The research tries to establish Wistar rat’s model of atherosclerosis for evaluating the antiatherosclerotic effect of hederagenin and exploring its antiatherosclerosis-related mechanisms. The statistical data have shown that hederagenin exhibits multiple pharmacological activities in the treatment of hyperlipidemia, antiplatelet aggregation, liver protection, and anti-inflammation, indicating that hederagenin may exert a protective effect on vascular walls by improving lipid metabolism disorders and lipid deposition. The results show that hederagenin can correct the imbalance of endothelial function by inhibiting the release of large amounts of iNOS and increasing eNOS contents and inhibits the IKK
Atherosclerosis (AS) is an inflammatory disease caused by the lesion-like deposition of lipids (primarily cholesterol and cholesterol ester), carbohydrates, and blood components on the vasculature intima or under the intima of the artery and its branches, in addition to the deposition of connective tissues and calcium. AS is accompanied by the migration of medial smooth muscle cells into the intima and the proliferation of these cells, causing intimal thickening and the formation of AS lesions or fibrous fatty plaque lesions. An inflammatory response is always present in AS. Deaths from cardiac and cerebrovascular accidents caused by AS account for the highest disease mortality in humans. Therefore, AS is referred to as the “number one killer” in western countries, and it is one of the most severe cardiovascular diseases that threaten human health. Hence, strategies for achieving early diagnosis and effective interventions for this disease are urgently required. Many studies have been conducted on the prevention and treatment of AS, both domestically and abroad. Most of these studies have investigated the pathogenesis of AS and attempted to delay the progression of the pathological changes associated with AS through drug interventions. Currently, hyperlipidemia is considered the primary factor involved in the occurrence and development of AS. Good control of blood lipids can significantly slow the progression of AS lesions and reduce morbidity and mortality associated with AS-related cardiovascular diseases. There have been drug studies on the prevention and treatment of atherosclerosis conducted to date. Statins can contribute to the prevention of atherosclerosis, but the liver damage they cause after long-term administration is drawing increasing attention [
Consider the following: hederagenin (purity 95%), Nanjing Spring and Autumn Biological Engineering Co., Ltd, batch number: 20131020; vitamin D3, specifications: 1 mL: 7.5 mg, Shanghai General Pharmaceutical Co., Ltd., batch number: 121123; atorvastatin calcium (Lipitor), specifications: 20 mg
Male, Specific Pathogen-Free- (SPF-) level Wistar rats with weights of 160–200 g, aged 5~6 weeks (provided by the Experimental Animal Center of Southern Medical University, license number: SCKK2011-0015, animal certificate number: 44002100002027), were used in this study. The animals were fed in animal experimental center of Jinan University SPF animal housing management and given free access to water and food; the feeding room temperature was set at 23°C–25°C, and the relative humidity was approximately 7%. After one week of feeding adaptation, the experiments were initiated. The high-fat diet was composed of 3% cholesterol, 0.5% sodium cholate, 0.2% propylthiouracil, 5% sugar, 10% lard, and 81.3% basic fodder, which were mixed well and irradiated with cobalt-60 (radiation dose 25.0 kGy) before feeding.
After one week of adaptive feeding with basic rat fodder, 40 quarantined Wistar rats were selected and weighed. The rats were randomly divided into four groups according to a random number table. These groups included a normal group, model group, atorvastatin calcium (Lipitor) group and hederagenin group, with 10 rats in each group. With the exception of the normal group, the rats in all other groups were administered vitamin D3 at 600,000 IU/kg/d via intraperitoneal injection, and they were also fed continuously with the high-fat diet. Furthermore, an additional 100,000 IU/kg of vitamin D3 was administered to these rats via intraperitoneal injection in the 3rd, 6th, and 9th weeks of the experiments. The rats in the normal group were administered saline through intraperitoneal injection and were fed with normal fodder.
At the end of 12 weeks, arteries were collected from the rats to perform HE staining and observe pathological changes under a light microscope and electron microscope. A Zeiss-Axioskop 20 microscope was used to observe the histological changes in HE-stained sections, and an Axiocan HRc camera was employed to obtain micrographs. Leica Qwin Image Processing and Analysis Software was used to analyze AS lesions and the cross-sectional area of the artery lumen and to calculate the relative area of atherosclerosis lesions (the ratio of the lesion area versus the lumen cross-sectional area), which is represented as a percentage (%). Blood lipids, liver lipids, blood rheology, inflammatory factors, and the gene expression and protein expression of eNOS, iNOS, IKK
The experimental data were analyzed using Social Sciences (SPSS, USA) 16.0 statistical software. Measured data were represented as the mean ± standard deviation (SD), and comparisons of the means between two groups were carried out with one-way ANOVA. When the variance was homogeneous, the SNK test and Tukey test were applied, whereas when the variance was not homogeneous, the T2 test was used and probability value (
Histological changes of aorta morphology in different groups (HE stain
Comparison of relative AS lesion areas in the rat aorta in each group.
