Edible Bird’s Nest Effectively Attenuates Atherosclerosis through Modulation of Cholesterol Metabolism via Activation of PPAR γ /LXR α Signaling Pathway In Vivo

Atherosclerosis is the underlying cause of the majority of cardiovascular diseases (CVDs) due to high-cholesterol deposition in the aorta, resulting in hardening and narrowing of blood vessels and subsequently leading to an obstructed blood fow. Tere are few previous publications showing the preventive efects of edible bird’s nest (EBN) on CVD


Introduction
Hypercholesterolemia condition leads to pathogenic accumulation of low-density lipoprotein cholesterol (LDL-c) in blood vessels, fatty streak initiation, and atherosclerotic plaque formation, which are highly associated with the development of cardiovascular diseases (CVDs) [1].Te maintenance of hepatic cholesterol homeostasis is very important which involves biosynthesis by hydroxy-3-methylglutrayl coenzyme A reductase (HMGCR), uptake by low-density lipoprotein receptors (LDLRs), lipoprotein release into the circulation, storage via esterifcation, and excretion as bile acids [2].Disturbances in hepatic cholesterol homeostasis may also result in nonalcoholic fatty liver disease (NAFLD), which is highly associated with the pathogenesis of atherosclerosis.
Te hallmark of atherosclerosis in the vascular wall happens when the oxidized LDL (oxLDL) is taken up by macrophages via scavenger receptors such as lectin-like old receptor-1 (LOX-1) and cell diferentiation 36 (CD36) and subsequently transformed into foam cells [3,4].Te imbalanced cholesterol uptake and efux result in increased macrophage cholesterol levels, leading to the massive production of foam cells and the initiation of the fatty streak in the aorta [5].Reverse cholesterol transport (RCT) is the primary pathway for cholesterol efux in order to maintain cellular cholesterol homeostasis.In RCT, activation of transcription factors, liver X receptor alpha (LXRα) and peroxisome proliferator-activated receptor gamma (PPARc), has been shown to reduce the production of macrophage foam cells and the progression of fatty streaks in the subendothelial space of the vascular wall via regulation of the LXR-dependent ATP-binding cassette (ABC) pathway [5,6].
Te PPARy/LXRα signaling pathway upregulates the ABC subfamily A member 1 (ABCA1) expression, enabling cholesterol efux in macrophages [7].Tis leads to the production of cholesterol ester via lecithin-cholesterol acyltransferase (LCAT), which transports intracellular cholesterol to the liver as high-density lipoprotein (HDL).Low-density lipoprotein receptor (LDLR) is responsible for taking up HDL in the liver, and the cholesterol will be excreted as bile acids through cholesterol 7-hydroxylase (CYP7A1) via the activation of LXRα [8].Inhibiting cholesterol uptake and promoting cholesterol efux ofer a therapeutic alternative for atherogenesis.
Statins are widely used to reduce the risk of atherosclerosis and NAFLD, but prolonged use can lead to side efects such as liver injury, muscle toxicity, and acute renal failure [9].Edible bird's nest (EBN) is a natural food supplement with cardioprotective properties, modulating serum lipid profles, coagulation status, cholesterol metabolism, and insulin sensitivity, thereby potentially aiding antiatherogenic therapeutic development [10][11][12].Terefore, the aim of this paper was to determine whether the EBN possess putative serum cholesterol-lowering and antiatherogenic efects in comparison to simvastatin (a lipid-lowering drug) in diet-induced hypercholesterolemia in New Zealand white rabbits.In addition, the efects of EBN on cholesterol metabolism were assessed at molecular levels in order to elucidate the underlying mechanism involved in hepatic and aortic cholesterol homeostasis.

EBN Sample Preparation.
Half-cup-shaped raw-cleaned house edible bird's nest (EBN) was obtained from Blossom View Sdn.Bhd in Terengganu (East Coast of Malaysia).Feathers and other impurities identifed during the screening process of the samples were manually removed using tweezers, and the cleaned EBN was kept at 4 °C until further usage.Te extraction of EBN was conducted by stewing process according to the previous method [13].Te soaked EBN was double-boiled at 100 °C for 30 minutes before being cooled to room temperature.Te full stew (FS) of EBN was produced from the entire sample of doubleboiled EBN without any fltration, while the stew extract (SE) was prepared from double-boiled EBN that had been fltered with a muslin cloth.

