Background
Cardiovascular diseases are the leading causes of morbidity and mortality among adult population throughout the world [
1,
2]. Atherosclerosis is the primary cause of cardiovascular disease, leading to the occlusion of the arteries, resulting in ischemia in vital organs such as heart and brain and subsequent death. The pathogenesis of atherosclerosis is a complex and chronic multifactorial process and remains a popular topic of research. Identified risk factors are mainly metabolic disorders such as dyslipidemia, obesity and diabetes, in which inflammation and oxidative stress seems to be the common routes to atherogenesis [
3]. The initial step of oxidative-stress induced damage to endothelium was extensively studied, which involves macrophage and oxidized low-density lipoprotein (ox-LDL) [
4,
5]. Atherosclerosis have well-characterized pathological changes include dysfunction of vascular endothelium, differentiation of monocyte into macrophages, conversion of macrophages into foam cells and proliferation of smooth muscle cell [
4,
5].
The investigation of contributing factors of atherosclerosis is an ongoing hot research topic. Hyperlipidemia, especially hypercholesterolemia was found to play an important role in the initiation and progression of atherosclerosis [
2]. Among the negative impacts of hyperlipidemias, it has been well characterized that hyperlipidemia could induce overproduction of reactive oxygen species in endothelial cells, smooth muscle cells and macrophages, subsequently resulting in oxidative stress and lipid peroxidation in both humans and animals [
6,
7]. Thus, oxidative stress has been implicated in the pathogenesis of atherosclerosis, as an independent stage of the disease progression [
8].
Arctium lappa L, commonly known as burdock or bardana, is a perennial plant of the Asteraceae (Compositae) family. It has been reported that the extract and fractions of
Arctium lappa L roots are used for its colitis prophylactic [
9], gastro-protective [
10], anti-sterility [
11], antimicrobial [
12] and anti-proliferative effects [
13,
14]. A recent study reported the anti-inflammatory and free radical scavenging activities of
Arctium lappa root extracts (AREs) [
15]. The anti-oxidant capability [
16] suggested the protective potential of AREs against atherosclerosis. The present study utilized high fat diet induced atherosclerosis model in quail and focused on the protective effects of AAE on high fat diet induced atherosclerosis. The high fat diet quail model ad been used in multiple studies [
17‐
19], features diet induced atherosclerosis and does not require genetic modifications, which is a good mimic of real-life atherosclerosis.
Methods
Materials
Arctium lappa L. Root was purchased from Anqiu vegetable farm center (Weifang, Shandong, China) and identified by Dr. Jingying Sun (Shandong Academy of Medicine Pharmacy Institute). Arctium lappa L. is a well established traditional Chinese medicine herb, since the material used in this study met the quality standards set by the Shandong Food and Drug Administration (SDFDA), no further specimen deposit was performed. Simvastatin was purchased from Hainan Pharmaceutical Factory Co., Ltd. (Hainan, China) (20 mg/Tablet, Batch number: 130406). 1,4-dioxane was purchased from Tianjin Ruijin Chemicals Co. Ltd. (Tianjin, China). Isopropyl alcohol (ISO) was obtained from Tianjin Fuyu Fine Chemical Co., LTD. (Tianjin, China). Nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione peroxidase (GSH-Px) assay kits were purchased from Jiancheng Institute of Biological Engineering (Nanjing, China).
Preparation of AREs
Preparation of the ethanol extract (AEE)
The dried roots of A. Lappa (0.6 kg) were soaked in 4 L of 80 % ethanol for 24 h, and then reflux extracted twice with 80 and 60 % ethanol for 5 h respectively. The resulting residue was dried and used for next extraction. The organic solutions were combined and concentrated under reduced pressure to give 301.8 g of the ethanol extract. 231.6 g starch was added to facilitate the formation of powder, resulting in a total of 533.4 g AEE.
Preparation of the aqueous extract (AAE)
The dried pretreated sample was dipped in distilled water (9 L) and boiled twice for 2 h. The combined filtrate was then concentrated under reduced pressure and evaporated to dryness and the yield of the aqueous extract is 59.1 g. 21.5 g starch was added, resulting in a total of 80.6 g AAE.
The dried roots of A. Lappa (10 kg) were reflux extracted twice with methanol for 5 h. The methanol solution was contration under reduced pressure to obtain a residue. The extract was suspended in water (6 L) and extracted with chloroform (6 L × 3 times). The resulting fraction was collected and then concentrated in vacuo to afford 48.3 g the chloroform fraction. 68.6 g starch was added to form powder. 116.9 g ACE was the final total yield.
