Zhao Gao, Xin-Rui Xu, Qiu-Shuo Jin, Hao Sun, Ke-Han Sun, Man-Fang Yang, Qing-Yue Zhang, Yang Li, Li-Xia Lou, Ai-Ming Wu, Bo Nie,?

1. Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing 100700, China

2. Department of Pathology, Dongzhimen Hospital of Beijing University of Chinese Medicine, Beijing 100700, China

3. School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China

Keywords:Simiao Yongan Decoction Atherosclerosis Lipid metabolism pathway Ox-LDL

ABSTRACT

1. Introduction

Atherosclerosis (AS) is the pathological basis of cardiovascular and cerebrovascular diseases[1]. AS is caused by the interaction of lipid metabolism disorders, oxidative stress factors, inflammatory stimulation, endothelial cell dysfunction and other factors[2].Oxidized low density lipoprotein (ox-LDL) aggravates AS by affecting lipid metabolism, which is an independent risk factor for AS lesions in the pathological process of AS[3, 4].In macrophages(Mφ), excess fatty acid binding protein 4 (FABP4) expression can aggravate the phagocytosis of Mφ, while lack of FABP4 can promote the outflow of cholesterol from Mφ [5, 6]. Peroxisome proliferators-activated receptor γ (PPARγ) is a key receptor that mediates the flow of cholesterol and free fatty acids from intracellular to extracellular. Its main purpose is to prevent and treat AS by promoting the reverse transport pathway of cholesterol[7]. Matrix Metalloproteinases (MMPs) play an important role in the whole process of AS lesions, especially in the increase of the instability of AS plaques. Among them, MMP2 can reduce collagen fibers at AS plaque, make the fiber cap thinner and increase instability. Besides, it is also a key factor used to evaluate the instability of AS plaque[8].

Recent studies have shown that ox-LDL can increase plaque instability by inducing FABP4 production [5, 6, 9]. Meanwhile,FABP4 can inhibit the expression of PPARγ, leading to the increase of lipid synthesis, leading to the disorder of lipid metabolism and the development of AS [10, 11]. The preliminary experiment of the research team found that Simiao Yongan Decoction (SMYA) could inhibit a variety of inflammatory factors to reduce the inflammatory response of the intima of blood vessels, reduce the area of AS plaque, and reduce the instability of plaque. However, it was also found that SMYA decoction did not have the effect of lowering blood lipids [12, 13], and it is not clear whether SMYA decoction can exert the effect of anti-AS by reducing ox-LDL. In this study,we observed its efficacy in the stability of carotid artery plaque of AS and explore its mechanism by regulating ox-LDL, FABP4 and PPARγ lipid metabolism pathways.

2.1 Materials

2.1.1 Experimental animals

Male (aged 7 weeks) ApoE-/-mice was purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (No. SCXK(Beijing) 2016-0006), weighing (20±2) g. All the mice were kept in a barrier animal house of Dongzhimen Hospital of Beijing University of Chinese Medicine (No. SYXK (Beijing) 2015-0001) with a food and water freely available, 12h light/dark, and a constant temperature environment of 50℃. The high-fat diet was based on literature [14],and the diet composition consisted of 15% fat, 0.25% cholesterol and 84.75% basal feed, which was purchased from Beijing HFK Bioscience CO., Ltd. Science and Technology Co., Ltd. (NO. SCXK(Beijing) 2014-0008).

2.1.2 Preparation of experimental drugs

The SMYA Decoction consists of honeysuckle(No.15051904)90g, radix scrophulariae (No.150181431) 90g, angelica sinensis(No.150313061) 60g, and liquorice(No.150280731) 30g, provided by Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine. Pioglitazone hydrochloride tablet, alias: Cassiping,(NO.H20050500), purchased from Zhongmei Huadong Co.,Ltd. Atorvastatin calcium tablet, alias: Lipitor, (No.H20051408),purchased from Pfizer Pharmaceutical Co., Ltd.

