The anti-inflammatory effect of kaempferol on early atherosclerosis in high cholesterol fed rabbits
© Kong et al.; licensee BioMed Central Ltd. 2013
Received: 2 May 2013
Accepted: 26 July 2013
Published: 29 July 2013
Atherosclerosis has been widely accepted as an inflammatory disease of vascular, adhesion molecules play an important role in the early progression of it. The aim of the present study was to evaluate the effect of kaempferol on the inflammatory molecules such as E-selectin (E-sel), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesionmolecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) in high cholesterol induced atherosclerosis rabbit models.
Thirty male New Zealand white (NZW) rabbits were randomly divided into five groups, control group, model group, fenofibrate (12mg/kg) group and kaempferol groups (150 mg/kg and 30 mg/kg). The rabbits were fed with a normal diet or a high cholesterol diet for 10 weeks. Levels of blood lipids, serum tumour-necrosis factor-alpha (TNF-α) and serum interleukin-1beta (IL-1β) were detected at the end of the sixth and tenth week. Malonaldehyde (MDA) level and superoxide dismutase (SOD) activity in serum were also determined. Lesion areas of the aorta were measured with morphometry analysis after ten weeks. Gene expression of E-sel, ICAM-1, VCAM-1 and MCP-1 in aortas was determined by RT-PCR (reverse transcription-polymerase chain reaction). Immunohistochemical staining was employed to measure protein expression of E-sel, ICAM-1, VCAM-1 and MCP-1.
Model rabbits fed with ten weeks of high-cholesterol diet developed significant progression of atherosclerosis. Compared with the control, levels of blood lipids, TNF-α, IL-1β and MDA increased markedly in serum of model rabbits, while SOD levels decreased. Gene and protein expressions of E-sel, ICAM-1, VCAM-1 and MCP-1 in atherosclerotic aortas increased remarkably in model group. However, comparing to the model rabbits, levels of TNF-α, IL-1β and MDA decreased significantly and serum SOD activity increased, gene and protein expressions of E-sel, ICAM-1, VCAM-1 and MCP-1 in aortas decreased significantly with the treatment of kaempferol.
Kaempferol shows anti-atherosclerotic effect by modulating the gene and protein expression of inflammatory molecules.
KeywordsKaempferol Atherosclerosis Inflammation Vascular adhesion molecule
Cardiovascular disease (CVD) continues to be the leading cause of death in developed countries nowadays. As the most important contributor of CVD, atherosclerosis is arousing great attention worldwide. With the development and progression of atherosclerosis, a great deal of lipid deposits and fibrous plaques accumulate in arteries of some critical organs, especially heart and brain, leading to the formation of thrombus, which is responsible for most of the breakouts of clinical vascular events .
Atherosclerosis is currently well accepted as a chronic inflammation of arteries. The lesions of atherosclerosis represent a protective and inflammatory response against different and multiple risk agents including cholesterol, elevated oxidated LDL, free radicals caused by cigarette smoking, hypertension, diabetes and infections . A recent report found some new risk factors in the pathogenesis of atherosclerosis such as homocysteinemia, elevated plasma levels of lipoprotein (a) [Lp(a)], fibrinogen, impaired fibrinolysis, increased platelet reactivity and hypercoagulability .
Inflammatory mechanisms play a critical role in the pathogenesis of atherosclerosis. In the early phase of atherosclerosis, dysfunction of endothelium leads to the increased permeability of endothelium and adhesiveness of leukocytes to artic wall. The production of procoagulant agents including vasoactive molecules, cytokines and growth factors can also be induced in endothelium. Blood monocytes adhere to epithelium, migrate into intima and become intimal macrophages, then change to foam cells by excessive ingestion of modified lipoprotein and gradually form the so called fatty streak .
