Tyloxapol – Stenabolic 0

Regulation of hepatic hydroXy methyl glutarate – CoA reductase for controlling hypercholesterolemia in rats

A B S T R A C T
Hypercholesterolemia is a major risk factor upon developing cardiovascular diseases. This study is aiming to investigate the inhibition role of quercetin on hydroXy methyl glutarate − CoA reductase activity and its gene for attenuating hypercholesterolemia. The kinetic characteristics of HMG-CoA reductase activity were evaluated on extracellular rat liver microsomes. For studying the eﬀect of quercetin by inducing hypercholesterolemia rats by TyloXapol (i.v.). In addition, rats were treated with diﬀerent doses of quercetin according to the inhibition constant of this inhibitor. Our results showed that in quercetin rats groups plasma cholesterol, triglycerides, LDL
−cholesterol and total lipids levels and hepatic (TBARS) level were signiﬁcantly decreased as compared with negative control. However, plasma HDL level, hepatic total thiol level, catalase activity and total protein level signiﬁcantly increased groups as compared with negative control. In addition, HMG-CoA reductase activity was decreased in quercetin groups and this conﬁrmed in gene expression that these groups caused downregulation for HMG-CoA reductase. However, LDL receptor (LDLr) gene expression was upregulated by quercetin. Moreover, histopathological examination of rat liver showed the ameliorative eﬀect of quercetin on hyperch- olesterolemic eﬀect of triton. In conclusion, quercetin may consider as a new saving candidate for the future development of hypocholesterolemia agents.

1.Introduction
Coronary heart diseases (CHD) are the premier cause of death. Among CHD, ischemic heart disease (IHD) which causes high mortality rate worldwide and is gradually increased. Epidemiological studies showed that relationship between plasma cholesterol level and evol- vement of (IHD), generally hypercholesterolemia is associated with increased plasma concentrations of LDL and VLDL. Through lowering
elevated level of LDL –Cholesterol can slower the progression of atherosclerotic lesions [1]. Hyperlipidemia shows sustained by eleva-
tion of serum cholesterol and triglyceride levels and it’s a secondary metabolic dysregulation usually associated with diabetes, elevated serum levels of total cholesterol (TC), triglycerides (TG), low density lipoprotein (LDL) along with decrease in high density lipoprotein (HDL), studies showed that hypercholesterolemia is a major risk factor for cardiovascular diseases like hypertension, coronary heart disease, and atherosclerosis. A massive number of synthetic drugs (collectively called statins) for hyperlipidemia are currently available in the market, but they lack the desired features such as safety on prolonged use, eﬀectiveness, cost and statin intolerance including muscle complaints and increased liver or muscle enzymes and various neurological symptoms. Therefore, attention is being directed toward plants as a medicinal source for lipid lowering activity [2].

3-HydroXy-3-methyl-CoA reductase (EC:1.1.1.34) catalyzes the rate limiting step in cholesterol biosynthesis via the mevalonic acid (MVA) [3]. The statins are eﬀectively used as an inhibitor for HMG-CoA re- ductase for lowering cholesterol in order to decrease cardiovascular risk [4]. Statins have been used for years since its discovery by Endo and Kuroda in 1970’S although it’s adverse eﬀects has limited its use in many patients leading to discontinuation in some patients and low adherence to therapy in others. Statin intolerance have been reported which is related to muscle complaints and increased liver or muscle enzymes and various neurological symptoms. Therefore, many ap- proach to ﬁnd new candidates for natural regulators of cholesterol metabolism. Attention was projected toward products from plant origin that possess antiatherosclerotic activity and is capable of promoting human’s health. Flavonoids which converges a broad family of sec- ondary plant metabolites have shown its eﬀects as Kaempferol,myrcetin, rutin, narginenin, catechin, ﬁstein, and gossypetin [5]. They all showed anti-hypercholesterolemic eﬀect through reduction of he- patic HMG-CoA reductase and inhibit enzyme activity in vitro, and anti- obesity, reducing oXidative stress, antioXidant, and cardioprotective eﬀects [5].

