|Year : 2018 | Volume
| Issue : 1 | Page : 58-65
Role of inflammation versus hypercholesterolemia in the development of atherosclerosis in male albino rats
Salah M Ibrahim, Randa S Gomaa, Safya I Ismail, Heba S.G Ibrahim
Department of Physiology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||18-Jun-2018|
|Date of Acceptance||15-Aug-2018|
|Date of Web Publication||20-Nov-2018|
Randa S Gomaa
Department of Physiology, Faculty of Medicine, Zagazig University, Zagazig, 44519
Source of Support: None, Conflict of Interest: None
Background Atherosclerosis (AS) is a chronic condition in which dyslipidemia had been contributed to its development, along with evidence proving an inflammatory cause.
Objective The aim was to determine the relation between dietary hypercholesterolemia and AS with a trial to evaluate the role of inflammation in development of AS in male albino rats.
Materials and methods A total of 30 adult male albino rats were divided into two main groups: control group (n=6) and high-cholesterol diet (HCD)-fed group (n=24), which was subdivided into four subgroups (n=6): HCD-fed, HCD-fed with methotrexate, HCD-fed with cholestyramine, and HCD-fed with statin groups. Serum total cholesterol (TC), triglycerides, high-density lipoprotein (HDL) cholesterol, non-HDL cholesterol, low-density lipoprotein (LDL)-cholesterol, very LDL-cholesterol, atherogenic index, LDL/HDL ratio, and inflammatory markers such as inerlukin-6, tumor necrotic factor-α, and highly sensitive C-reactive protein were estimated. Carotid artery histopathology was done.
Results HCD produced marked disturbance in lipid profile and increased inflammatory markers and atherosclerotic changes in carotid artery. Administration of statin and cholestyramine significantly improved this dyslipidemia. Elevated inflammatory markers were significantly decreased by administration of methotrexate and statin. Atherosclerotic changes in carotid artery decreased significantly in rats pretreated with methotrexate and statin.
Conclusion Atherosclerotic condition is closely associated with excessive intake of cholesterol-rich diet; however, inflammation has a central role in the pathogenesis of the atherosclerotic process, as atherosclerotic changes could be reduced despite disturbed lipid profile by anti-inflammatory medication. Further studies are recommended for more evaluation of the role of anti-inflammatory drugs in reduction of clinical outcomes in atherosclerotic conditions.
Keywords: atherosclerosis, dyslipidemia, high-cholesterol diet, inflammation
|How to cite this article:|
Ibrahim SM, Gomaa RS, Ismail SI, Ibrahim HS. Role of inflammation versus hypercholesterolemia in the development of atherosclerosis in male albino rats. Al-Azhar Assiut Med J 2018;16:58-65
|How to cite this URL:|
Ibrahim SM, Gomaa RS, Ismail SI, Ibrahim HS. Role of inflammation versus hypercholesterolemia in the development of atherosclerosis in male albino rats. Al-Azhar Assiut Med J [serial online] 2018 [cited 2019 Aug 20];16:58-65. Available from: http://www.azmj.eg.net/text.asp?2018/16/1/58/244150
| Introduction|| |
Cardiovascular diseases (CVDs) are still the leading cause of morbidity and mortality worldwide. Coronary heart disease is a serious health threat in the developed world and responsible for ∼20% of all deaths . Dietary cholesterol causes increase in blood cholesterol level, which leads to arterial wall lesions . The International Atherosclerosis Society recommends decreasing dietary cholesterol as a strategy for lowering low-density lipoprotein cholesterol (LDL-C) . It was reported that dietary cholesterol increases CVD risk . However, Ravnskov et al.  reported that there is a lack of an association or an inverse association between LDL-C level and cardiovascular mortality.
Atherosclerosis (AS) is a complex disease of the arterial wall characterized by the formation of atherosclerotic plaques, which lead to progressive occlusion of arteries. These lesions remain silent for decades but over time, they may cause stenosis or rupture. This can lead to distal ischemia and thrombosis, with clinical consequences such as myocardial infarction and stroke . Despite being a multifactorial disease, elevated concentrations of cholesterol, mainly transported by LDL-C particles, promote atherosclerotic lesions .