The initial stage of inflammation of atherosclerosis is usually silent and long with increased adhesion of monocytes in arterial endothelium . Adhesion molecules and monocyte chemotactic protein-1 (MCP-1) participate in the progress and play an important role. Fisrtly, selectin including E-selectin (E-sel), P-selectin(P-sel), and L-selectin (L-sel) facilitate the rolling of molecules on the surface of endothelium cells, then adhesion molecules, such as vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) mediate the advanced and firm adhesion of monocytes. Lastly, with the function of MCP-1, monocytes pass though endothelium and migrate into intima [5, 6]. Damaged endothelial cells, arterial lesions from atherosclerosis experiment model and human all show increased expression of these molecules including E-sel, ICAM-1, VCAM-1 and MCP-1 [7, 8]. Therefore, one possible mechanism for ameliorating atherosclerosis is the down-regulation of pro-atherogenic molecules such as E-sel, ICAM-1, VCAM-1 and MCP-1. Besides, in the inflammatory progress of atherosclerosis, activation of endothelial cells and mactophages leads to the release of various kinds of cytokines, chemokines, and growth factors, which in return regulate the continued and advanced accumulation and migration of leukocytes, thus induce further inflammation . Pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) participate in it by inducing E-sel, ICAM-1 and VCAM-1 expression in endothelial cells , releasing of other inflammatory mediators and inducing apoptosis of endothelial cells. Previous studies revealed that elevated levels of TNF-α and IL-1β in patients with CVD have been detected [10, 11], indicating that TNF-α and IL-1β are two important cytokines in vascular inflammation and highly associated with atherosclerosis. Recent studies also suggested that, inflammatory biomarkers such as TNF-α, IL-1β, E-sel, ICAM-1, VCAM-1 and MCP-1 may play a potential role for the prediction of risk for developing CVD and may correlate with the severity of CVD [12, 13].
Kaempferol(3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), a yellow compound with a low molecular weight (MW: 286.2 g/mol), is a common natural flavonoid. This flavonoid is abundant in many plant-derived foods and traditional medicine. It has been reported that kaempferol and its glycosides have many kinds of pharmacological activities, such as antioxidant, anti-inflammatory, anticancer, antimicrobial, neuroprotective, antidiabetic, analgesic and anti-allergic activities . And results of some studies suggested that the dietary intake of kaempferol-rich foods can reduce risk of developing several disorders including cardiovascular diseases [15, 16].
Therefore we considered kaempferol to be a potent and effective agent against atherosclerosis. So far, the researches about the antioxidant, anti-inflammatory and cardioprotective effect of kaempferol have been mostly centered on endothelium cells in vitro. However, in vivo studies related to the anti-atherosclerosis effect of kaempferol are so rare. So the aim of the present study was to investigate the protective effect of kaempferol on atherosclerosis rabbit models. And our study will be focused on the anti-inflammation effect and its influence on the expression of inflammatory molecules E-sel, ICAM-1, VCAM-1 and MCP-1 in the aorta of rabbits. Besides, Fenofibrate was designed as a positive control in our study.
As a commonly used and successful atherosclerosis animal model, New Zealand White rabbits were chosen in our experiment. The lipoprotein metabolism and cardiovascular system of rabbits are much similar to those of humans, and they have been proved as the excellent model for human atherosclerosis [17, 18].
Materials and methods
Animals and experimental procedures
This study was approved by the Animal Ethics Committee, Chongqing Medical University. Thirty male New Zealand White rabbits with average body weight between 2.0± 0.5 kg were purchased from Experimental Animal Center, Chongqing Medical University (Grade II, Certificated SCXKYu 2012–0001). High-cholesterol diet (normal diet supplemented with 1% cholesterol, 10% yolk powder, 5% animal oil) was supplied by Experimental Animal Center, Chongqing Medical University. The animals were housed separately in cages under standard animal house conditions with a 12 h light /dark cycle at the temperature of 25°C. All animals had free access to water.
