Introduction

Modern life is full of stress because of many factors. Emotional and other physical stress factors affect the normal physiological system of the body. Hans Selye¹ defined stress as the reaction of an organism to various stimuli. Stress can be considered as a state of a disharmony or threatened homeostasis. Stressful stimuli may influence the onset and progression of a number of disorders in human being leading to hypertension, diabetes, stroke, cancer depression etc.^2,3. Cardiovascular system is more prone to be affected by stress, either directly or indirectly⁴. Presently cardiovascular accidents are the major killer. There are various reports that stress can change the level of certain hormones like insulin, cortisol and epinephrine^5-7. All these hormone affect lipid profile of the body to a great extent. In animal studies it was found that stress raises serum and tissue cholesterol level of rats on normal diet⁸. Berger etal ⁹ had shown that serum cholesterol level increased after different psychological stress in rats. Prabhakaran etal ¹⁰ had also shown that acute noise as stress increased the serum cholesterol levels in rats. Studies in human had shown that serum lipid levels are increased during the examination period in students¹¹. The foregoing observations suggest that stress is linked with lipid metabolism, alteration of which increased cardiovascular morbidity and mortality. Hence with the prior approval from Institutional Ethical Committee, the present study was planned to evaluate the effect of hypolipidemic drugs, gemfibrozil and bezafibrate on stress induced alteration on lipid profile in rat.

Materials And Methods

Animal - Albino rats of either sex (150-200 g) were used. They were housed in plastic cages at an ambient temperature of 26±2⁰C and 45 to 55% relative humidity with a standard 12 h light or dark cycle. They had free access to food and water and were acclimatised for at least one week before experimentation. Each experimental group consisted of minimum of 6 animals. National Research Council guidelines for the care and use of laboratory animals were followed throughout the study.

Drugs - Gemfibrozil (LOPID-CIPLA^R) and Bezafibrate (Bezalip-Boehriger Mannhem^R) were used in the present study. Gemfibrozil 1000 mg/kg and bezafibrate 500 mg/kg were administered once daily orally for 22 days in suspension form through a rat feeding cannula on empty stomach in the morning. These doses were extrapolated from human dose on body surface area basis¹².

Stress - A 5 hours swimming stress^13,14 was given to all the animals on day 22. This include active swimming and immobility period. For Swimming, a plastic tub (24” in height and 40” in diameter) half filled with tap water and maintained at room temperature of 28^oC was used.

Blood Sample – The animals were anaesthetized with ether rapidly within 2 minutes according to stress free procedure of Feldman and Conforti¹⁵ which does not cause stress to the animal. The blood sample was collected from retrobulbar plexus immediately after anaesthetization. Serum was separated and was kept at 4⁰ C until use.

Observational Parameter – In this experimental study the following serum lipid levels were estimated. Total cholesterol (TC) and HDL cholesterol¹⁶, Triglyceride (TG)¹⁷. LDL & VLDL cholesterol was calculated on the basis of Friedwalds equation¹⁸ LDL cholesterol = TC-(HDLC + TG/5) ; VLDL cholesterol = TC-(HDLC + LDLC).

Procedure – Experiment was designed to study the effect of stress on lipid profile and effect of hypolipidemic drugs, gemfibrozil and bezafibrate on stress induced alteration on lipid profile in rats. For this experimental study rats were divided into three group of 6 each. The different treatment scheduled were as follows.

Group I – Control, Group II and Group III treated with gemfibrozil and bezafibrate respectively. In control group animals were treated with 0.5 ml normal saline (p.o.) daily from day 1 to day 22. Serum lipid levels were estimated on day O (blank control) and on day 22 after stress (positive control). In group II and III animals were treated with gemfibrozil 1000 mg/kg and bezafibrate 500 mg/kg body weight (p.o.) respectively daily from day 1 to day 22. Serum lipid levels were estimated on day 3,7,14 and 21 and on day 22 after stress.

Statistical analysis – Results were expressed as Mean ± SEM and student ‘t’ was applied for analysis of data. P value<0.05 were considered as significant.

Results

Gemfibrozil (1000 mg/kg p.o.) on normal diet reduced the TC from 84±1.87 to 73.68±1.33 p<0.01, raised the HDLC from 35.2±1.03 to 38.0±0.68 p<0.05, reduced the TG from 54.81±1.81 to 38.51±1.36 p<0.001, reduced the LDLC from 37.83±1.34 to 27.97±0.81 p<0.01, and reduced the VLDLC from 10.26±0.36 to 7.70±0.25 p<0.001 (table I group II versus group I). Similarly bezafibrate (500 mg/kg p.o.) reduced the TC from 84±1.87 to 71.88±0.53 p<0.001, raised the HDLC from 35.2±1.03 to 38.56±0.41 p<0.01, reduced the TG from 54.81±1.81 to 38.6±0.69 p<0.001, reduced the LDLC from 37.83±1.34 to 25.58±0.54 p<0.001 and reduced the VLDLC from 10.26±0.36 to 7.74±0.17 p<0.001 (table I group III versus group I). In group II and group III when compared to blank control gemfibrozil (1000 mg/kg po) and bezafibrate (500 mg/kg p.o) reduced the serum lipid levels in rats significantly except the HDL cholesterol which increased significantly.