The idea that AS is a predominantly lipid-driven disease has dominated the field of CVD. The concept of atherogenesis has changed owing to new evidence that AS is predominantly a chronic low-grade inflammatory disease of the vessel wall . Chronic inflammation has become recognized as a contributory factor in the development of atherosclerotic CVD and other diverse chronic diseases, with new evidence continually being added that supports AS being an inflammatory condition . In AS, inflammation starts and evolves in response to cholesterol accumulation in the arterial intima of the large and medium arteries. However, innate and adaptive immune responses play a pivotal role throughout the initiation, progression, and clinical consequences of atherosclerotic diseases .
On basis of these data, determining the relation between dietary hypercholesterolemia and AS with a trial to evaluate role of inflammation in atherosclerotic process in male albino rats is the objective of the current study.
| Materials and methods|| |
A total of 30 adult male albino rats 12 weeks old weighing 180–200 g were obtained from the Animal House Faculty of Veterinary Medicine, Zagazig University. They were kept in steel wire cages (six/cage) in the animal house in Faculty of Medicine of Zagazig University under hygienic conditions. Animals had free access to water, were kept at room temperature, and were maintained on a 12-h light/dark cycle . The rats were accommodated to animal house conditions for 1 : for 2 weeks before the experiments were carried out . The experimental protocols were approved by Physiology Department and by Institutional Review Board (IRB) Committee, Faculty of Medicine, Zagazig University, Egypt.
The animals were divided into two main groups:
- Group I (n=6) (control): the rats were fed on normal diet, which consisted of 25.8% protein, 62.8% carbohydrate, and 11.4% fat .
- Group II (n=24): the rats were fed on high-cholesterol diet (HCD) composed of cow fat (4%), cholic acid (0.2%), cholesterol (1%), egg yolk (7%), methyl thiouracil (0.2%), corn starch (35%) sodium chloride (1%), wheat bran (6.6%), and wheat flour (45%) .
These animals were subdivided into four subgroups, with 6 rats each:
- Group IIa: rats were fed HCD.
- Group IIb: rats were fed HCD with methotrexate (tablets 2.5 mg SANDOZ, Egypt) as anti-inflammatory drug by dose 0.3 mg/kg/twice per week with 3-day interval by oral route in the morning before eating .
- Group IIc: rats were fed HCD with cholestyramine (sachets 4 g SANDOZ) as lipid-lowering drug by dose 0.5 gm/kg/day by oral route in the morning before eating .
- Group IId: rats were fed HCD with simvastatin (tablets 40 mg Gulf Pharmaceutical Industries, Julphar, Egypt) as lipid-lowering and anti-inflammatory drug by dose 10 mg/kg/day by oral route in the morning before eating .
The experiment lasted for 6 weeks after acclimatization of the rats to the experimental conditions, and then the rats were killed by decapitation after 12 h of fasting under anesthesia (chloral hydrate) inhalation. Blood samples were obtained by exsanguination at the time of killing, collected and allowed to clot for 2 h at room temperature before centrifugation. Sera were stored at −20°C until analysis. Repeated freezing and thawing was avoided. Then, the carotid artery was rapidly removed, dissected from the connective tissues, and fixed in 10% neutral-buffered formalin. Sections of 5 μm were stained with hematoxylin and eosin and then were examined and photographed by light microscopy for histopathology. The thickness of tunica media was measured from photographs of 400× magnification using Digimizer 4.3.2. Image analysis software (Med Calc Software, Belgium). The values of the thickness from each sample were finally statistically compared.
The sera were examined for levels of TC by colorimetric method  and triglyceride levels (TG) and high-density lipoprotein-cholesterol (HDL-C) by an enzymatic assay . Calculation of non-high density lipoprotein cholesterol level (non-HDL-C) was done by the following formula: non-HDL-C=TC–HDL-C . Low-density lipoprotein-cholesterol (LDL-C) was calculated using Friedewald’s formula : LDL-C=[TC–(HDL–TG)/5]. Very low-density lipoprotein-cholesterol (VLDL-C) was calculated as follows: VLDL-C=TG/5 . LDL/HDL ratio was calculated . The atherogenic index (AI) was calculated from the formula: AI=(TC–HDL)/HDL .
