Yindanxinnaotong, a Chinese compound medicine, synergistically attenuates atherosclerosis progress

YD regulates the lipid profile in atherosclerotic rats

Comparing with normal-controlled group, cholesterol and TGs increased significantly in AS group; YD reduced such elevation (Table 1). In atherosclerotic rats, YD reduced serum concentrations of total cholesterol, LDL, and TGs. Thus, YD treatment mediated the lipid profile, especially reducing TGs, cholesterol, and LDL. Our data suggest that YD improves the serum lipid profile that associates with the pathogenesis of atherosclerosis. As expected, atorvastatin improved the serum lipid profile as well¹⁶.

YD elicits antioxidant action in vivo

Table 2 shows the parameters of redox behavior in rats. Compared to AS group, YD significantly (P < 0.05) modified oxidative stress markers after 12 weeks. Malondialdehyde (MDA), an end product of lipid peroxidation, increased significantly in atherosclerotic versus non-atherosclerotic controls.

YD and atorvastatin treatments significantly reduced MDA levels. Concentrations of glutathione (GHS), an antioxidant biomarker, were lower in atherosclerotic rats compared with normal-controlled group, and YD significantly increased GSH levels compared with AS group. Compared with normal-control group, antioxidant enzymes superoxide dismutase (SOD) and selenium-dependent glutathione peroxidase (GSH-px) were significantly less active (P < 0.05) in atherosclerotic rats. Moreover, YD treatment yielded significantly higher activity of antioxidant enzymes.

YD regulates the secretion of pro-inflammatory cytokines and vascular endothelial function markers in vivo

AS is a chronic inflammatory disease that results in increased production of inflammatory mediators^18,19,20. Our results show that YD and atorvastatin treatment significantly decreased plasma levels of C-reactive protein, tumor necrosis factor alpha (TNF-α), and IL-1β (Table 3), compared with AS group.

Endothelial function is chronically disturbed in atherosclerosis, and endothelial cell dysfunction is a critical step in the progression of the disease^21,22. Our data (Table 4) show significantly decreased concentrations of endothelium-derived relaxing factor (NO) in atherosclerotic rats. Importantly, YD and atorvastatin inhibited such decreases. On the other hand, YD and atorvastatin treatment mediated vasoconstrictor function markers (i.e., endothelin and TXB2).

Pretreatment with YD attenuated oxidative stress and increased cell viability in HUVECs

To confirm that YD protects against ox-LDL-induced injury, we conducted an MTT assay to check cell viability between groups. Compared with control Group, the ox-LDL-exposed group exhibited significantly decreased cell viability (100% vs. 49%, respectively). We also checked cell viability by pretreating with YD 1.5–100 mg/L. When exposed to ox-LDL-induced oxidative injury, cell viability in groups pretreated with YD (1.5 and 3.0 mg/L) differed little from control group (no pretreated group). Compared with control group, pretreatment with YD (6.25–100 mg/L) significantly increased cell viability (54% ~ 96% vs. 49%) after ox-LDL-induced injury. The maximum non-toxicity concentration was 100 mg/L.

Prediction analysis of pharmacological mechanism based on network pharmacology

YD mainly comprises ginkgo leaves, salvia miltiorrhiza, herba erigeromtis, gynostemma pentaphyllum, hawthorn, allium sativum, panax notoginseng and borneol. The active chemical components of YD were collected and extracted from the article database (Chinese Pharmacopoeia 2010 edition, web of science, http://www.wanfangdata.com.cn/, http://www.cnki.net/, www.ncbi.nlm.nih.gov/pubmed/). After reviewing the database, we tried to extract a higher relative content of various medicinal ingredients, taking into account the relevant activity reports. The 173 compounds we collected encompassed the main components of YD.

We put the 173 chemical compounds input TCMSP (http://sm.nwsuaf.edu.cn/lsp/index.php) database to simulate the drug-likeness results by ADME Sico TCMSP database model. Given the integral role of multi-components in TCM, we set at bioavailability (OB) >10%, drug-like >0.04 as the threshold for further extraction and optimization of the medicinal ingredients. To build a compound-medicine network (C-M network), we extracted 63 compounds covering the main drug-likeness component of the YD and put them into the Cytoscape 2.8 software.

