Astragaloside IV (AsIV) is the major effective component extracted from the Chinese herb
Huang qi (
Studies have suggested that platelets play a vital role in inflammatory reactions. Resting platelets can be activated when they are exposed to stimulants, damaged vessel wall, or extravascular tissue, accompanied by change in platelet shape and ability of adhesion, as well as aggregation. Platelet activation is an ordered sequence of events which begins with the interaction between the adhesive protein (agonist) and its receptor; the activation of platelets leads to secretion of a variety of mediators which can lead to progress of atherosclerosis, such as cytokines, chemokines, growth factors, adhesion molecules, and coagulation factors [
There are markers that can be used for platelet activation, such as integrin
In the present study, we measured the level of biomarkers of platelet activation, PAC-1, CD40L, and CXCR4 in platelet-rich plasma, so as to investigate the effect of AsIV on platelet activation. We also examined the expression of SDF-1/CXCR4 biological axis in apoE−/− mice. The effects of astragaloside IV on atherosclerosis in high-fat diet apoE−/− mice may be through platelet activation and SDF-1/CXCR4 biological axis.
30 healthy male apoE−/− mice at 8 weeks old were provided by Model Animal Research Center of Nanjing University, specific pathogen-free grade, weighing
Astragaloside IV (purity > 98%, CAS: 84687-43-4) was purchased from Nanjing Spring & Autumn Biological Engineering Co. (Nanjing, China). AMD3100 octahydrochloride hydrate (A5602-5MG, P-code: 1001580646–1001580659) was from Sigma-Aldrich Co. LLC (St. Louis, MO, USA).
Immunohistochemical kit was purchased from Beijing Zhongshan Reagent Co. Ltd. Kit of SABC (SA2002) and kit of DAB chromogenic (AR1022) were purchased from Wuhan Boster Company (Wuhan, China).
Mice in blank control group were given normal diet. All apoE−/− mice were given high-fat western diet whole diet (21% fat, cholesterol, 0.15%) till 2 weeks old, three of them were chosen randomly to be sacrificed in order to observe the success of modeling. apoE−/− mice were randomly divided into three groups: model group, AMD3100 groups, and AsIV group; C57BL/6 mice were used as the control group.
Mice in AsIV group were given 40 mg/kg·d−1 astragaloside by oral gavage. Mice in AMD3100 group were given 2.5 mg/kg·2d−1 AMD3100 by intraperitoneal injection. Mice of model group and the normal group were given 0.9% sodium chloride solution by oral gavage. Mice of each group continued to be fed following the original feeding, within all 12 weeks.
After 12 weeks of drug administration, mice were fasted for 12 hours, and venous blood was collected through the eyeball under sterile condition. Then open the chest and abdominal cavity quickly, and peel total length of the aorta along the aorta valve to the iliac artery branch.
After 30-minute standing, venous blood was obtained and centrifuged at 3000 rpm for 15 min. Serum was isolated. Total cholesterol [
Thoracic aorta was isolated, fixed, and embedded in paraffin for histopathological analysis. Hematoxylin and eosin (H&E) staining was performed. Imaging from aorta tissues was detected with microscope. Detection using Image Pro Plus: luminal area (LA), intimal thickness (IMT), plaque area (PA), fibromuscular component (FS), a lipid center area (CA), and minimum fibrous cap thickness (FCT).
Platelet-rich plasma was prepared from flesh blood of the mice by density gradient centrifugation as described by Hoffman’s report [
Immunohistochemical staining was performed as described [
Bone marrow-derived EPC were isolated from femurs of apoE−/− mouse bone marrow (Bm) progenitor cells and cultured in endothelial basal medium-2 (EBM-2) supplemented with growth factors (EBM-2; Lonza, catalogue number CC-3156) and 10% FCS as previously reported [
Total RNA of bone marrow-derived endothelial progenitor cells and aorta tissue were extracted with the TRIzol reagent (Invitrogen Corporation, California, USA) reverse transcription system (TaKaRa Biotechnology Ltd., Shandong, China). Real-time PCR was performed using SYBR Green SuperMix with an iCycler thermal cycler (Bio-Rad Laboratories Inc., California, USA). Primer sequences of SDF-1 and CXCR4 are listed (SDF-1: F: 5′-CCTGTGTGTCATGCCCTCTT-3′ and R: 5′-AGTCCAGCCTGCTATCCTCA-3′; CXCR4: F: 5′-GTCAACCTCTACAGCAGCGT-3′ and R: 5′-CTATCGGGGTAAAGGCGGTC-3′). The data were collected and analyzed using the comparative Ct (threshold cycle) method. GADPH RNA was used as internal control.