Estimation of serum highly sensitive C-reactive protein (HS-CRP) level was done by immune-enzymatic assay technique described by Buduneli et al. . Estimation of serum inerlukin-6 (IL-6) level was done by using rat ELISA kits (Bio Basic Inc., Ohio, USA) . Serum tumor necrotic factor-α (TNF-α) level was determined by using rat ELISA kits (Bio Basic Inc.) according to Fernando et al. .
Results were presented as mean±SD. Statistical analysis was performed using the statistical package for the social sciences, version 19.0 (SPSS; SPSS Inc., Chicago, Illinois, USA). Repeated measures of analysis of variance was applied followed by least significance differences for multiple comparisons. Levels of significance (P) were considered to be statistically significant when P value was less than 0.05 .
| Results|| |
Effect of HCD and HCD with medication on lipid profile
There was increase in serum levels of TC, TG, non-HDL-C, LDL-C, VLDL-C, HDL/LDL, and AI with decrease in HDL-C level in all HCD-fed groups in comparison with the control group. Administration of simvastatin and cholestyramine significantly increased serum HDL-C level and decreased serum levels of TC, TG, non-HDL-C, LDL-C, VLDL-C, HDL/LDL and AI, whereas methotrexate did not change serum lipid profile levels in comparison with HCD-fed rats ([Table 1]).
Effect of HCD and HCD with medication on inflammatory markers
There was an increase in serum levels of IL6, TNF-α, and HS-CRP in all HCD-fed groups in comparison with the control group. Administration of cholestyramine did not change serum inflammatory marker levels in comparison with HCD-fed rats whereas methotrexate and simvastatin administration significantly decreased the serum levels of IL6, TNF-α, and HS-CRP in comparison with HCD-fed and HCD-fed cholestyramine-treated rats ([Table 2]).
The wall of the carotid artery showed normal architecture with normal 3 layers: tunica intima with smooth and regular surface that is composed of a continuous layer of flat endothelial cells (I), tunica media that is formed of several layers of regularly arranged smooth muscle fibers (M), and tunica adventia that is formed of loose connective tissue (A) ([Figure 1]–slide I).
|Figure 1 Photograph of transverse sections (TC) of carotid artery. (Slide I): TC of carotid artery of control group (group I) showing normal histological structures of intima, media, and adventitia. (Slide IIa): TC of carotid artery of group IIa shows atherogenic changes in form of marked intimal thickening and subintimal atheromatous plaque (a) with atheromatous cap formation formed of foamy histocytes (b), fibrous bands, and scattered inflammatory cells (c) with ulceration of intimal lining (d). (Slide IIb): TC of carotid artery of group IIb shows that methotrexate administration prevented to a large extent the development of atherosclerotic changes as indicated by very thin atheromatous plaque with less inflammatory cellular infiltration and some foam cells formation with nearly normal thickness of media. (Slide IIc): TC of carotid artery of group IIc shows that cholestyramine produced slight improvement in the atherogenic changes in the form of slight reduction in the thickness of subintimal atheromatous plaque (↑) with needle-like cholesterol clefts formation, foamy histocytes, fibrous bands, and inflammatory cells infiltration with intimal ulceration (a), thickening, and protrusion in the lumen (b). (Slide IId): TC of carotid artery of group IId shows that simvastatin administration prevented to a large extent the development of atherosclerotic changes as indicated by minimal intimal thickening, less inflammatory cellular infiltration, less foam cells, and less cholesterol clefts with nearly normal thickness of media.|
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The wall of carotid artery showed atherogenic changes in form of marked intimal thickening and subintimal atheromatous plaque (a) with atheromatous cap formation formed of foamy histocytes (b), fibrous bands, and scattered inflammatory cells (c) with ulceration of intimal lining (d) ([Figure 1]–slide IIa).