2.1.3 Reagents and key instruments

Mouse ox-LDL serum enzyme-linked immunosorbent assay(ELISA) Kit purchased from Elabscience Biotechnology Co.,Ltd. (No.E-EL-M0066C); PPARγ, FABP4, MMP2 and the first antibody of GAPDH were all purchased from Abcam Company in USA (NO.ab45036, ab 92501, ab37150 and ab9485, respectively).Goat anti-rabbit IgG/HRP antibody, BCA protein assay kit, 5%BSA and enhanced RIPA lysate were purchased from BOSTER Biological Technology Co., Ltd. (NO.BA1054, AR1189, AR0004 and AR0102, respectively). splink detection kits (biotin-streptavidin hrp detection systems (NO.SP-9001), DAB Color solution (No.ZLI-9018) and neutral balsam (NO.ZLI-9555) were purchased from Beijing ZSGB-BIO Co., Ltd. AU5800 Automatic biochemical analyzer (Beckman-Coulter Co., LTD.); BX60 optical microscope(Olympus Corporation, Japan); RM2135 Tissue slicer (Arcadia,Germany); EG1150 tissue embedding machine (Germany Arcadia);MK3 automatic enzyme marker (Thermo Company); LAS-4000 MINI biomolecular imager (Fuji Corporation of Japan).

2.2 Methods

2.2.1 Establishment of Atherosclerosis ModelAfter 1 week of adaptive feeding, ApoE-/-cervical AS mouse model was prepared by Perivascular Common Carotid artery (right) vessel Placement (PCCP) with reference to literature [15], the AS model was established by a high-fat diet and carotid cannulation surgery in the ApoE-/-mouse. The surgery was operated after a 2-week highfat feeding (containing15% fat and 0.25% cholesterol). Firstly, all ApoE-/-mice were fasted for 12 hours. After anesthesia, the right common carotid artery was exposed, and a silicone cannula(length:2.5mm, inner diameter: 0.3mm) was fixed around the carotid artery.Penicillin was injected intraperitoneally for 3 consecutive days after surgery to prevent infection. From the day of PCCP, the diet was changed from general diet to high fat diet for 10 weeks. The study followed the national guidelines for laboratory animal welfare and was approved by the Animal Ethics Committee of Dongzhimen Hospital of Beijing University of Chinese Medicine (NO. (2016) 06)

2.2.2 Grouping and treatment administrationThe atherosclerosis mice were randomly divided into four group-the model group, the SMYA group, Pioglitazone group and Atorvastatin group. The mice in SMYA, Pioglitazone and Atorvastatin group received a high-fat diet plus SMYA decoction(34.9g kg-1),Pioglitazone(2.28g kg-1) and Atorvastatin(2.57g kg-1), once a day(8:30 am) for consecutive 8 weeks, respectively. While the mice in the sham operation and model group received high-fat diet plus deionized water, which was consistent with the cycle of each administration group.

2.2.3 Biochemical indicators assayAfter an 8-week intervention of SMYA decoction, blood was taken by removing eyeball collected. ①TC, ②TG, ③HDL-C and④LDL-C of serum were detected by automatic biochemical analyzer[12].

2.2.4 Enzyme-linked immunosorbent assay (ELISA)According to the instructions, the serum ox-LDL level of the mice was measured according to the experimental procedures attached to the ELISA kit. The OD values of serum ox-LDL in each group were read at 450 nm wavelength with a microplate reader, and then analyzed statistically.

2.2.5 Hematoxylin-eosin (HE) staining

For a histopathology study, the carotid artery was fixed in 4%paraformaldehyde for 24h and was embedded with paraffin after gradient-alcohol dehydration, xylene vitrification, and waxdip.Sections that were 3 um thick were used in HE staining , and the immunochemistry study.For 1h in the baking machine at 60℃, dewaxing to water according to the sequence of xylene and descending alcohol, washing under flowing water, dyeing according to the sequence of hematoxylin dyeing solution, 1%hydrochloric acid ethanol differentiation, flushing cyanidation under flowing water, eosin dyeing. Dehydrated with alcohol and xylene, transparent, using neutral gum to stick the cover glass on the slide. 1-3 visual fields were randomly selected under a light microscope (×200) and images were collected. Image Pro Plus 6.0 image processing program was used to measure vascular parameters. Measurement indexes: vascular intima thickness (IT),media thickness (MT), internal elastic plate surrounding lumen area(LA), plaque area (PA), intima to media thickness ratio (IT/MT) and plaque vascular lumen area ratio (PA/LA), and the mean values of IT, PA, IT/MT and PA/LA were analyzed statistically [12]. Figure 1.