At the end of the tenth week, all animals were fasted overnight and sacrificed by intravenous injection of pentobarbital (Laboratories, Urchem Ltd., Shanghai,China, 60 mg/kg body weight). Immediately after opening the chest, the aorta was excised, then dissected longitudinally and separated into three portions. The first portion, a one-centimeter segment between the first and the second intercostals artery was fixed in 10% neutral buffered formalin for 1 day and paraffin-embedded; and it was used for histological and immunohistochemical analysis. The second portion, a segment between the second and the third intercostals artery, was snap-frozen in liquid nitrogen and stored at −70°C until being used for PCR. The third portion, a segment between the third and the seventh intercostals artery was stained with oil red O (sigma, USA) for 30 minutes, then transferred to 70% alcohol for differentiation. 15 min later, it was washed and taken photo with a digital camera (Olympas, Janpan). The percentage of oil red O-positive area to the internal surface was measured with a computer-assisted morphometry system (Image Pro Plus 6.0, USA).
Weight and serum lipids determination
At the end of the sixth and tenth week, all the rabbits were weighted and 2.0 mL blood was taken via the marginal ear vein from each rabbit and centrifuged at 8,000 g for 10 min. Obtained serum was analyzed in an automatic blood chemical analyzer (Beckman Instruments, USA) and the serum concentrations of total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were determined.
Evaluation of serum TNF-α and IL-1β
At the end of tenth week, TNF-α and IL-1β concentrations in the collected serum were detected with commercially available kits (Rapidbio Lab Co., California, USA) under the manufacturer’s instructions by the method of Enzyme-linked immunosorbent assay (ELISA). Values of samples were calculated from a standard curve generated from standards of known concentrations. Absorbance at 450 nm was detected using microplate reader (Bio-Rad, USA).
Evaluation of serum SOD and MDA
After six and ten weeks, SOD and MDA levels in the collected serum of rabbits were detected by colorimetric method of xanthine oxidase and thiobarbituric acid with kits (Nanjingjiancheng Bioengineering Institute, Nanjing, China) respectively. The absorbance was measured with UV–vis spectrophotometer (Shimadzu,Japan) at the wavelength of 550 nm and 532 nm respectively, according to the instructions of the kits.
Histological and immunohistochemical analysis
The first aorta portion, the paraffin-embedded segment between the first and the second intercostals artery, was cut into five non-consecutive serials of 5-μm-thick sections. The first serial was stained with hematoxylin and eosin for observation under light microscopy (Olympas, Japan). The other four serials of sections were incubated with primary antibodies to E-sel (Bioss, Beijing, China; 1:100 dilution), ICAM-1 (Bioss, Beijing, China; 1:100 dilution), VCAM-1 (Abcam Inc., Cambridge, USA; 1:100 dilution) or MCP-1 (Bioss, Beijing, China;1:100 dilution) respectively. After washing, specific biotinylated secondary antibodies (biotinylated goat anti-mouse IgG for VCAM-1, and biotinylated goat anti-rabbit IgG for E-sel, ICAM-1 and MCP-1) were applied, followed by avidin-biotin peroxidase complexes (Histostain™-Plus Kits, Beijing Zhongshan Goldenbridge Company, China). Antibody binding was visualized with 3, 3-diaminobenzidin (DAB). Sections were counterstained with hematoxylin. After scanned to computer, percentage of immunoreactive cells and staining intensity of each section were measured with use of a computerized image analysis program (Image-Pro Plus 6.0, USA).
For the immunohistochemical semiquantitative assessment of E-sel, ICAM-1, VCAM-1 and MCP-1expression, immunohistochemical score (HIS, ranging from 0–12) was applied. It is calculated by multiplication of the percentage of immunoreactive cells (quantity score) with the staining intensity (staining intensity score). Quantity score was calculated based on the following scoring system: no staining is scored as 0, 1–10% of cells stained scored as 1, 11–50% as 2, 51–80% as 3, and 81–100% as 4. Staining intensity is rated on a scale of 0 to 3, with 0 = negative; 1 = weak; 2 = moderate, and 3 = strong.