Treatment of Experimentally-Induced Atherosclerosis in
Rabbits.Twenty-fve healthy male New Zealand white rabbits (8-10 weeks old, 1500-1700 g) were obtained from Goodtree Garden Sdn.Bhd.(Selangor, Malaysia) and housed individually in stainless steel mesh-bottomed cages (n � 1) under standard laboratory conditions with a 12-hour dark/light cycle, at 20-24 °C and 40-50% relative humidity.Te rabbits were randomly assigned to 5 groups (n � 5) consisting of two control groups and three treatment groups.Te control groups were divided into two groups, namely, normal control (NC): rabbits were maintained on standard rabbit pellet; and high-fat high-cholesterol diet (HFCD): rabbits were fed with a HFC diet containing 69% standard rabbit pellet, 20% palm oil, 10% starch, and 1.0% cholesterol, whereas the treatment groups were divided into three groups, namely, HFCD + FS: rabbits were fed with a similar diet to the HFCD group, with additional supplementation of FS of EBN at 500 mg/kg b.w./day; HFCD + SE: rabbits were fed with a similar diet to the HFCD group, with additional supplementation of SE of EBN at 500 mg/kg b.w./day; and HFCD + STATIN: rabbits were fed a HFC diet and treated with simvastatin at 10 mg/kg b.w./day.
Before starting the study, all rabbits were acclimatized for 1 week on a standard rabbit pellet (ad-libitum) with free access to water.Following acclimatization, the rabbits were fed with a high-fat high-cholesterol (HFC) diet for 12 weeks to induce hypercholesterolemia condition and atherosclerosis.Te HFC diet was formulated according to Yida et al. [11] with minor modifcations.Accordingly, every kg of HFC diet formulation contained 690 g of rabbit normal pellet (containing 40% carbohydrate, 17% protein, 3% fat, 15% fbre, 2% vitamin/mineral mix, 13% moisture, and 10% ash), 10 g cholesterol, 100 g palm oil, 100 g of margarine, and 100 g starch (to cement the pellet together).Te HFC diet pellet was dried in an incubator at 60 °C for 48 hr, cut into small equal-sized cubes, and fed to the rabbits.Doses for EBN supplementation were calculated based on the human equivalent dose (HED) which is about 10 g per day of human consumption as recommended by the EBN supplier.Te EBN dose for the experimental animal was determined by the method described by Shin et al. [14]; and 500 mg/kg b.w/day of EBN dosage was set for experimental purposes [14].Te food composition and animal groups are described in Table 1.
EBN and simvastatin were given to the rabbits orally on a daily basis.After 12 weeks of intervention, blood withdrawal (6 ml) through the ear marginal vein under anaesthesia (ketamine/xylazine (35 mg/5 kg b.w-IM)) before sacrifcing by pentobarbital (100 mg/kg b.w-IV) was performed on all experimental rabbits.Blood sera were collected after centrifugation of collected blood in serum clot activator tubes with a gel separator.Te liver and aorta were carefully excised, weighed, and cleaned.Te liver and aorta were preserved in RCL2 ® solution and stored at −80 °C before the RNA extraction protocol whereas the formalinfxed aorta tissue was kept at room temperature before the histological protocol for examination.All experiments and protocols described in the study were conducted at the experimental animal house of the Faculty of Medicine and Health Sciences, UPM, in accordance with the ethics protocol approved by the Institutional Animal Care and Use Committee of Universiti Putra Malaysia (Animal Ethic' approval no.UPM/IACUC/AUP-R009/2017).

Food Intake and Body
Weight.Te rabbits received 100 g per day of respective experimental diet, and the food intake of each rabbit was measured by subtracting the amount of leftover food in the food container from the 100 g of the food given.Te percentage of body weight gain for each rabbit was calculated as ((fnal b.w − baseline b.w.)/baseline b.w.) × 100).Food intake and body weight of all rabbits were recorded on a daily and weekly basis, respectively.

Lipid Profle, Atherogenic Parameters, and Coagulation
Function Test.Te collected serum and whole blood were sent for analysis of lipid profles (i.e., TC: total cholesterol, TG: triglycerides, LDL-c: low-density lipoprotein cholesterol, and HDL-c: high-density lipoprotein cholesterol) and the coagulation test (i.e., PT: prothrombin time, APTT: activated partial thromboplastin time, and fbrinogen).Atherogenic indices were calculated as described by Sujatha and Kavitha [15] as follows [15]: 2.6.Determination of Tissue Total Cholesterol Content and Hepatic HMGCR.Te total cholesterol of the aorta and liver was determined by using a total cholesterol assay kit following the manufacturer's instruction, and the results were expressed as mg/g wet weight of tissue.Immunoassays were performed by the measurement of hepatic HMGCR by using an ELISA kit according to the manufacturer's instructions, and the result was expressed as mg/g hepatic protein.
Te hepatic protein content was determined using the Bradford assay kit following the manufacturer's instructions.

Histopathology of Aorta and Liver Tissues.
Histological slides of the aorta and liver tissues were prepared according to the standard histological protocol as described by Andrés-Manzano et al. [16].Te trimmed aorta and liver tissues were placed into specimen cassettes prior to fxation, dehydration and clearing, parafn infltration, and tissue embedding.Ten, the tissue sectioning at a thickness of 5 μm thickness was conducted using a microtome (Leica Biosystem, Nussloch, Germany), followed by staining with Hematoxylin and Eosin (H&E) stain.To evaluate the histopathological changes, the slides were examined under a MOTIC BA410 light microscope (Motic Hong Kong Ltd., Kowloon, Hong Kong).Atherosclerotic plaque formation was evaluated by measuring the thickness of tunica intima and tunica media, and the ratio of tunica intima to tunica media was calculated.Te results are expressed as the ratio of the mRNA of interest to the GAPDH mRNA.