Most studies have focused on ﬂavonoids heterogeneous group as a natural occurring compounds for cholesterol reduction these clariﬁed by [6]. These compounds have diﬀerent biological and pharmacological importance. Also they have the ability to inhibit enzymes, anti-in- ﬂammatory, and antitumor [7]. Several studies showed that ﬂavonoids from diﬀerent sources exhibited signiﬁcant inhibition of hepatic HMG- CoA reductase in model animals and in vitro [8]. Quercetin (3′,4′,3,5,7-pentahydroXyﬂavone) as a ﬂavonoid has shown to have peripheral importance in treatment of CVDs, anti-in- ﬂammatory, antihypercholestermic and antiathersclerotic eﬀect [5]. Quercetin was inaugurated to be potent in hyperlipidemia through acceleration of beta oXidation of lipids [9]. Also, Quercetin also has weight reduction function in obese animal models [10].The aim of the present study is to evaluate the inhibition of ﬂavo- noid substance quercetin on microsomal liver HMG-COA reductase activity and gene. In addition, the hypocholesterolemic and antioXidant eﬀects of quercetin were assessed.

2.Materials and methods
TyloXapol (WR1339), quercetin, HMG-CoA, and NADPHH+ were purchased from Sigma-Aldrich (St. Louis Mo.,U.S.A), dimethylsulph- SiXty adult male Wistar rats weighted from 120 to 170 g were ob- tained from the Faculty of Agriculture, Alexandria University (Alexandria, Egypt). Rats were housed in plastic cages and maintained under standard conditions of temperature, humidity and 12 h light/ dark cycle along the experimental period and they were provided with a pellet concentrated diet containing all the necessary nutritive elements. Rats were acclimatized for two weeks; animal’s procedures were con- sistent with the guidelines of Ethics by Public Health Guide for the Care and Use of Laboratory Animals [18]. Rats were divided equally into 3 groups with diﬀerent subgroups; each subgroup contains 10 rats as following; Group Ia: (Negative control group) rats were treated with a vehicle injected with 1.5 ml saline intravenously (i.v.) via tail –and intraperitoneally (i.p.).Group Ib: (Quercetin control group) rats were injected intraperitoneally (i.p.)with quercetin [19]. The dose was (200 mg quercetin/kg body weight) three times/week for 2 weeks [20]. Group II: (Positive control group) rats were injected intravenously (i.v.) with triton WR 1339 (TyloXapol) dissolved in saline at 50 mg/ml concentration and the dose was ml/ 100 g body weight for 2 weeks (6 doses day after day). The dosing was performed using rat i.v. injection cage [21]. Group IIIa: (Quercetin treated group) rats were injected intravenously (i.v.) with triton WR 1339 (TyloXapol) as group II.

At the same time rats were injected in- traperitoneally (i.p.) with quercetin (50 mg/kg body weight) three oXide (DMSO), DTT, potassium dihydrogenphosphate, dipotassium times/week for 2 weeks. Group IIIb: (Quercetin treated group) rats hydrogen phosphate EDTA, KCL, sucrose, and commasie G-250 (Fisher scientiﬁc UK), and other chemicals of high analytical grade.Liver was excised from male rats and immediately placed in an ice- cold50 mM potassium phosphate buﬀer, pH 7.0 containing 0.2 M su- crose and 2 mM DTT, buﬀer 1(Homogenization buﬀer2 ml/g liver). The homogenization was performed in three strokes with a motor-driven Teﬂon pestle in a glass homogenizer. The homogenate was centrifuged for 10 min at 15,000 × g and the supernatant solution was centrifuged using Beckman coulter optima XE-100 ultracentrifuge at Institute of Graduate Studies and Research Alexandria University, Egypt at 100,000 × g for 75 min, at 4 °C. The precipitate was re-suspended in the same buﬀer (1) with 50 mM EDTA buﬀer 2 (Resuspension buﬀer) and then centrifuged at 100,000 × g for 60 min, for 4 °C to obtainmicrosomal pellet [11]. It was kept frozen at −20 °C until used. Afterthawing in an ice-bath, the microsomes were manually homogenized with a Teﬂon pestle and a glass homogenizer, solubilization buﬀer consisting of 0.1 M sucrose, 2 mM DTT, 50 m MKCl, and 30 mM EDTA in 50 mM potassium phosphate buﬀer, pH 7.0 (5 ml/g liver) [12]. The homogenates were quantiﬁed for their protein contents and further used for HMG-CoA reductase assays and kinetic studies.The HMG-CoA-dependent oXidation of NADPHH+ was monitored at 340 nm and 37 °C according to the method described by [13]. One unit of HMG CoA reductase is the amount of enzyme that oXidizes one na- nomole of NADPH to NADP for one minute under standard assay con- ditions.