Next, we used Target-Bank, DrugBank, BindingDB, and PDTD to validate 123 CVD targets. We put the 63 chemical compounds into Cytoscape 2.8 software to build a compound-target network (C-T network, Fig. 4).

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Figure 4

The Compound-Target network.

To explain target participation in cardiovascular activity, we used Cytoscape 2.8 to build a network between target and function (T-F network, Fig. 5). Multiple targets indicated an integral role of YD in CVD and cerebrovascular disease, sharing synergy targets of the different compounds.

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Figure 5

The Target-Function network.

The main 110 targets included the following functions: anti-inflammation, coagulation and thrombosis, anti-apoptotic, insulin mediation, glucose mediation, cardiac energy metabolism manipulation, ion channel mediation, vascular protection, blood lipids regulation, endothelium protection, and anti-oxidation. To verify this predication, the next experiment was performed accordingly.

Animal experiments for verification

Sprague Dawley rats (150–200 g) obtained from Beijing Vital River Laboratories Co., Ltd. (Beijing, China) were housed in a controlled environment (temperature 23° ± 2 °C, relative humidity 50% ± 10%, 12 h light/dark cycle) and allowed free access to standard diet. All animals were acclimatized for 5 days prior to the initiation of our experiment. All experimental procedures were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals. The Ethics Committee for Animal Experiments (Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, China) approved our experimental protocol.

We used an AS rat model to evaluate the attenuation of AS and reveal the possible mechanism of YD. Following a procedure established in related reports^31,32,33,34, we induced AS by injecting the rats with vitamin D3 and ovalbumin and feeding them a high-fat diet (HFD). normal control group rats consumed a regular diet for 21 weeks, whereas rats in AS groups consumed a HFD (1% cholesterol, 0.2% pig bile salts, 10% lard, 10% egg yolk powder, 78.8% basal diet). Initially, rats in the AS model received an intraperitoneal injection of vitamin D3 (600 K IU/kg), followed by a hypodermically injected antigen emulsion (ovalbumin [3 mg/kg] and complete Freund’s adjuvant) to their backs on day 2. After 3 weeks, atherosclerotic rats received weekly intraperitoneal booster injections of ovalbumin (2.5 mg/kg) for 3 consecutive weeks, whereas controls consumed a normal diet and received isovolumic saline injections. At week 9, we randomly divided atherosclerotic rats into five groups for a 12-week treatment regimen, using normal rats as control group (con group). con group and atherosclerotic model controls (AS group) received an equal volume of vehicles. The other atherosclerotic rats received atorvastatin (anti-hyperlipidemics, Ator, 0.01 g/kg b.w., i.g [Ator Group]) or YD treatment (YD 2.0 g/kg b.w. i.g. [YD-2.0 group]); YD 1.0 g/kg BW i.g. [YD-1.0 group]; or YD 0.5 g/kg b.w. i.g. [YD-0.5 group]). Normal control rats (con group) consumed a normal diet throughout the experiment.

After 21 weeks, we deprived all animals of food (but not water) overnight, and then anesthetized them. Next, we drew blood samples from the abdominal aorta, and obtained serum and plasma via blood centrifugation (3,000 rpm for 10 min at 4° C). Prior to histopathologic examination and immunohistochemical analysis, we harvested and weighed the abdominal aorta.

Histology lesion analysis and immunohistochemical staining

After taking blood samples from the abdominal aorta, we quickly removed the aorta between the aortic valve cusps and the iliac bifurcation, storing them in 4% paraformaldehyde for histological studies. Next, we cut consecutive cross-sections (4 μm thick) of the aorta, and stained with hematoxylin-eosin (HE). Pathological changes were observed with an optical microscope, and intima-media thickness (IMT) was measured using Image-Pro Express 5.0 software.

