caused significant increases in
serum TC (14% or 48%, respectively) and hepatic TC (25% or 33%) in mice (Figure 3).
Effects of bifendate on serum lipid profile in mice
Treatment with bifendate at oral daily doses of 0.25 and 1 g/kg for 4 d
caused significant increases in serum levels of Apo A-I (38%-48%), Apo B (14%-25%) and TG (56%-79%) in a
dose-dependent manner when compared with the vehicle control (Table 1). While bifendate treatment did not produce any
detectable changes in serum TC, HDL-C and LDL-C levels. CHO/bile salt treatment (2/0.5 g/kg×4 d) significantly
increased serum Apo A-I (38%), TC (48%), HDL-C (18%) and LDL-C (125%) levels. However, the serum TG level was
decreased (by 32%) in CHO/bile salt-treated mice. The hepatic TG level was also significantly increased in bifendate-treated
mice (data not shown).
Effects of lipid-lowering agents on bifendate-induced hypertriglyceridemia
Hypertriglyceridemia was induced by bifendate treatment (1 g/kg×4 d) in mice. Concurrent treatment with fenofibrate (0.03 and 0.1 g/kg×4 d) significantly reduced serum TG
levels in a dose-dependent manner in bifendate-treated mice. Treatment with inositol nicotinate (0.1 and 0.3 g/kg×4 d) slightly
decreased serum TG levels in bifendate-treated mice, but the differences did not show a statistical significance (Figure 4).
Discussion
Hyperlipidemia is characterized by elevated plasma/serum TC and TG levels, which are well over normal values in the
population. Based on the lipoprotein profile, hyperlipidemia can be classified into various subtypes in that patients suffering
from type I or IV hyperlipidemia exhibit a high plasma TG level, but little or no elevation in plasma TC level. In the present
study, bifendate was found to increase serum TG but not TC levels and thus the resultant hyper-triglyceridemic state
resembled that of type I or IV hyperlipidemia in humans. Experimental studies have demonstrated that the elevation of plasma
TC and TG concentration may produce lesions similar to atherosclerosis in human blood
vessels[13,14]. Whether or not hypertriglyceridemia induced by bifendate treatment can lead to the development of atherosclerosis remains to be determined.
While high-fat and excessive carbohydrate intake caused
hypertrigly-ceridemia, the inhibition of b-oxidation could lead to the
production of excessive TG from the esterification of fatty
acids[15,16]. Given the elevation of both serum and hepatic TG levels, it
is possible that bifendate can stimulate the esterification of fatty acids and/or inhibit
b-oxidation. Despite that fact that TG is just one type of lipid molecule for energy storage in the body, hypertriglyceridemia has been regarded as an independent
risk factor of cardiovascular
disease[17,18]. TG primarily exists in chylomicrons and very low density lipoproteins (VLDL).
While the former is formed by intestinal mucosal cells during the absorption of dietary fat, the latter is manufactured in the
liver in response to a high carbohydrate meal. Therefore, the amount of TG-rich lipoproteins, such as chylomicrons and
VLDL, is increased in hypertriglyceridemic
states[19]. Consistent with this, Apo A and Apo B, which are abundantly present
in chylomicron and VLDL, were also increased by bifendate treatment.
Bifendate is available in a formulation of 1.5-mg pill for oral use at daily dosages ranging from 75 to 150 mg in adult
(1.5-3 mg/kg for 50 kg body weight). However, the dosage (0.25-1 g/kg) adopted in the present animal study is about 83-667 fold
higher than the human dosage. Pharmacokinetic studies indicate that orally administered bifendate (1
g/kg) reached the peak plasma level in 12 h in rats and the absorption efficiency was approximately
20%[20]. However, the formulated bifendate pill was found to be absorbed more efficiently from the gastrointestinal tract. This is consistent with the
observation that the effective dosage of bifendate pill-induced hypertriglyceridemia is smaller than that of bifendate Fibric
(unformulated compound) (data not shown). In the present study, the maximal degree of hypertriglyceridemia induced by
bifendate pill was attained between 24 or 36 h following the intragastric administration of the drug.
Changes in plasma TG level reflect a dynamic process involving the removal of TG from the blood and
synthesis/secretion of TG from the liver. If a drug enhances the hepatic synthesis and/or secretion of TG, the blood TG level will be
increased. Results obtained from the present study indicate that, while bifendate treatment dose-dependently increased the
serum TG level at all time intervals post-dosing, the bifendate-induced change in hepatic TG levels showed an inverse
dose-response relationship at 24 h post-dosing and onwards. It is therefore conceivable that while low doses of bifendate
stimulate hepatic TG synthesis, high doses might also enhance the secretion of TG from the liver into the blood.
acid derivatives are a class of lipid-modifying drugs mainly used in patients with elevated TG levels. Fenofibrate, one of the
most widely used fibric acid derivatives, is a useful therapeutic option for patients with primary combined dyslipidemias or
secondary dyslipidemias[21-23]. Fenofibrate, when administered at 0.3 g per day in adults, is especially good at lowering
plasma TG. The drug increases lipolysis and the elimination of TG-rich particles from plasma by activating lipoprotein lipase
and reducing production of apolipoprotein C-III, an inhibitor of lipoprotein lipase activity. In addition, fenofibrate was found
to reduce body weight gain and adiposity in female sham-operated and ovariectomized
mice[24]. Inositol nicotinate consists
of six niacin molecules linked to inositol, and the niacin molecules are slowly cleaved and released from inositol in the body.
Niacin was first reported to be a hypolipidemic agent in 1955, and it is now most frequently prescribed for patients suffering
from dyslipidemia in an attempt to raise low HDL-C and to lower VLDL-C and LDL-C
levels [25,26]. In addition, the blood
vessel-widening effect of niacin may improve the circulation to the extremities, which is useful for the treatment of peripheral artery
diseases[27,28]. Our finding that fenofibrate is more effective than inositol nicotinate in lowering serum TG levels in
bifendate-treated mice is corroborated by a relatively higher clinical efficacy of fenofibrate than inositol nicotinate in lowering plasma
TG levels.
In conclusion, our results help substantiate the suggestion for the development of animal models of acute
hyper-triglyceridemia by oral administration of a single and high dose of bifendate in rabbits and mice. This will provide a
convenient in vivo screen for novel TG-lowering
agents.
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