Microsomal pellet total protein was assayed using Bradford assay [14]. In addition, the protein concentration in liver tissues was de- termined by [15] method as modiﬁed by [16]. were injected intravenously (i.v.) with triton WR 1339 (TyloXapol) as group II. At the same time rats were injected intraperitoneally (i.p.) with quercetin (100 mg/kg body weight) three times/week for 2 weeks. Group IIIc: (Quercetin treated group) rats were injected intravenously (i.v.) with triton WR 1339 (TyloXapol) as group II. At the same time rats were injected intraperitoneally (i.p.) with quercetin (200 mg/kg body weight) three times/week for 2 weeks.At the end of the experiments rats were euthanized after fasting for 12 h. Blood samples from each rat were collected from portal vein in heparinized tubes to prevent clotting. Plasma was obtained from blood by centrifugation at 3000 rpm for 15 min. Liver was directly separated and washed in ice-cold saline and dried on ﬁlter paper then weighted. In addition, some liver samples from rats were kept in 10% buﬀered formalin for histopathological examination and the rest were storedfrozen at −20 °C until assayed.Plasma total cholesterol level was determined by the colorimetric method using available commercial kits (Bio diagnostic, Egypt) ac- cording to the method described by [22]. Plasma triglyceride level was determined by the colorimetric method using available commercial kits (Bio diagnostic, Egypt) according to the method described by [23]. Plasma HDL cholesterol was determined by the colorimetric method using available commercial kits (Bio diagnostic, Egypt) according to the method described by [24].Determination of plasma LDL –C concentration was carried out byenzymatic colorimetric method according to [25]. The method was performed using kits obtained from Bio diagnostic, Egypt. Plasma total lipids were measured by the colorimetric method using available commercial kits (ABC diagnostics, Egypt) according to method de- scribed by [26]. colorimetrically by measuring thiobarbituric acid reactive substances (TBARS).

The method is based on the determination of malondialdehyde (MDA) an end product of lipid peroXidation, which can react with thiobarbituric acid to yield a pink colored complex exhibiting a maximum absorption at 532 nm [27].To quantify the concentration of RNA and cDNA to assure that the concentrations are pure enough to conduct real time PCR. For very pure samples (without signiﬁcant contamination from protein, phenol, free nucleic acids, organic solvents, carbohydrates, etc.), the absorption of Ultra-Violet (UV) light by the ring structure of purines and pyrimidines can Q5000/USA) automatically performs all necessary measurements and calculations [30].Real-time PCR with SYBR Green was used to measure expression of mRNAs of target genes in the liver, with β- actin as an internal re- ference. The isolated cDNA was ampliﬁed using 2X Maxima SYBR Green/ROX qPCR Master MiX following the manufacturer protocol (Thermo scientiﬁc, USA, # K0221) and gene speciﬁc primers [31]. Theprimers used in the ampliﬁcation, the web based tool, Primer 3 (http:// www-genome.wi.mit.edu/cgi-bin/primer/primer3) was used to design these primers based on published rat sequences. To ensure primer se- quence is unique for the template sequence; we checked similarity to other known sequences with BLAST (www.ncbi.nlm.nih.gov/blast/ Blast.cgi).Two primers were purchased from lab technology according to the manufacture instructions and primers were prepared as following: Lyophilized primer at −20 °C was equilibrated at room temperature.