Figure 1 Carotid artery IT, PA, IT/MT, PA/LA measurement methods ( 200)

2.2.6 Immunohistochemistry experiment

Film with HE method. y. The process is described briefly as follows: (a) Dewaxing with xylene, gradient-alcohol hydration,and antigen retrieval with citrate solution in microwave stove; (b)inactivation of peroxidase with the 3% hydrogen peroxide and blocking with goat serum; (c) incubated over night at 4 C refrigerator with first antibody(FABP4 dilution 1:200, PPARγ dilution 1:150 and MMP2 dilution 1: 200) (d) incubated for 30 min at 37 C with second antibody; (e) DAB coloration, hematoxylin staining,conventional dehydration, xylene vitrification. and sealing with gelatin; and (f) images were captured with microscope. The staining results showed that the nucleus was blue, PPARγ protein was stained in the nucleus of endothelial cells, smooth muscle cells and foam cells, and cytoplasm was occasional. FABP4 and MMP2 were stained in the cytoplasm of endothelial cells, smooth muscle cells and foam cells. The expression was judged according to the degree of staining. Under the microscope, yellow or brown-yellow color of different degrees was positive, while negative color was only blue staining [16].

2.2.7 Western blot assayIt's all on the ice. RIPA was added according to tissue weight, and the extracted protein was semi-quantified. 25 μg protein sample;SDS-PAGE was used for electrophoresis and electric transfer. After blocking for 1h, the blots were incubated overnight at 4℃ with first antibody FABP4 dilute 1:1000, PPARγ dilute 1:1000, MMP2 dilute 1:1000 and GAPDH dilute 1: 1000. After washing, the membranes were probed with secondary anti-rabbit IgG (1:5000 dilution) for 1h at room temperature. The bands were detected by using an enhanced chemiluminescence solution and followed by exposure to X-ray film. Using Image J image processing software to measure the gray values of the target band and the internal reference, and the ratio(target band/internal reference) was statistically analyzed [17].

2.2.8 Statistical analysisAll data were expressed as the mean ± the standard error of the mean (SEM). Statistical analyses were performed using SPSS 23.0.The measurement data between groups were compared by one-way ANOVA analysis, following which the least significant difference(LSD) method was used for comparisons among groups. P ＜ 0.05 indicates statistical significance.

3. Results

3.1 Effects of SMYA Decoction on serum lipid content in AS mice

Four items of blood lipids showed that compared with the sham operation group, the HDL-C of the model group was significantly increased(P ＜ 0.05) , and the level of TC, TG and LDL-C were not statistically different (P＞0.05). Compared with model group,TC and LDL-C in SMYA group and Atorvastatin group were increased (P ＜ 0.05), HDL-C was decreased (P ＜ 0.05), TG had no statistically different; There were no significant changes in TC and TG in Pioglitazone group, but HDL-C was decreased (P ＜ 0.05) and LDL-C was increased (P ＜ 0.05). Table 1.

Table 1 Effect of SMYA Decoction on serum lipid content in AS mice(n=8,±s)

Table 1 Effect of SMYA Decoction on serum lipid content in AS mice(n=8,±s)

Note: Compared with the sham operation group, *P ＜ 0.05; Compared with the model group, #P ＜ 0.05

Group TC TG HDL-C LDL-C The sham operation group 28.96±1.43 1.50±0.45 0.60±0.10 10.35±4.68 Model group 29.74±1.31 1.57±0.43 1.17±0.19* 7.92±1.15 SMYA Decoction group 43.41±10.94# 1.65±0.64 0.46±0.16# 15.60±0.71#Pioglitazone group 30.46±1.51 1.87±0.45 0.57±0.10# 14.72±2.14#Atorvastatin group 37.62±7.27# 1.69±0.35 0.72±0.21# 13.00±3.97#F 8.825 0.706 24.087 9.129 P 0.000 0.593 0.000 0.000

3.2 Effect of SMYA Decoction on ox-LDL in serum of AS mice

Compared with the sham operation group, the ox-LDL of the model group was increased (P ＜ 0.05); Compared with the model group,ox-LDL was decreased in SMYA Decoction group, Pioglitazone group and Atorvastatin group (P ＜ 0.05). Table 2.

Table 2 Effect of SMYA Decoction on serum ox-LDL of AS mice(n=6,`±s)

Table 2 Effect of SMYA Decoction on serum ox-LDL of AS mice(n=6,`±s)

Note: Compared with the sham operation group, *P ＜ 0.05; Compared with the model group, #P ＜ 0.05

Group Ox-LDL The sham operation group 60.58±10.79 Model group 103.53±33.99*SMYA Decoction group 51.06±12.05#Pioglitazone group 52.55±13.16#Atorvastatin group 40.61±5.88#F 11.045 P 0.000