Hepatic and Aortic Gene Expression
2.9.Statistical Analysis.Statistical analysis was performed using SPSS version 28.0.All results were expressed as the mean ± standard error of the mean (SEM).Te data were statistically treated by analysis of variance (ANOVA) and Tukey's test with p < 0.05 considered as statistically signifcant.

Characterization and Bioactive Compound of EBN.
In our previous studies, we found that EBN is high in nutritional value and bioactive components [13,17].Te nutritional composition analysis of the EBN had revealed that it comprises of high protein (>50%) and carbohydrates (>20%).In addition, the major bioactive compound detected in EBN is glycoprotein sialic acid with 7.91% (FS) and 8.47% (SE).Further exploration of its monomeric amino acid contents has identifed that the major amino acids in EBN were glutamic acid, aspartic acid, serine, and valine.Protein profling has confrmed that EBN from this study contains seven parent proteins, namely, 78 kDa glucose-regulated protein, lysyl oxidase-3, mucin-5AC-like, acidic mammalian chitinase-like (AMCase-like), 45 kDa calcium-binding protein, nucleobindin-2, and ovoinhibitor.Te fndings of   [18], the administration of a diet containing more than 18% of fat for 6 weeks resulted in an increase in body weight and visceral abdominal fat in rabbits.Terefore, the increase in body weight could have been due to increased lipid accumulation in the body tissue of the rabbits in the HFCD group.However, signifcantly the lowest body weight gain was shown by the HFCD + STATIN group with 20.14 ± 0.19%, which could be contributed by the weight-loss properties of simvastatin [19].
As shown in Table 3, rabbits in the NC group showed a high caloric intake in their diet as compared to other groups (p < 0.05).Tis is most likely due to the food being poorly accepted or other factors related to the level of activity or stress in rabbits receiving the HFC diet regime.Despite the similar caloric intake between treatment groups (except for the HFCD + STATIN group), rabbits supplemented with EBN showed signifcantly less weight gain than rabbits administered with the HFC diet alone (p < 0.05).Te presence of glycoprotein sialic acid, protein nucelobindin-2, and fbre components in FS and SE of EBN could probably deliver the potential of EBN in weight management [13,17].Te efects of these EBN components on body weight changes have been reported in the literature involving in vivo models [20][21][22].Apart from the nutrition components mentioned above, Hou et al. [23] have claimed that protein lactoferrin and ovotransferrin in EBN signifcantly reduced the food intake and body weight increment in ovariectomized-induced menopause in rats, as low level of estrogen in these rats could promote weight gain.Tese fndings support the weight-modulating properties of EBN in HFC diet-fed rabbits in this study.

Improved Lipid Profles and Atherogenic Indices with EBN
Supplementation.Table 4 shows the changes in serum TC, LDL-c, HDL-c, and TG values in rabbits supplemented with simvastatin and EBN.At the end of the intervention, the serum TC (22.90 ± 0.07 mmol/L) and LDL-c levels (16.43 ± 1.35 mmol/L) were markedly elevated in the HFCD group as compared to the NC group (p < 0.05).Te results suggested that hypercholesterolemia induced in the rabbit model had been successfully established.In comparison to the HFCD group, cosupplementation of EBN with the HFC diet efectively lowered both serum TC and LDL-c of hypercholesterolemic rabbits (p < 0.05).Te antihypercholesterolemic of FS and SE was comparable to the simvastatin treatment (HFCD + STATIN group) in reducing the serum TC level (p > 0.05).
Meanwhile, both the HFCD + SE and HFCD + STATIN groups reduced serum LDL-c levels in hypercholesterolemic rabbits in a similar manner (20.57% vs. 24.77%;p > 0.05).In addition, the serum HDL-c level in the HFCD + FS group was markedly increased as compared to that in the HFCD group (p < 0.05), and this enhancing property was comparable to simvastatin treatment (HFCD + STATIN group, p > 0.05).However, there was no signifcant diference in the serum HDL-c level observed between HFCD + SE and HFCD groups (p > 0.05), indicating that a combination of the water-soluble and insoluble fractions in FS of EBN is essential for HDL-c improvement.
Te level of serum TG in the HFCD + STATIN group was comparable to the NC group (p > 0.05), indicating that simvastatin treatment was able to maintain circulating TG at a normal level.Tis could be related to low-calorie intake (low percentage of fat) by the rabbits in the HFCD + STATIN group aforementioned.Interestingly, both HFCD + FS and HFCD + SE groups showed a marked reduction in serum TG levels as compared to other experimental groups (p < 0.05).Te increase in serum TG level in the HFCD group indicated that the HFC diet leads to the accumulation of fat in the blood, and EBN supplementation is able to minimize the circulating TG level.EBN groups tackled diferent lipid parameters, whereby SE and FS worked superior on reducing serum TC and HDL-c, respectively, than those achieved by the simvastatin drug.However, despite the fact that HDL-c increased with a slight statistical signifcance, the increasing fold of LDL-c was more prominent.It is hypothesized that there would be some compensatory increase in HDL-c to remove extra cholesterol, but the increase is signifcantly less than that of LDL-c.
Te Castelli risk index I (CRI) has been demonstrated to accurately refect the formation of coronary plaques and the thickness of the intima-media in the carotid arteries of young adults [24].Meanwhile, the CRI-II or LDL-c/HDL-c ratio has been related to predicting the coronary artery disease (CAD) risk [25].Te atherogenic index (AI) is a measurement of cholesterol in the LDL-c, VLDL-c, and IDL-c lipoprotein fractions in comparison to good cholesterol or HDL-c, whereby it may refect the atherogenic potential of the whole lipoprotein fraction.In this study, the HFC diet feeding increased the CRI-I, CRI-II, and AI values of the HFCD group by 38%, 211%, and 38%, respectively (p < 0.05), when compared to the NC group.In comparison to the HFCD group, EBN supplementation for 12 weeks produced a low risk of CHD by reducing the CRI-I (FS: 22.8%; SE: 21.3%), Journal of Food Biochemistry CRI-II (FS: 35.5%;SE: 25.8%), and AI (FS: 33.4%; SE: 19.4%) values in respective experimental groups.Tese data suggested that EBN potentially reduces the risk of cardiovascular disease and atherosclerosis by improvement in lipid profles, with an alteration in LDL-c and HDL-c possibly contributing to this process.A similar fnding was also reported that EBN was found to improve lipid profle by lowering serum TC and LDL-c, while increasing HDL-c levels in high-fat diet-fed rats, even better than simvastatin [11].