Equilibrated primer was spin down for 3 s. using spin-centrifuge-vortex.Lyophilized primer was diluted (both forward and reverse) with RNase free water (the volume was added to get 100 μM stock) and then the tube was gently invert for 2 min at room temperature. Stock primer was diluted with RNase free water buﬀer (pH 8.0) to get 5 μM and kept at−20 °C until used (www.ncbi.nlm.nih.gov/blast/Blast.cgi).Liver samples were embedded into 10% neutral buﬀer formalin as ﬁXative solution. The ﬁXed tissues were stored in 70% ethyl alcohol until they were processed. The ﬁXed tissues were dehydrated through a graded series of ethanol and embedded in paraﬃn according tostandard procedures [32]. Then slides were stained with HaematoXylin and Eosin stains as a routine method [33].All Data was presented as the mean ± SE. The data was evaluated by one-way ANOVA using GraphPad instat, and the diﬀerences between the means assessed using Dunett’s compare all to control. Statistical signiﬁcance was considered at p < 0.05., for PCR technique all the data were expressed as means ± S.E. The statistical signiﬁcance wasevaluated by one-way analysis of variance (ANOVA) using SPSS, 18.0 software 2011 and the individual comparisons were obtained by Duncan's multiple range test (DMRT). Values were considered statisti- cally signiﬁcant when p < 0.05. 3.Results In Fig. 1 it was observed that the type of inhibition for quercetin on microsomal rat liver HMG- CoA reductase was miXed inhibition with inhibition constant (Ki) of 100 μg Fig. 1a and b. According to the in-hibition constant (Ki) of quercetin (100 μg) on microsomal rat liverHMG- CoA reductase, three diﬀerent dose of quercetin were selected (50, 100 and 200 mg/kg body weight). These doses were half, equal and double the inhibition constant of quercetin on microsomal rat liver HMG- CoA reductase, respectively. These doses were used for rats treatment hypercholesterolmia. Table 1 showed lipid proﬁle results as following: plasma cholesterol, triglycerides, LDL – cholesterol levels showed insigniﬁcant change (P > 0.05) in quercetin control group (Ib) in comparison with negativecontrol group (Ia).On the other hand, there was a signiﬁcant increase in cholesterol, triglycerides, and LDL – cholesterol levels in triton group(II) with (P < 0.01) in comparison with negative control group (Ia).Rats have been treated with triton and quercetin groups (IIIa–IIIc) showed signiﬁcant increase in cholesterol, triglycerides, LDL –choles-terol levels with (P < 0.01) when compared to negative control group (Ia) and signiﬁcant decrease (P < 0.01) when compared to rats have been treated with triton only group (II). Plasma HDL-cholesterol levels showed insigniﬁcant increase (P > 0.05) in control group (Ib) in comparison with negative control group (Ia).On the other hand, there was a signiﬁcant decrease in rats have been treated with triton only group II positive control (P < 0.01) when compared with negative control group (Ia). Rats have been treated triton with diﬀerent concentrations of quercetin (groups: IIIa, IIIb, IIIc) showed signiﬁcant increase plasma HDL-cholesterol level (P < 0.01) when compared to negative (Ia).Plasma total lipids showed insigniﬁcant change (P > 0.05) in group Ib in comparison with negative control group (Ia). On the other hand, there was a signiﬁcant increase (P < 0.01) of total lipids in group II positive control (rats have been treated with triton only) when compared with negative control group (Ia). Rat groups treated with triton and diﬀerent concentrations of quercetin (group IIIa, IIIb, IIIc) showed signiﬁcant increase (P < 0.01) in total lipids when compared to negative control group (Ia) and signiﬁcant decrease when compared to positive control group II Table 1.Hepatic concentration of malondialdehyde (MDA) concentration showed a signiﬁcant increase (P < 0.01) of MDA in group II in com- parison with the negative control group (Ia). Groups of rats have been treated with triton and diﬀerent concentrations for quercetin (groups IIIa, IIIb, IIIc) showed signiﬁcant increase (P < 0.01) when compared to negative (Ia) and signiﬁcant decrease when compared to positive control group (II) Table 2. Hepatic total thiol levels showed a signiﬁcant decrease (P < 0.01) in group II when compared with negative control group (Ia). Groups IIIa, IIIb, IIIc showed signiﬁcant decrease (P < 0.01) when compared to negative group (Ia) and signiﬁcant in- crease (P < 0.01) when compared to positive control group (II) Table 2. Hepatic catalase enzyme activity showed a signiﬁcant decrease (P < 0.01) in group II when compared to the negative control group (Ia). Hepatic total protein levels in group II showed a signiﬁcant de- crease (P < 0.01) in comparison with negative control group (Ia) Table 2.In Table 3 the results showed that HMG-CoA reductase activity wasdetermined in liver of diﬀerent groups