In group IIb
Methotrexate administration prevented to a large extent the development of atherosclerotic changes as indicated by very thin atheromatous plaque with less inflammatory cellular infiltration and some foam cells formation with nearly normal thickness of media ([Figure 1]–slide IIb).
In group IIc
Cholestyramine produced slight improvement in the atherogenic changes in the form of slight reduction in the thickness of subintimal atheromatous plaque (↑) with needle-like cholesterol clefts formation, foamy histocytes, fibrous bands, and inflammatory cells infiltration with intimal ulceration (a), thickening, and protrusion in the lumen (b) ([Figure 1]–slide IIc).
In group IId
Simvastatin administration prevented to a large extent the development of atherosclerotic changes as indicated by minimal intimal thickening, less inflammatory cellular infiltration, less foam cells, and less cholesterol clefts with nearly normal thickness of media ([Figure 1]–slide IId).
Effect of HCD and HCD with medication on carotid intima media thickness (CIMT)
CIMT was 30.7±3.8 μm, 49.9±3 μm, 39.9±0.7 μm, 48.4±2.8 μm, and 38.9± 0.69 μm in groups I, IIa, IIb, IIc, and IId, respectively. There was a significant increase in CIMT in all HCD-fed groups in comparison with control group. Administration of cholestyramine did not change CIMT in comparison with HCD-fed rats whereas methotrexate and simvastatin administration significantly decreased CIMT in comparison with HCD-fed and HCD-fed cholestyramine-treated rats ([Figure 2]).
|Figure 2 Comparison of carotid intima media thickness in all studied groups. n=6 in each group − Data are represented as mean±standard deviation. CIMT, carotid intima media thickness. P<0.05. *Significant when compared with control group, (a) significant when compared with group IIa, (b) significant when compared with group IIb, (c) significant when compared with group IIc.|
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| Discussion|| |
Data of the present study revealed marked disturbance in lipid profile of rats fed with cholesterol-rich diet manifested by increased serum levels of TC, TG, LDL-C, VLDL-C as well as non-HDL-C, LDL/HDL, and AI parallel to a decrease in HDL-C level.
These results are in agreement with many other researchers who reported that feeding animals with cholesterol-rich diet is commonly used as a model for induction of hypercholesterolemia to study the etiology of hypercholesterolemia-related metabolic disorders and the efficiency of anti-hypercholesterolemia agents ,,.
Moreover, it was found that the serum TC, non-HDL-C, LDL-C, and LDL/HDL of animals fed high-cholesterol high-fat diet increased significantly , and it was reported that cholesterol is an undisputed risk factor in CVD, and the total lifelong cholesterol burden defines the risk .
Non-HDL-C contains more atherogenic cholesterol than LDL-C  and has been suggested to be a surrogate marker of small, dense LDL-C . Zhang et al.  showed that the use of non-HDL-C is ahead of LDL-C in predicting the severity of coronary AS .
LDL/HDL is an important parameter in detection of atherosclerotic diseases, and it was considered to be an indicator with greater predictive value than isolated parameters used independently .
In contrast, it was reported that epidemiologic studies have found no  or little  association between cholesterol intake and CVD risk.
The recently ACCELERATE trial that examined whether the addition of evacetrapib to standard medical therapy reduces the risk of cardiovascular (CV) morbidity and mortality in patients with high-risk vascular disease dumbfounded many experts by failing to demonstrate any cardiovascular benefit of evacetrapib, a novel cholesteryl ester transfer protein inhibitor, despite dramatically lowering LDL-C and raising HDL-C in high-risk patients with coronary disease. It is now clear that lowering cholesterol through diet or with different classes of drugs does not significantly prolong life or consistently prevent CVD, and cholesterol paradox is supported .
In addition, Dehghan et al.  found in their prospective urban rural epidemiology (PURE) study that high carbohydrate intake but not total fat and individual types of fat was associated with higher risk of total mortality. Total fat and types of fat were not associated with CVD mortality, whereas saturated fat had an inverse association with stroke. They recommended that global dietary guidelines should be reconsidered in light of these findings.
The results of the present study showed that administration of simvastatin and cholestyramine but not methotrexate significantly improved dyslipidemia produced by HCD.