Immunohistochemical staining were conducted using standard techniques, as described previously³⁵. Briefly, we inhibited endogenous peroxidase activity via incubation with 3% H₂O₂. After blocking the sections with 5% albumin from bovine serum and incubating them for 10 min, we added primary antibodies and incubated the sections for 60 min before preserving them overnight at 4° C. Following a PBS wash, we incubated the sections with a secondary antibody for 60 min at 37° C. Immunohistochemical staining was visualized using a DAB kit (Boster Biotechnology, LTD, Wuhan, China), according to the manufacturer’s instructions. Immunohistochemical staining (relative positive expression) was quantified using integral optic density

Biochemical analysis

We collected blood samples and measured serum levels of endothelium-derived relaxing factor (NO), cholesterol, triglycerides (TGs), LDL, and high-density lipoprotein (HDL) using chemical colorimetry assay kits (Nanjing Jiancheng Bioengineering Institute). Rat serum IL-1β, tumor necrosis factor alpha (TNF-α), endothelin, 6-keto-PGF1α, and thromboxane B₂ (TXB₂) concentrations were measured by radio-immunity assay, using commercial kits according to manufacturer instructions.

Measurement of the antioxidative activity in vivo

We used total superoxide dismutase (SOD, hydroxylamine method), glutathione peroxidase (GSH-PX, colorimetric method), lipid peroxidation malondialdehyde (MDA), and reduced GSH (spectrophotometric method) assay kits to determine anti-oxidative activity in serum. These commercially available assay kits were provided by Nanjing Jiancheng Bioengineering Institute.

Culture, proliferation and cytotoxity assay of human umbilical vein endothelial cells

For in vitro experiments, we used human umbilical vein endothelial cells (HUVECs) obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Peking Union Medical College/Chinese Academy of Medical Sciences (Beijing, China), and cultured them in a humidified atmosphere in Dulbecco’s Modified Eagle Medium (DMEM, Thermo US) with 20% fetal bovine serum (20% FBS) at 37° C under 5% CO₂. The 6th passage cells were used for experiments.

HUVECs (1 × 10⁵) were planted in a 96-well plate, then incubated for 24 h with YD (at the series concentration of 1.5, 3, 6.25, 12.5, 25, 50, 100, 200 mg/L) for protection. After 24 h pretreatment, the supernatant was discarded. Cells then were exposed to 100 mg/L ox-LDL for 24 h to cause oxidative injury, at 37 °C in a 5% CO₂ atmosphere^10,34,35. The viability of HUVEC cells was determined by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, sigma) assay to express the protection of YD. The culture medium was replaced with serum-free medium containing 0.5 g/L MTT tetrazolium salt and incubated at 37 °C for 4 hours^36,37. At the end of the incubation with MTT, the cells were scraped and solubilized into DMSO solution. The absorbance was measured at 570 nm by microplate reader (Thermo Scientific). The viability of the cells was expressed as the percentage of the absorbance measured in control cells.

Western blotting

Using lysis buffer (1%Triton X-100; 50 mmol/L Tris–HCl, pH 7.6; 150 mmol/L NaCl; and 1% protease inhibitor cocktail), we extracted tissue homogenates from the resected left common carotid artery. For western blot, proteins were separated by SDS–PAGE and transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated at room temperature for with the indicated antibodies, including anti-p65, anti-IkB, and anti-actin (1:1000 dilution). Next, we treated the membranes with horseradish peroxidase-conjugated secondary antibodies, diluted to 1:2000 (Santa Cruz Biotechnology)^38,39. The blots were developed using ECL western blotting reagents and quantified using optical densitometry. We quantified the bands according to the average ratios of integral optic density of anti-p65/β-actin and anti-IkB/β-actin.

The authors thank scientific editor Karen Williams (Kwills Editing Services, Weymouth, MA, USA) and Dr. Li Yong for providing professional English language editing of this paper. This work has been funded by China Ministry of Science and Technology (the National Science and Technology Projects No: 2012ZX09201201-003, 2013BAI11B01) and China Postdoctoral Science Foundation (No: 2014M552546XB).