Determination of E-sel, ICAM-1, VCAM-1 and MCP-1 gene expression
Primers sequence of target genes
Gene bank accession no.
Annealing temperature (°C)
5′ -TTCCAGCCCTCCTTCCT- 3′
5′ -GCCCGACTCGTCATACT- 3′
5′ - AATGGCAGATACAGAGAACT- 3′
5′ -TGGCTTGGAAGAGAATAACT- 3′
5′ -GACATTCTTGAACAGTGACAG- 3′
5′ -CGGACACAGCTCTCAGTA- 3′
5′ -GGAGACACTGTCATTATCTCCTG- 3′
5′ -TCCTTTCATGTTGGCTTTTCTTGC- 3′
5′ -GGTGTAAAGGCAGGTGTG- 3′
5′ -AGGATAGGAAAGGATGGG- 3′
Results were expressed as mean±SD. Statistic analysis was carried out using the SPSS statistical package version 10.0 (SPSS Inc., USA). Student’s t-test was performed to compare means between two groups. The p value<0.05 or 0.01 was considered to be statistically significant.
Body weight and serum lipids analysis
Changes of body weight and serum lipid levels after six and ten weeks in different groups (mean± SD, n=6)
2.49 ± 0.22
2.82 ± 0.36
2.65 ± 0.24
2.68 ± 0.28
2.66 ± 0.25
2.93 ± 0.15
3.24 ± 0.40
3.15 ± 0.33
3.10 ± 0.13
3.09 ± 0.24
1.13 ± 0.33
6.01 ± 2.48##
2.43 ± 0.57**
1.52 ± 0.24**
2.74 ± 0.83**
1.11 ± 0.24
9.36 ± 3.96##
4.26 ± 1.44**
2.97 ± 1.41**
3.70 ± 1.14**
0.58 ± 0.16
1.80 ± 0.47##
1.09 ± 0.32**
0.85 ± 0.10**
0.88 ± 0.27**
0.62 ± 0.1
2.26 ± 0.82##
0.88 ± 0.14**
1.39 ± 0.63*
1.30 ± 0.42*
0.65 ± 0.09
1.92 ± 0.57##
1.04 ± 0.24**
0.83 ± 0.16**
1.04 ± 0.21**
0.68 ± 0.14
2.51 ± 0.74##
1.76 ± 0.67*
1.29 ± 0.57**
1.64 ± 0.37*
0.44 ± 0.13
3.21 ± 1.85##
1.07 ± 0.12**
0.62 ± 0.20**
1.24 ± 0.48*
0.46 ± 0.13
3.79 ± 2.20##
1.76 ± 0.50**
1.07 ± 0.61**
1.69 ± 0.86*
Serum inflammatory factors analysis
Changes of inflammatory factors in serum (mean± SD, n=6)
59.18 ± 11.23
14.46 ± 1.91
143.93 ± 45.64##
27.66 ± 8.11#
92.82 ± 24.21*
17.16 ± 7.38*
80.84 ± 29.69*
19.29 ± 3.12*
83.76 ± 21.34*
20.02 ± 5.25*
Serum antioxidation analysis
Changes of serum SOD and MDA after six and ten weeks in different groups (mean± SD, n=6)
Morphologic and histologic studies
Gene expression of E-sel, ICAM-1, VCAM-1 and MCP-1
According to several epidemiological and experimental studies, natural or synthetic products having an anti-inflammatory action have been proven to have a strong preventive effect on the development of atherosclerosis [19, 20]. To investigate the potential anti-inflammatory activity of kaempferol as a new anti-atherogenic agent, the present study was designed to evaluate the role of kaempferol on the inflammatory progression of early atherosclerosis in rabbits. After ten weeks of kaempferol treatment together with high-cholesterol diet, blood lipids and arterial lesions of rabbits were significantly deduced compared to the untreated group. Levels of serum IL-1β and TNF-a dropped, mRNA and protein expression of E-sel, VCAM-1, ICAM-1 and MCP-1 in the aorta of rabbits were respectively down-regulated by the treatment of kaempferol, indicating that kaempferol can prevent atherosclerosis by alleviating vascular inflammation.