Simvastatin is commonly used to reduce blood lipids and to treat CVDs including myocardial infarction, stroke, and hypertension  and is useful for high-risk patients with hypercholesterolemia in primary prevention settings . It possesses additional pleiotropic effects, including antioxidant, anti-inflammatory, and immunemodulatory properties .
Although methotrexate directly targets the inflammatory process of atherogenesis, methotrexate not only attenuated systemic inflammation but additionally decreased cardiovascular events, suggesting that a direct treatment of inflammation may reduce the risk of CVD . Moreover, it was reported that methotrexate also may have direct anti-atherosclerotic effects; in the cholesterol-fed rabbit model, methotrexate reduced new atheroma formation by 75% and reduced multiple biomarkers of macrophage function within the vessel wall .
Cardiovascular inflammation reduction trial (CIRT) proposed that the reduction in CVD events in patients with prior MI and either type II diabetes or metabolic syndrome, conditions associated with persistent inflammation, by using methotrexate was derived from its effect on vascular inflammation .
However, cholestyramine is one of bile acid substrates that selectively bind and remove bile acid molecules from the gastrointestinal tract, decreasing plasma cholesterol levels .
The present results revealed that TNF-α, IL6, and HS-CRP are elevated in HCD-fed rats and HCD-fed rats pretreated with cholestyramine when compared with controls but significantly decreased in HCD-fed rats pretreated with methotrexate and simvastatin when compared with HCD group and HCD pretreated with cholestyramine group. Atherosclerotic changes in carotid artery decreased significantly in rats pretreated with methotrexate and simvastatin with minimal improvement in cholestyramine pretreated groups.
TNF-α has been implicated in the pathogenesis of chronic systemic inflammatory conditions and in the development of atherogenesis and vascular inflammation , whereas IL-6 was shown to be produced by macrophages in atherosclerotic mice . It has been reported that high IL-6 concentrations have been associated with increased risk of MI in healthy men and has an early peak at the acute phase of MI related to plaque instability .
Justification for the Use of Statins in Prevention; an Intervention Trial Evaluating Rosuvastatin (JUPITER) is a trial that was conducted to determine if patients with elevated CRP level without hyperlipidemia might benefit from statin therapy and confirmed the strong relationship between CRP and CVDs . It was concluded that estimation of HS-CRP can be considered as a better predictor for CVD than the serum LDL-cholesterol .It was reported that that during the AS, proinflammatory mediators (IL-6 and TNF-α) were activated. Consequently, for the treatment of atherogenesis, anti-inflammatory therapy was also needed . It was reported that atherosclerotic plaque initiation and progression associated with the roles of inflammatory cells and cytokines .
Moreover, the results of Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) trial showed that anti-inflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid level lowering .
In contrast, other studies considered AS a predominantly lipid-driven disease. They reported that the possible mechanisms implicated in the pathogenesis of AS have made considerable ‘response to lipoprotein retention’ hypothesis as the initiating process in AS ,,.
It was claimed that the augmented level of the serum cholesterol is the imperative feature of the atherogenesis, which was confirmed by an epidemiological study that showed correlation in the LDL, TC, and the severity of the AS . AS is considered as a clinical condition of the cholesterol. Wang et al.  considered the hardening and narrowing the arteries as a consequence of manufacturing of the fatty material.
Current treatment guidelines and recommendations target LDL-C and non-HDL-C reduction to decrease CVD risk . It was reported that control of LDL-C remains a cornerstone of CHD risk management and that the risk modification by cholesterol-lowering treatment is proved .
| Conclusion|| |
Atherosclerotic condition is closely associated with excessive intake of cholesterol-rich diet; however, inflammation has a central role in the pathogenesis of the atherosclerotic process as atherosclerotic changes could be reduced despite disturbed lipid profile by anti-inflammatory medication. Further studies are recommended for more evaluation of the role of anti-inflammatory drugs in reduction of the clinical outcomes in atherosclerotic conditions.
The histopathological assessment in the current study was done by Professor Dr Kamal Ahmed Al Kashishy, Professor of Pathology, Faculty of Medicine, Zagazig University.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]