EBN Reduced Hepatosteatosis and Stabilized the Atherosclerotic Plaque
Formation.Histological sections on the liver tissue of experimental animals are shown in Figure 1.Rabbits in the NC group displayed a normal structure of liver tissue with the hepatic cords surrounding central veins with regularly oriented sinusoids (Figure 1(a)).Te hepatic parenchyma was shown to exhibit focal macrovesicular steatosis, which is generally observed in the normal liver since the liver serves as a key gland for nutritional storage and metabolism [26].Te changes in the liver structure after 12 weeks of dietary intervention can be noticed microscopically, as shown in Figures 1(b)-1(e).Te administration of the HFC diet resulted in a remarkable change in liver histology of the HFCD group with the presence of multiple macrovesicular steatosis in hepatocytes, as well as signifcant ballooning of the liver cells (Figure 1(b)).Tese histopathological fndings are consistent with the observation by Ashry et al. [27], who captured the same hepatic architecture with the presence of large lipid vacuoles in hypercholesterolemic rabbits.Te signifcant appearance of macrovesicular steatosis in the HFCD group indicated that a cholesterol-enriched diet potentially caused fatty liver.
Te liver image from the HFCD + STATIN group was found to restore the normal histological appearance of the liver cords, central vein, portal areas, and sinusoids, as well as a signifcant reduction in steatosis (Figure 1(c)).A similar feature of the liver histology was exhibited by the HFCD + FS (Figure 1(d)) and HFCD + SE (Figure 1(e)) groups.Te hepatic steatosis could be due to lipid accumulation in the hepatocytes, and there are direct correlations between serum TG and NAFLD [28].Te sialic acid content and protein nucleobindin-2 identifed in the EBN from this study may contribute to the reduction of fat accumulation in the hepatocytes [13].Te administration of exogenous sialic acid has been shown to reduce 26.7% lipid deposition in the liver of atherosclerotic mice fed with a HFC diet [29].Meanwhile, nucleobindin-2 reduces hepatic lipid accumulation in diet-induced obese mice by directly acting on hepatocytes, and this efect is independent of food intake [30].Tus, it can be suggested that the administration of EBN protects the liver against hypercholesterolemia-induced hepatosteatosis.
Te images of H&E-stained rabbits' aorta are depicted in Figure 2, and the image of the NC group exhibited the presence of an intact and normal aortic tunica intima with orderly oriented endothelial cells (Figure 2(a)).Histological examination of the aortic tissue of the hypercholesterolemic rabbit groups revealed an apparent thickening of the tunica intima, which was flled with foam cells, and resulted in a signifcant reduction of the aortic vascular lumens (Figures 2(b)-2(f)).Te smooth muscle cells within tunica media were replaced by a granular white substance precisely known as a fatty streak in the hypercholesterolemic rabbits with diferent severity as follows: HFCD > HFCD + SE > HFCD + FS > HFCD + STATIN.cotreatment with SE on HFCD-induced atherosclerosis rabbits had a less signifcant efect on decreasing the size of atherosclerotic plaques developed in the aortic wall but might assist with plaques stabilization.
In comparison to the NC group, it can be observed that the I/M ratio of the aorta of experimental rabbits in the HFCD group signifcantly increased (0.07 ± 0.03 vs. 1.10 ± 0.16; p < 0.05) after 12 weeks of HFC diet feeding.Meanwhile, EBN supplementation markedly reduced the I/M ratio of the aorta section (FS: 0.62 ± 0.09; SE: 0.80 ± 0.23) in the hypercholesterolemic rabbits (p < 0.05).Te I/M ratio of the aorta in the HFCD + FS group was comparable with the HFCD + STATIN group (0.70 ± 0.36; p > 0.05).In agreement with the literature [31,32], histopathological examination in the present study conveyed that the HFC diet is responsible for the thickening of the tunica intima layer.Hypercholesterolemic rabbits supplemented with EBN and simvastatin confrmed the thinner aortic tunica and managed to stabilize plaque progression as compared to the HFCD group.Tis result suggests that 1.0% cholesterol incorporated into a high-fat diet promoted atherogenesis and EBN supplementation delayed the progression of atherosclerosis.4 reveals the efect of EBN on the coagulation status of experimental rabbits.Te PT (Figure 4(a)) and APTT (Figure 4(b)) were signifcantly shorter in the HFCD group with recorded times of 6.85 ± 0.50 sec and 20.30 ± 1.71 sec, respectively.In comparison to the HFCD group, there was a substantial increase in both PT and APTT as recorded for the HFCD + FS (PT: 8.90 ± 0.37 sec; APTT: 24.10 ± 3.22 sec) and HFCD + SE (PT: 9.20 ± 0.77 sec; APTT: 23.63 ± 0.68 sec) groups, which were comparable with the HFCD + STATIN group (p > 0.05).Te Fb level (Figure 4(c)) was markedly increased following the HFC diet feeding (5.00 ± 1.33 sec; p < 0.05) and was found to be reduced (p < 0.05) by EBN supplementation and simvastatin treatment on hypercholesterolemic rabbits (HFCD + FS: 2.00 ± 0.33 g/L; HFCD + SE: 3.00 ± 0.81 g/L; HFCD + STATIN: 3.00 ± 0.67 g/L), with no diference observed between HFCD + SE and HFCD + STATIN groups (p > 0.05).No diference (p > 0.05) was observed in the Fb level between HFCD + FS and NC groups (2.00 ± 0.00 g/L).