of rats, in triton control groups(II) showed the highest enzyme activity when compared with control groups (Ia, Ib).Real time PCR was used to detect the relative expression of 3-hy- droXy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) gene and low density lipoprotein receptor (LDL r) gene that reﬂects the changes in transcription levels of these genes in rat liver after admin- istration of triton and/or quercetin in comparison to control rats. After extraction, the quality and concentration of total RNA were assessed by Nanodrop which revealed presence of pure RNA with a considerablehigher concentration (ranged from 1100 to 1640 ng/μl) Fig. 2.Theisolated total RNA was revise transcribed into cDNA which was used as a template for qPCR. Throughout the whole real time PCR experiment, the housekeeping gene encoding β-actin was used as an internal re- ference for normalization and data was expressed as mean ± SEM (n = 10 per group). The expression level of the target gene in negativecontrol group (Ia) rat was considered the baseline.The data showed signiﬁcant (P ≤ 0.05) up regulation of HMG-CoA reductase gene expression level in liver following administration of triton (group II) as compared to negative control group (Ia) Table 4 andFig. 2. The expression of HMG-CoA reductase gene was signiﬁcantly and gradually down regulated in groups administrated quercetin (IIIa, IIIb, IIIc) showed highest down regulation as compared to positive control group (II). HMG-CoA reductase gene expression was sig- niﬁcantly down regulated in group (Ib) as compared to negative control group (Ia).Our results revealed a signiﬁcant (P ≤ 0.05) downregulation of LDLr gene expression level in triton control group (II) as compared tonegative control group (Ia) Table 4 and Fig. 3. The mRNA expression of LDLr gene was signiﬁcantly and gradually up regulated following ad- ministration of quercetin alone (Ib) and in groups (IIIa, IIIb, and IIIc) as compared to positive control group II.Histopathological examination of rat’s liver tissue in the diﬀerent studied groups showed the hepatocytes of the normal control groups (Ia, Ib) were normal structure and arranged in the form of anastomosingcords (strands) forming a network that extend from a central vein to the periphery of the hepatic lobules at which the portal tracts appeared Fig. 4A and B in these groups the hepatocytes are polygonal with eo- sinophilic granular cytoplasm and vesicular basophilic nuclei. The liver cords were separated from each other by narrow blood sinusoids lined with endothelial cells and kupﬀer cells, the hepatic lobules enclosed among them spaces of connective tissue called portal spaces. Each portal space contains a branch of the portal vein, a branch of the he- patic artery and a bileductule lined with cubical epithelium Fig. 4A and B. co-treated triton with quercetin 100 mg/kg group (IIIb) Fig. 4E showed revealed mild atrophied, mild cellular inﬁltrations, mild vacuolated hepatocytes. Finally, Liver sections in co-treated triton with 200 mg/kg quercetin group (IIIc) showed revealed moderate vacuolated hepato- cytes and moderate congestion of the central vein Fig. 4F. 4.Discussion The kinetic characteristics of HMG-CoA reductase activity were evaluated in extracellular rat liver microsomes. Line Weaver-Burk plots was drawn for enzyme activity in presence of variable concentrations of tested substance to determine the inhibition constant (Ki) of quercetin with miXed inhibition. Therefore, this ﬂavonoid could be also substrates of microsomal HMG-CoA reductase inhibition. This mechanism was similar to the inhibitory eﬀect of quercetin on cytochrome p450 reac- tion enzymes [34].The Ki of quercetin of the present study was 100 (0.33 mM), The Line Weaver-Burk plots predicted the type of inhibition for quercetin was miXed inhibition. The inhibition constant of quercetin on micro- somal HMG-CoA reductase extracted from human liver carcinoma HepG2 was 0.3 mMFrom the data of Inhibition constant of quercetin (0.33 mM) on microsomal rat liver HMG-CoA reductase, three doses of quercetin (0.165, 0.33 and 0. 49 mM) (half, equal and double inhibition constant) were chosen to treat hypercholesterolemia [35].