Proteins of bone marrow-derived endothelial progenitor cells and aorta tissue were extracted and separated in SDS-PAGE gels, and western blot analyses were performed according to standard procedures as previously described [
Data were denoted by mean value ± standard deviation (
The results of TC, TG, HDL-C, and LDL-C levels of mouse in model group, AMD3100 groups, and AsIV group were shown in Table
Comparison of blood lipid (mmol/L,
Group | TC | TAG | LDL-C | HDL-C |
---|---|---|---|---|
Control group | 4.16 ± 1.59 | 1.47 ± 0.18 | 1.11 ± 0.23 | 2.59 ± 0.12 |
Model group | 16.12 ± 0.95∗ | 12.75 ± 2.65∗ | 6.48 ± 0.81∗ | 1.12 ± 0.13∗ |
AMD3100 group | 16.10 ± 0.93 | 12.64 ± 2.25 | 6.28 ± 0.91 | 1.13 ± 0.13 |
AsIV group | 10.96 ± 1.32△▲ | 4.78 ± 0.86△▲ | 4.30 ± 0.76△▲ | 2.90 ± 0.39△▲ |
Note: compared with control group:
To assess the extent of atherosclerosis in thoracic aorta of high-fat diet apoE−/− mice after AMD3100 or AsIV treatment, aorta cross-section pathological damage was detected by HE staining. Histopathological specific data analysis (Tables
Comparison of LA, IMT, and PA (
Group | LA (mm2) | IMT ( |
PA (mm2) |
---|---|---|---|
Model group | 0.255 ± 0.007 | 0.159 ± 0.047 | 0.158 ± 0.016 |
AMD3100 group | 0.447 ± 0.042△ | 0.074 ± 0.013△ | 0.076 ± 0.018△ |
AsIV group | 0.532 ± 0.017△▲ | 0.028 ± 0.011△▲ | 0.012 ± 0.013△▲ |
Note: compared with model group: △
Comparison of FS, CA, and FCT (
Group | FS (mm2) | CA (mm2) | FCT ( |
---|---|---|---|
Model group | 0.065 ± 0.002 | 0.093 ± 0.012 | 13.724 ± 0.443 |
AMD3100 group | 0.031 ± 0.005△ | 0.041 ± 0.011△ | 4.136 ± 0.672△ |
AsIV group | 0.002 ± 0.003△▲ | 0.013 ± 0.012△▲ | 2.306 ± 0.558△▲ |
Note: compared with model group: △
Comparison of PA/LA, CA/PA, and CA/FS (
Group | PA/LA | CA/PA | CA/FS |
---|---|---|---|
Model group | 2.718 ± 0.386 | 0.581 ± 0.150 | 1.493 ± 0.290 |
AMD3100 group | 0.690 ± 0.130△ | 0.776 ± 0.070△ | 2.516 ± 0.317△ |
AsIV group | 0.041 ± 0.023△▲ | 0.797 ± 0.116△▲ | 3.546 ± 0.913△▲ |
Note: compared with model group: △
Hematoxylin and eosin stained histological sections. Note: C: control group; M: model group; A: AMD3100 group; Z: AsIV group (magnification ×200).
To investigate the effect of AsIV on the activation of platelet, biomarkers of platelet activation were measured by flow cytometry. Results showed that expression of PAC-1, CD40L, and CXCR4 was significantly higher in the model group than in the control group (
PAC-1, CD40L, and CXCR4 expression of platelet surface. Note: C: control group; M: model group; A: AMD3100 group; Z: AsIV group. Compared with control group: ∗
Immunohistochemical staining was applied to investigate the effect of AsIV on SDF-1/CXCR4 biological axis in aorta wall of the high-fat diet apoE−/− mice. Figure
(a) The expression of SDF-1 and CXCR4 (optical density value). Compared with control group: ∗
We further examined the expression of SDF-1 and CXCR4 in mRNA level and protein level in aorta by quantitative PCR and western blotting analysis. Quantitative real-time PCR demonstrated that mRNA level of SDF-1 and CXCR4 was significantly lower in AMD3100 group and AsIV group than in model group (Figure
(a) The expression of SDF-1, CXCR4 mRNA. (b, c) The expression of SDF-1, CXCR4 protein. Note: C: control group; M: model group; A: AMD3100 group; Z: AsIV group. Compared with control group: ∗
Moreover, western blotting demonstrated that proteins level of SDF-1 and CXCR4 was significantly lower in AMD3100 group and AsIV group than in model group. However, compared with the AMD3100 group, the proteins levels of SDF-1 and CXCR4 were higher in the AsIV group, but the difference was not statistically significant (
To further verify the effect of AsIV SDF-1/CXCR4 biological axis associated with atherosclerosis, bone marrow-derived EPC were isolated and cultured (Figure
Identification of bone marrow-derived EPC (magnification ×200, VEGF-R positive cells, white shot; PE labeled VEGF-R antibodies stimulate green shoot).