EBN Improved the Coagulation Status of Hypercholesterolemic Rabbits. Figure
Signifcant increases in clotting factors such as prothrombin, VII, and X were observed in high cholesterol-fed rabbits [33], and PT and APTT prolongation may also be caused by the suppression of these intrinsic coagulation factors.Findings from this study were in accordance with the previous literature that reported prolonged PT and APTT in HFD-induced coagulation in rats supplemented with EBN and its bioactive sialic acid [12,34].Moreover, sialic acid is found on the surface of vascular endothelium and erythrocytes, thereby increasing the negative charge that prevents blood clotting [35].
Te coagulation proteins are commonly present in early atherosclerotic lesions compared to advanced atherosclerotic lesions [36], thus suggesting that these coagulation proteins play a signifcant role in the initial development of atherosclerosis, rather than being involved exclusively in thrombus formation in unstable plaques.Furthermore, the elevated Fb serum concentration is considered one of the proatherogenic indicators that may accelerate the progression of atherosclerotic plaque formation through several mechanisms of action such as increasing the severity of infammation, platelet activation, facilitating LDL-c absorption on the vessel wall, and enhancing infltration of macrophage into the arterial intima [37,38].Terefore, improving coagulation status is one of the possible approaches to prevent atherosclerosis.It can be suggested that the HFC diet may promote hypercoagulation partially due to the induction of hypercholesterolemia and that EBN may have mitigated hypercoagulation partly by regulating cholesterol levels.