[36] demonstrated that triton WR1339 prevents the lipolysis of triglycerides-rich lipoproteins by inhibiting lipoprotein lipase (LPL) and blocking the removal of triglycerides from plasma. In consistence, we found a signiﬁcant increase in the plasma level of cholesterol, trigly- cerides, LDL –C, and total lipids in rats have been treated with triton(group II). However, triton caused a signiﬁcant decrease in serum HDL-C compared to the negative control group (Ia). These results were compatible with those published by [8,37]. In addition, [38], had re- ported that triton-treatment decreased the HDL-C level in plasma and decreased HDL-C particles for lecithin cholesterol acyltransferases ac- Quercetin reduced the serum cholesterol, triglycerides and LDL – cholesterol concentrations when compared with triton groups (II) and this was compatible with the results reported by previous studies[39,40]. In accordance with the data obtained from these study. [41] reported that quercetin decreases cholesterol, triglycerides, LDL –Cho- lesterol-C and increases HDL-C, as well as protecting LDL –Cholesterol- C from oXidative damage, which is eﬀective in preventing DNA damageinduced by hydrogen peroXide in human lymphocytes and cause de- crease in weight of obese adult males and females.Total plasma lipids signiﬁcantly increased in triton-treated group(II) when compared with negative control group (Ia) and these results were in agreement with the results published by [42]. In the present study, the elevated concentration of total plasma lipids was reduced following administration of quercetin. It was shown that quercetin re- duces lipid through modulation of lipase activities in obese rats [42]. Hypolipidemic and antioXidative eﬀects of the quercetin shed a light on its beneﬁcial use against obesity-related metabolic alterations.In the present study, administration of triton led to a signiﬁcant increase in the level of liver lipid peroXides (MDA). These data agree with the study reported by [43]. In atherosclerotic condition the in- creased level of lipids was responsible for the higher level of lipid tivity causing decrease in the activity of this enzyme. peroXidation and tissue injury. Lipid peroXides accelerate the incorporation of LDL –cholesterol, into arterial smooth muscle cells and promote the formation of lipid laden foam cells which contribute to the development of atheromatous plaques [44]. Assessment of TBARS isoften used to measure tissue concentrations of malondialdehyde (MDA), which formed during oXidative degeneration as a product of free oXygen radicals, which is accepted as an indicator of lipid peroX- idation. On the other hand, our results revealed that quercetin atte- nuated the triton eﬀect which induced increasing in MDA level through there active role as antioXidant thereby lowering the availability of li- pids for peroXidation [45].Quercetin is the most potent scavenger of free radicals within the ﬂavonoid family and its qualities as an antioXidant makes quercetin a strong lipid peroXidation inhibitor. The antioXidant properties of ﬂa- vonoids like quercetin are often correlated with the reduction of rates of several chronic diseases including coronary heart disease, stroke, and diabetes [46].The results of the present study demonstrated that the hepatic total thiol level was reduced in the rats treated with triton group (II) com- pared to the negative control group (Ia). These results were in ac- cordance with those of [47] who stated that the decreased antioXidant levels are possibly due to their increased utilization combating ex- cessive oXidative stress in hypercholesterolemic rats. Consequently, decreased total thiol level in the hyperlipidemic group might be result of the increase in peroXidation of the membrane lipids in this group since it causes reduction in the activity of the antioXidant enzymes. The disturbance between oXidants and antioXidants due to hyperlipidemia has been conﬁrmed by [48]. On the contrary, the present ﬁnding in- dicates a signiﬁcantly increased in hepatic total thiol in rats treated with quercetin (IIIa, IIIb, IIIc) compared to triton treated rats. The an- tioXidant activity of these ﬂavonoids may be attributed to a catechol group found in ring B.In the present study ROS, probably generated by triton induction,induced a rise of protein carbonyls (PCO) products (markers of protein oXidative injury), all that explain the decrease of protein content of liver tissue in triton-treated group when compared with control group. Administration of quercetin showed enhancement in total protein content, this is due to the role of quercetin in preventing lipid peroX- idation and protein oXidation induced by triton due to the antioXidant properties of quercetin. These results were in agreement with those of [41].The liver is the major site for the synthesis and net excretion of cholesterol, either directly as free cholesterolin the bile or after con- version into bile acid. The hepatic enzyme involved in the regulation of cholesterol metabolism is HMG-CoA reductase, a rate-limiting enzyme in the cholesterol biosynthetic pathway. Hepatic HMG-CoA reductase activity can normally be decreased under high-cholesterol feeding conditions using a negative feedback control [49]. In our study in groups treated with quercetin showed reduction in activity of hydroXylmethyl glutaratereductase. In addition, the intracellular concentration of LDL –cholesterol is maintained by a balance between endogenous biosynthesis and exogenous supply. This balance is regulated