Selectin belongs to one kinds of adhesion molecules. Selectin family is divided into three categories: E-selectin, P-selectin and L-selectin. They play an important role in vascular inflammation, thrombus and plaques formation, atherosclerosis, vascular vasodilation, metabolism of vascular endothelial cells. E-selectin (E-sel) is a cytokine select selectin and mainly located in the capillaries and endothelial cell membrane of venules. Expression of E-sel on non-activated endothelial cells is minimal, but it rise up greatly when stimulated by inflammatory cytokines like IL-1β, TNF-α and lipopolysaccharide, and this change is induced at the transcriptional level . ICAM-1 and VCAM-1 are two frequently studied adhesion molecules that are cytokine-inducible members of immunoglobulin gene superfamily, they are highly expressed by arterial endothelial cells at the atherosclerotic lesions . It is well established that E-sel, VCAM-1, ICAM-1 and MCP-1are key molecules involved in the initiation of inflammation pathogenesis of atherosclerosis, they collaboratively promote the adhesion of monocytes and then the migration to endothelium. Previous reports found that the expression of VCAM-1 and ICAM-1are up-regulated by a high cholesterol diet [22, 23]. The high expression of E-sel, ICAM-1 and VCAM-1 were also detected mainly in human atherosclerotic plaques . Levels of soluble E-sel, ICAM-1 and VCAM-1 in blood plasma were significantly higher in patients with unstable angina, acute coronary syndrome and atherosclerosis. These adhesion molecules are becoming potent clinical biomarkers of CVD . Monocyte chemoattractant protein-1 (MCP-1) is thought to play a central role in filtration of monocyte /macrophage to the arterial wall and the recruitment of these cells . It has been demonstrated that MCP-1 expression occurs in the arterial wall in response to hypercholesterolemia in rabbits and MCP-1 is highly expressed in the macrophage -rich area of the atherosclerotic lesions in human and animal models . Increases in MCP-1 levels in serum have also been observed in patients with acute coronary syndromes and expression of vascular MCP-1 can be used to predict atherosclerosis . Also, it was revealed that TNF-α, IL-1β as well as oxidized low density lipoprotein (LDL) were able to induce local vascular cells to produce MCP-1, which causes monocyte recruitment and promotes the release of lipids, thereby enhancing the progression of the atherosclerotic lesion [28, 29]. Another report found that the inhibition of MCP-1 expression by gene targeting could reduce atherogenesis in LDL receptor deficient mice .
According to some studies, flavonoids including phloretin , apigenin  and quercetin [33, 34] can inhibit E-sel, ICAM-1, VCAM-1 or MCP-1 expression in vitro and in vivo. In our study, mRNA and protein expressions of E-sel, ICAM-1, VCAM-1 and MCP-1 in aorta of rabbits were all up-regulated by a high-cholesterol diet and expressions of those three molecules were all down-regulated by kaempferol (30 mg/kg and 150 mg/kg). Therefore, similar to the other flavonoids mentioned above, kaempferol also showed a down-regulation effect on E-sel, ICAM-1, VCAM-1 or MCP-1 expression in vivo.