Electron microscopy observations of the rat aorta (12500x). (a) Normal control group. (b) Model group. (c) Lipitor group. (d) Hederagenin group.
Comparison of rat serum TC, TG, HDL-C, and LDL-C contents in each group.
Comparison of rat serum ALT and AST contents.
Comparison of rat hemorheological parameters in each group.
Group | Whole-blood viscosity/mpa·s | Hematocrit | Erythrocyte aggregation index | Platelet aggregation rate/% | |||
---|---|---|---|---|---|---|---|
Low shear | Mid shear | High shear | Viscosity | ||||
Normal | 10.76 ± 0.83 | 7.15 ± 0.72 | 5.18 ± 0.42 | 1.28 ± 0.12 | 0.41 ± 0.04 | 5.12 ± 0.33 | 39.22 ± 3.69 |
Model | 19.23 ± 2.51a | 9.66 ± 0.89a | 7.33 ± 0.76a | 1.81 ± 0.23a | 0.69 ± 0.12a | 6.51 ± 0.87a | 59.03 ± 5.17a |
Lipitor | 12.64 ± 0.97b | 7.32 ± 0.83b | 6.26 ± 0.58b | 1.32 ± 0.15b | 0.52 ± 0.05b | 5.28 ± 0.39b | 46.61 ± 4.09b |
Hederagenin | 12.58 ± 0.89b | 7.27 ± 0.71b | 5.66 ± 0.53bc | 1.37 ± 0.14b | 0.53 ± 0.03b | 5.37 ± 0.45b | 46.38 ± 4.19b |
Comparison of rat serum NO and ET-1 contents.
Comparison of rat aortic iNOS and eNOS protein expression levels.
Contents of the inflammatory cytokines IL-6, IFN-
Relative expression level of IKK
The expression levels of NF-
Atherosclerosis (AS) is a complicated pathological process resulting from interactions between various pathways and factors. The theory of AS pathogenesis primarily involves thrombosis theory, lipid filtration theory, endothelial dysfunction theory, oxidation stress theory, and inflammatory response theory. These theories correspondingly explain different aspects of the pathogenesis and progression of atherosclerosis. An increasing number of studies have shown that atherosclerosis is an inflammatory disease, and the inflammatory response is present throughout the process of the pathogenesis and progression of AS. During the early stage of atherosclerosis, when unstable plaques break, there are continuous activation and amplification of inflammation. Therefore, the early identification of unstable AS plaques, detecting sensitive and specific serological markers of these plaques and associated inflammation targets, in addition to reducing and blocking atherosclerosis and other vascular events through early anti-inflammation treatment, represent current research hot spots that will continue to direct future research.
Hyperlipidemia and hemorheological abnormalities usually occur at the same time and promote each other’s occurrence. Epidemiological studies have shown that, as an initiation factor of atherosclerosis, hyperlipidemia, accompanied by hemorheological abnormalities, is generally a risk factor for atherosclerosis. Studies have demonstrated that an increase of serum cholesterol levels is positively correlated with the occurrence of AS and can lead to abnormal plasma lipoproteins, thereby inducing artery wall lesions. Lipoproteins are the major form of lipids that exists in human plasma. Low density lipoprotein (LDL) plays an important role in the pathogenesis of atherosclerosis and is considered the major risk factor for atherosclerosis. Drug intervention experiments confirmed that lowering LDL levels can significantly reduce the risk of the occurrence of cardiovascular diseases for hypercholesterolemia patients and can also benefit patients with a normal level of LDL [
AS is an inflammatory disease, and the inflammatory response is observed throughout the process of the occurrence and progression of AS [
Thus, in the present study, we explored and evaluated the antiatherosclerosis function of hederagenin by establishing a Wistar rat AS model, and we analyzed the antiatherosclerosis mechanism of hederagenin from the perspective of lipid metabolism disorders, liver function, blood rheology, endothelial function, and inflammation signaling pathways. Studies have shown that, in AS rat models induced by a high-lipid diet plus VD3, hederagenin can effectively reduce serum lipid, ALT, and AST levels, in addition to improving liver function, relieving high blood coagulation, and slowing blood flow and stasis by improving blood rheology. Hederagenin can correct the imbalance of endothelial function by inhibiting the release of large amounts of iNOS and increasing eNOS contents. Hederagenin also inhibits the IKK
In conclusion, the experimental results showed that hederagenin can inhibit or ameliorate the pathological changes associated with AS, displaying an excellent preventive function against AS. The mechanism of hederagenin action may be related to the regulation of lipid metabolism disorders, protection of liver function, improvement of blood rheology, regulation of endothelial dysfunction, and inhibition of the IKK
All of the authors of this paper declare that they have no direct financial relation with the commercial identities mentioned in this paper. And all of the authors declare that they have no competing interests.
The present work was supported by a grant (no. 81173189) from Natural Science Foundation of China.