by HMG-CoA reductase (HMGR), a key regulatory enzyme in cholesterol bio- synthesis, and the LDL –cholesterol receptor synthesis which facilitates cholesterol uptake from the blood circulation [50].In our study and [51] study, the HMG-CoA reductase gene expres- sion in hypercholesterolaemic animals increased in parallel with serum total cholesterol concentration. We also found higher enzymatic ac- tivity of HMG-CoA reductase in liver of hypercholesterolaemic rats. This result agrees with previous reports where animals fed a high-fat diet had elevated HMG-CoA reductase [52]. This ﬁnding points out the potential role of exogenous cholesterol as a cause for the up-regulation of hepatic HMG-CoA reductase activity and expression.In the present study, the lipid lowering eﬀect of quercetin was ac- companied with signiﬁcant decrease in the expression of HMG-CoA reductase gene in liver of triton-induced hypercholesterolemic rat. This is similar to a previous study in which phytosterol ester supplementation increased HMG-CoA reductase mRNA expressions in mononuclear blood cells and livers of healthy humans [53]. This may be due to a compensatory mechanism by which the HMG-CoA reductase gene is up-regulated in response to reduction/inhibition of HMG-CoA reductase enzyme activity [54]. The phenomena whereby HMG-CoA reductase inhibitors trigger an over-expression of HMG-CoA reductase gene transcription are well documented [54]. This contradiction may be due to variation in species (human vs. rats) and treatments (rice bran oil and phytosterol ester vs. quercetin. In the present study, we used the active component instead of the whole plant extract used by [53,55]. Thus, our results are more speciﬁc when compared by these previous studies.Quercetin leads to signiﬁcant downregulation of HMG-CoA re- ductase gene as compared to negative control rats (Ia). Interestingly, quercetin lowers HMG-CoA reductase gene in rat of negative control group (Ib) and with higher rate in rat injected with triton. This in- dicated that in either absence or presence of cholesterol, the hy- pocholesterolemic eﬀect of quercetin could have possibly reduced cholesterol levels by reducing hepatic HMG-CoA reductase gene ex- pression.Elevated levels of LDL –cholesterol are an established risk factor for the development and progression of CVD. The metabolism of LDL–Cholesterol is greatly inﬂuenced by the number and activity of LDL receptors [56]. The liver plays an important role in the regulation of plasma LDL levels by LDL receptor-mediated LDL uptake. Plasma totalcholesterol levels were increased by ∼250% in LDLr knockout mice and hepatic cholesterol levels were augmented by ∼50% as compared with wild-type mice [57], suggesting a role for the hepatic LDLr in the up-take and clearance of atherogenic lipoproteins. Hence, functional LDL receptors are vital to take up LDL −cholesterol in order to decrease their circulating levels in the blood circulation which is one of the contributing factors towards the development of atherosclerosis. The eﬀect of quercetin on the expression of LDL receptor gene was studiedto ascertain if the cholesterol-lowering eﬀect of this ﬂavonoids occurred through increased production of LDL receptors. In the present study, administration of quercetin resulted in signiﬁcant increase in the ex- pression of LDL receptor gene in liver of rats injected with triton.However, the expression of LDL receptor gene was signiﬁcantly down-regulated in rats fed on the high-cholesterol diet. This signifying the possibility that high intracellular cholesterol reduced and sup- pressed synthesis of LDL receptors. LDL –colesterol in excess was not eﬃciently taken up by the limited amounts of LDL receptors, hence decreasing LDL –C clearance from peripheral tissues. This could explainthe elevation of serum LDL –cholesterol observed in the present study.This ﬁnding is consistent with another study in monkeys [58], mice [59], rats [60], hamsters [51], and where high-cholesterol diet caused down-regulation of LDL receptor expression.Quercetin lead to signiﬁcant upregulation of LDL receptor gene as compared to negative control rats (Ia). Interestingly, quercetin upre- gulates LDL receptor gene in rat fed on normal diet with a higher rate than rats injected with triton group (II). This indicated that in either absence or presence of cholesterol, the hypocholesterolaemic eﬀect of quercetin could possibly reduced cholesterol levels by increasing he- patic LDL receptor gene expression.From the current study, we ﬁrst provide evidence that quercetin can upregulate hepatic LDL receptor expression in vivo (in rat). In parallel, a previous study by [61] showed that quercetin promoted strong LDL receptor expression at both mRNA and protein levels in vitro (in HepG2 cells). Quercetin-driven LDL receptor expression can lower circulating LDL cholesterol and might