Efect of EBN on Cholesterol Accumulation and Gene
Expression in Liver and Aorta Tissues.In order to identify the mechanism underlying hypercholesterolemia and atherosclerotic plaque formation, the cholesterol accumulation and transcriptional analysis related to cholesterol metabolism were determined in the liver and aorta of rabbits.Te level of hepatic HMGCR (Figure 5(a)) and hepatic total cholesterol (TC) (Figure 5(b)) in the hypercholesterolemic rabbits was increased by 3.3-fold and 7.6-fold, respectively, as compared to the NC group (p < 0.05).Meanwhile, cosupplementation with EBNs was found to reduce the hepatic HMGCR (HFCD + FS: 1.3-fold; HFCD + SE: 1.4-fold) and hepatic TC (HFCD + FS: 1.6-fold; HFCD + SE: 1.7-fold) levels of the hypercholesterolemic rabbits and were comparable to simvastatin treatment (p > 0.05).A reduced HMGCR activity decreases TC production in the liver and lowers serum cholesterol levels [39].Tis is in line with the reduction in serum TC levels in experimental groups following the dietary interventions of EBN and simvastatin in this study.
A similar efect on cholesterol accumulation was observed in the aortic tissue of hypercholesterolemic rabbits (Figure 5(c)), where EBN reduced the aortic TC level by 59% and 44% in HFCD + FS and HFCD + SE groups, respectively (p < 0.05).However, rabbits supplemented with FS of EBN showed the lowest aortic TC among hypercholesterolemic rabbits which were signifcantly diferent from SE and simvastatin treatment (p < 0.05), and the efcacy of FS in lowering the aortic TC was coherent with the reduction of tunica intima/media thickness of the aorta in the HFCD + FS group (Figure 3(d)).In accordance with the previous fndings [40,41], administration of the HFC diet signifcantly increased hepatic TC content, which was strongly correlated with the aortic TC level (r � 0.963; p < 0.05) of the hypercholesterolemic rabbits in this study.Tus, EBN supplementation could exert a similar efect to that of simvastatin, by lowering hepatic TC via inhibiting the HMGCR activity and subsequently reducing cholesterol accumulation in the aorta.
As illustrated in Figure 6(a), HFC diet feeding altered the transcription of cholesterol-mediated receptors in the liver.In comparison to the NC group, HFC diet administration has suppressed hepatic LDLR expression by 1.7-fold.Meanwhile, EBN supplementation (both FS and SE) has increased the hepatic LDLR expression of hypercholesterolemic rabbits by 2.0-fold (p < 0.05), as compared to the HFCD group.As a standard drug used for hypercholesterolemia, simvastatin treatment showed a substantially higher hepatic LDLR expression than other hypercholesterolemic groups (p < 0.05).In contrast, there was no signifcant diference in the transcription of aortic LDLR (Figure 7(a)) between all experimental groups (p > 0.05).Tis implied that LDL-c transendothelial transport in the aorta is mediated by receptors other than LDLR [42].
In contrast, the CD36 mRNA expression was greatly expressed than the LOX-1 mRNA expression in the aorta of hypercholesterolemic rabbits.According to Schaefer et al. [43], LOX-1 knockout in macrophages did not result in substantial diferences in oxLDL uptake compared to wildtype cells.Tus, under normal settings, LOX-1 has a negligible efect on the intake and degradation of oxLDL by macrophages, most likely due to the large involvement of the CD36 receptor.Terefore, in this study, the major receptors involved in oxLDL uptake in the liver and aorta of hypercholesterolemic rabbits were LOX-1 and CD36, respectively.Te enhancement of hepatic LOX-1 by EBN remarkably increased the oxLDL clearance by the liver, subsequently reducing the oxLDL uptake by aortic CD36 receptor in hypercholesterolemic rabbits.
As depicted in Figures 6(b) and 7(b), rabbits administered with a HFC diet showed no changes in both hepatic and aortic ABCA1 expression in the HFCD group, when compared with the NC group (p < 0.05).However, upregulation of LCAT ).Te primary function of ABCA1 is to transfer intracellular free cholesterol and phospholipids to extracellular lipid-poor apolipoprotein (mainly ApoAI), where they are assembled to form nascent HDL [44].Meanwhile, LCAT has the ability to transform free cholesterol in nascent HDL into cholesterol ester (CE), which can then be converted into mature HDL as well as blocking cholesterol export from HDL [45].From the transcriptional analyses, ABCA1 and LCAT genes were highly expressed in the liver as compared to the aorta of experimental rabbits.Moreover, in mice with liver-specifc ABCA1 deletion, HDL levels decreased by over 80%, indicating that the liver is the principal source of HDL in the blood [46].Te modulation of the hepatic ABCA1 and LCAT expressions could be the major contributor to the augmentation of serum HDL-c levels.EBN and simvastatin enhanced reverse cholesterol transport (RCT) by upregulating ABCA1 and LCAT genes, thereby reducing cholesterol accumulation in the liver and aorta.RCT plays a signifcant role in atherogenesis as HDL-c transfers excess cholesterol from peripheral tissues to the liver for excretion [46,47].
CYP7A1 is a critical rate-limiting enzyme in cholesterolto-bile acid conversion.Te production of bile acids, which are the last products of cholesterol catabolism, and their excretion into the feces help reduce the amount of excess cholesterol in the liver [8].Terefore, the modulation of specifc hepatic CYP7A1 in the hypercholesterolemic model of this study was identifed to evaluate the potential of EBN in enhancing the hepatic cholesterol elimination.12 Journal of Food Biochemistry expression of the CYP7A1 level was signifcantly increased in the hypercholesterolemic rabbits in the following order: HCFD > HFCD + FS > HFCD + SE > HFCD + STATIN as compared to the normal group (p < 0.05).EBN and simvastatin enhanced the cholesterol excretion in the liver of hypercholesterolemic rabbits by upregulating the hepatic CYP7A1 expression (HFCD + FS: 1.2-fold; HFCD + SE: 1.3-fold; HFCD + STATIN: 2.4-fold).Meanwhile, an increase in the hepatic CYP7A1 gene transcription in hypercholesterolemic rabbits in the HFCD group may have been a compensatory response to hepatic cholesterol accumulation [48].Te enhancement of RCT following EBN and simvastatin intervention reduced cholesterol burden and lipotoxicity as well as prevented hepatosteatosis occurrence in hypercholesterolemic rabbits.