Preparation of the flavones extract (AFE)
The remaining solution from chloroform extract was heated to evaporate the organic solvent. Cooled solution was added into column loading-treated Macroporous resin DM-130 for absorption, and then washed with water and 10 % ethanol, respectively, to get rid of impurities. The total flavonoids in column was eluted with 80 % ethanol and dried in a vacuum condition until powder was formed (157.7 g). 17.1 g starch was added to form a total of 174.8 g AFE.
Identification of the AREs
Extracted AREs were analyzed in a previous study carried out by our group. Refer to this work for information about detailed compositions and identified specific chemicals in different AREs [
20].
Animal treatment and sample collection
Male quails (3 weeks old, body weight about 100 g) were purchased from Lanke Poultry Breeding Center (Jimo, Qingdao, China). All experimental protocols were approved by the Institutional Animal Use and Care Committee of Qingdao University (Qingdao, China). Quails were kept on a 12 h day/night lighting schedule and had access to standard quail chow and water ad libitum. After one week environment adaption, the quails were randomly divided into treatment groups. Due to limited handling capacity, two separated batches were used. First batch included: control group, model group, positive control group and AAE groups (AAE 0.75, 1.5 or 3 g/kg/day); second batch included: control group, model group, positive control group, AEE groups (AEE 1 or 2 g/kg/day), ACE groups (ACE 100 or 200 mg/kg/day) and AFE groups (150 or 300 mg/kg/day). Quails in the control group were fed standard chow (0 % cholesterol). Quails in all other groups were fed with high fat diet (1 % cholesterol and 14 % pork oil, w/w), along with specified treatments via gavage. After 4.5 weeks treatment, 2 mL blood were collected from quail right jugular vein. After 10 weeks treatment, terminal body weights were recorded, then venous blood sample (3 mL) was taken from the right jugular vein of each quail. Collected blood samples were incubated at 37 °C for 10 min and then centrifuged at 3000 rpm for 10 min, resulting serum samples were collected and archived at -80 °C until further analysis. The animals were then sacrificed and aorta were dissected for further analysis.
Serum lipid profile assessment
Serum collected at the beginning of the experiment, after 4.5 weeks treatment and after ten weeks treatment were subjected to automatic biochemistry analyzer Beckman AU5400 (Brea, CA, US) for serum lipid profile assessment. The levels of serum total cholesterol (TC), low density lipoprotein (LDL), and high density lipoprotein (HDL) were assessed.
Anti-oxidant and pro-oxidative status assessment in serum
All the biochemical parameters (NO, MDA, SOD, GSH, NADPH and GSH-Px) were measured with commercial kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) following manufacturer’s protocols. All the kits were based on colorimetric methods, and were carried out on a spectrophotometer 722E (Shanghai Spectrum Instruments, Shanhai, China).
Histology of quail aorta
Dissected aortas (aorta arch) were fixed in 4 % formaldehyde for 24 h, embedded in paraffin, and then sectioned at 6 μm on a rotary microtome (Leica RM2016, Wetzlar, Germany). Hematoxylin and eosin (Beyotime, Jiangsu, China) staining was performed following manufacturer’s protocol. Pictures were taken with a microscope (Olympus BX51, Tokyo, Japan) and analyzed with ImageJ (NIH, US). The ratio of atherosclerotic area to total aorta area was calculated as an assessment of atherosclerosis.
Transmission electronic microscopy on quail aorta
Dissected aortas were fixed in 5 % glutaraldehyde for 24 h, dehydrated with graded ethanol, embedded with epoxy resin 618, sectioned and observed under a transmission electronic microscope JEM-1200EX (JEOL, Tokyo, Japan).
Aorta lipid profile assessment
Aortas were homogenized in 1 % 1,4-dioxane. Samples were then incubated in a 37 oC incubator shaker for 72 h. For each sample, 1 mL of supernatant was collected and dried, and then dissolved in 50 μL of isopropanol. Lipid profile was then assessed by subjecting dissolved samples to automatic biochemical analyzer Beckman AU5400 (Brea, CA, US).
Statistical analysis
Drawing lots method was used to ensure that quails were randomly assigned into each treatment groups. SPSS 17.0 was used to perform statistical analysis. All data were expressed as mean ± standard derivation. Normal distribution was confirmed with Levene’s test, then one-way analysis of variance (ANOVA) was performed. When P-values were less than 0.05, the statistical significance was determined and post-hoc least significant difference (LSD) tests were performed for the differences among groups.
Discussion
Hypolipidemic effects of AREs
Hyperlipidemia, especially increased TC, TG and LDL levels, along with decreased HDL level, are major risk factors for atherosclerosis [
22]. Many current drugs, such as statins, mainly target the blood lipids to prevent/treat atherosclerosis. Among the four AREs tested in the current study, ACE does not possess significant hypolipidemic effect and AEE only possess limited effect, while AAE and AFE possess significant hypolipidemic effects, whose onset seems to be a bit slower comparing to simvastatin, but with enough administration duration, the hypolipidemic effects are comparable to the well-characterized hypolipidemic agent simvastatin, thus AAE or AFE administration has the potential to decrease the risk of atherosclerosis.