On the initiation of inflammation, inflammatory stimuli leads to increased secretion of pro-inflammatory cytokines such as TNF-α and IL-1β, which can significantly induce oxidation of LDL and promote aggregation of oxidized LDL and macrophages, thereby lead to the formation of foam cells, increase the binding of LDL on endothelial cells and the expression of LDL receptor of endothelial cells. Subsequently nuclear transcription factor-kappa B (NF-κB) pathway is activated in response to pro-inflammatory cytokines. This pathway plays an essential role in inflammation through the regulation of genes encoding pro-inflammatory cytokines, adhesion molecules, chemokines and growth factors. The activation of these genes will stimulating the migration and proliferation of smooth muscle cells, promoting the development of atherosclerotic plaques . Studies demonstrated the activation of gene expression of endothelial cell adhesion molecules including E-sel, ICAM-1 and VCAM-1 by IL-1β or TNF-α in endothelial cells is mediated by NF-κB pathway . Increased IL-1β protein  as well as TNF-α mRNA and protein expression  are found in injured arterial smooth muscle cells, and the inhibition of TNF-α treatment can accelerate endothelial recovery after injury . Besides, the disruption of TNF-α gene diminishes the development of atherosclerosis in ApoE-deficient mice . A resent study demonstrated that an antibody targeting IL-1β can inhibit the progression of atherosclerosis in vivo, highlighting the importance of this key cytokine in cardiovascular disease . Therefore, IL-1β and TNF-α are two important pro-inflammatory cytokines in the initiation and development of atherosclerosis.
Several studies on the anti-inflammation effect of kaempferol in vitro have also shown that kaempferol can down-regulate TNF-α and IL-1β expression [42, 43]. In our study, which centering on the in vivo anti-inflammatory activity of kaempferol, we demonstrated that kaempferol can inhibit inflammatory cell adhesion, migration and block the initiating processes of inflammatory response. The effect may be medicated by reducing the secretion and release of inflammatory mediators such as TNF-α and IL-1β and decreasing the expression of the E-sel, ICAM-1, VCAM-1 as well as MCP-1 in the aorta of rabbits. And both high and low dosage of kaempferol (150 mg/kg and 30 mg/kg) have a strong effect both on the gene and protein expression, but the effect was not dosage related, for there were no statistic difference between the two groups. Maybe the treatment dosages designed in our study were not in the linear efficacy range of kaempferol. In addition, the serum TNF-α and IL-1β levels were increased in atherosclerosis rabbits, and kaempferol groups showed lower TNF-α and IL-1β levels, indicating that the down-regulation of E-sel, ICAM-1, VCAM-1 and MCP-1 might be mediated by the reducing of TNF-α and IL-1β.
Besides, our results suggested that kaempferol have anti-oxidant effect on high cholesterol fed rabbits, for that increased SOD and reduced MDA were observed six and ten weeks post kaempferol administration. It has been known that free radicals is highly related with caediovascular disease and antioxidants including many natural agents can protect the developing of CVD [44, 45]. According to some studies, kaempferol is a potent and powerful superoxide scavenger . The ability of kaempferol to decrease superoxide levels by inhibiting the activity of enzymes that generate reactive oxygen species (ROS), such as the enzyme xanthine oxidase, may play a crucial role in its antioxidant activity . Many reports have shown that kaempferol and several kaempferol-containing plants have antioxidant activity not only in vitro, but also in vivo [48, 49]. Several epidemiological studies reported that fruit and vegetables rich in kaempferol can prevent cardiovascular diseases, and intake of them reduces the risk of coronary heart disease. The antioxidant effect of kaempferol observed in our experiment may be mediated by protecting LDL from oxidation, which is also one of the early processes involved in atherosclerosis .
In summary, the present study suggested that six and ten weeks of treatment of kaempferol (30 mg/kg, 150 mg/kg) effectively prevent atherosclerosis induced by high cholesterol in New Zealand White rabbits. The anti-atherogenic effect possibly works by improving antioxidant ability, reducing the release of TNF-α and IL-1β and down-regulation the gene and protein expression of pro-atherogenic molecules, such as E-sel, ICAM-1, VCAM-1 and MCP-1. Further studies related to the anti-inflammatory actions of keampferol may needed to provide more evidences for its anti-atherosclerotic effect.
Intercellular adhesion molecule-1
Vascular cell adhesionmolecule-1
Monocyte chemotactic protein-1
Reverse transcription-polymerase chain reaction
Low density lipoprotein
High density lipoprotein cholesterol
Low density lipoprotein cholesterol.
This study was supported by the Foundation of Chongqing Public Health Bureau (2009-1-6).
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