3.3 Effect of SMYA Decoction on the pathophysiology of carotid artery in ApoE-/-AS mice

Carotid pathology showed that the sham operation group was intact. Endothelial cells are smooth and neatly arranged, and the Vascular smooth muscle cells (VSMCs) of the middle membrane was no obvious proliferation migration observed. No lesions of AS were observed. Compared with the sham operation group, model group observed carotid intimal thickening obviously, subcutaneous gaps with foam cells within a large number of deposits, convex to the vascular cavity (P ＜ 0.05), and the VSMCs of the middle membrane was significantly thickened (P ＜ 0.05), the blood vessels obviously narrow (P ＜ 0.05), plaque area increases (P ＜ 0.05), a large number of plaques formed. Compared with the model group,SMYA Decoction group, Pioglitazone group and Atorvastatin group showed IT ecreased (P ＜ 0.05), smooth muscle arranged neatly, IT/MT decreased (P ＜ 0.05), the content of foam cells in carotid intima decreased, PA decreased significantly. Figure 2 and 3.

Figure 2. Effects of SMYA Decoction on the pathophysiology of carotid artery in ApoE-/-AS mice (HE staining, ×400)

Figure 3Effects ofSMYA Decoction on carotid artery IT, PA, PA/LA and IT/MT inApoE-/-ASmice(n=5,±s)

3.4 Effects of SMYA Decoction on protein expression of FABP4, PPARγ and MMP2 in carotid artery tissues of ApoE-/-AS mice

The results of immunohistochemical showed that the positive expression of FABP4 and MMP2 was significantly reduced, while PPARγwas increased in the shame operation group. Compared with the sham operation group, the positive expression of FABP4 and MMP2 in the model group was significantly increased, while PARγwas reduced. Compared with the model group, the positive expression of FABP4 and MMP2 in SMYA Decoction group,Pioglitazone group and Atorvastatin group was significantly reduced,while PPARγwas increased. Figure 4.

Figure 4 Effects of SMYA Decoction on the expression of FABP4,PPARγ and MMP2 protein in carotid artery of ApoE-/-AS mice(immunohistochemical staining, ×400)

3.5 Effects of SMYA Decoction on the expression levels of lipid metabolization-related proteins FABP4, PPARγ and plaque stabilization protein MMP2 in ApoE-/-AS mice

Western blot results showed that compared with the sham operation group, the expression of FABP4 and MMP2 in the model group was increased (P ＜ 0.05), and the expression of PPAR γ was decreased(P ＜ 0.05). Compared with the model group, the expression of FABP4 and MMP2 in SMYA Decoction group were decreased (P ＜0.05), and the expression of PPAR γ was increased (P ＜ 0.05); the expression of FABP4 and MMP2 in Pioglitazone and Atorvastatin groups were decreased (P ＜ 0.05), and the expression of PPAR γ was increased (P ＜ 0.05). Figure 5.

Figure 5 Effects of SMYA Decoction on FABP4, PPARγ and MMP2 levels of lipid metabolization-related proteins in ApoE-/-AS mice

4. Discussions

In the pathological process of AS, lipid metabolism disorders accompany the whole process of AS[18]. ox-LDL is a direct factor of abnormal lipid metabolism, with strong chemotaxis and cytotoxicity as its pathogenic characteristics, which can induce and accelerate its recognition and phagocytosis by macrophages, and is the direct cause of foam cell formation [19-21]. Chainika [22] confirmed that elevated serum ox-LDL could be regarded AS an independent risk factor for exacerbating AS disease. LDL-C undergoes oxidative modification and transforms into ox-LDL, which is an essential factor leading to the occurrence and development of AS. In this study, ApoE-/-mouse AS model was prepared by high-fat combined with PCCP, and SMYA Decoction was given AS drug intervention.It was found that SMYA Decoction could significantly reduce the serum ox-LDL level of mice with carotid artery plaque AS, reduce the plaque area of mice with AS without reducing TC and LDL-C,and the anti-AS effect was significant.

The measurement of vascular IT, PA, IT/MT and PA/LA is an important factor in predicting AS and cardiovascular and cerebrovascular diseases [12]. The carotid artery in the study of HE staining showed that the endothelial cells the model group was are thickened, and VSMCs proliferation, plaque area and vascular stenosis rate increased significantly, while after treatment with SMYA Decoction for 8 weeks can make IT, PA, IT/MT and PA/LA reduced, suggesting that SMYA Decoction intervention can make the endothelial cells thickness reduced, and inhibited VSMCs proliferation. It can effectively reduce the carotid plaque area and the rate of vascular stenosis, and increase the stability of plaque.