NO and antioxidant status
NO plays a key role in the pathogenesis of vascular diseases [
23,
24]. It is well documented that endothelial dysfunction caused by lack of endothelium-derived NO production and/or decreased nitric oxide (NO) bioavailability or activity has been linked to atherosclerosis initiation and progression [
25,
26] Moreover, NO deficiency can directly impair vascular function and structure thus promote arteriosclerosis [
27]. The results from current study demonstrated that, similar with simvastatin, AREs (except for 0.75 g/kg AAE) increases NO production significantly. Thus, the increase of NO production could also contribute to the anti-atherosclerosis effect of AAE.
It has been demonstrated that oxidative stress plays a key role in the initiation and progression of atherosclerosis. The generation of oxidative stress may induce vascular disorder and contribute to atherosclerotic plaque formation [
28]. One of the outcome of oxidative stress, the lipid peroxidation, could damage the cell plasma membrane and further leads to deletion of cytoplasmic components and cell death [
28]. MDA, an end product of lipid peroxidation, is considered a critical biomarker of oxidative stress [
29]. Enzymes including SOD, CAT and GPx, can reduce the ROS level [
30‐
32]. In current study, the levels of MDA were significantly increased after high fat diet, confirming high levels of oxidative stress. Meanwhile, the levels of anti-oxidant related enzymes SOD, CAT and GSH-Px, as well as endogenous reducing agents GSH and NADPH were significantly decreased, further confirming the induction of oxdative stress by high fat diet. AREs at all doses (except for the lowest dose on SOD levels) significantly reverted the changes, decreasing the levels of MDA and increasing the levels of SOD, CAT, GSH-Px, GSH and NADPH. Therefore, endogenous antioxidants regulation is a possible mechanism involved in the anti-atherosclerosis effects of AREs.
Atherosclerotic changes
Consistent with published results with atrovastatin [
33], both simvastatin and AREs significantly improved the mophology of high diet fed quail aortas. This is the direct confirmation that AREs could protect high fat diet fed quail against atherosclerosis formation in aorta. Among different AREs, high dose of AAE (3 g/kg) and low dose of AFE (150 mg/kg) exhibited comparable protective effects as simvastatin; high dose of AFE (300 mg/kg) treatment resulted in an even greater anti-atherosclerosis effect comparing to simvastatin. The protective effects were further confirmed with TEM results, in which AFE exerted protective effects for the microstructures of aorta. These data indicates great potential for the AFE fraction to be used as anti-atherosclerosis agents.
Aorta lipid profile
The lipid contents of aorta directly reflects the amount of lipid deposition. Technically, the lower aorta lipid profile is, the less lipid has deposited in the aorta. Our results indicates that AAE and AFE exerted best hypolipidemic effects among the four AREs tested, which is consistent with our morphological assessment. Interestingly, AAE seems to be more potent decreasing TC, with 1.5 and 3 g/kg decreased TC even further than simvastatin did. Meanwhile, high dose AAE (3 g/kg) are about as potent as simvastatin reducing TG and LDL. The differential response in TC and TG/LDL suggests that AAE are more effective reducing cholesterol other than LDL. Further mechanistic study is planned to explore this effect. Another point worth noting is that even ACE, which did not have any hypolipidemic effect, exhibited some antiatherosclerosis effect, suggesting the existence of hypolipidemic effect-independent mechanism, which might worth further exploration.
Conclusions
To evaluate the protective effects of AAE on high fat diet induced atherosclerosis, the high fat diet induced quail atherosclerosis model was successfully established and used for the investigation. The results revealed that AAE is effective in protection against high fat diet induced weight gain, improving serum lipid profile (decreasing serum LDL, TG and TC levels, increasing serum HDL level); protecting against oxidative stress (decreasing MDA level, while increasing SOD, GSH, GSH-Px and CAT levels), increasing NO levels in serum, decreasing lipid content in aorta, and decreasing atherosclerotic area in the aorta. All these effects indicate that AAE is a promising agent in the prevention of atherosclerosis. The underlying molecular mechanism is currently under investigation.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ZW, PL and CW performed the animal treatment work, participated in the histology and serum lipid profile work and drafted part of the manuscript. QJ carried out the histology work and drafted part of the manuscript. LZ performed the extraction of AREs. YC and WZ participated in the animal treatment work, serum lipid profile work, and helped with manuscript drafting. CW designed the study and performed statistical analysis. All authors read and approved the final manuscript.