EBN modulates cholesterol infux and efux in both the liver and aorta, which is in accordance with the results of the hepatic and aortic TC in this study.Increased oxLDL uptake and/or decreased cholesterol efux result in esterifed cholesterol deposition in macrophage cytoplasm and the formation of foam cells [49].In addition, the severity of atherosclerosis lesions in cholesterol-fed rabbits has also been associated with the cholesterol content of the aorta [50].In accordance with the total cholesterol levels in tissues measured in this study, tunica intima thickness of the aorta was highly related to the TC levels (liver: r � 0.904; aorta: r � 0.913; p < 0.05).Te improved serum lipid profle and tissue TC by EBN attenuate hepatic steatosis and atheromatous plaque formation in hypercholesterolemic rabbits.
PPARc is a transcription factor that belongs to the nuclear receptor superfamily that regulates the macrophage cholesterol efux by stimulating the expression of LXRα [6].Te activation of LXRα stimulates the expression of ABCA1 and CYP7A1, thus promoting RCT by increasing the HDL-c production, bile acid biogenesis, and cholesterol excretion [51].Meanwhile, the suppression of hepatic ABCA1 and CYP7A1 has accelerated aortic cholesterol accumulation in LXRα-defcient mice, indicating that both ABCA1 protein and CYP7A1 enzyme activity are LXRα-dependent [52,53].In fact, the suppression of LXRα/PPARc activation also downregulates ABCA1, LDLR, and HMGCR expressions in mice fed with the HFC diet [54].Terefore, the activation of the PPARc/LXRα signaling pathway by EBN provides a signifcant role in the modulation of cholesterol homeostasis in both the liver and the aorta tissues.
It can be suggested that the bioactive compounds in EBN may produce a favorable efect on cholesterol metabolism and atherosclerotic plaque stabilization.As an important metabolite in EBN, sialic acid was proven to improve cholesterol metabolism by upregulating LDLR via the suppression of HMGCR gene expression in HepG2 cell lines [10].Furthermore, a recent report by Kawanishi et al. [55] showed that the aortic atherosclerotic plaque development and lipid deposition in the liver were reduced by 18.9% and 26.7%, respectively, in HFD-fed ApoE knockout mice supplemented with sialic acid.According to Hou et al. [56], the incorporation of sialic acid into drinking water improved intracellular cholesterol transfer to plasma (42.9%), cholesterol transported to the liver for excretion (35.8%), and cholesterol excreted in the feces (50.4%) in mice fed high-fat diet enriched with 1% cholesterol.However, the authors have claimed that this exogenous supplementation of sialic acid did not show any changes in ABCA1 and CYP7A1 expressions, suggesting that the presence of other bioactive in EBN may result in the alteration of these RCTrelated genes.
Te hypocholesterolemic efects of EBN in the liver were executed via four major mechanisms: increasing the clearance of circulating LDL-c and oxLDL in the blood through upregulation of the LDLR and LOX-1 genes, prevention of cholesterol synthesis by lowering the activity of the hepatic HMGCR enzyme, promoting hepatic cholesterol balance via upregulation of hepatic ABCA1 and LCAT genes which are activated by PPARc/LXRα signaling, and increasing the removal of excess hepatic cholesterol through cholesterolto-bile acid conversion via upregulation of the hepatic CYP7A1 gene.Given the potential of FS and SE to suppress cholesterol synthesis while also expressing hepatic LDLR activity, EBN may prove to be efective as a cholesterollowering agent.
As EBN regulates hepatic cholesterol metabolism, the reduction in atheromatous plaque formation was attained.Te activation of PPARc/LXRα initiates a negative feedback loop to inhibit the intake of additional cholesterol as foam cells become overloaded with cholesterol.Findings from this study demonstrated the modulatory efects of EBN on aortic cholesterol metabolism through two major mechanisms as follows: (1) controlled cholesterol uptake by downregulation of aortic LOX-1 and CD36 expression and (2) increased cholesterol efux through RCT mechanism via upregulating aortic ABCA1 and LCAT genes which were activated by PPARc/LXRα signaling.Given these fndings, the fact that the HFC diet increases CD36 and ABCA1 expressions in the aorta may provide useful information for understanding the mechanism underlying the infuence of dietary cholesterol on atherosclerotic plaque formation.
Te regulation of cholesterol metabolism is not only activated by PPARc alone, but it could be triggered by other peroxisome proliferator-activated receptors (PPARs) such as PPARα and PPARβ/δ.Te activation of PPARα by fbrates consistently showed positive efects on plasma levels of HDL-c and decreased levels of "atherogenic lipids," such as triglycerides and LDL-c [57].In fact, the expression of the CYP7A1 gene in the liver tissue was suppressed by Journal of Food Biochemistry ciprofbrate, a PPARα agonist [58].Te action of PPARβ/δ agonists was found to exhibit similar efects as PPARα and PPARc agonists, by increasing the plasma level of HDL-c and decreasing the plasma level of LDL-c.Diferent rodent and primate models have supported these benefcial benefts [59][60][61].In additiona, it has been demonstrated that PPARβ/ δ decreases cholesterol absorption and enhances transintestinal cholesterol efux in the intestine [62].In cholesterol-sensing signaling, PPARs in conjunction with other nuclear receptors such as LXR are vital for balancing cholesterol metabolism homeostasis.