After stress in control group TC increased from 84±1.87 to 113.33±1.36 p<0.001, HDLC reduced from 35.2±1.03 to 34.83±0.74 p>0.05, TG increased from 54.81±1.81 to 77.03±1.81 p<0.001, LDLC increased from 37.83±1.34 to 63.09±1.14 p<0.001 and VLDLC increased from 10.96±0.36 to 15.40±0.36 p<0.001 (table I and table II control group). In control group stress increased the lipid levels in rats significantly except HDL cholesterol which reduced insignificantly.

When drug treated animals subjected to stress on day 22, their serum lipid levels increased significantly except HDL cholesterol which reduced significantly (table I and table II). After stress in gemfibrozil treated rats TC increased from 73.68±1.33 to 96.11±1.37 p<0.001, HDLC reduced from 38.0±0.68 to 34.22±0.56 p<0.01, TG increased from 38.51±1.36 to 53.33±1.13 p<0.001, LDLC increased from 27.97±0.81 to 51.23±1.14 p<.001, and VLDLC increased from 7.70±0.25 to 10.67±0.18 p<0.001 (table I and table II group II). Similarly after stress in bezafibrate treated rats TC increased from 71.88±0.53 to 94.36±0.51 p<0.001, HDLC reduced from 38.56±0.41 to 37.22±0.40 p<0.05, TG increased from 38.6±0.69 to 52.26±0.92 p<0.001, LDLC increased from 25.58±0.54 to 47.7±0.86 p<0.001 and VLDLC increased from 7.74±0.17 to 10.45±0.19 p<0.001 (table I and table II group III).

When compared to positive control serum lipid levels were significantly less in gemfibrozil treated rats and bezafibrate treated rats except HDL cholesterol which reduced insignificantly in gemfibrozil treated rats and increased significantly in bezafibrate treated rats (table II group II & III versus group I).

Discussion

Hans Selye¹ defined stress as the reaction of an organism to various stimuli. Stressful stimuli may influence the onset and progression of a number of disorders in human being leading to hypertension, diabetes, stroke, cancer depression etc.^2,3. Swimming and force water swimming in small laboratory animals has been widely used for studying the physiological changes and the capacity of the organism in response to stress^19.20. Swimming is not always a simple exercise stress because emotional factors are difficult to eliminate²¹.

In the present experimental study our findings were comparable to the observations of certain other workers^8-10 with regard to effect of stress on serum lipid levels. Hypolipidemic drugs gemfibrozil and bezafibrate which are fibric acid derivative and act by increasing lipoprotein lipase activity. Other mechanism as well²² not only reduced raised lipid levels but also reduced normal lipid levels. Observations also suggest that bezafibrate alter serum lipid levels more significantly than gemfibrozil. During treatment with hypolipidemic drugs serum lipid levels were estimated on day 3, 7, 14 and on day 21. The serum lipid values estimated on day 14 and on day 21 were statistically similar. Therefore serum lipid levels estimated on day 21 were included for analysis. Serum lipid levels were increased after physical stress of 5 hours swimming on day 22 in all the rats (group I, II and III). This confirmed the previous reports by Berger et al ⁹ and Prabhakaran et al ¹⁰. But the hypercholesterolemia after stress observe in this study is in contradiction to the report by Tsopanakis and Tesseromatis²³,who found a decline in total cholesterol level when rats were exposed to cold swim stress for a period of 20 and 60 days. Change in lipid levels to stress is rather contradictory and may depend on situational, environmental and inter individual factors. Stress affect hypothalamic-pituitary-adrenal axis and affect various neurotransmission system like dopamenergic, cholinergic, 5 Hydroxydrytaminergic gabaminergic and benzodiazepine. Various hormone secretion also altered by stress like CRH, GH, Insulin, epinephrine and cortisol^5-7,24. The physiological mechanism of stress induced changes in lipid levels remains largely unelucidated. It appears like that the hypothalamic–pituitary–adrenal (HPA) axis contributes to the stress induced cholesterol changes²⁵. Stress increased serum lipid levels in all the three groups except HDLC which reduced insignificantly in control group, but reduced significantly in gemfibrozil group (p<0.01) and bezafibrate group (p<0.05). When the rise in serum lipid levels after stress in group II and III were compared with positive control observation (table II) it was noticed that the rise in serum lipid levels in gemfibrozil and bezafibrate treated rats after stress were significantly less than that of control rats after stress except HDLC which reduced insignificantly in gemfilbrozil treated rat and increased significantly in bezafibrate treated rat since bezafibrate is a more effective hypolipidemic drug.

It can be concluded that gemfibrozil (1000mg/kg p.o.) and bezafibrate (500mg/kg p.o.) are not only hypolipidemic but also effective in attenuating stress induced dyslipidemia in rats.

Figure 1

Table I. Serum lipid levels in control rats and drug treated rats. (before stress)

Figure 2

Table II. Serum lipid levels in control rats and drug treated rats. (after stress)