The expression CXCR4 on EPC. Note: C: control group; M: model group; A: AMD3100 group; Z: AsIV group. Compared with control group: ∗
The pathology of atherosclerosis development is a comprehensive long-term process. Due to its complex pathogenesis, there is no existing preventive and control strategy. “Injury-response theory” and the “inflammatory reaction theory” are the most popular explanation theories [
Targeting the key component in the atherosclerosis inflammatory response network can interrupt the formation of atherosclerosis and reduce the degree of injury. Recently, an increasing number of reports show that Chinese medicine has extraordinary effects on the treatment of atherosclerosis, which gradually draw people’s attention to Chinese medicine. Huang qi (
Recently, the role of CD40/CD40 ligand (CD40L) interactions in atherothrombosis, in the response of the immune system to pathogens and in thrombosis is now widely accepted. CD40-CD40L interactions have been identified in atherosclerosis, and such interactions can destabilize atherosclerotic plaques by inducing the expression of cytokines, chemokines, growth factors, matrix metalloproteinases, and procoagulant factors. Many literatures report that activated platelets can lead to overexpression of SDF-1; moreover, conglutination between SDF-1 and chemokine receptor CXCR4 can regulate cell migration, tissue targeting, and homing [
A latest study shows that atherosclerosis was related to SDF-1/CXCR4 biological axis. SDF-1, also known as CXCL12, belonged to CXC subfamily of chemokines. It was first discovered in cytokines secreted by mouse bone marrow stromal cells. After platelet activation, SDF-1 can be abundantly expressed and combined with chemokine receptors, CXCR4. SDF-1/CXCR4 biological axis consisting of SDF-1 and its receptor CXCR4 induced CD34+ stem cells to differentiate into macrophages and foam cells, which later caused the atherosclerosis. CXCR4 is widely expressed on surfaces of hematopoietic stem cells and bone marrow stromal cells; it is also expressed in AS lesions. Interaction of SDF-1 and CXCR4 control cell migration and issue-selective homing, which cause blood cells adhere to endothelial cells more conducive. Treating endothelium with chemokines suggests that SDF-1/CXCR4 axis plays an important role in the development of atherosclerosis [
apoE knockout (apoE−/−) mice were used as the animal model in our study. apoE−/− mice fed by high-fat diet had the lesion characteristics of serious dyslipidemia and atherosclerosis, which were relatively good animal model for the research on atherosclerosis. In this research, C57BL mice had the characteristics of short life time and generation time, which were a kind of experimental animal commonly used in geriatric medicine.
In our research, we observed the effects of AsIV on blood lipids, CD40-CD40L signal system, and SDF-1/CXCR4 biological axis in high-fat diet apoE−/− mice; the research results showed that TG, TC, HDL-C, and LDL-C levels in AsIV group were significantly better than those in model group (
By above results, we can clearly know that platelet activation can induce SDF-1/CXCR4 biological axis imbalance. The protective effects of AsIV in atherosclerosis injury may be related to AsIV downregulation of CD40L, PAC-1, and CXCR4 expression by blocking the CD40-CD40L system. In addition, AsIV significantly down-regulated mRNA and protein level of SDF-1, CXCR4 in thoracic aorta. SDF-1/CXCR4 biological axis is probably one of the main targets of intervening atherosclerosis. Therefore, astragaloside IV plays a role in atherosclerosis of high-fat diet apoE−/− mice by regulating blood lipids, CD40-CD40L system, and SDF-1/CXCR4 biological axis probably. The research provides new approach for treatment of atherosclerosis and related diseases.
The authors declare that they have no conflict of interests.
The study was financially supported by 2012 Project of Shanghai outstanding academic leaders (no. 12XD1404700), Doctoral Foundation of Ministry of Education (no. 20133107110003), and Shanghai Advanced Personnel Training Project of Combined Traditional Chinese and Western Medicine (no. ZYSNXD012-RC-ZXY008).