As a free fatty acid binding chaperone, FABP4 plays an important role in transport and targeting during lipid metabolism[17]. Studies have shown [23] that FABP4 plays a significant regulatory role in cholesterol metabolism, and the increase of ox-LDL content can induce the expression of FABP4 and cholesterol content increased in Mφ, and lead to the generation and accumulation of foam cells in AS process. The activation of PPARγ depends on ligand activation and belongs to the nuclear receptor superfamily, which participates in the regulation of lipid metabolism and can improve the disorder of lipid metabolism in mice and intervene in the pathological process of AS[24]. Studies have shown that activation of PPARγ antagonizes ox-LDL-mediated lipid metabolism disorders [25],reducing the expression of MMP2, and increasing the stability of plaque[26]. MMP2 is one of the proteolytic enzymes. In the process of AS, collagen fibers of AS plaques are cracked and the thickness of fibrous caps is reduced by degrading various collagen and basement membrane components, and the increase of MMP2 content is an important evaluation factor for the increase of instability of AS plaques[27, 28]. Immunohistochemical and WB results showed that SMYA Decoction could increase the expression of PPARγ and significantly reduce the expression of FABP4 and MMP2. Therefore,we speculated that SMYA Decoction could inhibit ox-LDL production, inhibit FABP4 expression, activate PPARγ, increase plaque stability, decrease the expression of MMP-2, and play an anti-AS role.

Studies have shown that statins can improve endothelial cell function and prevent AS through anti-inflammatory and other ways[29]. However, Zadelaar[30] found that ApoE-/-mice had no cholesterol-lowering effect due to lack of receptors for cholesterolassociated synthetic protein LDL. Zhang[31] reported that statins could increase cholesterol in ApoE-/-AS mice. In this study, it was found that although Atorvastatin group increased serum TC and LDL-C contents in ApoE-/-AS mice, however, the expression of FABP4 and MMP-2 reduced. Besides, it was also have the ability to increase plaque stability by inhibiting ox-LDL production. This is consistent with the experimental results reported above that statins have no cholesterol-lowering effect in ApoE-/-AS mice. Claesen[32] reported that statins interfere with AS through multidirectional effects independent of cholesterol reduction in ApoE deficient mice. Pioglitazone, a PPARγ agonist, can play an anti-AS role by regulating glucose and lipid metabolism, improving vascular endothelial function, inhibiting VSMCs proliferation and other ways[33]. In this study, Pioglitazone, a PPARγ agonist, was established as a positive drug, because previous studies have shown that SMYA Decoction can activate PPARγ expression in blood vessels and liver[14, 34]. This study further proves that SMYA Decoction can increase the expression of PPARγ in carotid artery, and play a role in the prevention and treatment of AS, and its anti-AS effect is consistent with Pioglitazone. The sham operation group and model group ApoE-/-mice were fed with high-fat diet, so the level of TC, LDL-C was increased, however, no lesions of AS plaque were observed from the HE staining, while the model group AS plaque was formed obviously. It is verified the truth that PCCP accelerating the formation of plaque, and the sham operation group only stripped the vessels without cannula no plaque formation was observed, which was significantly different from the model group.

In conclusion, SMYA Decoction antagonizes ox-LDL mediated vascular wall injury in ApoE-/-AS mice, increases PPARγ expression and inhibits FABP4 expression [23, 25]. At the same time,the expression of PPARγ increased can reduce ox-LDL induced vascular injury, reduce MMP2, increase plaque stability, and play a role in stabilizing AS plaques [35, 36]. The molecular mechanism of increasing plaque stability may be related to the inhibition of ox-LDL and its lipid metabolism pathway. Figure 6.

Figure 6 The mechanism of SMYA Decoction regulating ox-LDL lipid metabolism pathway to increase the stability of AS plaques in ApoE-/- mice

In this study, the mechanism of SMYA Decoction in regulating ox-LDL lipid metabolism to stabilize AS plaques was preliminarily discussed from the overall animal level, and further experimental verification will be carried out from the cellular level.

Conflicts of interest

All authors declare no conflict of interest

Author contributions Corresponding author Nie Bo designed, modeled and reviewed the experiment. Sun Hao and Wu Aiming provided technical support for pathological index detection and immunohistochemical experiment;Jin Qiushuo and Lou Lixia provided technical guidance for Western blot; Gao Zhao, Xu Xinrui, Sun Kehan, Yang Manfang, Zhang Qingyue, Li Yang were responsible for drug intervention, sampling,index detection and data analysis; Gao Zhao wrote the paper.