participate in the prevention or amelioration of atherosclerosis and cardiovascular disease.LDL receptor expression in the liver is known to be regulated pre- dominantly at the transcriptional level through sterol regulatory ele- ment binding protein (SREBP) binding to the sterol regulatory element (SRE) of the LDL receptor promoter [62]. Intracellular cholesterol levels regulate LDL receptor transcription through a negative feedback mechanism. When cellular cholesterol levels are low, SREBP2 activates LDL receptor transcription after being proteolytically processed to a mature and nuclear form of SREBP2 in the endoplasmic reticulum to the Golgi apparatus. [61] have shown that quercetin increased the nuclear form of SREBP2 and subsequently increased LDL receptor and protein levels in HepGs cells. These results clearly indicated that quercetin promotes SREBP2 maturation in presence of JNK and ERK signals, thereby leading to LDL receptor expression. To lower blood LDL–C levels, statins have been widely used to inhibit the enzymatic ac- tivity of HMGR which results in a decrease in the cellular cholesterolpool, subsequent up-regulation of the LDL receptor gene, and an in- crease in blood LDL –C removal.Since the majority of plasma LDL –C is removed by the liver through LDL r-mediated uptake [50], LDL –C clearance from peripheral cells tothe liver is considered to be crucial to avoid the formation of athero- sclerotic lesions. When intracellular cholesterol concentration in- creased, the transcriptional activity of these two genes become down- regulated by SREBP. However, in the case of severe hyperlipidemia that does not respond well to single statin therapy, additional combinationsof other drugs or agents to lower plasma LDL –C may be necessary [63].Our results suggested that the blood cholesterol-lowering eﬀect of quercetin was due to increase of cholesterol uptake in addition to in- hibition of cholesterol biosynthesis. These ﬁndings showed that quer- cetin- lowered plasma LDL –cholesterol levels via LDL receptor gene induction and HMG CoA reductase gene and enzyme inhibition. Thedownregulation of expression of HMG-CoA reductase gene suggesting decreased cholesterol synthesis by an alternative mechanism to that of statins which inhibit not only the enzyme’s activity but also the geneexpression.Lipidemia is one of the major risk factors for atherosclerosis that considered as a chronic inﬂammatory disease initiated by monocyte and lymphocyte adhesion to activated endothelial cells, underlies im- portant adverse vascular events such as coronary artery disease, stroke and peripheral artery disease, responsible for most of the cardiovascular morbidity and mortality today. In the current study; liver sections in triton group II showed many histopathological abnormalities as, marked cytoplasmic vacuolization of hepatocytes, moderate ﬁbrosis, congestion of the central veins, hepatic sinusoids with the presence of massive intracytoplasmic vacuolations and marked inﬂammatory cells. Quercetin revealed good degree of improvement with normal he- patocyte architecture. Quercetin had protective and ameliorative ef- fects on decreasing atherosclerosis, but when combined showed higher level of improvement and more protection in concordance with results of [64] that suggested the additive eﬀect of antioXidants as restoration of plasma total protein, albumin, globulin and bilirub in and the he- patoprotective eﬀect might be a consequence of the stabilization in the redoX state and maintenance of the antioXidant capacity oﬀered by these antioXidants. Use of combinations of quercetin is recommended for normal people to decrease the free radicals and toXicity as we ex-posed daily for many polluted factors in air, water and food. 5.Conclusion In conclusion, this study showed that quercetinis natural ﬂavonoids with hypocholesterolaemic and antioXidant properties. They exert their potential hypocholesterolaemic action by increasing hepatic gene ex- pression of LDL receptor while suppressing HMG-CoA gene expressions. Hence, quercetin could potentially inhibit cholesterol biosynthesis, increased uptake and clearance of LDL –cholesterol from peripheral tissues and suppress triglycerides accumulation in the liver. Quercetin can also prevent oXidative damage, particularly oXidation of LDL choles- terol which is one of the risk factors for atherosclerosis. The ﬁndings from this study thus suggested that quercetin could be considered as an eﬀective HMG-CoA reductase inhibitor and strong LDL –cholesterol receptor inducer that might be developed into a new hypocholester- olemic agent with antioXidant properties in the future. However,further work is needed to conﬁrm the eﬃcacy and proper dosage of the quercetin as a potential cholesterol-lowering agent including human trials to gain further insight into its therapeutic Tyloxapol eﬀect.