Conclusion
Te efcacy of FS of EBN in ameliorating hypercholesterolemia and atherosclerosis is on par with simvastatin drugs as compared to SE, suggesting that the combination of both soluble and insoluble fractions of EBN produced better efects on cholesterol homeostasis than soluble fractions alone.Tese fndings narrate the use of EBN as a new therapeutic approach targeting cholesterol fux in the liver and aorta.In fact, EBN potentially alleviates the severity of NAFLD by reducing hepatic steatosis caused by hypercholesterolemia. Te efects of EBN on improving serum lipid profles and stabilizing atherosclerotic plaque in hypercholesterolemic rabbits suggested that EBN may represent a promising candidate for a therapeutic agent for the treatment or prevention of atherosclerosis.

6
Journal of Food Biochemistry As expected, a substantial lipid core (Figure 2(b)) and calcifcation (Figure 2(c)) in the intimal layer of the aorta would be highly expressed in the HFCD group.In comparison to the NC group, the HFCD + FS (Figure 2(d)) and HFCD + STATIN (Figure 2(f )) groups resulted in mild atheromatic lesions with a thin layer of plaque in the aorta.Meanwhile, the HFCD + SE group resulted in more profound plaques (Figure 2(e)).Microscopic examinations of the aorta wall indicated the superiority of FS in reducing plaque formation as compared to SE of EBN while

Figure 4 :
Figure 4: Efects of EBN on (a) prothrombin time, (b) activated partial thromboplastin time, and (c) fbrinogen levels in the hypercholesterolemic rabbits.Values shown are expressed as the mean ± standard error of the mean (SEM, n � 5).Diferent letters mean signifcant diferences between groups (p < 0.05, a > b > c).NC: normal control; HFCD: high-fat high-cholesterol diet; STATIN: simvastatin; FS: full stew of EBN; SE: stew extract of EBN.

Figure 6 :Figure 7 :
Figure 6: Gene expressions in the liver tissue of experimental rabbits after 12 weeks of dietary intervention.Modulation of hepatic mRNA expression by EBN on key genes involved in (a) receptor-mediated uptake of cholesterol, (b) cholesterol efux, and (c) cholesterol-sensing signaling.Values shown are expressed as the mean ± standard error of the mean (SEM, n � 5).* p < 0.05 compared to the HFCD group and * * p < 0.05 compared to the NC group.NC: normal control; HFCD: high-fat high-cholesterol diet; STATIN: simvastatin; FS: full stew of EBN; SE: stew extract of EBN.

Table 2 :
Gene name, accession number, and primer sequences used in the GeXP multiplex analysis of rabbit hepatic and aortic genes.

Table 1 :
Food composition and animal groups.composition,proteinprofle,andsialic acid content show that EBN is a good source of protein and glycoprotein, potentially exhibiting nutritional and medicinal properties.Based on the protein and carbohydrate composition, FS and SE of half-cup EBN were selected and used in this study.3.2.Caloric Intake and BodyWeight Gain following EBN Supplementation.Te efects of FS and SE of EBN on weight changes and caloric intake of HFC diet-fed rabbits are shown in Table3.Tere was no signifcant intragroup change in basal body weights prior to the intervention (p > 0.05).Te HFCD group gained signifcantly more weight (35.85 ± 0.22%) as compared to other experimental groups after 12 weeks of intervention on the respective diet (p < 0.05).In addition, there was no diference in weight gain between the NC, HFCD + FS, and HFCD + SE groups with the increment in body weight ranging from 23.89 to 25.29% NC, normal control; HFCD, high-fat high-cholesterol diet; EBN, edible bird's nest; FS, full stew; SE, stew extract; mg/kg b.w./day, milligram per kilogram of body weight per day.aDose at 10 mg/kg b.w.b Dose at 500 mg/kg b.w.nutritional (p > 0.05).Compared to the NC group, the increment of body weight in the HFCD group could be explained by the diference in diet composition between HFCD (carbohydrate: 38%; fat: 23%) and standard rabbit pellet (carbohydrate: 42%; fat: 3%).According to Alarcon et al.

Table 3 :
Bodyweight gain and caloric intake of experimental rabbits after 12 weeks of intervention.